1: Oncogene. 2005 Oct 13;24(45):6765-72. 

Promoter CpG hypomethylation and transcription factor EGR1 hyperactivate
heparanase expression in bladder cancer.

Ogishima T, Shiina H, Breault JE, Terashima M, Honda S, Enokida H, Urakami S,
Tokizane T, Kawakami T, Ribeiro-Filho LA, Fujime M, Kane CJ, Carroll PR, Igawa
M, Dahiya R.

Department of Urology, Veterans Affairs Medical Center, San Francisco and
University of California, San Francisco, 4150 Clement Street, CA 94121, USA.

Heparanase plays a critical role in the degradation of extracellular matrix and
cell membrane and is frequently upregulated in malignant tumors. Transcription
factor, early growth response 1 (EGR1), is closely associated with inducible
transcription of the heparanase gene. We hypothesized that promoter CpG
hypomethylation with increased EGR1 expression could determine heparanase
expression during the pathogenesis of bladder cancer. Bladder cancer cell lines
(J82, T24 and transitional cell carcinoma) significantly restored heparanase
expression after 5-Aza-dC treatment. Transfection of EGR1 siRNA with T24 bladder
cancer cell line significantly downregulated heparanase expression compared to
the control siRNA transfection. In 54 bladder cancer and paired normal bladder
samples, heparanase expression was significantly higher in bladder cancer than
in normal bladder (P<0.01). We performed methylation-specific PCR targeting the
CpG sites within the core-binding consensus motifs of EGR1 (GGCG) and Sp1
(GGGCGG). Methylation prevalence was significantly higher in normal bladder than
in bladder cancer (P<0.05) and inversely correlated with heparanase expression
(P=0.055). In the total series of bladder cancer and normal bladder samples, the
combination of promoter CpG methylation and EGR1 expression regulated heparanase
expression in a stepwise manner, where heparanase expression was the lowest in
methylation-positive and EGR1-negative samples and the highest in
methylation-negative and EGR1-positive samples. To our knowledge, this is the
first study demonstrating that increased heparanase expression during the
pathogenesis of bladder cancer is due to promoter hypomethylation and
transcription factor EGR1.

PMID: 16007175 [PubMed - in process]


---------------------------------------------------------------


2: Allergy. 2005 Jun;60(6):760-5. 

Polymorphism of tandem repeat in promoter of 5-lipoxygenase in ASA-intolerant
asthma: a positive association with airway hyperresponsiveness.

Kim SH, Bae JS, Suh CH, Nahm DH, Holloway JW, Park HS.

Department of Allergy and Rheumatology, Ajou University School of Medicine,
Suwon, Korea.

BACKGROUND: 5-Lipooxygenase (ALOX5) and 5-lipoxygenase-activating protein
(ALOX5AP) are known as key enzymes in cysteinyl-leukotriene (cys-LT) production,
critical mediators in aspirin acetylsalicyclic acid (ASA)-intolerant asthma
(AIA). To date, studies of the promoter region of ALOX5 gene has revealed the
potential influence of a variable number of tandem repeats of a Sp1- and
Egr1-binding motif, on the transcription rate. METHODS: To understand the
pathological process that arises from cys-LT overproduction in AIA, we genotyped
ALOX5 Sp1 and ALOX5AP poly(A) repeat promoter polymorphism by fluorescent-based
capillary electrophoresis in the Korean population. RESULTS: No significant
differences in allele and genotype frequencies of the ALOX5 and ALOX5AP promoter
polymorphisms were observed between the three groups. However, there was a
strong association of the ALOX5 Sp1 repeat polymorphism with airway
hyperresponsiveness (AHR; PC20 methacholine); AIA patients carrying a mutant
allele (n > 5 or n < 5 repeats) showed increased AHR compared to AIA patients
with wild-type genotype (P=0.003). CONCLUSION: Although the alleles of the ALOX5
and ALOX5AP promoter cannot be considered as a prominent risk factor in the
development of AIA, the genetic variant of tandem repeat (GGGCGG; Sp1-binding
motif) in ALOX5 promoter is associated with the severity of airway
hyperresponsiveness in AIA patients.

PMID: 15876305 [PubMed - in process]


---------------------------------------------------------------


3: Biochem Biophys Res Commun. 2005 Apr 15;329(3):1094-101. 

Transcriptional regulation of the mouse interleukin-2 receptor beta chain gene
by Ets and Egr-1.

Ye SK, Kim TJ, Won SS, Yoon TJ, Park TK, Yoo YC, Kim YN, Lee HC, Ikuta K, Chung
MH, Lee KH.

Department of Pharmacology, Seoul National University College of Medicine, 28
Yongon-dong, Chongno-gu, Seoul 110-799, Republic of Korea.

To clarify the mechanisms and factors involved in the regulation of mouse
IL-2Rbeta gene expression, we isolated the 5'-flanking region of IL-2Rbeta gene
and investigated the promoter activity. Here we elucidated the positive
regulatory regions, the most potent of which are located between -50 to -30bp
and -164 to -135bp. These regions contain a potentially functional Ets and
Egr-1-binding sites whose mutations abrogate promoter activity. Data from
electrophoretic mobility shift assay indicate that Ets and Egr-1, but not Sp1,
bind to the positive regulatory regions, -50 to -30bp and -164 to -135bp,
respectively. Furthermore, recruitment of Ets and Egr-1 at endogenous IL-2Rbeta
promoter segments in an IL-2-dependent F7 cells was verified by the chromatin
immunoprecipitation assay. This study for the first time delineates the
molecular mechanisms underlying regulation of mouse IL-2Rbeta gene transcription
by Ets family proteins, partially with Egr-1, and thereby further elucidates the
molecular basis of lymphocyte activation and differentiation.

PMID: 15752766 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


4: Mol Cell Endocrinol. 2005 Mar 31;232(1-2):9-19. 

Prolactin-induced expression of vascular endothelial growth factor via Egr-1.

Goldhar AS, Vonderhaar BK, Trott JF, Hovey RC.

Mammary Biology and Tumorigenesis Laboratory, National Cancer Institute,
National Institutes of Health, Bethesda, MD 20892-1402, USA.

Angiogenesis is a dynamic process regulated by both local and systemic factors.
Among these is vascular endothelial growth factor (VEGF), a potent effector of
angiogenesis and vascular permeability. Previously we showed that VEGF is
temporally and spatially regulated in the mouse mammary gland during development
and lactation. Given the functions of prolactin (PRL) during these stages and
the supporting role of the vasculature, we investigated the regulation of VEGF
by PRL. Treatment of HC11 mouse mammary epithelial and Nb2 rat lymphoma cells
with PRL induced VEGF expression. Deletion and mutation analysis identified a
GC-rich region in the proximal region of the VEGF promoter that constitutively
bound Sp1 and PRL-induced Egr-1. These sites conferred PRL-responsiveness
leading to increased VEGF transcription. The induction of VEGF by PRL was PRL
receptor-, Jak2- and MAP kinase kinase-dependent. Our results indicate that PRL
induces VEGF expression through Egr-1, and implicates VEGF as an intermediary of
PRL-regulated angiogenesis.

PMID: 15737464 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


5: Clin Cancer Res. 2005 Feb 1;11(3):1028-36. 

Increased heparanase expression is caused by promoter hypomethylation and
up-regulation of transcriptional factor early growth response-1 in human
prostate cancer.

Ogishima T, Shiina H, Breault JE, Tabatabai L, Bassett WW, Enokida H, Li LC,
Kawakami T, Urakami S, Ribeiro-Filho LA, Terashima M, Fujime M, Igawa M, Dahiya
R.

Department of Urology, University of California-San Francisco and Veterans
Affairs Medical Center, 4150 Clement Street, San Francisco, CA 94121, USA.

PURPOSE: Heparanase degrades heparan sulfate and has been implicated in tumor
invasion and metastasis. The transcription factor, early growth response 1
(EGR1), is associated with the inducible transcription of the heparanase gene.
We hypothesize that CpG hypomethylation in the heparanase promoter coupled with
up-regulation of EGR1 levels may induce heparanase expression in human prostate
cancer. EXPERIMENTAL DESIGN: Cultured prostate cancer cell lines (Du145, DuPro,
LNCaP, and PC-3) with and without 5'-aza-2-deoxycytidine treatment, 177 prostate
cancer samples, and 69 benign prostatic hyperplasia (BPH) samples were used. The
frequency and level of heparanase promoter methylation were analyzed by
methylation-specific primers which covered the core binding motif of EGR1 (GGCG)
or SP1 (GGGCGG) or both. RESULTS: In cultured Du145, DuPro, LNCaP, and PC-3 cell
lines, mRNA transcripts of heparanase were significantly increased after
5'-aza-2-deoxycytidine treatment, suggesting that promoter methylation was
involved in the regulation of heparanase mRNA transcript. Significantly higher
methylation was found in BPH samples than in prostate cancer samples (P <
0.0001), whereas mRNA transcripts of the heparanase gene were inversely lower in
BPH samples than in prostate cancer samples (P < 0.01). EGR1 expression in
prostate cancer tissues was significantly higher than in BPH tissues (P < 0.001)
and correlated with heparanase expression (P < 0.0001). Moreover, multiple
regression analysis revealed that up-regulation of EGR1 contributed
significantly more to heparanase expression than did promoter CpG
hypomethylation in prostate cancer samples (P < 0.0001). CONCLUSIONS: To our
knowledge this is the first comprehensive study demonstrating that increased
heparanase expression in prostate cancer tissues is due to promoter
hypomethylation and up-regulation of transcription factor EGR1.

PMID: 15709168 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


6: J Mol Biol. 2005 Feb 18;346(2):411-22. Epub 2004 Dec 22. 

Transcriptional regulator CTCF controls human interleukin 1 receptor-associated
kinase 2 promoter.

Kuzmin I, Geil L, Gibson L, Cavinato T, Loukinov D, Lobanenkov V, Lerman MI.

Basic Research Program, SAIC-Frederick, Inc., Frederick, MD 21702, USA.

Immune responses to invading pathogens are mediated largely through a family of
transmembrane Toll-like receptors and modulated by a number of downstream
effectors. In particular, a family of four interleukin 1 receptor-associated
kinases (IRAK) regulates responsiveness to bacterial endotoxins. Pharmacological
targeting of particular IRAK components may be beneficial for treatment of
bacterial infections. Here, we studied transcriptional regulation of the human
IRAK2 gene. Analysis of the IRAK2 promoter region reveals putative binding sites
for several transcriptional factors, including ZIP (EGR1 and SP1), CTCF and
AP-2beta. Deletion of the ZIP or AP-2 sites did not significantly affect IRAK2
promoter activity in naive and endotoxin-treated mononuclear cells, in dormant
and activated Jurkat T-cells, in lung and kidney cells. In contrast, we found
that CTCF plays a major role in IRAK2 transcription. An electrophoretic mobility
shift assay of the DNA fragments containing the IRAK2 CpG island, revealed a
single high-affinity binding site for the transcriptional regulator and a
chromatin insulator protein, CTCF. This assay revealed a CTCF-binding site
within the mouse Irak2 promoter. The presence of the CTCF protein in human IRAK2
promoter was confirmed by chromatin immunoprecipitation assay. Specific residues
that interacted with the CTCF protein, were identified by methylation
interference assay. In all cell lines analyzed, including cells of lung, renal,
monocytic and T-cell origin, the IRAK2 luciferase reporter construct, containing
an intact CTCF-binding site, showed strong promoter activity. However, IRAK2
promoter activity was decreased dramatically for the constructs with a mutated
CTCF-binding site.

PMID: 15670593 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


7: J Appl Physiol. 2004 Dec;97(6):2207-13. Epub 2004 Aug 13. 

Regulation of Egr-1, SRF, and Sp1 mRNA expression in contracting skeletal muscle
cells.

Irrcher I, Hood DA.

Dept. of Kinesiology and Health Science, York University, Toronto, Ontario,
Canada M3J 1P3.

The early cellular signals associated with contractile activity initiate the
activation and induction of transcription factors that regulate changes in
skeletal muscle phenotype. The transcription factors Egr-1, Sp1, and serum
response factor (SRF) are potentially important mediators of mitochondrial
biogenesis based on the prevalence of binding sites for them in the promoter
regions of genes encoding mitochondrial proteins, including PGC-1 alpha, the
important regulator of mitochondrial biogenesis. Thus, to further define a role
for transcription factors at the onset of contractile activity, we examined the
time-dependent alterations in Egr-1, Sp1, and SRF mRNA and the levels in
electrically stimulated mouse C(2)C(12) skeletal muscle cells. Early transient
increases in Egr-1 mRNA levels within 30 min (P < 0.05) of contractile activity
led to threefold increases (P < 0.05) in Egr-1 protein by 60 min. The increase
in Egr-1 mRNA was not because of increased stability, as Egr-1 mRNA half-life
after 30 min of stimulation showed only a 58% decline. Stimulation of muscle
cells had no effect on Sp1 mRNA but led to progressive increases (P < 0.05) in
SRF mRNA by 30 and 60 min. This was not matched by increases in SRF protein but
occurred coincident with increases (P < 0.05) in SRF-serum response element DNA
binding at 30 and 60 min as a result of SRF phosphorylation on serine-103. To
assess the importance of the recovery period, 12 h of continuous contractile
activity was compared with four successive 3-h bouts, with an intervening 21-h
recovery period after each bout. Continuous contractile activity led to a
twofold increase (P < 0.05) in Egr-1 mRNA, no change in SRF mRNA, and a 43%
decrease in Sp1 mRNA expression. The recovery period prevented the decline in
Sp1 mRNA, produced a decrease in Egr-1 mRNA, and had no effect on SRF mRNA. Thus
continuous and intermittent contractile activity evoked different specific
transcription factor expression patterns, which may ultimately contribute to
divergent qualitative, or temporal patterns of, phenotypic adaptation in muscle.

PMID: 15310743 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


8: J Biol Chem. 2004 Sep 24;279(39):40289-95. Epub 2004 Jul 9. 

Fibroblast growth factor-2 induction of platelet-derived growth factor-C chain
transcription in vascular smooth muscle cells is ERK-dependent but not
JNK-dependent and mediated by Egr-1.

Midgley VC, Khachigian LM.

Centre for Vascular Research, The University of New South Wales, Department of
Haematology, The Prince of Wales Hospital, Sydney, New South Wales 2052,
Australia.

Platelet-derived growth factors (PDGFs) play an integral role in normal tissue
growth and maintenance as well as many human pathological states including
atherosclerosis, fibrosis, and tumorigenesis. The PDGF family of ligands is
comprised of A, B, C, and D chains. Here, we provide the first functional
characterization of the PDGF-C promoter. We examined 797 bp of the human PDGF-C
promoter and identified several putative recognition elements for Sp1, Ets
Egr-1, and Smad. The proximal region of the PDGF-C promoter bears a remarkable
resemblance to a comparable region of the PDGF-A promoter (1). Binding and
transient transfection analysis in primary vascular smooth muscle cells revealed
that PDGF-C, like PDGF-A, is under the transcriptional control of the zinc
finger nuclear protein Egr-1 (early growth response-1). Electrophoretic mobility
shift analysis using both smooth muscle cell nuclear extracts and recombinant
protein revealed that Egr-1 and Sp1 bind this region of the PDGF-C promoter
(Oligo C, -35 to -1). Egr-1 competes with Sp1 for overlapping binding sites even
when the former is at a stoichiometric disadvantage. Reverse transcriptase PCR
and supershift analysis demonstrate that fibroblast growth factor-2 (FGF-2)
stimulates both Egr-1 and PDGF-C mRNA expression in a time-dependent and
transient manner and that FGF-2-inducible Egr-1 binds the proximal PDGF-C
promoter. FGF-2-inducible PDGF-C expression was completely abrogated using
catalytic DNA (DNAzymes) targeting Egr-1 but not by its scrambled counterpart.
Moreover, using pharmacological inhibitors we demonstrate the critical role of
ERK but not JNK in FGF-2-inducible PDGF-C expression. These findings thus
demonstrate that PDGF-C transcription, activated by FGF-2, is mediated by Egr-1
and its upstream kinase ERK. Copyright 2004 American Society for Biochemistry
and Molecular Biology, Inc.

PMID: 15247255 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


9: Gene. 2004 Apr 14;330:133-42. 

Regulation of KLF5 involves the Sp1 transcription factor in human epithelial
cells.

Chen C, Zhou Y, Zhou Z, Sun X, Otto KB, Uht RM, Dong JT.

Department of Oncology and Hematology, Winship Cancer Institute, Emory
University School of Medicine, 1365-C Clifton Road, Atlanta, GA 30322, USA.

Human Kruppel-like factor 5 (hKLF5) is a transcription factor with a potential
tumor suppressor function in prostate and breast cancers. In the majority of
cancer samples examined, a significant loss of expression for KLF5 has been
detected. Whereas hemizygous deletion appears to be responsible for KLF5's
reduced expression in about half of the cases, the mechanism for reduction is
unknown in the remaining half; gene promoter methylation does not appear to be
involved. In this report, we studied the regulation of KLF5 and cloned and
functionally characterized a 1944-bp fragment of the 5'-flanking region of the
hKLF5 gene. Several mitogens as well as global demethylation induced the
expression of KLF5, implicating multiple factors in the regulation of KLF5.
KLF5's promoter lacks a TATA box and has a GC-rich region. Deletion mapping in
combination with promoter activity assay showed that multiple cis-elements are
involved in the transcriptional regulation of KLF5, some of which may play a
repressor role whereas some others play an enhancer role. The Sp1 site between
position -239 and -219 is essential for a basal promoter activity. Deletion or
mutations of this Sp1 site significantly reduced promoter activity in several
epithelial cell lines. Electrophoretic mobility shift assays (EMSAs) revealed
that the Sp1 site binds Sp1 protein in nucleic extracts of different cell lines.
In addition, overexpression of Sp1 protein transactivates KLF5 promoter
activity. These findings suggest that Sp1 is a key transcription factor in
KLF5's dynamic transcriptional regulation.

PMID: 15087132 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


10: Am J Physiol Cell Physiol. 2004 May;286(5):C1053-61. Epub 2004 Jan 7. 

Calcium-regulated changes in mitochondrial phenotype in skeletal muscle cells.

Freyssenet D, Irrcher I, Connor MK, Di Carlo M, Hood DA.

Dept. of Biology, York University, Toronto, Ontario, M3J 1P3 Canada.

Cytochrome c expression and mitochondrial biogenesis can be invoked by elevated
intracellular Ca(2+) in muscle cells. To characterize the potential role of
Ca(2+) as a messenger involved in mitochondrial biogenesis in muscle, we
determined the effects of the Ca(2+) ionophore A-23187 on the expression of
nuclear- and mitochondrially encoded genes. Treatment of myotubes with 1 microM
A-23187 for 48-96 h increased nuclear-encoded beta-subunit F(1)ATPase and malate
dehydrogenase (MDH) mRNA levels by 50-100% (P < 0.05) but decreased mRNA levels
of glutamate dehydrogenase (GDH) by 19% (P < 0.05). mRNA levels of the
cytochrome c oxidase (COX) nuclear-encoded subunits IV, Vb, and VIc were
unchanged, whereas the mitochondrially encoded subunits COX II and COX III were
decreased by 30 and 70%, respectively (P < 0.05). This was paralleled by a 20%
decrease (P < 0.05) in COX activity. These data suggest that cytoplasmic Ca(2+)
differentially regulates the mRNA level of nuclear and mitochondrial genes. The
decline in COX II and III mRNA may be mediated by Tfam, because A-23187 modestly
reduced Tfam levels by 48 h. A-23187 induced time-dependent increases in Egr-1
mRNA, along with the activation of ERK1/2 and AMP-activated protein kinase. MEK
inhibition with PD-98059 attenuated the increase in Egr-1 mRNA. A-23187 also
increased Egr-1, serum response factor, and Sp1 protein expression,
transcription factors implicated in mitochondrial biogenesis. Egr-1
overexpression increased nuclear-encoded cytochrome c transcriptional activation
by 1.5-fold (P < 0.05) and reduced GDH mRNA by 37% (P < 0.05) but had no effect
on MDH or beta-subunit F(1)ATPase mRNA. These results indicate that changes in
intracellular Ca(2+) can modify mitochondrial phenotype, in part via the
involvement of Egr-1.

PMID: 15075204 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


11: Am J Physiol Lung Cell Mol Physiol. 2004 Apr;286(4):L817-25. 

Induction of early growth-response factor 1 by platelet-derived growth factor in
human airway smooth muscle.

Hjoberg J, Le L, Imrich A, Subramaniam V, Mathew SI, Vallone J, Haley KJ, Green
FH, Shore SA, Silverman ES.

Physiology Program, Dept. of Environmental Health, Harvard School of Public
Health, 665 Huntington Ave., Boston, MA 02115-6021, USA.

Platelet-derived growth factors (PDGF) may contribute to the activation and
growth of smooth muscle that is characteristic of airway remodeling in asthmatic
patients. Early growth response 1 (EGR-1) is a transcription factor that is
induced in several cell types by PDGF and may mediate some of the effects of
PDGF. We show that human airway smooth muscle cells in cell culture express
EGR-1 1 h after addition of PDGF. Analysis of the EGR-1 promoter indicates that
a serum response element located between 663 and 654 bp 5' to the ATG start site
is essential for this induction. Serum response factor, E26 transcription
factor-like protein 1, and serum protein 1 bind to this region. PDGF causes
phosphorylation of ERK1/2 and is temporally associated with E26 transcription
factor-like protein 1 phosphorylation. Finally, the specific ERK1/2 inhibitor
U-0126 abolishes PDGF-induced expression of EGR-1 in these cells. On the basis
of these data, we speculated that EGR-1 would be increased in airway smooth
muscle of asthmatic patients compared with nonasthmatic controls. Using
immunohistochemistry, we found that EGR-1 protein was expressed in airway smooth
muscle cells and epithelial cells of asthmatic patients and nonasthmatic
controls; however, there was no significant difference in the intensity of
staining between groups. EGR-1 was similarly expressed in the lungs of mice with
and without ovalbumin-induced airway inflammation; however, there was no
difference between groups by immunohistochemistry and quantitative PCR. Although
EGR-1 is induced by PDGF in human airway smooth muscle cells in cell culture,
the role of EGR-1 in airway remodeling and asthma remains to be established.

PMID: 15003938 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


12: Biochem J. 2004 Jun 15;380(Pt 3):735-47. 

Co-operative interactions between NFAT (nuclear factor of activated T cells) c1
and the zinc finger transcription factors Sp1/Sp3 and Egr-1 regulate MT1-MMP
(membrane type 1 matrix metalloproteinase) transcription by glomerular mesangial
cells.

Alfonso-Jaume MA, Mahimkar R, Lovett DH.

The Department of Medicine, San Francisco VAMC/University of California, 111J
Medical Service, 4150 Clement Street, San Francisco, CA 94121, USA.

The transition of normally quiescent glomerular MCs (mesangial cells) to a
highly proliferative phenotype with characteristics of myofibroblasts is a
process commonly observed in inflammatory diseases affecting the renal
glomerulus, the ultimate result of which is glomerulosclerosis. Generation of
proteolytically active MMP (matrix metalloproteinase)-2 by the
membrane-associated membrane type 1 (MT1)-MMP is responsible for the transition
of mesangial cells to the myofibroblast phenotype [Turck, Pollock, Lee, Marti
and Lovett (1996) J. Biol. Chem. 271, 15074-15083]. In the present study, we
show that the expression of MT1-MMP within the context of MCs is mediated by
three discrete cis -acting elements: a proximal non-canonical Sp1 site that
preferentially binds Sp1; an overlapping Sp1/Egr-1-binding site that
preferentially binds Egr-1; and a more distal binding site for the NFAT (nuclear
factor of activated T cells) that binds the NFAT c1 isoform present in MC
nuclear extracts. Transfection with an NFAT c1 expression plasmid, or activation
of calcineurin with a calcium ionophore, yielded major increases in NFAT c1
nuclear DNA-binding activity, MT1-MMP transcription and protein synthesis, which
were additive with the lower levels of transactivation provided by the proximal
Sp1 and the overlapping Sp1/Egr-1 sites. Specific binding of NFAT c1 to the
MT1-MMP promoter was confirmed by chromatin immunoprecipitation studies, while
MT1-MMP expression was suppressed by treatment with the calcineurin inhibitor,
cyclosporin A. These studies are the first demonstration that a specific NFAT
isoform enhances transcription of an MMP (MT1-MMP) that plays a major role in
the proteolytic events that are a dominant feature of acute glomerular
inflammation. Suppression of MT1-MMP by commonly used calcineurin inhibitors may
play a role in the development of renal fibrosis following renal
transplantation.

PMID: 14979875 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


13: J Biol Chem. 2004 Mar 5;279(10):8558-66. Epub 2003 Dec 11. 

The dominant role of Sp1 in regulating the cystathionine beta-synthase -1a and
-1b promoters facilitates potential tissue-specific regulation by Kruppel-like
factors.

Maclean KN, Kraus E, Kraus JP.

Department of Pediatrics, University of Colorado School of Medicine, Denver,
Colorado 80262, USA. ken.maclean@uchsc.edu

Cystathionine beta-synthase (CBS) catalyzes the condensation of serine with
homocysteine to form cystathionine and occupies a crucial regulatory position
between the methionine cycle and transsulfuration. The human cystathionine
beta-synthase gene promoters -1a and -1b are expressed in a limited number of
tissues and are coordinately regulated with proliferation through a
redox-sensitive mechanism. Site-directed mutagenesis, DNase I footprinting and
deletion analysis of 5276 bp of 5' proximal -1b flanking sequence revealed that
this region does not confer tissue-specific expression and that 210 bp of
proximal sequence is sufficient for maximal promoter activity. As little as 32
bp of the -1b proximal promoter region is capable of driving transcription in
HepG2 cells, and this activity is entirely dependent upon the presence of a
single overlapping Sp1/Egr1 binding site. Co-transfection studies in Drosophila
SL2 cells indicated that both promoters are transactivated by Sp1 and Sp3 but
only the -1b promoter is subject to a site-specific synergistic regulatory
interaction between Sp1 and Sp3. Sp1-deficient fibroblasts expressing both Sp3
and NF-Y were negative for CBS activity. Transfection of these cells with a
mammalian Sp1 expression construct induced high levels of CBS activity
indicating that Sp1 has a critical and indispensable role in the regulation of
cystathionine beta-synthase. Sp1 binding to both CBS promoters is sensitive to
proliferation status and is negatively regulated by Kruppel-like factors in
co-transfection experiments suggesting a possible mechanism for the tissue
specific regulation of cystathionine beta-synthase.

PMID: 14670973 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


14: Biochem Biophys Res Commun. 2003 Oct 3;309(4):910-6. 

Effect of cysteamine on redox-sensitive thiol-containing proteins in the
duodenal mucosa.

Khomenko T, Deng X, Jadus MR, Szabo S.

Pathology and Laboratory Medicine Service, Diagnostic and Molecular Medicine
Health Care Group, VA Medical Center, Long Beach, CA 90822, USA.

Recent studies from our laboratory demonstrated that Egr-1 is upregulated in the
rat duodenal mucosa during cysteamine-induced duodenal ulceration and that
antisense egr-1 oligonucleotide aggravates the duodenal ulcers. This study was
aimed to determine the effects of cysteamine on redox-sensitive Egr-1
transcriptional activity and on other thiol-containing proteins such as redox
factor-1 (Ref-1) and thioredoxin (Trx). Here we demonstrate for the first time
that cysteamine increases the expression and nuclear translocation of Egr-1,
Ref-1, and Trx, and activates binding of Egr-1 to DNA. Moreover, we also show
that Egr-1 forms a complex with other redox-sensitive transcription factors
(e.g., AP-1, AP-2, NFATc, Sp1, PAX-5, MTF-1, c-Myb, and CREB) in rat duodenal
mucosa and that cysteamine enhances the formation of these complexes. The
antioxidant ebselen markedly elevated the nuclear Ref-1 expression and Egr-1/DNA
binding, and decreased the ulcerogenic effect of cysteamine as did catalase.
Thus, redox-sensitive signaling systems seem to play an important role in
cysteamine-induced duodenal ulceration.

PMID: 13679060 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


15: J Neurochem. 2003 May;85(3):816-29. 

Protein kinase A and protein kinase C signaling pathway interaction in
phenylethanolamine N-methyltransferase gene regulation.

Tai TC, Wong DL.

Department of Psychiatry, Harvard Medical School, Laboratory of Molecular and
Developmental Neurobiology, McLean Hospital, Belmont, Massachusetts 02478, USA.

The protein kinase A (PKA) and protein kinase C (PKC) signaling pathways appear
to interact in regulating phenylethanolamine N-methyltransferase (PNMT)
promoter-driven gene transcription in PC12 cells. Forskolin treatment of cells
transfected with the rat PNMT promoter-luciferase reporter gene construct
pGL3RP893 increased promoter activity approximately two-fold whereas
phorbol-12-myristate-13 acetate (PMA) treatment had no effect. However,
simultaneous forskolin and PMA treatment synergistically activated the PNMT
promoter approximately four-fold, suggesting that PKC stimulation requires prior
induction of the PKA pathway. Consistent with this possibility the adenylate
cyclase inhibitor MDL12,330A, and the PKA inhibitor H-89 prevented PNMT promoter
stimulation by the combination of forskolin and PMA. PKA and PKC regulation
seems to be mediated in part by Egr-1 and Sp1 through their consensus elements
in the PNMT promoter. Forskolin and PMA treatment of PC12 cells increased Egr-1
protein and phosphorylated Egr-1/DNA-binding complex formation to the same
extent but only increased phosphorylated Sp1/DNA binding complex formation
without altering Sp1 protein levels. Mutation of the - 165 bp Egr-1 and - 48 bp
Sp1 sites, respectively, attenuated and abolished combined forskolin and
PMA-mediated promoter activation. PNMT promoter analysis further showed that
synergistic stimulation by PKA and PKC involves DNA sequences between - 442 and
- 392 bp, and potentially a GCM binding element lying within this region.

PMID: 12694408 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


16: J Mol Biol. 2003 Apr 18;328(1):9-32. 

Assessment by molecular dynamics simulations of the structural determinants of
DNA-binding specificity for transcription factor Sp1.

Marco E, Garcia-Nieto R, Gago F.

Departamento de Farmacologia, Universidad de Alcala Alcala de Henares, E-28871,
Madrid, Spain.

The DNA-binding domain (DBD) of the ubiquituous transcription factor Sp1
consists of three consecutive zinc fingers that recognize a number of nucleotide
sequences different from, but related to and sometimes overlapping, those
recognized by the structurally better characterized early growth response
protein 1 (EGR1, also known as Zif268, Krox-24, and NGFI-A). The accepted
consensus binding sequence for Sp1 is usually defined by the asymmetric
hexanucleotide core GGGCGG but this sequence does not include, among others, the
GAG (=CTC) repeat that constitutes a high-affinity site for Sp1 binding to the
wt1 promoter. Since no 3D structure of the whole DBD of Sp1 is available, either
alone or in complex with DNA, a homology-based model was built and its
interaction with two DNA 14-mers was studied using nanosecond molecular dynamics
simulations in the presence of explicit water molecules. These oligonucleotides
represent Sp1 target sites that are present in the promoters of the mdr1 and wt1
genes. For comparative purposes and validation of the protocol, the complex
between the DBD of EGR1 and its DNA target site within the proximal mdr1
promoter was simulated under the same conditions. Some water molecules were seen
to play an important role in recognition and stabilization of the protein-DNA
complexes. Our results, which are supported by the available experimental
evidence, suggest that the accuracy in the prediction of putative Sp1-binding
sites can be improved by interpreting a set of rules, which are a blend of both
stringency and tolerance, for the juxtaposed triplet subsites to which each zinc
finger binds. Our approach can be extrapolated to WT1 and other related natural
or artificial zinc-finger-containing DNA-binding proteins and may aid in the
assignment of particular DNA stretches as allowed or disallowed-binding sites.
Copyright 2003 Elsevier Science Ltd.

PMID: 12683994 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


17: Brain Res Mol Brain Res. 2003 Apr 10;112(1-2):61-9. 

Interactions between Egr1 and AP1 factors in regulation of tyrosine hydroxylase
transcription.

Nakashima A, Ota A, Sabban EL.

Department of Biochemistry and Molecular Biology, New York Medical College,
Valhalla, NY 10595, USA.

Several treatments which regulate tyrosine hydroxylase (TH) transcription, such
as stress in vivo, or 12-O-tetradecanoylphorbol-13-acetate (TPA) in cell
culture, induce both Egr1 and AP1 factors. Previously, we identified a
functional Egr1 motif overlapping with Sp1 site in the rat TH promoter. Its
response to Egr1 also required the presence of an AP1/Ebox motif. Here, we
further examined the cross-talk between these sites. Insertion of 10- or 20-bp
between the Sp1/Egr1 and AP1/Ebox elements, reduced the ability of Egr1 to
upregulate luciferase reporter activity controlled by the proximal 272
nucleotides of the rat TH promoter in PC12 cells. Electrophoretic mobility shift
assays with nuclear extracts from TPA treated cells were used to identify the
composition of the factors which bound the AP1/Ebox motif and whether there is
competition with factors which bind the Sp1/Egr1 motif. The complexes formed
with labeled AP1/E box oligonucleotide were reduced or supershifted with
antisera to Fos family, c-Fos, Fra-2, and Jun D. Excess Sp1/Egr1 oligonucleotide
or anti Egr1 antisera did not compete. Fra-2 was a major component of the
complex after 2-4 h TPA. Transfection of PC12 cells with Fra-2 induced reporter
activity requiring the AP1, but not the Egr1 motif. However, when cotransfected
with Fra-2, Egr1 expression plasmids elicited lower induction of luciferase
activity than observed with Egr1 alone. Our results suggest that although it
does not compete for binding to the promoter, Egr1 can modulate the regulation
of TH transcription by AP1 factors.

PMID: 12670703 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


18: J Biol Chem. 2003 May 16;278(20):17688-700. Epub 2003 Mar 11. 

Egr-1 mediates transcriptional repression of COL2A1 promoter activity by
interleukin-1beta.

Tan L, Peng H, Osaki M, Choy BK, Auron PE, Sandell LJ, Goldring MB.

Rheumatology Division, Beth Israel Deaconess Medical Center and New England
Baptist Bone & Joint Institute, Harvard Institutes of Medicine, Boston,
Massachusetts 02115, USA.

Following induction and activation of the early growth response (Egr)-1
transcription factor in human chondrocytes, interleukin-1beta (IL-1beta)
suppresses the expression of the type II collagen gene (COL2A1), associated with
induction of Egr-1 binding activity in nuclear extracts. The COL2A1 proximal
promoter contains overlapping binding sites for Egr-1 and Sp1 family members at
-119/-112 bp and -81/-74 bp. Mutations that block binding of Sp1 and Sp3 to
either site markedly reduce constitutive expression of the core promoter.
IL-1beta-induced Egr-1 binds strongly to the -119/-112 bp site, and mutations
that block Egr-1 binding prevent inhibition by IL-1beta. Cotransfection with
pCMV-Egr1 potentiates the inhibition of COL2A1 promoter activity by IL-1beta,
whereas overexpression of dominant-negative Egr-1 mutant, Wilm's tumor-1
(WT1)/Egr1, Sp1, or Sp3 reverses the inhibition by IL-1beta. Cotransfection of
pGL2-COL2/Gal4, in which we substituted the critical residue for Egr-1 binding
with a Gal4 binding domain and a pCMV-Gal4-Egr1 chimera permits an inhibitory
response to IL-1beta that is reversed by overexpression of Gal4-CBP. Our results
indicate that IL-1beta-induced activation of Egr-1 binding is required for
inhibition of COL2A1 proximal promoter activity and suggest that Egr-1 acts as a
repressor of a constitutively expressed collagen gene by preventing interactions
between Sp1 and the general transcriptional machinery.

PMID: 12637574 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


19: Am J Physiol Renal Physiol. 2003 Jun;284(6):F1216-25. Epub 2003 Feb 4. 

Suppression of HGF receptor gene expression by oxidative stress is mediated
through the interplay between Sp1 and Egr-1.

Zhang X, Liu Y.

Department of Pathology, University of Pittsburgh School of Medicine,
Pittsburgh, Pennsylvania 15261, USA.

Hepatocyte growth factor (HGF) receptor, the product of the c-met protooncogene,
is transcriptionally regulated by a wide variety of cytokines as well as
extracellular environmental cues. In this report, we demonstrate that c-met
expression was significantly suppressed by oxidative stress. Treatment of mouse
renal inner medullary collecting duct epithelial cells with 0.5 mM H(2)O(2)
inhibited c-met mRNA and protein expression, which was concomitant with
induction of Egr-1 transcription factor. Ectopic expression of Egr-1 in renal
epithelial cells markedly inhibited endogenous c-met expression in a
dose-dependent fashion, suggesting a causative effect of Egr-1 in mediating
c-met suppression. The cis-acting element responsible for H(2)O(2)-induced c-met
inhibition was localized at nucleotide position -223 to -68 of c-met promoter,
in which reside an imperfect Egr-1 and three Sp1-binding sites. Egr-1 markedly
suppressed c-met promoter activity but did not directly bind to its cis-acting
element in the c-met gene. Induction of Egr-1 by oxidative stress attenuated the
binding of Sp1 to its cognate sites, but it did not affect Sp1 abundance in
renal epithelial cells. Immunoprecipitation uncovered that Egr-1 physically
interacted with Sp1 by forming the Sp1/Egr-1 complex, which presumably resulted
in a decreased availability of unbound Sp1 as a transcriptional activator for
the c-met gene. Thus it appears that inhibition of c-met expression by oxidative
stress is mediated by the interplay between Sp1 and Egr-1 transcription factors.
Our findings reveal a novel transcriptional regulatory mechanism by which Egr-1
sequesters Sp1 as a transcriptional activator of c-met via physical interaction.

PMID: 12569082 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


20: Mol Endocrinol. 2003 Apr;17(4):520-33. Epub 2003 Jan 23. 

Egr-1 induction in rat granulosa cells by follicle-stimulating hormone and
luteinizing hormone: combinatorial regulation by transcription factors cyclic
adenosine 3',5'-monophosphate regulatory element binding protein, serum response
factor, sp1, and early growth response factor-1.

Russell DL, Doyle KM, Gonzales-Robayna I, Pipaon C, Richards JS.

Department of Molecular and Cellular Biology, Baylor College of Medicine, One
Baylor Plaza, Houston, Texas 77030, USA.

Early growth response factor (Egr-1) is an inducible zinc finger transcription
factor that binds specific GC-rich enhancer elements and impacts female
reproduction. These studies document for the first time that FSH rapidly induces
Egr-1 expression in granulosa cells of small growing follicles. This response is
transient but is reinitiated in preovulatory follicles exposed to the LH analog,
human chorionic gonadotropin. Immunohistochemical analysis also showed
gonadotropin induced Egr-1 in theca cells. The Egr-1 gene regulatory region
responsive to gonadotropin signaling was localized within -164 bp of the
transcription initiation site. Binding of Sp1/Sp3 to a proximal GC-box at
-64/-46 bp was enhanced by FSH in immature granulosa cells but reduced after
human chorionic gonadotropin stimulation of preovulatory follicles despite
constant protein expression. This dynamic regulation of Sp1 binding was
dependent on gonadotropin-regulated mechanisms that modulate Sp1/3-DNA binding
activity. Serum response factor was active in granulosa cells and bound a
consensus CArG-box/serum response element site, whereas two putative cAMP
response elements within the -164-bp region bound cAMP regulatory element (CRE)
binding protein (CREB) and a second cAMP-inducible protein immunologically
related to CREB. Transient transfection analyses using Egr-1 promoter-luciferase
constructs and site-specific mutations show that the serum response element,
GC-box, and CRE-131 are involved in gonadotropin regulation of Egr-1 expression
in granulosa cells. Specific kinase inhibitors of Erk or protein kinase A
antagonized this induction while exogenously expressed Egr-1 enhanced reporter
expression. These observations indicate that the Egr-1 gene is a target of both
FSH and LH action that may mediate molecular programs of proliferation and/or
differentiation during follicle growth, ovulation, and luteinization.

PMID: 12554779 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


21: Mol Cell Biol. 2003 Jan;23(2):526-33. 

Regulation of tumor necrosis factor alpha gene expression by mycobacteria
involves the assembly of a unique enhanceosome dependent on the coactivator
proteins CBP/p300.

Barthel R, Tsytsykova AV, Barczak AK, Tsai EY, Dascher CC, Brenner MB, Goldfeld
AE.

The Center for Blood Research. Department of Medicine, Harvard Medical School.
The Brigham and Women's Hospital, Boston, Massachusetts 02115, USA.

Tumor necrosis factor alpha (TNF-alpha) plays an important role in host
containment of infection by Mycobacterium tuberculosis, one of the leading
causes of death by an infectious agent globally. Using the pathogenic M.
tuberculosis strain H37Rv, we present evidence that upon stimulation of
monocytic cells by M. tuberculosis a unique TNF-alpha enhanceosome is formed,
and it is distinct from the TNF-alpha enhanceosome that forms in T cells
stimulated by antigen engagement or virus infection. A distinct set of
activators including ATF-2, c-jun, Ets, Sp1, Egr-1 and the coactivator proteins
CBP/p300 are recruited to the TNF-alpha promoter after stimulation with M.
tuberculosis. Furthermore, the formation of this enhanceosome is dependent on
inducer-specific helical phasing relationships between transcription factor
binding sites. We also show that the transcriptional activity of CBP/p300 is
potentiated by mycobacterial stimulation of monocytes. The identification of
TNF-alpha regulatory elements and coactivators involved in M.
tuberculosis-stimulated gene expression thus provides potential selective
molecular targets in the modulation of TNF-alpha gene expression in the setting
of mycobacterial infection.

PMID: 12509451 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


22: Graefes Arch Clin Exp Ophthalmol. 2002 Dec;240(12):1003-10. Epub 2002 Nov 6.


Functional role of Egr-1 mediating VEGF-induced tissue factor expression in the
retinal capillary endothelium.

Sassa Y, Hata Y, Murata T, Yamanaka I, Honda M, Hisatomi T, Fujisawa K, Sakamoto
T, Kubota T, Nakagawa K, Sueishi K, Ishibashi T.

Department of Ophthalmology, Graduate School of Medical Sciences, Kyushu
University, 3-1-1, Maidashi, Higashi-ku, Fukuoka, 812-8582 Japan.

PURPOSE: To investigate the causal relationship between VEGF and tissue factor
(TF) expression, and its intracellular signaling in the retinal capillary
endothelium both in vitro and in vivo. METHODS: TF mRNA and protein expression
in cultured bovine retinal capillary endothelial cells (BRECs) were detected by
RT-PCR and western blotting. The expression and subcellular localization of
Egr-1 were analyzed by immunocytochemistry and western blotting. Involvement of
p44/p42 MAPK pathway in this signaling was assessed using PD98059.
Electrophoretic mobility shift assay (EMSA) was performed using human TF
Egr-1/Sp-1 overlapping promoter region (-85 to -70). Decoy oligonucleotide was
transfected into BRECs to clarify the critical transcription factor mediating
VEGF-induced TF gene expression. To evaluate the importance of GC rich region in
VEGF-induced TF protein expression in rat retinas, Mithramycin was
intraperitoneally administered. RESULTS: VEGF stimulated TF mRNA and protein
expression in cultured BRECs, reaching maximal effect after 4 h and 10 h,
respectively. VEGF activated transcription factor Egr-1 within 60 min.
Inactivation of Egr-1 by PD98059 resulted in the prohibition of VEGF-induced TF
gene expression. EMSA revealed the increment of Egr-1 binding with TF promoter
region by displacing Sp1 after treatment with VEGF. Transfection of the
Egr-1/Sp-1 overlapping decoy into BRECs inhibited VEGF-dependent TF gene
expression. Mithramycin almost completely suppressed VEGF-induced TF protein
expression in retinal capillary system in vivo (80%, p<0.01). CONCLUSION:
Transcription factor Egr-1, which lies downstream of p44/p42 MAPK, critically
mediates VEGF-dependent TF expression in the retinal capillary endothelium.

PMID: 12483323 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


23: J Biol Chem. 2003 Jan 24;278(4):2571-80. Epub 2002 Nov 8. 

Regulation of expression of the phospholipid hydroperoxide/sperm nucleus
glutathione peroxidase gene. Tissue-specific expression pattern and
identification of functional cis- and trans-regulatory elements.

Borchert A, Savaskan NE, Kuhn H.

Institute of Biochemistry, Humboldt University Medical School Charite,
Monbijoustrasse 2, 10117 Berlin, Germany.

A sperm nucleus glutathione peroxidase (snGPx), which is closely related to the
phospholipid hydroperoxide glutathione peroxidase (phGPx), was recently
discovered in late spermatids. Both GPx isoforms originate from a joint ph/snGPx
gene, but their N-terminal peptides are encoded by alternative first exons. The
expression of the two enzymes is differentially regulated in various cells, but
little is known about the regulatory mechanisms. To explore the tissue-specific
regulation of expression of the two isoenzymes, we first investigated their
tissue distribution. Whereas phGPx is expressed at low levels in many organs,
snGPx was only detected in testis, kidney, and in the human embryonic kidney
cell line HEK293. Subcellular fractionation studies and immunoelectron
microscopy revealed a cytosolic localization. To explore the mechanistic reasons
for the differential expression pattern, we first tested the activity of the
putative phGPx and snGPx promoters. The 5'-flanking region of the joint ph/snGPx
gene exhibits strong promoter activity. In contrast, the putative snGPx
promoter, which comprises 334 bp of intronic sequences, lacks major promoter
activity. However, it strongly suppresses the activity of the ph/snGPx promoter.
These data suggest negative regulatory elements in the first intron of the
ph/snGPx gene, and DNase protection assays revealed the existence of several
protein-binding sites. The corresponding trans-regulatory proteins (SP1, ERG1,
GATA1, SREBP1, USF1, and CREBP1) were identified, and in vivo binding of EGR1
and SREBP1 was shown by chromatin immunoprecipitation. These data indicate for
the first time somatic expression of the snGPx and provide evidence for the
existence of intronic negative cis-regulatory elements in the ph/snGPx gene. Our
failure to detect an alternative snGPx promoter suggests that transcription of
the ph/snGPx gene may be regulated by a joint basic promoter. The decision,
which GPx isoform is expressed in a given cell, appears to be made by
alternative splicing of a joint primary transcript.

PMID: 12427732 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


24: J Neurochem. 2002 Nov;83(4):784-96. 

Activation of the rat dopamine D2 receptor promoter by mitogen-activated protein
kinase and Ca2+/calmodulin-dependent protein kinase II pathways.

Takeuchi Y, Miyamoto E, Fukunaga K.

Department of Pharmacology, Kumamoto University School of Medicine, Kumamoto,
Japan. yusuket@gpo.kumamoto-u.ac.jp

To investigate regulation of D2 receptor (D2R) gene expression by protein
kinases, we evaluated effects of constitutively active MAPK kinase kinase
(MEKK), Ca2+/calmodulin-dependent protein kinase (CaMK) II, CaMKIV and cyclic
AMP-dependent protein kinase (PKA) on D2R promoter activity using luciferase
reporter gene assays. A 1.5-kbp fragment containing the rat D2R promoter was
cloned upstream of the reporter and transfected into D2R-expressing NB2A cells
or nonexpressing NG108-15 and C6 glioma cells. MEKK and CaMKII, but not CaMKIV
and PKA, increased promoter activity 4.5- and 1.5-fold, respectively, in NB2A
cells. Inhibitory effects of a MEK inhibitor and lack of effect by dominant
negative (DN)-JNK1 or DN-p38MAPK revealed that ERK but not JNK and p38MAPK is
involved in MEKK-induced promoter activation. Deletion and mutation of the
promoter revealed that the MEKK-responsive region was Sp1 site B between
nucleotides -56 and -47. Overexpression of Sp1 suppressed promoter activity
without affecting MEKK-induced activation. Interestingly, overexpression of
Zif268 increased promoter activity through the region between nucleotides -56
and -36. Increased activity by Zif268 was additive with CaMKII-induced
activation but not with activation induced by MEKK. Co-transfection with CaMKII
stimulated nuclear translocation of Zif268. These results suggest that ERK and
CaMKII positively regulate the D2R promoter and that Zif268 is a potential
transcription factor for the CaMKII-dependent pathway.

PMID: 12421350 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


25: Gene Ther. 2002 Oct;9(20):1396-402. 

Optimizing radiation-responsive gene promoters for radiogenetic cancer therapy.

Scott SD, Joiner MC, Marples B.

Department of Experimental Radiation Oncology, Gray Cancer Institute, Northwood,
Middlesex, UK.

We have been developing synthetic gene promoters responsive to clinical doses of
ionizing radiation (IR) for use in suicide gene therapy vectors. The crucial DNA
sequences utilized are units with the consensus motif CC(A/T)(6)GG, known as
CArG elements, derived from the IR-responsive Egr1 gene. In this study we have
investigated the parameters needed to enhance promoter activation to radiation.
A series of plasmid vectors containing different enhancer/promoters were
constructed, transiently transfected into tumor cells (MCF-7 breast
adenocarcinoma and U-373MG glioblastoma) and expression of a downstream reporter
assayed. Results revealed that increasing the number of CArG elements, up to a
certain level, increased promoter radiation-response; from a fold-induction of
1.95 +/- 0.17 for four elements to 2.74 +/- 0.17 for nine CArGs of the same
sequence (for MCF-7 cells). Specific alteration of the core A/T sequences caused
an even greater positive response, with fold-inductions of 1.71 +/- 0.23 for six
elements of prototype sequence compared with 2.96 +/- 0.52 for one of the new
sequences following irradiation. Alteration of spacing (from six to 18
nucleotides) between elements had little effect, as did the addition of an
adjacent Sp1 binding site. Combining the optimum number and sequence of CArG
elements in an additional enhancer was found to produce the best IR induction
levels. Furthermore, the improved enhancers also performed better than the
previously reported prototype when used in in vitro and in vivo experimental
GDEPT. We envisage such enhancers will be used to drive suicide gene expression
from vectors delivered to a tumor within an irradiated field. The modest, but
tight expression described in the present study could be amplified using a
molecular 'switch' system as previously described using Cre/LoxP. In combination
with targeted delivery, this strategy has great potential for significantly
improving the efficacy of cancer treatment in the large number of cases where
radiotherapy is currently employed.

PMID: 12365005 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


26: J Biol Chem. 2002 Sep 20;277(38):34808-14. Epub 2002 Jul 1. 

Transcription factor Sp1 phosphorylation induced by shear stress inhibits
membrane type 1-matrix metalloproteinase expression in endothelium.

Yun S, Dardik A, Haga M, Yamashita A, Yamaguchi S, Koh Y, Madri JA, Sumpio BE.

Department of Surgery, Yale University School of Medicine, New Haven,
Connecticut 06510, USA.

Membrane type 1-matrix metalloproteinase (MT1-MMP) plays a key role in
endothelial cell migration, matrix remodeling, and angiogenesis. Previous
studies demonstrated that a mechanical force, cyclic strain, increases MT1-MMP
expression by displacing Sp1 with increased Egr-1 expression and binding to the
promoter site. However, the effect of shear stress (SS) on MT1-MMP expression is
poorly understood. Although Egr-1 mRNA transcription and protein was induced
(7.6-fold) in response to SS (n = 5, 0-8 h, p < 0.05), SS decreased MT1-MMP mRNA
transcription and protein levels in a time-dependent fashion (10, 50, and 90%
reduction at 1, 4, and 8 h, respectively; n = 5, p < 0.05). Egr-1 protein was
increased after SS and cyclic strain, but Sp1 was serine-phosphorylated only
after SS. SS increased Sp1 DNA binding (3.8-, 5.8-, and 2.4-fold increase at 1,
4, and 8 h, respectively; n = 5, p < 0.05) that was inhibitable by calf
intestinal phosphatase. Thus, SS inhibits MT1-MMP expression despite Egr-1
up-regulation by inducing the serine phosphorylation of Sp1, which in turn
increases its binding affinity for its site on the MT1-MMP promoter, reducing
the ability of Egr-1 to displace it. These data illustrate the complex control
of microvascular endothelial cell MT1-MMP expression in response to distinct
environmental stimuli (cyclic strain versus shear stress), consisting of both
the modulation of specific transcription factor expression (Egr-1) as well as
transcription factor post-translational modification (serine phosphorylation of
Sp1).

PMID: 12093818 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


27: Mol Cell Endocrinol. 2002 Mar 28;189(1-2):85-96. 

Species differences in GnRH activation of the LHbeta promoter: role of Egr1 and
Sp1.

Call GB, Wolfe MW.

Department of Molecular and Integrative Physiology, University of Kansas Medical
Center, 3901 Rainbow Blvd, Kansas City, KS 66160-7401, USA.

Activation of the luteinizing hormone beta (LHbeta) promoter by
gonadotropin-releasing hormone (GnRH) via the transcription factor early growth
response protein-1 (Egr1) has been well characterized. To determine the
mechanisms affecting Egr1 regulation of LHbeta, we analyzed five different
species of LHbeta promoters (equine, mouse, rat, bovine and human).
Electrophoretic mobility shift assays (EMSAs) identified multiple transcription
factors binding to the Egr regions on the LHbeta promoter. Species-specific
differences existed in the binding affinity for Sp1, Sp3, steroidogenic factor-1
(SF-1) and Egr1. Upon mutation of the Egr elements, competition for the binding
of all zinc finger proteins was lost, suggesting that the Sp proteins compete
for binding to the same site that Egr1 occupies. In addition, the promoters from
species that had the highest affinity for Sp1 also had the lowest activation by
Egr1 and GnRH. Thus we hypothesize that Sp1 competes for Egr1 binding to the Egr
elements on the LHbeta promoter and thus inhibits the ability of GnRH and Egr1
to activate the LHbeta promoter.

PMID: 12039067 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


28: Biol Reprod. 2002 Mar;66(3):675-84. 

Sp1 and Egr1 regulate transcription of the Dmrt1 gene in Sertoli cells.

Lei N, Heckert LL.

Department of Molecular and Integrative Physiology, University of Kansas Medical
Center, Kansas City, Kansas 66160, USA.

Dmrt1 is a recently described gene that is specifically expressed in the gonads
and is required for postnatal testis differentiation. Here, we describe the
transcriptional mechanisms regulating the Dmrt1 proximal promoter in testicular
Sertoli cells. A genomic clone containing exon 1 of the rat Dmrt1 gene and more
than 9 kilobases of 5' flanking sequence was isolated and characterized. Several
prominent transcriptional start sites were identified, with the major site
located 102 bases from the translational start. The Dmrt1 5' flanking region
from -5000 to +74 was transcriptionally active in primary Sertoli cells, and
deletion analysis of this fragment identified 2 major regions needed for full
Dmrt1 promoter function. These regions were located between -3200 and -2000 base
pairs (bp) and downstream of -150 bp relative to the major transcriptional start
site. DNase I footprint analysis of the region downstream of -150 bp revealed 3
regions that are bound by proteins from Sertoli cell nuclear extracts.
Site-directed mutagenesis of these regions identified 2 elements that activate
the Dmrt1 promoter and 2 that repress it. The positive elements bind the
transcription factors Sp1, Sp3, and Egr1, suggesting that these transcription
factors play a critical role in Dmrt1 regulation in the testis.

PMID: 11870074 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


29: Mol Pharmacol. 2002 Feb;61(2):379-90. 

Reciprocal regulation of beta(1)-adrenergic receptor gene transcription by Sp1
and early growth response gene 1: induction of EGR-1 inhibits the expression of
the beta(1)-adrenergic receptor gene.

Bahouth SW, Beauchamp MJ, Vu KN.

Department of Pharmacology, College of Medicine, the University of Tennessee
Health Sciences Center, Memphis, Tennessee 38163, USA. sbahouth@utmem.edu

The beta(1)-adrenergic receptor (beta(1)-AR) plays a key role in regulating
heart rate and contractility in response to catecholamines. Our studies have
focused on defining the factors that regulate the expression of the beta(1)-AR
gene. We determined that a 65-base-pair (bp) region in the beta(1)-AR promoter
between bp -394 and bp -330 directs basal transcription. An element located
between -377 and -365 can bind Sp1 and Sp3. In Drosophila melanogaster SL2
cells, Sp1 stimulated the expression of the beta(1)-AR promoter, whereas Sp3 was
unable to activate transcription. Site-directed mutagenesis indicated that an
intact Sp1-binding site is essential for maintaining the activity of the basal
promoter. In addition to binding Sp family members, the nucleotides between -381
and -367 can bind the zinc-finger transcription factor Egr-1. The Egr-1 and Sp1
binding sites are partially overlapping and their binding sequence is conserved
among mammalian beta(1)-AR genes. The induction of Egr-1 in rat neonatal
ventricular myocytes with phorbol-12-myristate-13-acetate or in HeLa S3 cells by
regulated expression of Egr-1 in a tetracycline-responsive promoter, suppressed
expression from the beta(1)-AR promoter. Overexpression of Sp1 in SK-N-MC cells
increased beta(1)-AR mRNA by 2.4-fold, whereas overexpression of Egr-1 reduced
beta(1)-AR mRNA by 40%. Coexpression of Egr-1 with Sp1 reduced Sp1-mediated
up-regulation of beta(1)-AR mRNA by 60%. Mutagenesis revealed that an intact
Sp1-binding site is essential for observing transcriptional repression by Egr-1
and that Egr-1 suppressed the transcription of the beta(1)-AR gene by competing
with Sp1 for binding to their overlapping sites. These results reveal a novel
physiologically relevant transcriptional mechanism for reciprocal regulation of
beta(1)-AR gene expression.

PMID: 11809863 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


30: J Virol. 2002 Jan;76(1):303-12. 

In vivo analysis of retroviral enhancer mutations in hematopoietic cells:
SP1/EGR1 and ETS/GATA motifs contribute to long terminal repeat specificity.

Wahlers A, Zipfel PF, Schwieger M, Ostertag W, Baum C.

Department of Cell and Virus Genetics, Heinrich Pette Institute, D-20251
Hamburg, Germany.

The objective of this work was to identify, in the context of chromosomally
integrated DNA, the contribution of defined transcription factor binding motifs
to the function of a complex retrovirus enhancer in hematopoietic cells in vivo.
Repopulating murine hematopoietic cells were transduced with equal gene dosages
of replication-incompetent retrovirus vectors encoding enhanced green
fluorescent protein. Enhancer sequences were derived from mouse spleen
focus-forming virus. Destruction of GC-rich sites representing overlapping
targets for SP1 or EGR1 uniformly attenuated gene expression (approximately 25
to 70% of wild-type levels) in all hematopoietic lineages, as shown by
multicolor flow cytometry of peripheral blood and bone marrow cells at various
time points posttransplantation. In contrast, a point mutation within a dual
ETS/GATA motif that abolished transactivation by ETS factors but not by GATA-1
slightly increased activity in erythroid cells and significantly attenuated
enhancer function in T lymphocytes. This study shows that controlled gene
transfer in transplantable hematopoietic cells allows a functional analysis of
distinct cis elements within a complex retrovirus enhancer, as required for the
characterization and engineering of various cellular and viral regulatory
sequences in basic research and gene therapy.

PMID: 11739695 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


31: Kidney Int. 2001 Dec;60(6):2097-108. 

Comment in:
    Kidney Int. 2001 Dec;60(6):2415-6.

Protein kinase CK2 mediates TGF-beta1-stimulated type IV collagen gene
transcription and its reversal by estradiol.

Zdunek M, Silbiger S, Lei J, Neugarten J.

Nephrology Division, Department of Medicine, Montefiore Medical Center, Albert
Einstein College of Medicine, Bronx, New York 10467, USA.

BACKGROUND: We have previously shown that the transcription factor Sp1 mediates
the stimulatory effects of transforming growth factor-beta1 (TGF-beta1) on type
IV collagen gene transcription and protein synthesis, and that estradiol
reverses these effects by down-regulating Sp1 activity. Protein kinase casein
kinase II (CK2) phosphorylates Egr-1 and prevents its binding to Sp1. We
hypothesized that TGF-beta1 stimulates CK2 activity, which in turn activates
type IV collagen gene transcription via increased availability of free Sp1.
METHODS: The effects of TGF-beta1 and of estradiol on murine mesangial cell type
IV collagen gene transcription were measured using a reporter mini gene
construct and on collagen IV protein synthesis by Western blotting. Nuclear
Egr-1, phosphorylated Egr-1, Sp1, Egr-1/Sp1 complexes and unbound Sp1 were
measured using co-immunoprecipitation and Western blotting techniques. RESULTS:
TGF-beta1 stimulated CK2 activity in murine mesangial cells. Although TGF-beta1
failed to alter total Egr-1 protein, it increased phosphorylated Egr-1. This led
to decreased Egr-1/Sp1 complex formation, increased unbound Sp1, increased
binding of nuclear extracts to the collagen IV promoter, and increased type IV
collagen gene transcription and protein synthesis. Physiologic concentrations of
estradiol reversed these effects. CONCLUSIONS: These studies suggest that
activation of CK2 mediates the stimulatory effect of TGF-beta1 on type IV
collagen gene transcription. Moreover, the ability of estradiol to reverse
TGF-beta1-stimulated type IV collagen synthesis is mediated by down-regulating
CK2 activity, which ultimately limits the availability of unbound Sp1 to
activate gene transcription.

PMID: 11737584 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


32: FASEB J. 2001 Sep;15(11):2025-6. Epub 2001 Jul 9. 

Early growth-responsive-1-dependent manganese superoxide dismutase gene
transcription mediated by platelet-derived growth factor.

Maehara K, Oh-Hashi K, Isobe KI.

Department of Basic Gerontology, National Institute for Longevity Sciences, 36-3
Gengo, Morioka-cho, Obu, Aichi 474-8522, Japan.

Manganese superoxide dismutase Mn-SOD plays a major role in protecting
mitochondria from oxidative damage. Overexpression of Mn-SOD maintains cell
survival under conditions that lead to apoptotic death. In addition to the
antioxidative enzyme, platelet-derived growth factor (PDGF) is a principal
survival factor that inhibits apoptosis and promotes proliferation by activating
survival signaling pathways in various cells. Here we show that PDGF induced the
expression of the Mn-SOD gene in NIH3T3 cells, and its induction was associated
with early growth response-1 (Egr-1), a transcription factor. An electrophoretic
mobility shift assay demonstrated that Egr-1 bound to the proximal promoter of
the Mn-SOD gene in response to PDGF. The proximal promoter region of Mn-SOD was
shown to be transcriptionally responsive to both basal and PDGF stimulation by
transfection studies. Forced expression of Egr-1 in the cells activated Mn-SOD
transcription in a dose-dependent manner. The pathway by which PDGF induced
Egr-1 involved the mitogen-activated protein kinase kinase-1 (MEK1) and
extracellular signal-regulated kinases 1 and 2 (ERK1/2), because the effect of
PDGF on the induction of Egr-1 was blocked by U0126, a specific MEK1 inhibitor.
These findings indicate that the induction of Mn-SOD is part of the
anti-apoptotic properties mediated by PDGF.

PMID: 11511524 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


33: J Biol Chem. 2001 Oct 19;276(42):39394-403. Epub 2001 Aug 13. 

Angiotensin II-induced transcriptional activation of the cyclin D1 gene is
mediated by Egr-1 in CHO-AT(1A) cells.

Guillemot L, Levy A, Raymondjean M, Rothhut B.

UMR Physiologie et Physiopathologie, Universite Pierre et Marie Curie, Case
Courrier 256, Batiment A, 5eme etage, 7 Quai St-Bernard, Paris 75005, France.

Cyclin D1 protein expression is regulated by mitogenic stimuli and is a critical
component in the regulation of G(1) to S phase progression of the cell cycle.
Angiotensin II (Ang II) binds to specific G protein-coupled receptors and is
mitogenic in Chinese hamster ovary cells stably expressing the rat vascular Ang
II type 1A receptor (CHO-AT(1A)). We recently reported that in these cells, Ang
II induced cyclin D1 promoter activation and protein expression in a
phosphatidylinositol 3-kinase (PI3K)-, SHP-2-, and mitogen-activated protein
kinase/extracellular signal-regulated kinase (MAPK/ERK)-dependent manner
(Guillemot, L., Levy, A., Zhao, Z. J., Bereziat, G., and Rothhut, B. (2000) J.
Biol. Chem. 275, 26349-26358). In this report, transfection studies using a
series of deleted cyclin D1 promoters revealed that two regions between base
pairs (bp) -136 and -96 and between bp -29 and +139 of the human cyclin D1
promoter contained regulatory elements required for Ang II-mediated induction.
Mutational analysis in the -136 to -96 bp region provided evidence that a
Sp1/early growth response protein (Egr) motif was responsible for cyclin D1
promoter activation by Ang II. Gel shift and supershift studies showed that Ang
II-induced Egr-1 binding involved de novo protein synthesis and correlated well
with Egr-1 promoter activation. Both U0126 (an inhibitor of the MAPK/ERK kinase
MEK) and wortmannin (an inhibitor of PI3K) abrogated Egr-1 endogenous expression
and Egr-1 promoter activity induced by Ang II. Moreover, using a co-transfection
approach, we found that Ang II induction of Egr-1 promoter activity was blocked
by dominant-negative p21(ras), Raf-1, and tyrosine phosphatase SHP-2 mutants.
Identical effects were obtained when inhibitors and dominant negative mutants
were tested on the -29 to +139 bp region of the cyclin D1 promoter. Taken
together, these findings demonstrate that Ang II-induced cyclin D1 up-regulation
is mediated by the activation and specific interaction of Egr-1 with the -136 to
-96 bp region of the cyclin D1 promoter and by activation of the -29 to +139 bp
region, both in a p21(ras)/Raf-1/MEK/ERK-dependent manner, and also involves
PI3K and SHP-2.

PMID: 11502738 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


34: Can J Physiol Pharmacol. 2001 Jun;79(6):533-44. 

Diminished molecular response to doxorubicin and loss of cardioprotective effect
of dexrazoxane in Egr-1 deficient female mice.

Saadane N, Yue P, Alpert L, Mitmaker B, Kirby GM, Chalifour LE.

Lady Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish General
Hospital, Montreal, QC, Canada.

Doxorubicin (DOX) and VP16 are DNA topoisomerase II inhibitors yet only DOX
induces an irreversible cardiotoxicity, likely through DOX-induced oxidative
stress. Egr-1 is overexpressed after many stimuli that increase oxidative stress
in vitro and after DOX-injection into adult mice in vivo. To investigate Egr-1
function in the heart, we compared the molecular and histological responses of
wild type (+/+) and Egr-1 deficient (-/-) female mice to saline, DOX, VP16, the
cardioprotectant dexrazoxane (DZR), or DOX+DZR injection. DOX, and to a lesser
extent VP16, induced characteristic increases in cardiac muscle and non-muscle
genes typical of cardiac damage in +/+ mice, whereas only beta-MHC and Sp1 were
increased in -/- mice. DZR-alone treated +/+ mice showed increased cardiomyocyte
transnuclear width without a change to the heart to body weight (HW/BW) ratio.
However, DZR-alone treated -/- mice had an increased HW/BW, increased
cardiomyocyte transnuclear width, and gene expression changes similar to
DOX-injected +/+ mice. DZR pre-injection alleviated DOX-induced gene changes in
+/+ mice; in DZR+DOX injected -/- mice the increases in cardiac and non-muscle
gene expression were equal to, or exceeded that, detected after DOX-alone or
DZR-alone injections. We conclude that Egr-1 is required for DOX-induced
molecular changes and for DZR-mediated cardioprotection.

PMID: 11430591 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


35: Virology. 2001 May 10;283(2):167-77. 

Hepatitis C virus core protein transactivates insulin-like growth factor II gene
transcription through acting concurrently on Egr1 and Sp1 sites.

Lee S, Park U, Lee YI.

Liver Cell Signal Transduction Laboratory, Bioscience Research Division, Korea
Research Institute of Bioscience and Biotechnology, Taejon, Korea 305-606.

The possibility that hepatitis C virus core gene product (HCV-core) acts as a
transactivator in insulin-like growth factor II (IGF-II) gene transcription was
tested. HCV-core protein increases endogenous IGF-II expression from promoter 4
(P4) of the IGF-II gene through two cis-acting elements: Sp1 and Egr1 binding
sites. Sp1 and Egr1 both bind to IGF-II P4 and functionally cooperate in
mediating the maximal activity of IGF-II P4. HCV-core protein induced the
binding of Sp1 and Egr1 on its binding sites on IGF-II P4. In addition, Sp1 and
Egr1 were stimulated to phosphorylate by HCV-core, and its DNA binding activity
was up-regulated upon HCV-core transfection. Transfection with HCV-core in HepG2
cells stimulated the membrane translocation of protein kinase C (PKC) and the
treatment of HCV-core transfected cells with calphostin C, a PKC inhibitor,
blocked induction of Sp1 and Egr1 DNA binding activity, and eventually
transcriptional transactivations of the IGF-II gene. Increasing the DNA binding
activity of the phosphorylated form of Sp1 and Egr1 might be an important
mechanism for regulating IGF-II gene expression and for promoting cell division
during hepatic carcinogenesis. These results indicate that HCV-core functions as
a positive regulator of IGF-II transcription through the PKC pathway and that
Sp1 and Egr1 are direct targets of the transcriptional regulation of the IGF-II
gene which plays an important role in hepatitis C virus pathogenesis during the
formation of hepatocellular carcinoma (HCC). Copyright 2001 Academic Press.

PMID: 11336542 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


36: J Biol Chem. 2001 May 18;276(20):16749-57. Epub 2001 Feb 7. 

Characterization of a rat neuronal nicotinic acetylcholine receptor alpha7
promoter.

Nagavarapu U, Danthi S, Boyd RT.

Department of Neuroscience, The Ohio State University College of Medicine and
Public Health, Columbus, Ohio 43210, USA.

Neuronal nicotinic acetylcholine receptors (nAChRs) containing the alpha7
subunit are expressed in the central nervous system, autonomic nervous system,
retina, adrenal medulla, and PC12 cells. alpha7 nAChRs have been implicated in
several important biological activities apart from synaptic transmission such as
mediating neurite growth and presynaptic control of neurotransmitter release. A
178-base pair promoter was sufficient to drive high level expression of the
alpha7 gene in PC12 cells. The alpha7 promoter was also cell-specific,
expressing in PC12 cells but not in L6 rat muscle cells. Within our minimal rat
alpha7 nAChR promoter we identified two sequences important for basal level
expression. Mutation of a GC-rich sequence at -172 relative to the translational
start site led to an increase in activity of the promoter, indicating the
presence of a negative regulatory element. Upstream stimulatory factor-1 acted
to regulate alpha7 expression positively by binding to an E-box at -116. A site
directly adjacent to the upstream stimulatory factor-1 binding site was shown to
bind Egr-1. Sp1 and Sp3 binding also occurred downstream from or overlapping the
Egr-1 binding site in the rat alpha7 promoter. Several transcription factors
interact in close proximity to control expression of the rat alpha7 nicotinic
receptor gene.

PMID: 11278551 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


37: J Immunol. 2001 Apr 1;166(7):4534-42. 

Differential expression of Fas ligand in Th1 and Th2 cells is regulated by early
growth response gene and NF-AT family members.

Dzialo-Hatton R, Milbrandt J, Hockett RD Jr, Weaver CT.

Department of Pathology, University of Alabama, Birmingham, AL 35294, USA.

Inducible expression of Fas ligand (CD95 ligand) by activated T cells and the
resulting apoptosis of CD95-bearing cells is a critical component of peripheral
T cell homeostasis and cytotoxic effector mechanisms. Transcriptional control of
the expression of Fas ligand has been attributed to a number of factors,
including early growth response gene 2 (Egr2), Egr3, Sp1, and NF-AT, although a
direct contribution of NF-AT is controversial. The present study confirms a role
for Egr factors and indicates that NF-AT is essential for optimal expression of
murine Fas ligand through a direct interaction with an NF-AT consensus element.
The role of these factors was further defined by studying the differential
expression of Fas ligand in Th1 and Th2 lines derived from DO11.10 TCR
transgenic mice. EMSA analyses of a composite Egr/NF-AT site showed recruitment
of Sp1 to this site in Th2 cells, but not in Th1 cells. Furthermore, gel-shift
analyses demonstrated the binding of Egr1, 2, and 3 in Th2 cells and Egr1 and 2,
but not Egr3 in Th1 cells at a known Egr site. Northern analysis corroborated
the lack of Egr3 in Th1 cells. Differential usage of these transcription factors
by Th1 and Th2 cells suggests a potential mechanism underlying the differential
expression of Fas ligand by distinct T cell lineages.

PMID: 11254710 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


38: Int J Dev Neurosci. 2000 Dec;18(8):791-5. 

Lead exposure alters Egr-1 DNA-binding in the neonatal rat brain.

Reddy GR, Zawia NH.

Department of Biology and Life Sciences, Savannah State University, GA 31404,
USA.

Zinc finger proteins (ZFP) contain a structural motif (Cys-2 His-2) found in a
large family of eukaryotic transcriptional regulatory proteins, such as Sp1.
Previous studies have shown that Sp1 DNA-binding was disrupted by exposure to
lead (Pb), due to action on its zinc finger domain. In this paper, we discuss
the results of studies with another ZFP, Egr-1, an early growth response gene,
which is functionally involved in cell proliferation and differentiation. Egr-1
DNA-binding was studied by gel shift mobility assays in several brain regions of
developing rat pups. We observed a distinct developmental profile of Egr-1
DNA-binding with a gradual increase from the early to late postnatal days in all
the brain regions examined. Lactational exposure to Pb resulted in a modulation
of Egr-1 DNA binding manifested by premature peaks in DNA-binding reminiscent of
the in vivo changes previously reported for Sp1. These data are consistent with
earlier findings that exposure to Pb both in vivo and in vitro causes a
modulation in the DNA-binding of ZFP such as Sp1, Egr-1 and TFIIIA. The
commonality by which Pb exposure alters the DNA-binding patterns of ZFP suggests
that divalent Pb may be interacting directly with the Zn moiety of these
proteins.

PMID: 11154848 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


39: Br J Pharmacol. 2000 Dec;131(8):1592-6. 

Angiotensin-converting enzyme (ACE) inhibition attenuates insulin-like growth
factor-I (IGF-I) induced cardiac fibroblast proliferation.

van Eickels M, Vetter H, Grohe C.

Medizinische Universitats-Poliklinik, University of Bonn, Wilhelmstrasse 35-37,
53111 Bonn, Germany.

The effects of angiotensin-converting enzyme (ACE) inhibition and angiotensin
type 1 (AT(1)) receptor blockade on insulin-like growth factor-I (IGF-I) induced
proliferation and immediate-early-gene expression of neonatal rat cardiac
fibroblasts were investigated. Moreover the role of the IGF-I receptor (IGF-IR)
in this process was evaluated. IGF-I (10(-9) - 10(-7) M) stimulated neonatal rat
cardiac fibroblast growth in a dose-dependent fashion (maximum: 3.5+/-0.1 fold,
10(-7) M), as determined by 5-bromo-2'-deoxyuridine (BrdU) incorporation. ACE
inhibition or AT(1) receptor blockade attenuated the IGF-I (10(-7) M) induced
neonatal rat cardiac fibroblast growth in a concentration-dependent fashion
(moexiprilat: 50+/-2%, enalaprilat: 31+/-2%, CV11974; 58+/-1%, all: 10(-7) M).
IGF-I stimulated cellular growth was accompanied by an upregulation of the
immediate early genes c-Fos (2.4+/-0.3 fold), Egr-1 (4.7+/-1.1 fold) and Sp1
(6.2+/-0.7 fold). IGF-I induced expression was completely inhibited by ACE
inhibition or AT(1) receptor blockade. Stimulation with IGF-I or Ang II (10(-7)
M) increased IGF-IR expression 5.7+/-0. 5 fold and 3.6+/-0.5 fold respectively.
The IGF-I induced overexpression of the IGF-IR was reduced by ACE inhibition
with moexiprilat (10(-7) M) by 79+/-7% and by AT(1) receptor blockade with
CV11974 (10(-7) M) by 79+/-5%. These data demonstrate that the mitogenic action
of IGF-I in neonatal rat cardiac fibroblasts is in part mediated by activation
of the renin-angiotensin system (RAS) with subsequent upregulation of IGF-IR
expression. This observation has important implications for the treatment of
cardiac diseases with ACE inhibitors alone and their combination with IGF-I or
growth hormone.

PMID: 11139436 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


40: Vitam Horm. 2000;60:195-227. 

Transcriptional regulation of the LH beta gene by gonadotropin-releasing hormone
and the protein kinase C system.

Halvorson LM.

Division of Reproductive Endocrinology, New England Medical Center, Tufts
University School of Medicine, Boston, Massachusetts 02111, USA.

Publication Types:
    Review
    Review, Tutorial

PMID: 11037625 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


41: Mol Endocrinol. 2000 Aug;14(8):1235-45. 

Sp1, steroidogenic factor 1 (SF-1), and early growth response protein 1 (egr-1)
binding sites form a tripartite gonadotropin-releasing hormone response element
in the rat luteinizing hormone-beta gene promoter: an integral role for SF-1.

Kaiser UB, Halvorson LM, Chen MT.

Department of Medicine, Brigham and Women's Hospital and Harvard Medical School,
Boston, Massachusetts 02115, USA. ukaiser@rics.bwh.harvard.edu

Recently, several cis-regulatory elements that play roles in LHbeta gene
expression, and their cognate DNA-binding transcription factors, have been
identified. These factors include Sp1, steroidogenic factor-1 (SF-1), and early
growth response protein 1 (Egr-1). Using the GH3 pituitary cell line (which
lacks SF-1) as a model, we demonstrate that expression of SF-1 or Egr-1
increases rat LHbeta gene promoter activity but has little effect on the fold
response to GnRH. However, expression of both SF-1 and Egr-1 synergistically
enhances LHbeta gene promoter activity and prevents further stimulation of
activity by GnRH. Mutations in the Sp1 binding sites of the rat LHbeta gene
promoter decrease GnRH responsiveness, whereas mutations in the SF-1 and/or
Egr-1 binding sites alone have little effect on the GnRH response. Combinatorial
mutations in both the Sp1 and Egr-1 binding elements result in almost complete
loss of the GnRH response. In contrast, in GH3 cells cotransfected with SF-1,
mutations in the Sp1, SF-1, or Egr-1 binding elements independently decrease
GnRH responsiveness. In LbetaT2 cells, a gonadotrope-derived cell line that
expresses SF-1 endogenously, mutations in either the Sp1 or Egr-1 binding
elements decrease GnRH responsiveness. These data suggest that the Sp1, SF-1,
and Egr-1 binding sites form a tripartite GnRH response element in the rat
LHbeta gene promoter. Changes in the spacing between the upstream Sp1 binding
sites and the downstream SF-1/Egr-1 binding elements reduce the response to
GnRH. SF-1, while having little direct effect on GnRH responsiveness, has a
critical role in integrating the effects of Sp1 and Egr-1.

PMID: 10935547 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


42: Neurochem Int. 2000 Nov-Dec;37(5-6):473-82. 

Chronic ethanol has region-selective effects on Egr-1 and Egr-3 DNA-binding
activity and protein expression in the rat brain.

Depaz IM, Goodenough S, Wilce PA.

Alcohol Research Unit, Department of Biochemistry, University of Queensland, St.
Lucia, Qld 4072, Brisbane, Australia. iris.depaz@mailbox.up.edu.au

This study focused on the DNA-binding activity and protein expression of the
transcription factors Egr-1 and Egr-3 in the rat brain cortex and hippocampus
after chronic or acute ethanol exposure. DNA-binding activity was reduced in
both regions after chronic ethanol exposure and was restored to the level of the
pair-fed group at 16 h of withdrawal. Cortical Egr-1 protein levels were not
altered by chronic ethanol exposure but increased 16 h after withdrawal, thus
mirroring DNA-binding activity. In contrast, Egr-3 protein levels did not
undergo any change. There was no change in the level of either protein in the
hippocampus. Immunohistochemistry revealed a region-selective change in
immunopositive cells in the cortex and hippocampus. Finally, an acute bolus dose
of ethanol did not affect Egr DNA-binding activity and ethanol treatment did not
alter the DNA-binding activity or protein levels of the transcription factor
Sp1. These observations suggest that chronic exposure to ethanol has
region-selective effects on the DNA-binding activity and protein expression of
Egr-1 and Egr-3 transcription factors in the rat brain. These changes occur
after prolonged ethanol exposure and may thus reflect neuroadaptive changes
associated with physical dependency and withdrawal. These effects are also
transcription factor-selective. Clearly, protein expression is not the sole
mediator of the changes in DNA-binding activity and chronic ethanol exposure
must have effects on modulatory agents of Egr DNA-binding activity.

PMID: 10871699 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


43: Mol Pharmacol. 2000 Jul;58(1):1-10. 

Regulation of the MDR1 gene by transcriptional repressors selected using peptide
combinatorial libraries.

Bartsevich VV, Juliano RL.

Department of Pharmacology, School of Medicine, University of North Carolina,
Chapel Hill 27599-7365, USA.

The ability to selectively regulate the expression of genes implicated in cancer
or other diseases could have important ramifications for both basic research and
for therapy. Using peptide combinatorial libraries expressed in yeast, we have
screened for novel zinc finger proteins that selectively bind to an overlapping
EGR1/SP1/WT1 regulatory site in the promoter of the MDR1 multidrug resistance
gene. The novel proteins were only moderately effective in blocking
transcription by simple masking of the target site. However, when coupled to
mammalian transactivator or repressor domains, they could selectively modulate
the expression of reporter genes having promoters containing the MDR1 target
site. Moreover, they could also regulate transcription of the chromosomal MDR1
gene. Thus, in K562 cells, 12-O-tetradecanoylphorbol-13-acetate-inducible
expression of P-glycoprotein, the product of MDR1 gene, was strongly and
selectively inhibited by the presence of a repressor protein targeted to the
MDR1 promoter. These studies potentially provide a novel alternative approach to
the control of multidrug resistance. They also provide important insights into
strategies for developing selective regulators of gene expression.

PMID: 10860921 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


44: J Biol Chem. 2000 Sep 1;275(35):26683-9. 

Ability of Egr1 to activate tyrosine hydroxylase transcription in PC12 cells.
Cross-talk with AP-1 factors.

Papanikolaou NA, Sabban EL.

Department of Biochemistry and Molecular Biology, New York Medical College,
Valhalla, New York 10595, USA.

We have recently identified an Egr1 motif that overlaps with the Sp1 element in
the tyrosine hydroxylase (TH) promoter. Here we examine whether this motif has a
functional role in the regulation of TH transcription in PC12 cells. In nuclear
extracts from control PC12 cells, an oligonucleotide containing the TH Sp1/Egr1
motif binds Sp1-containing complexes. Treatment of PC12 cells with phorbol ester
(2 micrometer 12-O-tetradecanoylphorbol-13-acetate (TPA)) gives rise to a new
Egr1-containing complex. TPA treatment reduces the steady-state levels of the
Sp1 protein and leads to the appearance of immunoreactive Egr1 protein within
30-60 min. Expression of the Egr1 protein in PC12 cells stimulates the
chloramphenicol acetyltransferase reporter gene placed under the control of the
first 272 nucleotides of the rat TH promoter. Site-directed mutagenesis of
either the Sp1/Egr1 motif or of an upstream AP-1 motif or both abolishes the
Egr1-mediated induction of chloramphenicol acetyltransferase activity. An
oligonucleotide encompassing the AP-1/E-box sequence of the rat TH promoter
competes in electrophoretic mobility shift assays for binding of nuclear
extracts from control and TPA-treated cells to an oligonucleotide containing the
Sp1/Egr1 element, indicating that these two enhancers may interact. The results
show that Egr1 can activate TH transcription and reveals cross-talk between
Sp1/Egr1 and AP-1 factors.

PMID: 10842166 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


45: Brain Res Mol Brain Res. 2000 May 5;77(2):258-66. 

Sequential increase in Egr-1 and AP-1 DNA binding activity in the dentate gyrus
following the induction of long-term potentiation.

Williams JM, Beckmann AM, Mason-Parker SE, Abraham WC, Wilce PA, Tate WP.

Department of Biochemistry and Centre for Gene Research, University of Otago,
Dunedin, New Zealand. joanna.williams@stonebow.otago.ac.nz

Establishment of long-term potentiation (LTP) at perforant path synapses is
highly correlated with increased expression of Egr and AP-1 transcription
factors in rat dentate gyrus granule cells. We have investigated whether
increased transcription factor levels are reflected in increased transcription
factor activity by assessing Egr and AP-1 DNA binding activity using gel shift
assays. LTP produced an increase in binding to the Egr element, which was NMDA
receptor-dependent and correlated closely with our previously reported increase
in Egr-1 (zif/268) protein levels. Supershift analysis confirmed involvement of
Egr-1, but not Egr-2 in the DNA binding activity. AP-1 DNA binding was also
rapidly elevated in parallel with protein levels, however, the peak increase in
activity was delayed until 4 h, a time point when we have previously shown that
only jun-D protein was elevated. These data indicate that binding of Egr-1 and
AP-1 to their response elements is increased in two phases. This may result in
activation of distinct banks of target genes which contribute to the
establishment of persistent LTP.

PMID: 10837920 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


46: Mol Cell Endocrinol. 2000 Feb 25;160(1-2):193-202. 

Transcriptional regulation of the expression of macrophage colony stimulating
factor.

Rubin J, Fan D, Wade A, Murphy TC, Gewant H, Nanes MS, Fan X, Moerenhout M,
Hofstetter W.

Department of Medicine, Veterans Affairs Medical Center and Emory University
School of Medicine, VAMC-151, Dcatur, GA 30033, USA. jrubi02@emory.edu

The regulatory regions for transcriptional control of the MCSF gene are unknown.
We examined regulatory control in a 774-bp murine MCSF promoter transfected into
MC3T3-E1 osteoblast-like and COS-7 cells. Deletion of upstream sequence from
-635 increased basal activity of the promoter by at least four-fold, an increase
that was maintained when PU.1, NFkappaB and Egr1/Sp1 consensus sequences were
subsequently removed. Mutagenesis identified a suppressor element between -635
and -642 from the transcriptional start site and an oligonucleotide representing
this sequence was retarded by nuclear cell protein. TNFalpha (1 ng/ml), PTH
(5x10(-8) M), and IL-1alpha (100 pg/ml), which increased MCSF protein secretion,
failed to enhance the transcriptional rate of the full-length promoter. TNFalpha
was able to stimulate transcription of a heterologous reporter transfected into
COS-7 containing multiple copies of the murine MCSF NFkappaB site inserted
before a minimal promoter. In contrast, deletion of the same NFkappaB response
element increased basal activity in the native promoter. Thus, the NFkappaB
sequence may act as a negative regulator in the context of the endogenous
promoter. Our results indicate that constitutive transcriptional activity
conferred by the MCSF promoter may be damped by a suppressor protein.
Transcriptional regulation, however, does not appear to be a major stimulatory
mechanism for MCSF secretion.

PMID: 10715553 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


47: Am J Physiol Heart Circ Physiol. 2000 Mar;278(3):H796-805. 

Altered molecular response to adrenoreceptor-induced cardiac hypertrophy in
Egr-1-deficient mice.

Saadane N, Alpert L, Chalifour LE.

Lady Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish General
Hospital, Montreal H3T 1E2, Quebec, Canada H3A 1A3.

Unmanipulated early growth response-1 (Egr-1)-deficient -/- mice have similar
heart-to-body weight ratios but express lower amounts of atrial natriuretic
factor (ANF), beta-myosin heavy chain (beta-MHC), skeletal actin, NGF1-A binding
protein (NAB)-2, Sp1, c-fos, c-jun, GATA-4, and Nkx2.5 than +/+ or +/- mice.
alpha-MHC, tubulin, and NAB-1 expression was similar. Isoproterenol (Iso) and
phenylephrine (PE) infusion into +/+ and -/- mice increased heart weight, ANF,
beta-MHC, skeletal actin, Sp1, NAB-2, c-fos, and c-jun expression, but induction
in -/- mice was lower. Only Iso + PE-treated +/+ mice showed induction of NAB-1,
GATA-4, and Nkx2.5. Foci of fibrosis were found in Iso + PE-treated -/- and +/+
mice. Surprisingly, vehicle-treated -/- mice displayed fibrosis and increased
Sp1, skeletal actin, Nkx2.5, and GATA-4 expression without hypertrophy. Minipump
removal caused the agonist-treated hearts and gene expression to regress to
control or near-control levels. Thus Egr-1 deficiency caused a blunted
catecholamine-induced hypertrophy response and increased sensitivity to stress.

PMID: 10710348 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


48: Oncogene. 1999 Dec 2;18(51):7319-27. 

Expression of the SRF gene occurs through a Ras/Sp/SRF-mediated-mechanism in
response to serum growth signals.

Spencer JA, Misra RP.

Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin,
WI 53226, USA.

Serum Response Factor (SRF) plays a central role in the transcriptional response
of mammalian cells to a variety of extracellular signals. It is a key regulator
of many cellular early response genes which are believed to be involved in cell
growth, differentiation, and development. The mechanism by which SRF activates
transcription in response to mitogenic agents has been extensively studied,
however, less is known about regulation of the SRF gene itself. Previously, we
identified distinct regulatory elements in the SRF promoter that play a role in
activation, including an ETS domain binding site, an overlapping Sp1/Egr-1
binding site, and two SRF binding sites. We further showed that serum induces
the SRF gene by a mechanism that requires an intact SRF binding site, also
termed a CArG box. In the present study we demonstrate that in response to
stimulation by cells by lysophosphatidic acid (LPA) or whole serum, the SRF
promoter is upregulated by a bipartite pathway that requires both an Sp1 factor
binding site and the CArG motifs for maximal stimulation. The CArG box-dependent
component of this pathway is targeted by Rho mediated signals, and the Sp1
binding site dependent component is targeted by Ras mediated signals.

PMID: 10602487 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


49: Am J Pathol. 1999 Sep;155(3):897-905. 

Vascular smooth muscle cell proliferation and regrowth after mechanical injury
in vitro are Egr-1/NGFI-A-dependent.

Santiago FS, Atkins DG, Khachigian LM.

Centre for Thrombosis and Vascular Research, The University of New South Wales,
Sydney, Australia Sydney, Australia.

Smooth muscle cell (SMC) proliferation is a key event in renarrowing of blood
vessels after balloon angioplasty. Mechanical injury imparted to the arterial
wall in experimental models induces the expression of the immediate-early gene,
egr-1. Egr-1 binds to and activates expression from the proximal promoters of
multiple genes whose products can, in turn, influence the vascular response to
injury. Here, we used antisense strategies in vitro to inhibit rat vascular SMC
proliferation by directly targeting Egr-1. A series of phosphorothioate
antisense oligonucleotides of 15 base length and complementary to various
theoretically accessible regions within Egr-1 mRNA were synthesized and assessed
for their ability to selectively inhibit SMC proliferation in an Egr-1-dependent
manner. Western blot analysis revealed that two oligonucleotides, AS2 and E11,
inhibited Egr-1 synthesis in cells exposed to serum without affecting levels of
the zinc finger protein Sp1. AS2 and E11 inhibited serum-inducible
[(3)H]thymidine incorporation into DNA, as well as serum stimulation of total
cell numbers. Size-matched phosphorothioate oligonucleotides with random,
scrambled, sense or mismatch sequences failed to inhibit. Antisense Egr-1
inhibition was nontoxic and reversible. These oligonucleotides also inhibited
SMC regrowth after mechanical injury in vitro. Egr-1 thus plays a key regulatory
role in SMC proliferation and repair following injury.

PMID: 10487847 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


50: J Biol Chem. 1999 Aug 6;274(32):22679-85. 

Egr-1 mediates extracellular matrix-driven transcription of membrane type 1
matrix metalloproteinase in endothelium.

Haas TL, Stitelman D, Davis SJ, Apte SS, Madri JA.

Department of Pathology, Yale University Medical School, New Haven, Connecticut
06520, USA.

Matrix metalloproteinase activity is instrumental in processes of cellular
invasion. The interstitial invasion of endothelial cells during angiogenesis is
accompanied by up-regulation of several matrix metalloproteinases, including
membrane type 1 matrix metalloproteinase (MT1-MMP). In this study, we show that
endothelial cells stimulated to undergo angiogenesis by a three-dimensional
extracellular matrix environment increase production of the transcription factor
Egr-1. Increased binding of Egr-1 to the MT1-MMP promoter correlates with
enhanced transcriptional activity, whereas mutations in the Egr-1 binding site
abrogate the increased transcription of MT1-MMP in the stimulated cells. These
data identify Egr-1-mediated transcription of MT1-MMP as a mechanism by which
endothelial cells can initiate an invasive phenotype in response to an
alteration in extracellular matrix environment, thus functionally associating
MT1-MMP with a growing number of proteins known to be up-regulated by Egr-1 in
response to tissue injury or mechanical stress.

PMID: 10428849 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


51: J Neurochem. 1999 Jul;73(1):433-6. 

Sp1/Egr1 motif: a new candidate in the regulation of rat tyrosine hydroxylase
gene transcription by immobilization stress.

Papanikolaou NA, Sabban EL.

Department of Biochemistry and Molecular Biology, New York Medical College,
Valhalla 10595, USA.

The rat tyrosine hydroxylase (TH) promoter contains a GC box (Sp1 motif) whose
function is currently unclear. In this study, we examined the effect of
immobilization (IMO) stress on binding of adrenomedullary transcription factors
to that site. DNase I footprinting analysis reveals the binding of proteins to
the Sp1 motif. Extracts from the adrenal medulla of IMO-treated rats generate a
footprint on the Sp1 motif that is smaller than that generated by control
extracts. Electrophoretic mobility shift assays using an oligonucleotide
containing the Sp1 region show that two Sp1 protein-containing complexes (I and
III) form with control extracts. In contrast, extracts from the adrenal medulla
of IMO-treated rats form a novel complex (II) that contains the Egr1 protein.
This is the first report to reveal changes in the binding pattern on the TH Sp1
motif in response to stress and to identify an overlapping Egr1 site.

PMID: 10386997 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


52: J Biol Chem. 1999 Jun 18;274(25):17997-8004. 

Insulin-induced early growth response gene (Egr-1) mediates a short term
repression of rat malic enzyme gene transcription.

Barroso I, Santisteban P.

Instituto de Investigaciones Biomedicas "Alberto Sols," Consejo Superior de
Investigaciones Cientificas, Universidad Autonoma de Madrid, Arturo Duperier 4,
Madrid E-28029, Spain.

In this report we have studied insulin regulation of malic enzyme (ME) gene
transcription in rat H-35 hepatoma cells and localized the insulin-responsive
region of the ME promoter between positions -177 and -102. This region contains
a putative insulin response element (IRE-II). When nuclear extracts from
untreated or insulin-treated H-35 cells were incubated with IRE-II,
transcription factors Sp1 and Sp3 were observed to bind constitutively to this
element, whereas insulin induces the quick and transient binding of an insulin
response factor. This induction requires de novo protein synthesis. Competition
and supershift assays demonstrated that the insulin response factor is the
immediate-early gene Egr-1. In vitro assays revealed that Egr-1 displaces Sp1
from its binding site in IRE-II. Insulin induces Egr-1 mRNA, with a time course
pattern that corresponds perfectly to the Egr-1 binding to IRE-II. This
induction depends on the activation of mitogen-activated protein (MAP) kinase,
and it is phosphatidylinositol 3-kinase-independent, as demonstrated with
specific inhibitors for both pathways. By cotransfecting the wild-type or a
dominant negative Ras, an upstream regulator of MAP kinase, we show that Ras
inhibits ME promoter activity. Furthermore, overexpression of Egr-1 in H-35
cells represses the ME gene promoter in a dose-dependent manner. These results
suggest that insulin induces a quick, transient, and Ras/MAP kinase-dependent
activation of Egr-1 which leads to a transient repression of ME gene
transcription. On a late phase, insulin would activate a different,
Egr-1-independent pathway, which would result in activation of the ME gene.

PMID: 10364249 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


53: Oncogene. 1999 May 20;18(20):3143-51. 

The Wilms' tumor gene product represses the transcription of thrombospondin 1 in
response to overexpression of c-Jun.

Dejong V, Degeorges A, Filleur S, Ait-Si-Ali S, Mettouchi A, Bornstein P,
Binetruy B, Cabon F.

CNRS UPR9079, Oncogenese, Differenciation et Transduction du Signal, Villejuif,
France.

Thrombospondin 1 (TSP1) is known for its significant anti-angiogenic properties.
In a previous study, we have shown that transient or stable overexpression of
the transcription factor c-Jun, in rat fibroblasts, leads to repression of TSP1.
We now demonstrate that the c-Jun-induced repression of TSP1 does not occur
directly and does not require binding of c-Jun to the TSP1 promoter. Instead,
repression involves a factor secreted by c-Jun-overexpressing cells. This
secreted factor triggers a signal transduction pathway from the membrane to the
nucleus, and these signals lead to the binding of the product of the Wilms'
tumor suppressor gene, WT1, to the -210 region of the TSP1 promoter. This region
binds WT1 and SP1, but not EGR1, although its sequence fits the consensus
binding site for this transcription factor. WT1 overexpression in transfected
cells inhibits endogenous TSP1 gene expression and TSP1 transcription in
experiments using TSP1 promoter-reporter constructs. The WT1 - KTS isoform is
more active in repressing TSP1 transcription than WT1 + KTS, while EGR1 is
inactive. Enhancement of WT1 binding to DNA in response to c-Jun does not
require de novo protein synthesis. The above mechanism for TSP1 repression could
apply to other genes, thus coordinating their regulation in the vicinity of a
c-Jun-overexpressing cell. We conclude that WT1, which was discovered as a
result of its tumor suppressor properties, may also possess oncogenic
characteristics in the c-Jun transformation process, and thus repress the
anti-angiogenic protein, TSP1.

PMID: 10340386 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


54: Mol Cell Biol. 1999 Apr;19(4):2734-45. 

COUP-TF upregulates NGFI-A gene expression through an Sp1 binding site.

Pipaon C, Tsai SY, Tsai MJ.

Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030,
USA.

The formation of various tissues requires close communication between two groups
of cells, epithelial and mesenchymal cells. COUP-TFs are transcription factors
which have been shown to have functions in embryonic development. COUP-TFI is
expressed mainly in the nervous system, and its targeted deletion leads to
defects in the central and peripheral nervous systems. COUP-TFII is highly
expressed in the mesenchymal component of the developing organs. A null mutation
of COUP-TFII results in the malformation of the heart and blood vessels. From
their expression pattern, we proposed that COUP-TFs regulate paracrine signals
important for mesenchymal cell-epithelial cell interactions. In order to
identify genes regulated by COUP-TF in this process, a rat urogenital
mesenchymal cell line was stably transfected with a COUP-TFI expression vector.
We found that NGFI-A, a gene with important functions in brain, organ, and
vasculature development, has elevated mRNA and protein levels upon
overexpression of COUP-TFI in these cells. A study of the promoter region of
this gene identified a COUP-TF-responsive element between positions -64 and -46.
Surprisingly, this region includes binding sites for members of the Sp1 family
of transcription factors but no COUP-TF binding site. Mutations that abolish the
Sp1 binding activity also impair the transactivation of the NGFI-A promoter by
COUP-TF. Two regions of the COUP-TF molecule are shown to be important for
NGFI-A activation: the DNA binding domain and the extreme C terminus of the
putative ligand binding domain. The C-terminal region is likely to be important
for interaction with coactivators. In fact, the coactivators p300 and steroid
receptor activator 1 can enhance the transactivation of the NGFI-A promoter
induced by COUP-TFI. Finally, we demonstrated that COUP-TF can directly interact
with Sp1. Taken together, these results suggest that NGFI-A is a target gene for
COUP-TFs and that the Sp1 family of transcription factors mediates its
regulation by COUP-TFs.

PMID: 10082539 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


55: J Biol Chem. 1999 Jan 29;274(5):3199-206. 

Sp1 and egr-1 have opposing effects on the regulation of the rat Pgp2/mdr1b
gene.

Thottassery JV, Sun D, Zambetti GP, Troutman A, Sukhatme VP, Schuetz EG, Schuetz
JD.

Department of Pharmaceutical Sciences, St. Jude Children's Research Hospital,
Memphis, Tennessee 38105, USA.

The promoter of the rat pgp2/mdr1b gene has a GC-rich region (pgp2GC) that is
highly conserved in mdr genes and contains an consensus Sp1 site. Sp1's role in
transactivation of the pgp2/mdr1b promoter was tested in Drosophila Schneider
cells. The pgp2/mdr1b promoter was strongly activated by co-transfected wild
type Sp1 but not mutant Sp1 and mutation of the Sp1 site abrogated Sp1-dependent
transactivation. In gel shift assays, the same mutations abolished Sp1-DNA
complex formation. Moreover, basal activity of the pgp2/mdr1b Sp1 mutant
promoter was dramatically lower. Enforced ectopic overexpression of Sp1 in H35
rat hepatoma cells revealed that cell lines overexpressing Sp1 had increased
endogenous pgp2/mdr1b mRNA, demonstrating that Sp1 activates the endogenous
pgp2/mdr1b gene. Pgp2GC oligonucleotide also bound Egr-1 in gel shift assays and
Egr-1 competitively displaced bound Sp1. In transient transfections of H35 cells
(and human LS180 and HepG2 cells) Egr-1 potently and specifically suppressed
pgp2/mdr1b promoter activity and mutations in the Egr-1 site decreased Egr-1
binding and correlated with pgp2/mdr1b up-regulation. Ectopic overexpression of
Egr-1 in H35 cells decreased Pgp expression and selectively increased
vinblastine sensitivity. In conclusion, Sp1 positively regulates while Egr-1
negatively regulates the rat pgp2/mdr1b gene. Moreover, competitive interactions
between Sp1 and Egr-1 in all likelihood determine the constitutive expression of
the pgp2/mdr1b gene in H35 cells.

PMID: 9915860 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


56: J Biol Chem. 1999 Jan 15;274(3):1801-13. 

Regulation of MCL1 through a serum response factor/Elk-1-mediated mechanism
links expression of a viability-promoting member of the BCL2 family to the
induction of hematopoietic cell differentiation.

Townsend KJ, Zhou P, Qian L, Bieszczad CK, Lowrey CH, Yen A, Craig RW.

Departments of Pharmacology and Toxicology, Dartmouth Medical School, Hanover,
New Hampshire 03755-3835, USA.

Proliferation, differentiation, and apoptosis are tightly regulated during
hematopoiesis, allowing amplification along specific lineages while preventing
excessive proliferation of immature cells. The MCL1 member of the BCL2 family is
up-regulated during the induction of monocytic differentiation (approximately
10-fold with 12-O-tetradecanoylphorbol 13-acetate (TPA)). MCL1 has effects
similar to those of BCL2, up-regulation promoting viability, but differs from
BCL2 in its rapid inducibility and its pattern of expression. Nuclear factors
that regulate MCL1 transcription have now been identified, extending the
previous demonstration of signal transduction through mitogen-activated protein
kinase. A 162-base pair segment of the human MCL1 5'-flank was found to direct
luciferase reporter activity, allowing approximately 10-fold induction with TPA
that was suppressible upon inhibition of the extracellular signal-regulated
kinase (ERK) pathway. Serum response factor (SRF), Elk-1, and Sp1 bound to
cognate sites within this segment, SRF and Elk-1 acting coordinately to affect
both basal activity and TPA inducibility, whereas Sp1 affected basal activity
only. Thus, the mechanism of the TPA-induced increase in MCL1 expression seen in
myelomonocytic cells at early stages of differentiation involves signal
transduction through ERKs and transcriptional activation through SRF/Elk-1. This
finding provides a parallel to early response genes (e.g. c-FOS and EGR1) that
affect maturation commitment in these cells and therefore suggests a means
through which enhancement of cell viability may be linked to the induction of
differentiation.

PMID: 9880563 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


57: J Biol Chem. 1998 Dec 18;273(51):34000-7. 

Sp1 and CREB mediate gastrin-dependent regulation of chromogranin A promoter
activity in gastric carcinoma cells.

Hocker M, Raychowdhury R, Plath T, Wu H, O'Connor DT, Wiedenmann B, Rosewicz S,
Wang TC.

Medizinische Klink mit Schwerpunkt Gastroenterologie und Hepatologie,
Universitatsklinikum Charite, Campus Virchow-Klinikum, Humboldt Universitat
Berlin, Germany.

Chromogranin A (CgA) is a multifunctional acidic protein that in the stomach is
expressed predominantly in enterochromaffin-like cells (ECL cells) where it is
regulated by gastrin. In order to investigate the transcriptional response of
the mouse CgA (mCgA) promoter to gastrin stimulation, we studied a 4.8-kilobase
mCgA promoter-luciferase reporter gene construct in transiently transfected
AGS-B cells. 5'-Deletion analysis and scanning mutagenesis of mCgA 5'-flanking
DNA showed that a Sp1/Egr-1 site spanning -88 to -77 base pairs (bp) and a
cyclic AMP-responsive element (CRE) at -71 to -64 bp are essential for
gastrin-dependent mCgA transactivation. Gastrin stimulation increased cellular
Sp1 protein levels and Sp1-binding to the mCgA -88 to -77 bp element, as well as
binding of CREB to its consensus motif at -71 to -64 bp. Gastrin also stimulated
CREB Ser-133 phosphorylation, and abundance of cellular CREB protein levels.
Overexpression of either Sp1 or phosphorylated CREB transactivated the mCgA
promoter dose dependently, while coexpression of both transcription factors
resulted in an additive mCgA promoter response. mCgA -92 to -64 bp, comprising
the Sp1/Egr-1 site and the CRE motif, conferred gastrin responsiveness to a
heterologous thymidine kinase promoter system, and therefore functions as a
"true" enhancer element. This report demonstrates that Sp1 and CREB mediate
CCK-B/gastrin receptor-dependent gene regulation, and that the effect of gastrin
on the CgA gene is brought about by cooperative action of both transcription
factors.

PMID: 9852054 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


58: J Clin Invest. 1998 Nov 15;102(10):1850-9. 

Estrogen blocks M-CSF gene expression and osteoclast formation by regulating
phosphorylation of Egr-1 and its interaction with Sp-1.

Srivastava S, Weitzmann MN, Kimble RB, Rizzo M, Zahner M, Milbrandt J, Ross FP,
Pacifici R.

Division of Bone and Mineral Diseases, Washington University School of Medicine,
and Barnes-Jewish Hospital, St. Louis, Missouri 63110, USA.

Central to the pathogenesis of osteoporosis is the ability of estrogen
deficiency to increase osteoclast formation by enhancing stromal cell production
of the osteoclastogenic cytokine macrophage colony-stimulating factor (M-CSF).
We report that stromal cells from ovariectomized mice exhibit increased casein
kinase II-dependent phosphorylation of the nuclear protein Egr-1. Phosphorylated
Egr-1 binds less avidly to the transcriptional activator Sp-1 and the resulting
higher levels of free Sp-1 stimulate transactivation of the M-CSF gene. Estrogen
replacement fails to block M-CSF mRNA expression and osteoclast formation in
ovariectomized mice lacking Egr-1, confirming the critical role played by this
transcription factor in mediating the antiosteoclastogenic effects of estrogen.
Thus, by downregulating formation of a novel Egr-1/Sp-1 complex in stromal
cells, estrogen deficiency results in enhanced levels of free Sp-1 and increased
M-CSF gene expression and osteoclast formation.

PMID: 9819371 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


59: Brain Res. 1998 Aug 24;803(1-2):95-104. 

Novel structure of the human GDNF gene.

Woodbury D, Schaar DG, Ramakrishnan L, Black IB.

Department of Neuroscience and Cell Biology, UMDNJ-Robert Wood Johnson Medical
School, 675 Hoes Lane, Piscataway, NJ 08854, USA. woodburydl@aol.com

Glial cell line-derived neurotrophic factor (GDNF) is the most potent known
survival factor for substantia nigra neurons, which degenerate in Parkinson's
disease, for spinal motoneurons, which die in Lou Gehrig's disease (ALS), and
for Purkinje neurons, the critical outflow cells of the cerebellum. Moreover,
targeted deletion of the GDNF gene results in renal dysgenesis and abnormal
development of the enteric nervous system. GDNF mRNA is expressed in a complex
temporospatial pattern in the central nervous system and the periphery,
consistent with these observations. To begin elucidating mechanisms regulating
the pattern of expression of GDNF, we have cloned the human gene, and
characterized the promoter. The promoter is highly GC rich, and lacks canonical
CCAT-box and TATA-box motifs. It contains more than 12 binding sites for known
transcription factors. These cis-elements have the potential to interact with
factors regulating constitutive expression (Sp1) and developmental expression
(bHLH). Moreover, the promoter contains sites for binding transcription factors
which respond to environmental signals, including CREB, AP2, Zif/268, NFkB, and
MRE-BP. Combinatorial actions of these transcription factors may account for the
extraordinarily complex expression patterns of the GDNF gene. Importantly, we
demonstrate that the hGDNF gene utilizes a promoter distinct from that
identified in the rodent GDNF gene, a finding with ramifications for Parkinson's
disease and ALS research. Copyright 1998 Published by Elsevier Science B.V.

PMID: 9729303 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


60: Genomics. 1998 Aug 1;51(3):408-16. 

TGFbeta-inducible early gene (TIEG) also codes for early growth response alpha
(EGRalpha): evidence of multiple transcripts from alternate promoters.

Fautsch MP, Vrabel A, Subramaniam M, Hefferen TE, Spelsberg TC, Wieben ED.

Department of Biochemistry and Molecular Biology, Mayo Clinic/Foundation,
Rochester, Minnesota 55905, USA.

TGFbeta-inducible early gene (TIEG) and early growth response alpha (EGRalpha)
are putative transcription factors based on homology to known zinc finger
proteins SP1, EGR1, BTEB, and Wilm tumor. Here we report that TIEG and EGRalpha
are expressed from alternative promoters of the same gene. The TIEG/EGRalpha
gene spans 8 kb and contains five exons. Use of alternative first exons results
in TIEG having 12 unique amino acids on its N-terminus. Computer analysis of the
5' upstream regions of either TIEG (exon 1a) or EGRalpha (exon 1b) does not
identify a TATA box or initiator sequencebut shows consensus sequence
similarities to binding sites for several transcription factors including
SP1,JunB, and aromatic hydrocarbon/receptor-ligand complexes. Analysis of
constructs containing 5'-flanking regions show that both the TIEG and the
EGRalpha promoters have significant activity in human fetal osteoblast cells.
Northern analysis of mRNA from various human tissues and several cell lines
reveals that TIEG is the predominant transcript produced and regulated by growth
factors from the TIEG/EGRalpha gene. Copyright 1998 Academic Press.

PMID: 9721211 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


61: Proc Natl Acad Sci U S A. 1998 Jul 7;95(14):8298-303. 

Tissue factor transcription driven by Egr-1 is a critical mechanism of murine
pulmonary fibrin deposition in hypoxia.

Yan SF, Zou YS, Gao Y, Zhai C, Mackman N, Lee SL, Milbrandt J, Pinsky D, Kisiel
W, Stern D.

Departments of Physiology and Cellular Biophysics, Surgery, and Medicine,
Columbia University, College of Physicians and Surgeons, New York, NY 10032,
USA. sy18@columbia.edu

Local hypoxemia and stasis trigger thrombosis. We have demonstrated previously
that in a murine model of normobaric hypoxia pulmonary fibrin deposition is a
result of expression of tissue factor, especially in oxygen-deprived mononuclear
phagocytes (MPs). We now show that transcription factor early-growth-response
gene product (Egr-1) is rapidly activated in hypoxia, both in vitro and in vivo,
and is responsible for transcription and expression of tissue factor in hypoxic
lung. MPs and HeLa cells subjected to hypoxia (pO2 approximately 13 torr) had
increased levels of tissue factor transcripts (approximately 18-fold) and an
increased rate of transcription (approximately 15-fold), based on nuclear run-on
analysis. Gel-shift analysis of nuclear extracts from hypoxic MPs and HeLa cells
demonstrated increased DNA-binding activity at the serum response region (SRR;
-111/+14 bp) of the tissue factor promoter at Egr-1 motifs. Using 32P-labeled
Egr consensus oligonucleotide, we observed induction of DNA-binding activity in
nuclear extracts from hypoxic lung and HeLa cells because of activation of
Egr-1, by means of supershift analysis. Transient transfection of HeLa cells
with chimeric plasmids containing wild-type or mutant SRR from the tissue factor
promoter showed that intact Sp1 sites are necessary for basal promoter activity,
whereas the integrity of Egr-1 sites was required for hypoxia-enhanced
expression. A central role for Egr-1 in hypoxia-mediated tissue factor
expression was confirmed by experiments with homozygous Egr-1 null mice;
wild-type mice subjected to oxygen deprivation expressed tissue factor and
showed fibrin deposition, but hypoxic homozygous Egr-1 null mice displayed
neither tissue factor nor fibrin. These data delineate a novel biology for
hypoxia-induced fibrin deposition, in which oxygen deprivation-induced
activation of Egr-1, resulting in expression of tissue factor, has an unexpected
and central role.

PMID: 9653181 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


62: J Korean Med Sci. 1998 Apr;13(2):171-8. 

Different protein-binding patterns in the P3 promoter region of the human
insulin-like growth factor II gene in the human liver cirrhosis and
hepatocellular carcinoma tissues.

Seo JH, Kim KW, Park BC.

Department of Molecular Biology, Pusan National University, Korea.

The P3 promoter of the human insulin-like growth factor II (IGF-II) is the major
IGF-II promoter in fetal liver (FL) and hepatocellular carcinoma (HCC). However,
little information is available on the transcriptional factors (TFs) controlling
IGF-II gene expression in human liver cirrhosis (LC) and HCC tissues. To
evaluate the protein-binding patterns in the P3 promoter region, we performed
electromobility shift assay (EMSA) and DNase I footprinting assay using nuclear
extracts from human FL, LC and HCC tissues. EMSA showed considerable differences
in binding patterns of proteins to P3 promoter region according to different
nuclear extracts used in this study. By footprinting assay, eight footprints
were observed in extracts. In addition, LC extract showed two specific binding
at L1 [-80:+30] and L2 [-126:-80] regions, and HCC showed two specific binding
at H1 [-176:-120] and H2 [-210:-177] as well as two liver specific binding (L1
and L2). Footprinting after immunoprecipitation indicates that Egr1, Egr2 and
Sp1 could bind to P3 promoter directly, while c-jun and c-fos could not bind to
these region directly. Further study is required to determine the function of
these proteins.

PMID: 9610618 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


63: J Biol Chem. 1998 Mar 6;273(10):5758-64. 

Zinc finger transcription factors mediate high constitutive platelet-derived
growth factor-B expression in smooth muscle cells derived from aortae of newborn
rats.

Rafty LA, Khachigian LM.

The Centre for Thrombosis and Vascular Research, School of Pathology, The
University of New South Wales, Sydney, New South Wales 2052, Australia.

Platelet-derived growth factor (PDGF) B-chain gene is differentially expressed
in smooth muscle cells (SMCs) derived from the rat aortic wall. SMCs cultured
from two week-old rats (pups) express high levels of PDGF-B mRNA, whereas cells
isolated from three month-old rats (adults) express low levels of PDGF-B.
Nuclear run-off experiments indicate that increased PDGF-B gene expression in
pups is mediated, at least in part, at the transcriptional level. We used
electrophoretic mobility shift assays and Western blot analysis to demonstrate
that levels of Sp1 and Sp3, two zinc finger transcription factors which mediate
basal expression of the PDGF-B gene, are elevated in pup nuclei compared with
adult nuclei. The immediate-early transcription factor, Egr-1, which footprints
the PDGF-B promoter, is also constitutively expressed in these cells. Transient
transfection and binding studies show that these factors interact with a region
in the proximal PDGF-B promoter key for basal expression in pup cells. Mutation
of this proximal element in transfected pup cells attenuates reporter gene
expression to levels observed in adult cells. Conversely, overexpression of Sp1
in adult cells augments PDGF-B promoter-dependent expression. Elevated PDGF-B
expression in cultured newborn rat SMCs may therefore require high constitutive
expression of a number of zinc finger transcription factors and their specific
interactions with the proximal PDGF-B promoter.

PMID: 9488709 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


64: Hypertension. 1998 Jan;31(1 Pt 2):170-5. 

Strain-responsive regions in the platelet-derived growth factor-A gene promoter.

Wilson E, Vives F, Collins T, Ives HE.

Cardiovascular Research Institute, Division of Nephrology, University of
California, San Francisco 94143, USA.

Proliferation of cultured neonatal vascular smooth muscle (VSM) cells is
enhanced by exposure to cyclic mechanical strain, in part through autocrine
action of secreted platelet-derived growth factor (PDGF). We examined
transcription factors and DNA response elements that may participate in the
induction of PDGF-A gene transcription by mechanical strain. PDGF-A mRNA
increased gradually over 4 to 24 hours exposure to cyclic (1 Hz) strain. This
was due, at least in part, to increased transcription since a full length (890
bp) PDGF-A promoter reporter construct was induced 3.5-fold in transfected VSM
cells exposed to strain for 24 hours. A series of PDGF-A promoter truncation
reporter constructs was used to identify potential regions of the promoter
involved in regulation by strain. Strain-responsive regions were found between
-262 bp and -92 bp and between -92 bp and -41 bp of the promoter. Since these
regions are GC-rich and contain response elements for Egr-1 and Sp-1, we
examined expression of these transcription factors in response to strain. mRNA
for both factors increased over 0.5 to 4 hours of strain, while protein
expression for both increased gradually over a 24 hours period. Gel shift assays
with a probe specific for Egr-1 demonstrated at least 1 prominent new shifted
band after 4 to 12 hours exposure to strain. An Sp-1 probe demonstrated
constitutive shifted bands that did not change in response to strain. Thus,
GC-rich regions in the proximal 92 bp of the PDGF-A promoter contain mechanical
strain-responsive elements that bind Egr-1 and possibly Sp-1.

PMID: 9453298 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


65: Immunobiology. 1997 Dec;198(1-3):179-91. 

Coordinate expression and distinct DNA-binding characteristics of the four
EGR-zinc finger proteins in Jurkat T lymphocytes.

Skerka C, Decker EL, Zipfel PF.

Department of Molecular Biology, Bernhard Nocht Institute for Tropical Medicine,
Hamburg, Germany.

The Early Growth Response Genes (EGR-1 to AT133/EGR-4) encode a family of
proteins that are composed of three homologous consecutive zinc fingers of the
Cys2-His2 type and different flanking sequence. Upon growth stimulation of
resting cells the four EGR-genes are simultaneously transcribed. We have
analyzed the expression of the four EGR-proteins in Jurkat T cells and show by
Western blot analysis that the four EGR-proteins are coordinately induced upon
treatment with a combination of PHA and PMA. As the individual proteins are
reported to bind to identical target sequences, we have analyzed the DNA-binding
of the native proteins. Using nuclear extract in which we have demonstrated
expression of all four EGR-proteins, only EGR-1, but no other member of this
protein family is found to bind to the EGR-consensus site (GCG GGG GCG). In
addition, DNA-binding of both native EGR-1 and of recombinant EGR-1 and
AT133/EGR-4 proteins expressed in insect cells was analyzed. This comparison
revealed distinct binding properties of recombinant EGR-1 and AT133/EGR-4 to
oligonucleotides that include the EGR-consensus sites. The distinct binding
affinities suggest that in vivo EGR-proteins bind to different target sequences
and that each EGR-protein regulates distinct target genes. This is underlined by
demonstrating that EGR-1 but not AT133/EGR-4 binds to a related G-rich promoter
element with the sequence GGG GTG GGG. This G-rich sequence serves as an
overlapping binding site for the two zinc finger proteins EGR-1 and Sp1. As
similar overlapping binding sites for EGR-1 and Sp1 have been identified in
several human and mouse gene promoters, we raise the question whether the Sp1
binding sites described in a large number of eukaryotic gene promoters also
represent binding sites for EGR-1.

PMID: 9442390 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


66: Adv Exp Med Biol. 1997;430:155-64. 

Endothelial gene regulation by laminar shear stress.

Resnick N, Yahav H, Khachigian LM, Collins T, Anderson KR, Dewey FC, Gimbrone MA
Jr.

Department of Morphological Sciences, Bruce Rappaport Research Institute, Bruce
Rappaport Faculty of Medicine, Technion, Haifa, Israel.

Endothelial cells, because of their unique localization, are constantly exposed
to fluid mechanical forces derived by the flowing blood. These forces, and more
specifically shear stresses; affect endothelial structure and function, both in
vivo and in vitro, and are implicated as contributing factors in the development
of cardiovascular diseases. We have demonstrated earlier that the shear stress
selectively induces the transcription of several endothelial genes, and have
defined a shear stress response element (SSRE) in the promoter of
platelet-derived-growth-factor B (PDGF-B), that is shared by additional
endothelial shear stress responsive genes. Here we further characterize this
SSRE and the nuclear factors that bind to it, and imply the possible role of the
endothelium cytoskeleton in transducing shear stress, leading to the expression
of PDGF-B/SSRE constructs in transfected endothelial cells exposed to shear
stress. We also present, yet a new shear stress response element in the Platelet
Derived Growth Factor A promoter, that contains a binding site to the
transcription factors egr1/sp1. These results further demonstrate the complexity
of gene regulation by hemodynamic forces, and support the important part that
these forces have in the physiology and pathophysiology of the vessel wall.

PMID: 9330726 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


67: J Cell Biochem. 1997 Sep 15;66(4):489-99. 

Reciprocal modulation between Sp1 and Egr-1.

Huang RP, Fan Y, Ni Z, Mercola D, Adamson ED.

Burnham Institute, La Jolla Cancer Research Center, California 92037, USA.

Many ubiquitously expressed genes, including oncogenes, lack a proximal TATA or
CAAT box but have a region of G + C-rich sequences that appears to replace the
usual promoter initiation site. The zinc-finger protein Sp1 is one of the
prevalent activators of these genes. The Egr-1 zinc-finger protein has a similar
binding site and if the two sites occur in the same region, a variety of
activation or inhibitory responses may be obtained. We show that competition
between the two factors for overlapping sites on growth-promoting genes could
explain why the overexpression of Egr-1 suppresses transformed growth in a
number of cell types [Huang et al. (1995): Cancer Res 55:5054-5062; Huang et al.
(1997): Int J Cancer]. We demonstrate here that Egr-1 and Sp1 can bind to the
same G + C-rich sites and that Egr-1 can displace Sp1 and hence inhibit its
activity. We measured the responses of synthetic consensus binding sites and
natural promoter sequences linked to a reporter gene and showed that Egr-1
inhibited the activation of transcription by Sp1 on overlapping Sp1/Egr-1 sites.
In contrast, Sp1 activity could be augmented by Egr-1 at nonoverlapping sites in
the Egr-1 gene promoter, in transient reporter gene studies in Drosophila SL2
cells. In addition, over-expression of exogenous Sp1 in mammalian cells, also
leads to increased Egr-1 protein expression, which further inhibits Sp1
transactivation of numerous genes. Therefore, we can account for some of the
complex responses of G + C-rich enhancer/promoters by a form of "facilitated
inhibition" of Sp1 by Egr-1 at overlapping sites.

PMID: 9282327 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


68: Oncogene. 1997 Aug 7;15(6):669-76. 

Sp1 recognition sites in the proximal promoter of the human vascular endothelial
growth factor gene are essential for platelet-derived growth factor-induced gene
expression.

Finkenzeller G, Sparacio A, Technau A, Marme D, Siemeister G.

Institute of Molecular Medicine, Tumor Biology Center, Freiburg, Germany.

Stimulation of NIH3T3 cells with platelet-derived growth factor (PDGF)-BB
enhances expression of vascular endothelial growth factor (VEGF), an endothelial
cell-specific mitogen and a key mediator of tumor angiogenesis. Here, we
identified cis-acting VEGF promoter elements and trans-acting factors which are
involved in PDGF-stimulated VEGF expression. By 5'-deletion and transient
transfection analysis, a G + C-rich region at -85 to -50 of the human VEGF
promoter was shown to be necessary and sufficient for both PDGF inducible and
basal expression. The region contains three potential recognition sites for Sp1
transcription factors, which overlap with two Egr-1 sites. Mutations that
abolish the ability of Sp1 to interact with the VEGF promoter element also
abrogate expression induced by PDGF. Mutations of the potential Egr-1 binding
sites did not affect PDGF responsiveness. Gel shift and antibody supershift
analyses showed that Sp1 and Sp3 interact constitutively with the VEGF promoter
element. Our data strongly suggest that enhanced VEGF gene expression in
PDGF-induced NIH3T3 cells is mediated by Sp1 and/or Sp3 transcription factors
bound to the -85 to -50 promoter region of the VEGF gene.

PMID: 9264407 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


69: Proc Natl Acad Sci U S A. 1997 May 27;94(11):5525-30. 

Design of polydactyl zinc-finger proteins for unique addressing within complex
genomes.

Liu Q, Segal DJ, Ghiara JB, Barbas CF 3rd.

The Skaggs Institute for Chemical Biology and the Department of Molecular
Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La
Jolla, CA 92037, USA.

Zinc-finger proteins of the Cys2-His2 type represent a class of malleable
DNA-binding proteins that may be selected to bind diverse sequences. Typically,
zinc-finger proteins containing three zinc-finger domains, like the murine
transcription factor Zif268 and the human transcription factor Sp1, bind nine
contiguous base pairs. To create a class of proteins that would be generally
applicable to target unique sites within complex genomes, we have utilized
structure-based modeling to design a polypeptide linker that fuses two
three-finger proteins. Two six-fingered proteins were created and demonstrated
to bind 18 contiguous bp of DNA in a sequence-specific fashion. Expression of
these proteins as fusions to activation or repression domains allows
transcription to be specifically up- or down-modulated within human cells.
Polydactyl zinc-finger proteins should be broadly applicable as genome-specific
transcriptional switches in gene therapy strategies and the development of novel
transgenic plants and animals.

PMID: 9159105 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


70: J Immunol. 1996 Dec 15;157(12):5411-21. 

Cloning of the human platelet endothelial cell adhesion molecule-1 promoter and
its tissue-specific expression. Structural and functional characterization.

Almendro N, Bellon T, Rius C, Lastres P, Langa C, Corbi A, Bernabeu C.

Department of Immunology, Center of Biological Investigations, High Council of
Scientific Investigations, Madrid, Spain.

Platelet endothelial cell adhesion molecule-1 (PECAM-1; CD31) is a cell adhesion
molecule involved in transendothelial migration and expressed by hemopoietic and
endothelial cells. To understand the mechanisms underlying its regulated
expression, a genomic clone containing 1555 bp of the 5'-flanking region and the
first exon of the human PECAM-1 gene has been isolated. The 5'-flanking region
of the PECAM-1 gene lacks a consensus TATA box, but contains consensus motifs
for Sp1, EGR1, ets, helix-loop-helix (HLH) box, GATA, AP-2, C/EBP, YY1, CCACC,
LyF-1, imperfect octamer, heptamer, high mobility group proteins (HMG) box, and
nuclear factor-kappaB, as well as shear stress-, retinoic acid-,
glucocorticoid-, and acute phase-responsive elements, and an Alu sequence.
Successive 5' to 3' or 3' to 5' deletions revealed tissue-specific promoter
activity within the two contiguous 0.22-kb NheI/BglII and 0.44-kb BglII/PstI
fragments. The transcriptional activity displayed by the 0.22-kb NheI/BglII
fragment was specific for the myeloid lineage, whereas the promoter activity of
the 0.44-kb BglII/PstI fragment was apparently restricted to endothelial cells.
The transcriptional activity of the 0.22-kb NheI/BglII fragment was confirmed by
5' RACE (rapid amplification of 5' cDNA ends) and S1 nuclease protection
experiments that revealed previously unidentified transcription start sites. The
0.22-kb NheI/BglII promoter exhibited PMA inducibility in myeloid cells and
contained a PMA-responsive element recognized by Sp1 and EGR-1 transcription
factors. Isolation and characterization of the human PECAM-1 promoter represent
an initial step in elucidating the controlled expression of the PECAM-1 gene.

PMID: 8955189 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


71: J Biol Chem. 1996 Aug 2;271(31):18576-81. 

Egr-1 activates basic fibroblast growth factor transcription. Mechanistic
implications for astrocyte proliferation.

Biesiada E, Razandi M, Levin ER.

Department of Medicine, University of California, Irvine, Irvine, California
92716, USA.

The mechanisms controlling the proliferation of astrocytes are of great interest
but are not well defined. We have previously shown that the endogenous
neuropeptides, endothelin-3 (ET-3), and atrial natriuretic peptide (ANP),
modulate the proliferation of astrocytes through positively and negatively
regulating the transcription of the immediate-early gene egr-1 which
transactivates basic fibroblast growth factor (bFGF) by unknown mechanisms. In
these studies, we determined the involvement of MAP kinase (Erk) activation by
ET-3 in the transcription of egr-1, and the molecular determinants by which
Egr-1 transactivates bFGF. Transfection of astrocytes with a mitogen-activated
protein (MAP) kinase (MAPK) expression vector increased the transcription of a
cotransfected egr-chloramphenicol acetyltransferase (CAT) construct 3-fold. This
induction was totally abolished by a dominant negative MAPK mutant. A 3-fold
induction of egr-CAT expression by ET-3 was significantly reduced by treatment
with ANP, or a cotransfected dominant negative MAPK plasmid. Using mobility
shift assays, we showed that ET-3 induced the expression of Egr-1 protein which
bound specifically to several early growth-related protein (Egr-1) binding sites
on the bFGF promoter, and that this effect was significantly reversed by
treatment with ANP. We also found that the Sp1 transcriptional factor was bound
at these same sites, but was not stimulated by ET-3. Deletion experiments
indicated that only the site at -160 bp of the bFGF promoter was significant for
bFGF transactivation by Egr-1. We conclude that the astrocyte mitogen, ET-3,
stimulates egr-1 transcription through a MAP kinase (Erk) related mechanism, and
that Egr-1 transactivates bFGF through a specific noncanonical, Egr-1 site on
the promoter. ANP inhibits each of these steps, providing a pathway for its
anti-proliferative action.

PMID: 8702507 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


72: Eur J Biochem. 1996 Apr 1;237(1):311-7. 

A (G+C)-rich motif in the aldolase C promoter functions as a constitutive
transcriptional enhancer element.

Cibelli G, Schoch S, Pajunk H, Brand IA, Thiel G.

Institute for Genetics, University of Cologne, Germany.

The enzyme fructose-1,6-bisphosphate aldolase consists of three isozymes that
are expressed in a tissue-specific manner. Using antibodies against aldolase B
and C, it is shown that aldolase C is expressed in virtually all neuronal cell
lines derived from the central and peripheral nervous system. Recently,
experiments with transgenic mice indicated that a (G+C)-rich region of the
aldolase C promoter might function as a neuron-specific control element of the
rat aldolase C gene [Thomas, M., Makeh, I., Briand, P., Kahn, A. & Skala, H.
(1993) Eur. J. Biochem. 218, 143-151). To functionally analyse this element, a
plasmid consisting of four copies of this (G+C)-rich sequence, a TATA box, and
the rabbit beta-globin gene as reporter was constructed. This plasmid was
transfected into neuronal and nonneuronal cell lines and transcription was
monitored by RNase protection mapping of the beta-globin mRNA. It is shown that
the (G+C)-rich element of the aldolase C promoter directs transcription in
neuronal as well as in nonneuronal cells. In contrast, the synapsin I promoter,
used as a control for neuron-specific gene expression, directed transcription
only in neuronal cells. In gel-retardation assays, two major DNA-protein
complexes were detected with the (G+C)-rich element of the aldolase C promoter
used as a DNA probe and nuclear extracts from brain and liver as a source for
DNA-binding proteins. These DNA-proteins interactions could be impaired by a DNA
probe that contained an Sp1-binding site, indicating that Sp1 or an Sp1-related
factor binds to the aldolase C promoter (G+C)-rich element. This was confirmed
by supershift analysis with antibodies specific for Sp1. The zinc finger
transcription factor zif268/egr-1, also known to recognize a (G+C)-rich
consensus site, did not, however, bind to the (G+C)-rich motif of the aldolase C
promoter, nor could it stimulate transcription in transactivation assays from
this control region. From these data, we conclude that the (G+C)-rich element of
the aldolase C promoter functions as a constitutive transcriptional response
element mediated by Sp1 and Sp1-related transcription factors.

PMID: 8620889 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


73: Mol Cell Endocrinol. 1996 Mar 25;117(2):167-73. 

The same sequence mediates activation of the human urokinase promoter by cAMP in
mouse Sertoli cells and by SV40 large T antigen in COS cells.

Grimaldi P, Geremia R, Albanesi C, Rossi P.

Dipartimento di Sanita Pubblica e Biologia Cellulare, Universita di Roma Tor
Vergata, Italy.

Cell-specific activation by follicle-stimulating hormone and its intracellular
mediator, cAMP, of the human urokinase promoter in mouse Sertoli cells requires
overlapping purine-rich and GC-rich sequences between -54 and -42 from the
transcriptional start site. We have previously shown that binding of
unidentified nuclear factors to these sequences is induced by cAMP stimulation,
and that sequences from the enhancerless SV40 replication origin can interfere
with the binding, whereas consensus Sp1 binding sites are ineffective. We now
show that sequences within the SV40 origin able to compete for the formation of
cAMP-induced DNA-protein complexes in Sertoli cell nuclear extracts are binding
sites for the SV40 large T antigen. Large T antigen expressed in COS cells binds
the cAMP-responsive sequences of the human urokinase gene and transactivates the
proximal promoter, thus mimicking the effect of nuclear factors induced by cAMP
in Sertoli cells. We show that Egr-1 is one of the factors present in
cAMP-induced DNA-protein complexes formed between the human urokinase promoter
and Sertoli cell nuclear extracts. However, Egr-1 levels are similar in
unstimulated and cAMP-treated Sertoli cells, suggesting that this factor
interacts with a different GC-box binding factor, that we have previously shown
to be strongly induced by cAMP treatment of Sertoli cells. We propose that SV40
large T antigen in COS cells can mimick the action of heterodimers formed in
cAMP stimulated Sertoli cells between Egr-1 and a cell specific cAMP-induced
GC-box binding factor.

PMID: 8737376 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


74: Genomics. 1996 Mar 15;32(3):413-24. 

Genomic organization of the human beta-catenin gene (CTNNB1).

Nollet F, Berx G, Molemans F, van Roy F.

Section of Molecular Cell Biology, University of Ghent, Belgium.

The cytoplasmic beta-catenin protein is implicated in signal transduction and
associates with both the cell-cell adhesion protein E-cadherin and the tumor
suppressor gene product APC. We determined the primary structure of the human
beta-catenin gene (CTNNB1) by analysis of cDNA and genomic clones. The size of
the complete gene was determined to be 23.2 kb. Restriction mapping and partial
sequence analysis revealed 16 exons. All splice donor and acceptor sites were
conformable to the GT/AG rule. The exon size ranged from 61 to 790 bp. Half of
the introns were smaller than 550 bp, with the smallest being 84 bp and the
longest being 6700 bp. The intron-exon boundaries did not coincide either with
conserved sites in the 12 armadillo repeat sequences of beta-catenin or with
intron-exon boundaries in the armadillo gene of Drosophila. A major site for
transcription initiation was identified as an A residue 214 nucleotides upstream
of the ATG initiation codon. The resulting transcript is 3362 nucleotides long.
Compared to the previously published mRNA sequence, additional residues were
identified, 16 at the 5' end and 766 at the 3' end of the mRNA. An alternative
splice acceptor site within exon 16 reduced the 3' UTR sequence by 159 bp.
Polymerase chain reaction on cDNA from 14 human cell lines demonstrated the
general occurrence of both splice variants. The 5'-flanking region is highly
GC-rich and lacks a CCAAT box, but contains a TATA box and potential binding
sites for several transcription factors, such as NF kappa B, SP1, AP2, and EGR1.
Both a 437-bp fragment and a 6-kb fragment, containing about 4.7 kb of the
5'-flanking region in addition to the noncoding exon 1 and 1 kb of intron 1,
showed clear promoter activity when these fragments were linked to a secreted
alkaline phosphatase reporter gene and transfected into a mouse epithelial cell
line.

PMID: 8838805 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


75: Nucleic Acids Res. 1996 Mar 15;24(6):1149-57. 

Regulation of ornithine decarboxylase gene expression by the Wilms' tumor
suppressor WT1.

Moshier JA, Skunca M, Wu W, Boppana SM, Rauscher FJ 3rd, Dosescu J.

Department of Internal Medicine, Wayne State University School of Medicine,
Detroit, MI 48201 USA.

The importance of ornithine decarboxylase (ODC) to cell proliferation is
underscored by the complex array of cell-specific mechanisms invoked to regulate
its synthesis and activity. Misregulation of ODC has severe negative
consequences on normal cell function, including the acquisition of tumorigenic
growth properties by cells overexpressing ODC. We hypothesize that ODC gene
expression is a candidate target for the anti-proliferative function of certain
tumor suppressors. Here we show that the Wilms' tumor suppressor WT1 binds to
multiple sites within the human ODC promoter, as determined by DNase I
protection and methylation interference assays. The expression of WT1 in
transfected HCT 116, NIH/3T3 and HepG2 cells represses activity of the ODC
promoter controlling expression of a luciferase reporter gene. In contrast WT1
expression enhances ODC promoter activity in SV40-transfected HepG2 cells. Both
the extent of modulation of ODC gene expression and the mediating WT1 binding
elements are cell specific. Constructs expressing WT1 deletion mutants implicate
two regions required for repressor function, as well as an intrinsic activation
domain. Understanding the regulation of ODC gene expression by WT1 may provide
valuable insights into the roles of both WT1 and ODC in development and
tumorigenesis.

PMID: 8604351 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


76: Science. 1996 Mar 8;271(5254):1427-31. 

Egr-1-induced endothelial gene expression: a common theme in vascular injury.

Khachigian LM, Lindner V, Williams AJ, Collins T.

Vascular Research Division, Department of Pathology, Brigham and Women's
Hospital, Boston, MA 02115, USA.

A number of pathophysiologically relevant genes, including platelet-derived
growth factor B-chain (PDGF-B), are induced in the vasculature after acute
mechanical injury. In rat aorta, the activated expression of these genes was
preceded by a marked increase in the amount of the early-growth-response gene
product Egr-1 at the endothelial wound edge. Egr-1 interacts with a novel
element in the proximal PDGF-B promoter, as well as with consensus elements in
the promoters of other genes induced by endothelial injury. This interaction is
crucial for injury-induced PDGF-B promoter-dependent expression. Sp1, whose
binding site in the PDGF-B promoter overlaps that of Egr-1, occupies this
element in unstimulated cells and is displaced by increasing amounts of Egr-1.
These findings implicate Egr-1 in the up-regulated expression of PDGF-B and
other potent mediators in mechanically injured arterial endothelial cells.

PMID: 8596917 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


77: Mol Cell Biol. 1995 Nov;15(11):6100-8. 

12-O-tetradecanoylphorbol-13-acetate activation of the MDR1 promoter is mediated
by EGR1.

McCoy C, Smith DE, Cornwell MM.

Clinical Division, Fred Hutchinson Cancer Research Center, Seattle, Washington
98104, USA.

P-glycoprotein, the product of the MDR1 gene (multidrug resistance gene 1), is
an energy-dependent efflux pump associated with treatment failure in some
hematopoietic malignancies. Its expression is regulated during normal
hematopoietic differentiation, although its function in normal hematopoietic
cells is unknown. To identify cellular factors that regulate the expression of
MDR1 in hematopoietic cells, we characterized the cis- and trans-acting factors
mediating 12-O-tetradecanoylphorbol-13-acetate (TPA) activation of the MDR1
promoter in K562 cells. Transient-transfection assays demonstrated that an MDR1
promoter construct containing nucleotides -69 to +20 conferred a TPA response
equal to that of a construct containing nucleotides -434 to +105. TPA induced
EGR1 binding to the -69/+20 promoter sequences over a time course which
correlated with increased MDR1 promoter activity and increased steady-state MDR1
RNA levels. The -69/+20 promoter region contains an overlapping SP1/EGR site.
The TPA-responsive element was localized to the overlapping SP1/EGR site by
using a synthetic reporter construct. A mutation in this site that inhibited EGR
protein binding blocked the -69/+20 MDR1 promoter response to TPA. The
expression of a dominant negative EGR protein also blocked the TPA response of
the -69/+20 promoter construct. Finally, the expression of EGR1 was sufficient
to activate a construct containing tandem MDR1 promoter SP1/EGR sites. These
data suggest a role for EGR1 in modulating MDR1 promoter activity in
hematopoietic cells.

PMID: 7565762 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


78: J Biol Chem. 1995 Oct 20;270(42):25266-72. 

Promoter analysis of Zfp-36, the mitogen-inducible gene encoding the zinc finger
protein tristetraprolin.

Lai WS, Thompson MJ, Taylor GA, Liu Y, Blackshear PJ.

Howard Hughes Medical Institute Laboratories, Duke University Medical Center,
Durham, North Carolina 27710, USA.

The gene encoding the putative zinc finger protein tristetraprolin (TTP),
Zfp-36, is rapidly induced by a variety of mitogens and growth factors. We show
here that 77 base pairs 5' of the transcription start site are sufficient for
full serum inducibility of the mouse Zfp-36 promoter. This region of the
promoter includes consensus sequences for the binding of the transcription
factors EGR-1, AP2, and Sp1. In addition, we have identified a previously
undescribed element, TTP promoter element 1 (TPE1); this 10-base pair sequence
includes a palindrome and is identical in the human, bovine, and mouse genes.
Each of the three binding elements, EGR-1, AP2, and TPE1, contribute to the
serum induction of Zfp-36 and can confer serum-inducible expression on a
heterologous minimal promoter. Gel mobility shift assays demonstrated the
formation of complexes consisting of this region of the promoter and cellular
nuclear proteins and demonstrated that the extent of complex formation could be
altered by treatment of the cells with serum or insulin. These results suggest
that the response of Zfp-36 to serum and other mitogens is mediated by a series
of cis-acting elements acting in concert to confer full inducible transcription
of this gene.

PMID: 7559666 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


79: J Biol Chem. 1995 Sep 22;270(38):22500-6. 

A regulatory element in the human interleukin 2 gene promoter is a binding site
for the zinc finger proteins Sp1 and EGR-1.

Skerka C, Decker EL, Zipfel PF.

Department of Molecular Biology, Bernhard Nocht Institute for Tropical Medicine,
Hamburg, Germany.

Activation of the interleukin 2 (IL-2) gene after antigen recognition is a
critical event for T cell proliferation and effector function. Prior studies
have identified several transcription factors that contribute to the activity of
the IL-2 promoter in stimulated T lymphocytes. Here we describe a novel
regulatory element within the IL-2 promoter located immediately upstream of the
nuclear factor of activated T cell (NFAT) domain. This region (termed the zinc
finger protein binding region (ZIP)) serves as binding site for two differently
regulated zinc finger proteins: the constitutively expressed transcription
factor Sp1 and the inducible early growth response protein EGR-1. In
unstimulated cells which do not secrete IL-2, only Sp1 binds to this region,
while in stimulated IL-2 secreting cells the inducible EGR-1 protein recognizes
this element. In Jurkat T cells, the ZIP site serves as an activator for IL-2
gene expression, and a combination of ZIP and NFAT binding sites is required for
maximal IL-2 promoter activity. These results suggest a critical role of the ZIP
site for IL-2 promoter activity.

PMID: 7673240 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


80: J Biol Chem. 1995 Jul 21;270(29):17299-305. 

Differential activation of the rat phenylethanolamine N-methyltransferase gene
by Sp1 and Egr-1.

Ebert SN, Wong DL.

Nancy Pritzker Laboratory of Developmental and Molecular Neurobiology,
Department of Psychiatry and Behavioral Sciences, Stanford University School of
Medicine, California 94305-5485, USA.

The rat phenylethanolamine N-methyltransferase (PNMT) gene contains overlapping
consensus elements for the Sp1 and Egr-1 transcription factors located at -45 bp
and -165 bp in the PNMT promoter. In the present study, we show that Sp1 and
Egr-1 can specifically bind to these overlapping elements, that this binding
appears to be mutually exclusive, and that binding site occupancy is dependent
upon the concentration of each factor and its binding affinity for each site.
Egr-1 binds to the -165 bp site with relatively high affinity (IC50 = 14 nM) and
to the -45 bp site with relatively low affinity (IC50 = 1360 nM), whereas Sp1
binds to both sites with intermediate affinities (IC50 = 210 and 140 nM,
respectively). Consistent with the DNA-binding data, Egr-1 stimulates PNMT
promoter activity primarily through interaction with the -165 bp site, while Sp1
stimulates PNMT promoter activity by interacting with both the -45 bp and the
-165 bp sites. These results show that Sp1 and Egr-1 are capable of
differentially activating PNMT gene expression, thereby suggesting that
different stimuli may control the activity of the PNMT gene by selectively
regulating Sp1 and/or Egr-1.

PMID: 7615530 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


81: J Biol Chem. 1995 Jan 27;270(4):1866-72. 

Promoter elements of the mouse acetylcholinesterase gene. Transcriptional
regulation during muscle differentiation.

Mutero A, Camp S, Taylor P.

Department of Pharmacology, University of California, San Diego, La Jolla
92093-0636.

The increase in acetylcholinesterase expression during muscle differentiation
from myoblasts to myotubes was shown previously to reflect primarily a greater
stability of the messenger RNA (mRNA). Here, we investigate the regulation of
the acetylcholinesterase gene during early determination of the muscle
phenotype. (i) We employ myogenic transcription factors to transform non-muscle
cells into myoblasts in order to assess the role of the myogenic transcription
factors in this regulation. (ii) We analyze the Ache promoter region by deletion
analysis, point mutagenesis, and gel mobility shift assays. The myogenic
transcription factors do not accelerate transcription of the Ache gene in spite
of the presence of E-boxes at -335 base pairs from the start of transcription
and in the first intron, and they are not able to trigger stabilization of the
Ache mRNA when constitutively expressed in 10T1/2 fibroblasts. A GC-rich region
(at -105 to -59 base pairs from the start of transcription) containing
overlapping binding sites for the transcription factors Sp1 and Egr-1 is
essential for promoter activity. Mutation of the Sp1 sites dramatically reduces
the promoter activity while mutation of the Egr-1 sites has little effect. Sp1
and Egr-1 compete for binding to overlapping sites and an increase in Egr-1
decreases the expression of the Ache gene.

PMID: 7829523 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


82: J Biol Chem. 1994 Dec 23;269(51):32551-7. 

Overlapping Egr-1 and Sp1 sites function in the regulation of transcription of
the mouse thrombospondin 1 gene.

Shingu T, Bornstein P.

Department of Biochemistry, University of Washington, Seattle 98195.

We have evaluated the basis for the constitutive and serum-regulated expression
of the mouse thrombospondin (TSP) gene in both transiently and stably
transfected NIH-3T3 cells. Experiments with deleted and mutated mouse
promoter/CAT constructs and gel mobility assays demonstrated that an Egr-1
binding site in the proximal promoter, flanked by overlapping GC boxes and an
adjacent GC-rich region, functioned to positively regulate the constitutive
activity of the gene. These motifs, and their cognate transcription factors,
appear to act in concert, with partial redundancy, so that discrete mutations
were only partially effective in reducing transcriptional activity. The Egr-1
site corresponds in position to an NF-Y binding site which functions
synergistically with a distal serum-response element to mediate the serum
response of the human TSP1 gene. However, neither the Egr-1 motif nor the
surrounding proximal promoter region upstream from the TATA box participates in
the serum response of mouse TSP1. These experiments add support to the growing
realization that similar physiologic responses of homologous genes in mouse and
man need not utilize similarly placed cis-acting elements.

PMID: 7798257 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


83: Cell Growth Differ. 1994 Feb;5(2):187-96. 

AP-1 and Krox-24 transcription factors activate the neurofilament light gene
promoter in P19 embryonal carcinoma cells.

Pospelov VA, Pospelova TV, Julien JP.

Institute of Cytology, Russian Academy of Sciences, St. Petersburg.

Changes in the neuronal content of neurofilament proteins occur in some
neuropathological conditions, but little is known about the molecular mechanisms
that control both the cell type specificity and the levels of expression of
neurofilament genes. In addition to TATA and Sp1 elements, we report here the
presence in the neurofilament light (NF-L) promoter region of other regulatory
elements, namely, an AP-1 element TGCGTCAG, a Krox-24 element GCACCCCGC, and an
Ets-like element AGCAAGCAGGAATTT. These elements constitute binding sites for
specific nuclear factors present in aggregated P19 embryonal carcinoma cells.
Using cotransfection assays in P19 embryonal carcinoma cells, we show that NF-L
promoter fragments fused to the reporter chloramphenicol acetyltransferase gene
can be trans-activated by expression vectors encoding FOS and JUN (AP-1) and by
Krox-24 protein. The finding of functional elements for immediate early gene
products in the NF-L promoter suggests molecular pathways by which the
modulation of neurofilament expression can be coupled to growth factors and
other external stimuli.

PMID: 8180132 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


84: J Biol Chem. 1993 Sep 15;268(26):19505-11. 

SP1 activates the MDR1 promoter through one of two distinct G-rich regions that
modulate promoter activity.

Cornwell MM, Smith DE.

Clinical Division, Fred Hutchinson Cancer Research Center, Seattle, Washington
98104.

To define sequences in the human multidrug resistance (MDR1) promoter that
influence transcription, we measured the activity of MDR1 promoter constructs
using luciferase as a reporter gene. Deletion of promoter sequences to -121
(relative to the transcription initiation site) had very little effect on
promoter activity in transiently transfected cells. Further deletion to -88
increased activity about 4-fold, while deletion to -51 decreased activity about
10-fold. The data indicated that between -121 and -88 and between -73 and -51
were sequences that modulated promoter activity. Because these two regions
contain G-rich sequences, which are important for function in other promoters,
the effect of mutation of the G-rich regions (-110 to -103 and -61 to -43) on
MDR1 promoter activity was measured. Mutation of the -110 G-box (-110 to -103)
resulted in a 6-fold increase in promoter activity and inhibited the formation
of a specific nuclear protein complex, suggesting that this region functions as
a transcriptional "repressor" binding site in cycling cells. In contrast,
mutation of the -50 G-box (-61 to -43) reduced promoter activity 5-fold. DNase-I
footprinting and electrophoretic mobility shift analysis indicated that specific
but distinct protein-DNA complexes formed at each of these two G-rich regions.
The -50 G-box contains overlapping sites to which both SP1 and EGR-1 bind
specifically. Co-transfection of MDR1 promoter constructs with SP1 into cells
that lack SP1 (Drosophila Schneider 2 cells) activated equivalently both the
wild type MDR1 promoter and a synthetic promoter containing the MDR1 -50 GC box
and MDR1 initiator element.

PMID: 8103518 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


85: Biochem Biophys Res Commun. 1993 Aug 16;194(3):1475-82. 

Sp1 interacts with the consensus sequence for Egr-1 gene product with a cellular
factor(s) and activates the transcription through this element.

Kutoh E, Schwander J.

Dept. Innere Medizin, Kantonsspital Basel, Switzerland.

We report that Sp1 from nuclear extracts of BRL-3A cells interacts with the
consensus DNA sequence for the Egr-1 gene product in an overlapping manner.
Purified Sp1 failed to bind to this sequence and with the addition of
sub-saturating level of nuclear extracts, the binding activity appeared. In
Drosophila cells (SL2), exogenously expressed Sp1 activated the transcription
through the Egr-1 site. These findings suggest that Sp1 can be targeted to a
non-Sp1 (Egr-1) site with a cellular factor(s) and can activate the
transcription through this element.

PMID: 8352806 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


86: J Biol Chem. 1993 Aug 15;268(23):16949-57. 

Detection and characterization of cellular EGR-1 binding to its recognition
site.

Cao X, Mahendran R, Guy GR, Tan YH.

Signal Transduction Laboratory Institute of Molecular and Cell Biology, National
University of Singapore.

Most of what is known of the Egr-1 DNA binding site GCGGGGGCG was originally
identified by experiments using DNA sequences and bacterially expressed or in
vitro translated EGR-1 protein. Here we report the binding of cellular EGR-1
protein derived from HeLa, mouse and human fibroblasts to its consensus
sequence. Binding is strongly but transiently stimulated in these cells by
serum, phorbol ester, or by okadaic acid, an inhibitor of protein
serine/threonine phosphatases 1 and 2A, suggesting the regulation of this gene
expression and its DNA binding activity to be under the control of protein
kinase(s) and phosphatase(s). When EGR-1 synthesis is stimulated under the above
conditions, binding of the transcription factor Sp1 to its recognition site in
the Egr-1 promoter is reduced with a concomitant appearance of EGR-1 DNA
binding. This is likely a result of competition between Sp1 and the newly
synthesized EGR-1, since there is a partial overlap in the binding sequences
recognized by these proteins. In cotransfection experiments EGR-1 activated
transcription through multiple copies of GCGGGGGCG 5' to a minimum promoter of
c-fos. Interestingly, EGR-1 is shown to down-regulate the transcription of its
own gene expression, whereas Sp1 activated Egr-1 gene expression. The detection
of cellular EGR-1 binding to the Egr-1 consensus sequence in the different cell
types provides a model for studying the mechanism by which an immediate-early
gene is regulated by various ligands.

PMID: 8349585 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


87: Proc Natl Acad Sci U S A. 1993 Mar 15;90(6):2471-5. 

Expression of a human acetylcholinesterase promoter-reporter construct in
developing neuromuscular junctions of Xenopus embryos.

Ben Aziz-Aloya R, Seidman S, Timberg R, Sternfeld M, Zakut H, Soreq H.

Department of Biological Chemistry, Hebrew University of Jerusalem, Israel.

We have employed Xenopus embryos to express human acetylcholinesterase
(AcChoEase; EC 3.1.1.7) in developing synapses. Transcription of AcChoEase mRNA
was driven by a 2.2-kb sequence upstream from the initiator AUG in the ACHE gene
encoding AcChoEase, with multiple potential sites for binding universal and
tissue-specific transcription factors. These included clustered MyoD elements,
E-box, SP1, EGR1, AP-2, and the development-related GAGA motif. A DNA construct
composed of this sequence linked to a 2.1-kb sequence encoding human AcChoEase
was designated human AcChoEase promoter-reporter (HpACHE). HpACHE but none of
its several 5'-truncated derivatives was transcriptionally active in developing
Xenopus embryos. Furthermore, PCR analysis using chimeric PCR primers revealed
usage of the same 1.5-kb intron and 74-bp exon within the HpACHE sequence in
microinjected embryos and various human tissues. Cytochemical staining revealed
conspicuous accumulation of overexpressed AcChoEase in neuromuscular junctions
and within muscle fibers of apparently normal 2-day Xenopus embryos injected
with HpACHE. The same reporter driven by the cytomegalovirus promoter was
similarly efficient in directing the heterologous human enzyme toward
neuromuscular junctions, attributing the evolutionary conservation of AcChoEase
targeting to the coding sequence. Our findings demonstrate that a short DNA
sequence is sufficient to promote the exogenous transcription and faithful
splicing of human AcChoEase mRNA in developing Xenopus embryos and foreshadow
their use for integrative studies of cholinergic signaling and synapse
formation.

PMID: 8460160 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


88: Mol Endocrinol. 1993 Mar;7(3):365-79. 

Nerve growth factor induces transcription of NGFIA through complex regulatory
elements that are also sensitive to serum and phorbol 12-myristate 13-acetate.

DeFranco C, Damon DH, Endoh M, Wagner JA.

Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical
School, Boston, Massachusetts.

The expression of NGFIA (also known as egr1, zif268, TIS8, krox24, and d2) is
rapidly and transiently increased by nerve growth factor (NGF) in PC12 cells.
The 5'-region of this gene includes four serum response elements (SREs), a
cAMP-like response element, an AP1-like response element, and an SP1-binding
site. From deletion analysis of chloramphenicol acetyltransferase reporter
constructs, we have established that the first 106 basepairs 5' of the
transcriptional start site are sufficient for induction of NGFIA by NGF in PC12
cells; deletion beyond this point results in dramatically reduced induction of
the gene. Using defined mutations in the NGFIA promoter and NGFIA-thymidine
kinase hybrid promoters, we have defined three elements (SRE1, SRE2, and
AP1-like) in the first 106 basepairs of upstream DNA, each of which contributes
to induction of NGFIA by NGF. Cooperation by two of these elements (i.e. the two
SREs or one SRE and the AP1-like element) is sufficient to confer
transcriptional induction by NGF, but the combination of all three elements
increased induction by NGF more effectively than a pair of elements. This
suggests that the response of NGFIA to NGF is mediated by a cis-acting sequence
that is composed of at least three distinct elements. An oligonucleotide
composed of SRE1 and SRE2 that can confer the ability for NGF induction to
heterologous promoter constructs complexes with proteins in PC12 cell nuclear
extracts, but the protein-DNA complexes do not appear to be altered by NGF
treatment, as measured by DNA mobility shift assays. We have also established
that the regulatory region of NGFIA that mediates NGF induction also mediates
the induction by serum and phorbol 12-myristate 13-acetate, suggesting that
multiple signal transduction pathways must converge on these sequences to
regulate the expression of this gene.

PMID: 8483478 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


89: Proc Natl Acad Sci U S A. 1991 Sep 1;88(17):7523-7. 

Functional significance of an overlapping consensus binding motif for Sp1 and
Zif268 in the murine adenosine deaminase gene promoter.

Ackerman SL, Minden AG, Williams GT, Bobonis C, Yeung CY.

Department of Genetics, University of Illinois College of Medicine, Chicago
60612.

The murine adenosine deaminase (ADA) gene has a (G + C)-rich promoter that can
support diverse tissue-specific gene expression. By using deletion and mutation
analyses, we have identified a cis-acting "repressor" element located
immediately upstream of the proximal Sp1 binding site in the ADA gene promoter.
This repressor element was localized to a binding site for the immediate-early,
serum-responsive, DNA binding factor Zif268. This Zif268 binding site partially
overlaps a binding site for the general transcription activator Sp1. Disruption
of the Zif268 binding site without disturbing the Sp1 binding motif abolished
Zif268 binding and resulted in significantly elevated promoter function.
Conversely, disruption of the proximal consensus Sp1 binding motif without
disturbing the Zif268 binding site resulted in a loss of Sp1 binding at that
region and greatly reduced promoter activity. Our results suggest that one
function of Zif268 may be to down-regulate this type of mammalian gene promoter
by competing with Sp1 for binding to the overlapping binding motif.

PMID: 1881892 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


90: Oncogene. 1991 May;6(5):867-71. 

5' upstream sequence and genomic structure of the human primary response gene,
EGR-1/TIS8.

Sakamoto KM, Bardeleben C, Yates KE, Raines MA, Golde DW, Gasson JC.

Children's Hospital of Los Angeles, Department of Hematology/Oncology,
California 90054-0700.

EGR-1/TIS8 is a primary response gene that encodes a zinc finger containing
protein and is induced in a number of cell types by a variety of ligands. We
have isolated and mapped the human EGR-1/TIS8 gene and sequenced the 5' upstream
flanking region. A 'TATA' homology and several putative regulatory elements,
including two Sp1 sites, five serum response-like elements, two cAMP
response-like elements, an EGR-1 binding site (EBS), and a tetra-decanoyl
phorbol acetate (TPA)-responsive element have been identified within 700
nucleotides of the upstream region. We demonstrated that a 500-base pair
fragment, which includes several of these possible regulatory sequences, is
functional and responsive to TPA in transient transfection assays. A further
understanding of the regulation and function of human EGR-1/TIS8 gene expression
may provide insight into the mechanisms that control normal and neoplastic
proliferation of human cells.

PMID: 2052361 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


91: Mol Cell Biol. 1990 Jul;10(7):3456-67. 

The serum-inducible mouse gene Krox-24 encodes a sequence-specific
transcriptional activator.

Lemaire P, Vesque C, Schmitt J, Stunnenberg H, Frank R, Charnay P.

Laboratoire de Genetique Moleculaire, Centre National de la Recherche
Scientifique D 1302, Ecole Normale Superieure, Paris, France.

The mouse gene Krox-24 is transiently activated during cell cycle reentry. It
encodes a protein with three zinc fingers similar to those of the transcription
factor Sp1. Here we present a biochemical characterization of the gene products.
Krox-24 mRNA is translated into two proteins of 82 and 88 kilodaltons,
designated p82Krox-24 and p88Krox-24, respectively. p82Krox-24 is initiated at
the first AUG codon of the open reading frame, whereas synthesis of p88Krox-24
starts at a non-AUG codon located upstream. Both proteins were synthesized in
HeLa cells infected with recombinant vaccinia viruses expressing Krox-24 cDNAs.
Under these conditions, they were found phosphorylated on serine residues and
glycosylated. The availability of the proteins made possible the determination
of the DNA recognition sequence. In vitro, Krox-24 bound specifically to the
sequence 5'-GCG(C/G)GGGCG-3'. This sequence is similar but not identical to the
Sp1 target sequence. Insertion of an oligomer for the binding site in cis, close
to the herpes simplex virus thymidine kinase promoter, rendered this promoter
responsive to Krox-24. Krox-24 is therefore a sequence-specific transcriptional
activator. Krox-24-binding sites were found upstream of several serum-inducible
genes, raising the possibility that Krox-24 is involved in the regulation of
these genes.

PMID: 2113174 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


92: Proc Natl Acad Sci U S A. 1988 Jul;85(13):4691-5. 

Two mouse genes encoding potential transcription factors with identical
DNA-binding domains are activated by growth factors in cultured cells.

Lemaire P, Revelant O, Bravo R, Charnay P.

Differentiation Programme, European Molecular Biology Laboratory, Heidelberg,
Federal Republic of Germany.

We previously reported the identification of a mouse gene, Krox-20, encoding a
protein with three "zinc fingers" (DNA-binding domains with coordinated zinc
ions) whose expression is regulated during G0/G1 transition (cell-cycle
reentry). We now have isolated cDNAs corresponding to a related gene, Krox-24.
Krox-24 encodes a protein with zinc fingers nearly identical to those encoded by
Krox-20 and similar to those of transcription factor Sp1. Similarity between
Krox-20 and Krox-24 proteins also extends to several blocks of amino acid
sequence located upstream of the finger region. Like Krox-20, Krox-24 is
transiently activated in quiescent cells after treatment with fetal bovine serum
or purified growth factors. The kinetics of activation are similar to those of
the protooncogene c-fos. The induction does not require de novo protein
synthesis, and cycloheximide treatment of the cells leads to superinduction due,
at least in part, to mRNA stabilization. In the mouse, the two genes are
expressed in a tissue-specific manner, with slightly different patterns. These
properties suggest that Krox-20 and Krox-24 may encode transcription factors
with identical DNA target sequences and that these factors may be involved in
the modulation of cell proliferation and differentiation.

PMID: 3133658 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------