1: Biol Reprod. 2005 Sep;73(3):500-9. Epub 2005 May 11. 

A placenta-specific enhancer of the human syncytin gene.

Cheng YH, Handwerger S.

Department of Pediatrics, University of Cincinnati College of Medicine and
Division of Endocrinology, Cincinnati Children's Hospital Medical Center,
Cincinnati, Ohio 45229-3039, USA.

The cis- and trans-acting factors that are critical for placenta-specific
expression of the human syncytin gene are unknown. We identified a 146-base pair
(bp) region of the 5'-flanking region of the human syncytin gene from nt-294 to
-148 that is essential for basal gene expression in human BeWo and JEG3
choriocarcinoma cell lines but not in hepatoblastoma and kidney cell lines.
Ligation of the 146-bp fragment to a SV40 promoter or a human beta-globin
minimal promoter markedly enhanced promoter activity in the placenta cells but
not in the liver and kidney cells. DNase I footprint assays indicated that
nuclear extracts from BeWo cells but not HepG2 cells protected four regions
(FP1-FP4) of the 146-bp fragment. Site-directed mutagenesis of an SP1-binding
site in FP3 and a GATA-binding site in FP4 significantly repressed promoter
activity in the placenta cells. Overexpression of SP1 (Sp1 transcription factor)
and GATA2 (GATA binding protein 2) and GATA3 induced syncytin promoter activity
but had little or no effect on the activities of syncytin promoter fragments
containing mutations in the SP1- and GATA-binding sites. GATA2 and -3 mRNA
levels increased markedly during spontaneous in vitro differentiation of human
cytotrophoblast cells when the cytotrophoblast cells fused to form a syncytium.
These findings strongly suggest that the 146-bp region of the 5'-flanking region
(nt-294/-148) of the human syncytin gene acts as a placenta-specific enhancer.
Binding of SP1 and GATA family members to this enhancer is critical for
cell-specific expression of the syncytin gene.

PMID: 15888734 [PubMed - indexed for MEDLINE]


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2: Cell Mol Life Sci. 2005 Jan;62(2):214-26. 

The transcription factor profile of human mast cells in comparison with
monocytes and granulocytes.

Babina M, Schulke Y, Kirchhof L, Guhl S, Franke R, Bohm S, Zuberbier T, Henz BM,
Gombart AF.

Department of Dermatology and Allergy, University Hospital Charite Campus Mitte,
Schumannstr. 20/21, 10117, Berlin, Germany. magda.babina@charite.de

Expression profiles of mRNAs and proteins for various transcription factors were
determined for human skin mast cells (sMCs), leukemic HMC-1 MCs, monocytes and
granulocytes. By quantitative RT-PCR, sMCs expressed lower levels of c-fos,
PU.1, C/EBPalpha, and C/EBPepsilon than monocytes and granulocytes, but higher
levels of MITF, SCL, GATA-1 and GATA-2. At the protein level, MITF, SCL, GATA-2,
Elf-1 and c-fos were clearly detectable in sMCs. With the exception of c-fos,
these proteins were absent or expressed only slightly in monocytes and
granulocytes. The expression of NF-E2p45, GATA-1, PU.1, Ets-1, C/EBPalpha and
C/EBPepsilon was below the detection limit in sMCs, but detectable in other
myelocytes. The high expression of SCL and GATA-2 in sMCs is reminiscent of stem
cells. The absence of C/EBPvarepsilon in sMCs, but strong expression in HMC-1,
suggests it may impair MC maturation. In summary, mature human MCs can be
characterized as C/EBPalpha(low), C/EBPvarepsilon-, PU.1(low), GATA-1(low),
GATA-2+, SCL+, MITF(high).

PMID: 15666093 [PubMed - indexed for MEDLINE]


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3: J Hypertens. 2003 Nov;21(11):2111-24. 

Comment in:
    J Hypertens. 2003 Nov;21(11):2013-4.

Regulation of the major isoform of human endothelin-converting enzyme-1 by a
strong housekeeping promoter modulated by polymorphic microsatellites.

Funke-Kaiser H, Thomas A, Bremer J, Kovacevic SD, Scheuch K, Bolbrinker J, Theis
S, Lemmer J, Zimmermann A, Zollmann FS, Herrmann SM, Paul M, Orzechowski HD.

Institute of Clinical Pharmacology and Toxicology, Charite - Campus Benjamin
Franklin, Berlin, Germany.

BACKGROUND: Human endothelin-converting enzyme (ECE)-1, the key enzyme in
endothelin biosynthesis, shows broad cell and tissue expression within the
cardiovascular system. Expression of ECE-1c, which represents the major ECE-1
isoform, is directed by an alternative promoter, but the mechanisms of ECE-1c
promoter regulation are largely unknown. As ECE-1c transcription is initiated
from several start sites, we hypothesized that the ECE-1c promoter functions as
a housekeeping promoter. OBJECTIVE: To investigate the putative housekeeping
function of the ECE-1c promoter in vascular endothelial cells, which represent a
main site of its expression. RESULTS: Using promoter reporter assays, gel shift
and supershift assays, we have demonstrated, in human endothelial EA.hy926
cells, functionality of cis-acting elements for binding of the CAAT-box binding
protein NF-YB, GATA-2) E2F-2, and a GC-box binding factor, which are spatially
associated with transcriptional start sites of ECE-1c. In the more upstream
promoter region we have identified three highly polymorphic dinucleotide
repeats, 5'-(CA)n, (CG)n and 3'-(CA)n, which strongly affected promoter function
in endothelial EA.hy926 cells (2.7-fold activation comparing the most active to
the least active allele) and, in a similar manner, in human neuronal KELLY
cells. Finally, by in-vitro methylation, we were able to achieve strong
suppression of the ECE-1c promoter activity in endothelial cells. CONCLUSION:
Our results provide a molecular explanation for constitutive expression of
ECE-1c mRNA. Modulation by genetic and epigenetic mechanisms as revealed in our
study may account for interindividual variation of the constitutive endothelin
system activity in humans and thus influence individual predisposition to
cardiovascular disease.

PMID: 14597855 [PubMed - indexed for MEDLINE]


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4: J Biol Chem. 2000 Mar 17;275(11):8183-9. 

Molecular basis of cell-specific endothelial nitric-oxide synthase expression in
airway epithelium.

German Z, Chambliss KL, Pace MC, Arnet UA, Lowenstein CJ, Shaul PW.

Department of Pediatrics, University of Texas Southwestern Medical Center,
Dallas, Texas 75235, USA.

Nitric oxide (NO) plays an important role in airway function, and endothelial NO
synthase (eNOS) is expressed in airway epithelium. To determine the basis of
cell-specific eNOS expression in airway epithelium, studies were performed in
NCI-H441 human bronchiolar epithelial cells transfected with the human eNOS
promoter fused to luciferase. Transfection with 1624 base pairs of sequence 5'
to the initiation ATG (position -1624) yielded a 19-fold increase in promoter
activity versus vector alone. No activity was found in lung fibroblasts, which
do not express eNOS. 5' deletions from -1624 to -279 had modest effects on
promoter activity in H441 cells. Further deletion to -248 reduced activity by
65%, and activity was lost with deletion to -79. Point mutations revealed that
the GATA binding motif at -254 is mandatory for promoter activity and that the
positive regulatory element between -248 and -79 is the Sp1 binding motif at
-125. Electrophoretic mobility shift assays yielded two complexes with the GATA
site and three with the Sp1 site. Immunodepletion with antiserum to GATA-2
prevented formation of the slowest migrating GATA complex, and antiserum to Sp1
supershifted the slowest migrating Sp1 complex. An electrophoretic mobility
shift assay with H441 versus fibroblast nuclei revealed that the slowest
migrating GATA complex is unique to airway epithelium. Thus, cell-specific eNOS
expression in airway epithelium is dependent on the interaction of GATA-2 with
the core eNOS promoter, and the proximal Sp1 binding site is also an important
positive regulatory element.

PMID: 10713142 [PubMed - indexed for MEDLINE]


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5: J Biol Chem. 1998 May 8;273(19):11737-44. 

The human ICAM-2 promoter is endothelial cell-specific in vitro and in vivo and
contains critical Sp1 and GATA binding sites.

Cowan PJ, Tsang D, Pedic CM, Abbott LR, Shinkel TA, d'Apice AJ, Pearse MJ.

Immunology Research Center, St Vincent's Hospital, Fitzroy 3065, Victoria,
Australia.

The expression of intercellular adhesion molecule 2 (ICAM-2) in adult tissues is
restricted to vascular endothelial cells and megakaryocytes. We have previously
shown that the endothelial-specific in vivo activity of the human ICAM-2
promoter is contained within a small (0.33-kilobase (kbp)) 5'-flanking region of
the gene. Here we describe the in vitro characterization of this region. The
ICAM-2 promoter is TATA-less, and transcription in endothelial cells initiates
at four sites. Reporter gene expression directed by the promoter was 125-fold
greater than vector alone in bovine aortic endothelial cells but less than
2-fold vector alone in non-endothelial (COS) cells, confirming that specificity
in vivo was paralleled in vitro. The addition of 2.7 kbp of 5'-flanking region
to the 0.33-kbp fragment had no effect on promoter activity or specificity. The
mutation of an Sp1 motif centered at base pair -194 or an eight-base pair
palindrome at -268 each reduced promoter activity by 70%. Mutation of GATA
motifs at -145 and -53 reduced promoter activity by 78 and 61%, respectively.
Specific binding of bovine aortic endothelial cells nuclear proteins to the Sp1
and GATA sites was demonstrated by gel shift analysis. Promoter activity in COS
cells was transactivated 3-4-fold by overexpression of GATA-2. The results
presented here suggest that transcription from the ICAM-2 promoter in
endothelial cells is regulated by the interplay of several positive-acting
factors and provide the basis for further analysis of endothelial-specific gene
expression.

PMID: 9565596 [PubMed - indexed for MEDLINE]


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6: Endocrinology. 1997 Aug;138(8):3417-25. 

The proximal promoter region of the gene encoding human 17beta-hydroxysteroid
dehydrogenase type 1 contains GATA, AP-2, and Sp1 response elements: analysis of
promoter function in choriocarcinoma cells.

Piao YS, Peltoketo H, Vihko P, Vihko R.

Biocenter Oulu and Department of Clinical Chemistry, University of Oulu,
Finland.

The 5'-flanking region from -78 to +9 in the HSD17B1 gene serves as a promoter,
and an HSD17B1 silencer element is located in position -113 to -78. In the
present studies, we have characterized three regulatory elements in the proximal
5'-flanking regions of the gene, using electrophoretic mobility shift assays and
reporter gene analysis. First, nuclear factors recognized by antibodies against
Sp1 and Sp3 were found to bind the Sp1 motif in the region from -52 to -43.
Mutation of the Sp1-binding site decreased the promoter activity to 30% in JEG-3
cells and to 60% in JAR cells, suggesting that binding to the Sp1 motif has a
substantial role in the complete functioning of the HSD17B1 promoter. Second,
the binding of AP-2 to its motif in the region from -62 to -53 led to reduced
binding of Sp1 and Sp3, and furthermore, mutation of the AP-2 element increased
promoter activity to 260% in JEG-3 cells. The data thus implied that AP-2 can
repress the function of the HSD17B1 promoter by preventing binding to the Sp1
motif. Finally, GATA factors, GATA-3 in particular, were demonstrated to bind
their cognate sequence in the HSD17B1 silencer region, and mutations introduced
into the GATA-binding site increased transcriptional activity to the level seen
in constructs not containing the silencer element. Thus, GATA-3 seems to prevent
transcription in the constructs, and hence, the GATA motif also may operate as a
negative control element for HSD17B1 transcription.

PMID: 9231796 [PubMed - indexed for MEDLINE]


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7: Blood. 1997 Feb 15;89(4):1260-9. 

Characterization of the human platelet/endothelial cell adhesion molecule-1
promoter: identification of a GATA-2 binding element required for optimal
transcriptional activity.

Gumina RJ, Kirschbaum NE, Piotrowski K, Newman PJ.

Blood Research Institute, The Blood Center of Southeastern Wisconsin, Milwaukee
53201-2178, USA.

Platelet/endothelial cell adhesion molecule-1 (PECAM-1) is a 130-kD member of
the Ig gene superfamily that is expressed on platelets, endothelial cells, and
certain leukocyte subsets. To examine the factors controlling vascular-specific
expression of PECAM-1, we cloned the 5'-flanking region of the PECAM-1 gene and
analyzed its transcriptional activity. 5'-Rapid amplification of cDNA ends
(5'-RACE) analysis showed that transcription initiation occurred at several
closely spaced nearby sites originating approximately 204 bp upstream from the
translation start site. Analysis of the sequence immediately upstream from the
transcription initiation site (TIS) showed no canonical TATA or CAAT elements,
however an initiator element commonly found in TATA-less promoters encompassed
the TIS. 5'-serially truncated PECAM-1 promoter segments cloned in front of a
luciferase reporter drove transcription in both a lineage- and
orientation-specific manner. Putative cis-acting control elements present within
a 300-bp core promoter included two ets sites, an Sp1 site, tandem E-box
domains, two GATA-associated sites (CACCC), an AP-2 binding site, and a GATA
element at -24. Mutational analysis showed that optimal transcriptional activity
required the GATA sequence at position -24, and gel-shift assays further showed
that the GATA-2 transcription factor, but not GATA-1, bound to this region of
the PECAM-1 promoter. Understanding the cis- and transacting factors that
regulate the tissue-specific expression of PECAM-1 should increase our
understanding of the mechanisms by which vascular-specific gene expression is
achieved.

PMID: 9028949 [PubMed - indexed for MEDLINE]


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8: J Biol Chem. 1995 Jun 23;270(25):15320-6. 

Functional analysis of the human endothelial nitric oxide synthase promoter. Sp1
and GATA factors are necessary for basal transcription in endothelial cells.

Zhang R, Min W, Sessa WC.

Department of Pharmacology, Boyer Center for Molecular Medicine, Yale University
School of Medicine, New Haven, Connecticut 06536, USA.

To gain insights into the mechanisms of endothelial nitric oxide synthase (eNOS)
gene expression, we have cloned the eNOS promoter and fused it to a luciferase
reporter gene to map regions of the promoter important for basal transcription
in bovine aortic endothelial cells (BAEC). Transfection of BAEC with F1
luciferase (LUC) (-1600 to +22 nucleotides) yielded a 35-fold increase in
promoter. Progressive deletion from -1600 to -1033 (F2 and F3 LUC) did not
significantly influence eNOS promoter activity. Further deletion from -1033 to
-779 (F4 LUC) resulted in an approximate 40% reduction in basal promoter
activity, and still further deletion from -779 to -494 (F5 LUC) did not markedly
influence activity. Deletion from -494 to -166 (F6 LUC) reduced eNOS promoter
activity by 40-50%. Specific mutation of the consensus GATA site (-230) in the
F3 LUC construct reduced luciferase activity (by 25-30%). Gel shift analysis and
antibody depletion using BAEC nuclear extracts demonstrated in vitro binding of
GATA-2 to the oligonucleotide sequence containing the -230 GATA site. Next, we
mutated the Sp1 site (-103) in the F3 and F6 LUC constructs and in the F3 GATA
mutant construct. Expression of these Sp1 mutants in BAEC resulted in a 85-90%
reduction in normalized luciferase activity. Gel shift and antibody supershift
analysis using a BAEC nuclear extracts demonstrated four specific, DNA-protein
complexes binding to the eNOS Sp-1 site, with the slowest migrating form
composed of Sp1 and another nuclear protein. These data demonstrate that the Sp1
site is an important cis-element in the core eNOS promoter.

PMID: 7541039 [PubMed - indexed for MEDLINE]


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9: Bone. 1995 May;16(5):587-93. 

Characterization of the 5'-flanking region of the human tartrate-resistant acid
phosphatase (TRAP) gene.

Reddy SV, Kuzhandaivelu N, Acosta LG, Roodman GD.

Department of Medicine/Hematology, Audie Murphy Veterans Administration
Hospital, University of Texas Health Science Center, San Antonio 78284, USA.

Tartrate-resistant acid phosphatase (TRAP) is expressed at high levels in
osteoclasts and may play an important role in the bone resorptive process.
However, factors regulating human TRAP gene expression have not been clearly
defined. Therefore, we isolated a genomic clone (CL-9) for TRAP containing a
14-kb insert. A restriction map was generated for this insert, and a 2.6-kb ApaI
fragment containing the 5'-flanking region was subcloned. Sequence analysis of
this fragment revealed the presence of candidate transcription factor-binding
sequences for H-APF-1, SP1, GATA2, and the c-Myc proto-oncogene. PCR analysis of
RNA isolated from human osteoclastomas and pagetic bone revealed a 276-bp intron
at -1 bp to -276 bp relative to the ATG and a transcript originating from this
intron. Rapid amplification of the 5' end of the human TRAP mRNA by PCR
indicated the presence of a 93-bp untranslated region 5' from the intron.
Promoter activity was detected in the DNA fragment from +1 bp to -1903 bp
relative to the ATG initiation codon, which drove the transient expression of a
luciferase reporter gene when transfected into HRE H9 rabbit endometrial cells.
Comparison of the human TRAP 5'-flanking region with mouse TRAP and uteroferrin
revealed 41% and 47% homology, respectively. This suggests that regulation of
human TRAP gene expression may differ from that for the murine TRAP gene.

PMID: 7654474 [PubMed - indexed for MEDLINE]


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10: Oncogene. 1994 Sep;9(9):2623-32. 

GATA-and SP1-binding sites are required for the full activity of the
tissue-specific promoter of the tal-1 gene.

Lecointe N, Bernard O, Naert K, Joulin V, Larsen CJ, Romeo PH, Mathieu-Mahul D.

CNRS UMR 9942, Institut de Genetique Moleculaire de Montpellier, France.

The tal-1 gene, which is frequently activated in human T cell acute leukemias
(T-ALLs), codes for a protein of the basic helix-loop-helix family (b-HLH) and
potentially a transcription factor. In human and murine hematopoiesis tal-1 is
expressed during the differentiation of the erythroid, megakaryocytic and
mastocytic cell lineages. The expression of tal-1 appears to be comodulated with
that of the transcription factor GATA-1 gene, suggesting that the GATA-1 protein
may regulate the tal-1 gene activity in these hematopoietic lineages. To get
further insights into the molecular mechanisms that control tal-1 expression, we
have isolated 5' sequences of the murine gene and compared them to their human
counterparts. The 5' flanking sequences from the two genes show several regions
of high homology. The alignment of both sequences enabled us to predict that
similarly, to the human, the mouse gene contains two alternative first exons (Ia
and Ib). Remarkably, in both species, the proximal region of the tissue-specific
exon Ia (i.e. gene segment -122 to +1) contains two GATA-motifs (at -65 and -33)
and one SP-1 consensus binding site (-59). Mobility shift assays demonstrate
that GATA proteins are able to interact with both GATA-motifs in a sequence
specific fashion, but with different efficiencies. Moreover transfection studies
show that the GATA-1 protein directly mediates tal-1 transcription by
interacting with the -122/+1 fragment, defined as a minimal promoter in
erythroid cells. Mutagenesis of the promoter establishes that the -33
GATA-binding site present in this fragment is critical for tal-1 expression in
erythroid cells, but by itself does not lead to full promoter activity. Indeed,
further mutations show that the second -65 GATA-binding site and the binding
motif for SP1 (-59) significantly contribute to the overall activity of the
proximal tal-1 promoter. Altogether, our data provide evidence that GATA-1
cooperates with the transcription factor SP1 to mediate the erythroid-specific
expression of the tal-1 gene.

PMID: 8058326 [PubMed - indexed for MEDLINE]


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11: Proc Natl Acad Sci U S A. 1994 Aug 2;91(16):7737-41. 

PU.1 and GATA: components of a mast cell-specific interleukin 4 intronic
enhancer.

Henkel G, Brown MA.

Department of Microbiology/Immunology, Oregon Health Sciences University,
Portland 97201.

Interleukin 4 (IL-4), a critical immunoregulatory cytokine, is produced by a
subset of T lymphocytes and cells of the mast cell/basophil lineage. There are
cell-specific differences in the regulatory elements that control IL-4
transcription in these two cell types. A 683-bp Bgl II fragment, located within
the second intron of the murine IL-4 gene, was previously shown to exhibit mast
cell-specific enhancer activity. To define critical cis-acting elements that
regulate this enhancer, a series of deletions from the 5' and 3' ends of the Bgl
II fragment were generated. Their effect on enhancer activity was assessed in
IL-4-producing mast cell lines in transient transfection assays. Two
functionally independent subregions, E1 and E2, were defined in this analysis.
Both are required for full enhancer activity. Sequences identical to previously
defined DNA-binding sites for SP1 and GATA are present within E1, and an ets
binding site is located within E2. Although mutation of the SP1 sites had no
effect on enhancer function, alteration of either the GATA or ets site reduced
enhancer activity by 50-60%. Proteins that associate with the IL-4 intronic GATA
and ets sites were detected in mast cell nuclear extracts by mobility-shift
assays. Specific antibodies identified these factors as GATA-1 and GATA-2 and
the ets family member PU.1. GATA-1, GATA-2, and PU.1 exhibit cell-specific
expression, suggesting that these proteins play a critical role in the
lineage-restricted activity of the IL-4 intronic enhancer in mast cells.

PMID: 8052653 [PubMed - indexed for MEDLINE]


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