1: Toxicol Sci. 2005 Sep 28; [Epub ahead of print] 

The Aryl Hydrocarbon Receptor Directly Regulates Expression of the Potent
Mitogen Epiregulin.

Patel RD, Kim DJ, Peters JM, Perdew GH.

Graduate Programs in Molecular Medicine and Molecular Toxicology, The Huck
Institutes of the Life Sciences, Department of Veterinary Science and The Center
for Molecular Toxicology and Carcinogenesis, The Pennsylvania State University,
University Park, PA 16802, USA.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is known to cause a large number of
adverse effects, mediated largely by its binding to the aryl-hydrocarbon
receptor (AhR) and subsequent modulation of gene expression. It is thought that
AhR mediates these effects through the untimely and disproportionate expression
of specific genes. However, the exact mechanism, or the genes involved, through
which TCDD leads to these effects is still unknown. This study reports the
discovery of a novel target gene, epiregulin, which is regulated by
TCDD-activated AhR. Epiregulin is a growth regulator which belongs to the
epidermal growth factor (EGF) family. Using real time quantitative PCR (qPCR),
it was established that TCDD upregulates epiregulin gene expression. The
promoter region of epiregulin has a dioxin responsive element (DRE) 56
nucleotides upstream of the transcription start site, along with three potential
Sp1 binding sites. Chromatin immunoprecipitation (ChIP) assays with an anti-AhR
antibody showed promoter occupancy upon TCDD treatment. Luciferase reporter
assays using a vector harboring the first 125 base pairs of the epiregulin rat
promoter revealed an increase in signal on TCDD treatment, which was lost upon
mutation of the DRE. Epiregulin and TCDD treatment mediated a dose-dependent
increase in primary mouse keratinocyte growth. These results demonstrate that
AhR directly increases epiregulin expression, which could play an important role
in TCDD mediated tumor promotion observed in rodent models.

PMID: 16192470 [PubMed - as supplied by publisher]


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2: Clin Cancer Res. 2005 Aug 15;11(16):5793-801. 

Cytochrome P450 1B1 is overexpressed and regulated by hypomethylation in
prostate cancer.

Tokizane T, Shiina H, Igawa M, Enokida H, Urakami S, Kawakami T, Ogishima T,
Okino ST, Li LC, Tanaka Y, Nonomura N, Okuyama A, Dahiya R.

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

PURPOSE: Cytochrome P450 1B1 (CYP1B1), a dioxin inducible member of the CYP
supergene family, is overexpressed in various human malignancies including
prostate cancer. We hypothesized that promoter/enhancer CpG methylation
contributes to the regulation of CYP1B1 expression in human prostate tissue.
EXPERIMENTAL DESIGN: Expression and induction of the CYP1B1 gene in clinical
prostate tissues and prostate cancer cell lines were investigated. The
methylation status of the CYP1B1 gene was analyzed in 175 prostate cancer and 96
benign prostatic hyperplasia samples using methylation-specific PCR (MSP) and
bisulfite-modified DNA sequencing. MSP primers covered dioxin response elements
(DRE) and Sp1 sites that are important for the expression of CYP1B1. RESULTS:
Expressions of CYP1B1 mRNA and protein were increased in prostate cancer. The
aryl hydrocarbon receptor (AhR)/AhR nuclear translocator (ARNT) heterodimer
complex activates gene transcription by binding to the DREs of CYP1B1. In
prostate cancer cells, CYP1B1 mRNA was induced by
2,3,7,8-tetrachlorodigenzo-p-dioxin (TCDD) and/or demethylation agent
(5-aza-2-deoxycytidine). There was no change in the expressions of AhR and ARNT.
Methylation of promoter/enhancer regions was significantly higher in benign
prostatic hyperplasia compared with prostate cancer. MSP-positive patients had
significantly lower risk for prostate cancer as compared with MSP-negative
patients. There was no correlation between CYP1B1 methylation status and
clinicopathologic features.CONCLUSIONS: CYP1B1 is overexpressed in prostate
cancer and regulated by hypomethylation of its promoter/enhancer region. This is
the first report about CYP1B1 regulation in human clinical prostate samples
showing that hypomethylation of the CYP1B1 gene may play an important role in
prostate cancer.

PMID: 16115918 [PubMed - in process]


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3: 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]


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4: Arch Biochem Biophys. 2004 Jan 1;421(1):91-8. 

Single nucleotide polymorphism analysis and functional characterization of the
human Ah receptor (AhR) gene promoter.

Racky J, Schmitz HJ, Kauffmann HM, Schrenk D.

Food Chemistry and Environmental Toxicology, Technical University of
Kaiserslautern, Erwin-Schroedinger-Strasse, D-67663 Kaiserslautern, Germany.

The aryl hydrocarbon receptor (AhR) mediates biological and toxicological
actions of e.g., halogenated aromatic hydrocarbons such as
2,3,7,8-tetrachlorodibenzo-p-dioxin. Although much is known about the
biochemical and molecular mechanisms of AhR action, little is known about the
control of the expression of the AhR gene itself. Therefore, we aimed at the
identification and characterization of regions important for constitutive AhR
gene expression. First, we screened 2.6 kb of the 5(')-flanking region of the
AhR gene in 91 healthy Caucasian volunteers for naturally occurring genetic
variants. Seven variants were detected. However, they do not seem to influence
AhR gene expression in lymphocytes. Using a 2.7 kb AhR promoter luciferase
reporter gene construct and various deletion constructs, a putative regulatory
region was identified and characterized further by electrophoretic mobility
shift assays and site-directed mutagenesis. These investigations were confirmed
by cotransfection experiments in Drosophila SL2 cells. The obtained results
prove an involvement of Sp1 in AhR gene regulation.

PMID: 14678789 [PubMed - indexed for MEDLINE]


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5: J Mol Biol. 2003 Oct 17;333(2):249-60. 

Proteasome inhibition induces nuclear translocation of the dioxin receptor
through an Sp1 and protein kinase C-dependent pathway.

Santiago-Josefat B, Fernandez-Salguero PM.

Departamento de Bioquimica y Biologia Molecular, Facultad de Ciencias,
Universidad de Extremadura, Avenida de Elvas s/n, 06071 Badajoz, Spain.

The dioxin receptor (AhR), in addition to its role in xenobiotic-induced
carcinogenesis, appears to participate in cell proliferation, differentiation
and organ homeostasis. Understanding potential mechanisms of activation of this
receptor in the absence of exogenous ligands is therefore important to study its
contribution to endogenous cellular functions. Using mouse embryo primary
fibroblasts, we have previously shown that proteasome inhibition increased AhR
transcriptional activity in the absence of xenobiotics. We suggested that
proteasome inhibition-dependent AhR activation could involve an increase in the
expression of the partner protein dioxin receptor nuclear translocator (ARNT).
Since ARNT over-expression induced nuclear translocation of the AhR, and
ARNT-deficient cells were unable to translocate this receptor to the nucleus
upon proteasome inhibition, we have analyzed the effect of proteasome inhibition
on the expression of regulatory proteins controlling ARNT levels. Treatment with
the proteasome inhibitor MG132 increased endogenous Sp1 phosphorylation and its
DNA-binding activity to the ARNT promoter. Sp1 phosphorylation and binding to
the ARNT promoter, ARNT over-expression and AhR nuclear translocation were
inhibited by GF109203X, a protein kinase C-specific inhibitor. In addition,
MG132 stimulated protein kinase C activity in MEF cells with a pattern similar
to that observed for ARNT expression. These data suggest that cellular control
of protein kinase C activity, through Sp1 and ARNT, could regulate AhR
transcriptional activity in the absence of xenobiotics.

PMID: 14529614 [PubMed - indexed for MEDLINE]


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6: J Biochem (Tokyo). 2003 May;133(5):583-92. 

Critical enhancer region to which AhR/ARNT and Sp1 bind in the human CYP1B1
gene.

Tsuchiya Y, Nakajima M, Yokoi T.

Division of Drug Metabolism, Faculty of Pharmaceutical Sciences, Kanazawa
University, 13-1 Takara-machi, Kanazawa 920-0934, Japan.

Cytochrome P450 (CYP) 1B1 is known to be induced by polycyclic aromatic
hydrocarbons including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The
constitutive and TCDD-inducible transcriptional expression of human CYP1B1 is
known to be cell-specific. In order to identify the cis-elements that
cell-specifically regulate the constitutive and TCDD-inducible transcription of
CYP1B1, we constructed luciferase reporter plasmids containing a series of
deletions of the XRE core sequence in the 5'-flanking region of the human CYP1B1
gene. Luciferase assays were performed with MCF-7 (breast carcinoma), HepG2
(hepatocellular carcinoma), LS-180 (colon carcinoma), and OMC-3 (ovarian
carcinoma) cells. Although there were large differences in the relative
luciferase activity and inducibility between these four cell lines, the
contribution of each reporter construct was similar. Constitutive expression
increased with the regulatory elements that are present at -910 to -852 and
-1652 to -1243. Potential enhancer elements for TCDD-induction were located from
-1022 to -852 including three XREs, XRE3 at -853, XRE4 at -940, and XRE5 at
-989. Gel shift analyses revealed binding of the AhR/ARNT heterodimer to XRE2 at
-834, XRE3 at -853, XRE6 at -1024, and XRE7 at -1490. In addition, the binding
of a nuclear transcriptional factor, Sp1, near XRE2 and XRE8 was observed. It
was suggested that mutual interaction of XRE2 and XRE3 is important for
transcriptional regulation, and that the Sp1 binding to the Sp1-like motif
(-824) enhances both the constitutive and inducible transcriptional activities
of the human CYP1B1 gene.

PMID: 12801909 [PubMed - indexed for MEDLINE]


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7: J Biol Chem. 2001 Aug 31;276(35):33101-10. Epub 2001 Jun 21. 

Structure and expression of the Ah receptor repressor gene.

Baba T, Mimura J, Gradin K, Kuroiwa A, Watanabe T, Matsuda Y, Inazawa J, Sogawa
K, Fujii-Kuriyama Y.

Department of Biomolecular Science, Graduate School of Life Science, Tohoku
University, Sendai 980-8578, Japan.

The aryl hydrocarbon receptor (AhR) repressor (AhRR) gene has been isolated and
characterized from a mouse genomic library. The gene is distributed as 11 exons
in a total length of about 60 kilobase pairs. Fluorescence in situ hybridization
analysis has shown that the AhRR gene is located at mouse chromosome 13C2, at
rat chromosome 1p11.2, and at human chromosome 5p15.3. The AhRR gene has a
TATA-less promoter and several transcription start sites. In addition, putative
regulatory DNA sequences such as xenobiotic responsive element (XRE), GC box,
and NF-kappaB-binding sites have been identified in the 5'-upstream region of
the AhRR gene. Transient transfection analyses of HeLa cells with reporter genes
that contain deletions and point mutations in the AhRR promoter revealed that
all three XREs mediated the inducible expression of the AhRR gene by
3-methylcholanthrene treatment, and furthermore, GC box sequences were
indispensable for a high level of inducible expression and for constitutive
expression. Moreover, by using gel mobility shift assays we were able to show
that the AhR/Arnt heterodimer binds to the XREs with very low affinity, which is
due to three varied nucleotides outside the XRE core sequence. We have also
shown that Sp1 and Sp3 can bind to the GC boxes. Finally, both transient
transfection analysis and gel mobility shift assay revealed that the AhRR gene
is up-regulated by a p65/p50 heterodimer that binds to the NF-kappaB site when
the cells has been exposed to 12-O-tetradecanoylphorbol-13-acetate, and this
inducible expression was further enhanced by cotreatment of
12-O-tetradecanoylphorbol-13-acetate and 3-methylcholanthrene.

PMID: 11423533 [PubMed - indexed for MEDLINE]


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8: Mol Cell Endocrinol. 2001 Feb 14;172(1-2):91-103. 

Transcriptional activation of cathepsin D gene expression by 17beta-estradiol:
mechanism of aryl hydrocarbon receptor-mediated inhibition.

Wang F, Samudio I, Safe S.

Department of Veterinary Physiology and Pharmacology, Texas A & M University,
College Station, TX 77843-4466, USA.

17beta-estradiol (E2) induces cathepsin D gene expression in MCF-7 human breast
cancer cells and this response is inhibited by aryl hydrocarbon receptor (AhR)
agonists, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Analysis of the
cathepsin D gene promoter initially identified a pentanucleotide GCGTG core
dioxin responsive element (DRE) that blocked E2 action by inhibiting formation
of a transcriptionally active estrogen receptor (ER)-Sp1 complex. A second
functional downstream inhibitory DRE (iDRE2) (-130 to -126) has now been
identified in the cathepsin D gene promoter and inhibition of E2-induced
transactivation involves inhibitory AhR crosstalk with the E2-responsive
adenovirus major late promoter element (MLPE) at -124 to -104 in the cathepsin D
gene promoter. The MLPE site primarily binds USF1/USF2 and ERalpha, and gel
mobility shift and DNA footprinting assays show that the AhR complex decreases
binding of these transcription factors to the MLPE.

PMID: 11165043 [PubMed - indexed for MEDLINE]


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9: Mol Endocrinol. 1999 Sep;13(9):1511-21. 

Transcriptional activation of c-fos protooncogene by 17beta-estradiol: mechanism
of aryl hydrocarbon receptor-mediated inhibition.

Duan R, Porter W, Samudio I, Vyhlidal C, Kladde M, Safe S.

Department of Veterinary Physiology and Pharmacology, Texas A&M University,
College Station 77843-4466, USA.

17Beta-estradiol (E2) induced c-fos protooncogene mRNA levels in MCF-7 human
breast cancer cells, and maximal induction was observed within 1 h after
treatment. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) inhibited the E2-induced
response within 2 h. The molecular mechanism of this response was further
investigated using pFC2-CAT, a construct containing a -1400 to +41 sequence from
the human c-fos protooncogene linked to a bacterial chloramphenicol
acetyltransferase (CAT) reporter gene. In MCF-7 cells transiently transfected
with pFC2-CAT, 10 nM E2 induced an 8.5-fold increase of CAT activity, and
cotreatment with 10 nM TCDD decreased this response by more than 45%.
Alpha-Naphthoflavone, an aryl hydrocarbon receptor (AhR) antagonist, blocked the
inhibitory effects of TCDD; moreover, the inhibitory response was not observed
in variant Ah-nonresponsive MCF-7 cells, suggesting that the AhR complex was
required for estrogen receptor cross-talk. The E2-responsive sequence (-1220 to
-1155) in the c-fos gene promoter contains two putative core pentanucleotide
dioxin-responsive elements (DREs) at -1206 to -1202 and -1163 to -1159. In
transient transfection assays using wild-type and core DRE mutant constructs,
the downstream core DRE (at -1163 to -1159) was identified as a functional
inhibitory DRE. The results of photo-induced cross-linking, gel mobility shift,
and in vitro DNA footprinting assays showed that the AhR complex interacted with
the core DRE that also overlapped the E2-responsive GC-rich site (-1168 to
-1161), suggesting that the mechanism for AhR-mediated inhibitory effects may be
due to quenching or masking at the Sp1-binding site.

PMID: 10478842 [PubMed - indexed for MEDLINE]


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10: Biochemistry. 1999 Aug 31;38(35):11490-500. 

Regulation of constitutive gene expression through interactions of Sp1 protein
with the nuclear aryl hydrocarbon receptor complex.

Wang F, Wang W, Safe S.

Department of Veterinary Physiology and Pharmacology, Texas A&M University,
College Station 77843-4466, USA.

The region of residues -145 to -119 (CD/L) of the cathepsin D gene promoter
contains a GC-rich motif that binds Sp1 protein and an adjacent pentanucleotide
(CACGC) that corresponds to the core sequence of a dioxin responsive element
(DRE) and binds the aryl hydrocarbon receptor (AhR)-AhR nuclear translocator
(Arnt) complex. This Sp1(N)(4)DRE(core) motif has been identified in promoters
of several genes in which Sp1 plays an important role in basal gene expression.
In transient transfection assays with MCF-7 human breast cancer cells using
wild-type pCD/L and constructs mutated in the core DRE (pCD/L(m1)) and Sp1
(pCD/L(m2)) sites, it was shown that both motifs were required for maximal basal
activity. The requirements for AhR-Arnt interactions with Sp1 protein for
maximal activity of pCD/L were confirmed in wild-type MCF-7 and Hepa 1c1c7 cells
and Arnt-deficient Hepa 1c1c7 cells using antisense Arnt and Arnt expression
plasmids. The functional interactions of Sp1 with AhR-Arnt were paralleled by
physical interactions showing that AhR-Arnt and Sp1 proteins were
co-immunoprecipitated and AhR-Arnt enhanced Sp1-[(32)P]CD/L binding in
electrophoretic mobility shift assays. The physical and functional interactions
of Sp1 with AhR-Arnt proteins bound to the Sp1(N)(4)DRE(core) motif were also
dependent on the proximity of these sites, and both the activity and the extent
of Sp1-DNA binding decreased as the number of intervening nucleotides increased
from 4 to 20. These studies show that regulation of basal expression of some
genes by Sp1 may also require interactions with AhR-Arnt.

PMID: 10471301 [PubMed - indexed for MEDLINE]


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11: DNA Cell Biol. 1998 Sep;17(9):811-22. 

Regulation of mouse Ah receptor (Ahr) gene basal expression by members of the Sp
family of transcription factors.

Fitzgerald CT, Nebert DW, Puga A.

Center for Environmental Genetics and Department of Environmental Health,
University of Cincinnati Medical Center, OH 45267-0925, USA.

The aromatic hydrocarbon receptor (AHR) is a ligand-activated transcription
factor that regulates the expression of several drug-metabolizing enzymes and
has been implicated in immunosuppression, teratogenesis, cell-specific
hyperplasia, and certain types of malignancies and toxicities. The mouse Ahr
gene 5' proximal promoter region, which contains four potential Sp1 motifs, is
required for efficient basal expression. Using a fragment spanning the region
from nt -174 to +70 of the Ahr promoter, we found that four regions
corresponding to four Sp1 sites were protected from DNase I digestion using
nuclear extracts from MLE-12 (lung), F9 (embryonal carcinoma), Hepa-1
(hepatoma), and 41-5a (epidermal) cells. The Hepa-1 and F9 cell lines were shown
by reverse transcriptase-polymerase chain reaction and Western blot to contain
mRNA and protein for Sp1 and Sp3, but not Sp2 and Sp4. In electrophoretic
mobility shift assays using oligonucleotide probes corresponding to the four Ahr
Sp1 sites, nuclear extracts from Hepa-1 and F9 cells formed complexes that were
determined immunologically to contain both Sp1 and Sp3 protein. The two Ahr
proximal Sp1 sites (A and B) were shown to bind both Sp1 and Sp3 proteins,
whereas the more distal sites (C and D) bound only Sp1. Competition gel shift
experiments showed that sites A and B had 10-fold higher affinity for Sp factors
than did sites C and D. To determine the transactivation potential of each of
the four Ahr Sp1 sites, we fused the Ahr promoter to a luciferase (LUC) reporter
gene and transfected the construct into the Drosophila cell line Schneider-2,
which contains no Sp1 or Sp1-like factors. Cotransfection of this construct with
expression plasmids for each of the Sp factors revealed that Sp3 was
approximately 1.6-fold more efficient than Sp1 in Ahr transactivation. Mutation
of the four Sp1 sites individually and in combination demonstrated that each
site contributes to the overall level of expression of the reporter gene and
that interactions between these sites play a minor role in regulation of the
Ahr-LUC construct. These results suggest that basal Ahr expression may be
regulated by the expression and distribution of Sp1-like factors.

PMID: 9778040 [PubMed - indexed for MEDLINE]


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12: J Biol Chem. 1998 Sep 18;273(38):24867-73. 

Structure and expression of the mouse AhR nuclear translocator (mArnt) gene.

Wang F, Gao JX, Mimura J, Kobayashi A, Sogawa K, Fujii-Kuriyama Y.

Department of Chemistry, Graduate School of Science, Tohoku University, Sendai
980-8578, Japan.

Aryl hydrocarbon receptor (AhR) nuclear translocator (Arnt) gene has been
isolated and characterized from a mouse genomic DNA library. The gene is about
60 kilobases long and split into 22 exons. An unusual exon/intron junctional
sequence was found in the 11th intron of the gene that begins with GC at its
5'-end. The exon/intron arrangement of mArnt gene differs greatly from those of
the other members of the same basic-helix-loop-helix/PAS family. The gene is
TATA-less and has several transcription start sites. The promoter region of the
mArnt gene is GC-rich and contains a number of putative regulatory DNA sequences
such as two GC-boxes, a cAMP-responsive element, E-box, AP-1 site, and CAAT-box.
Deletion experiments revealed that all these DNA elements made substantial
contributions to a high level of expression of the gene, except for the
cAMP-responsive element. Of all, two GC-boxes displayed the most dominant
enhancing effects. It was demonstrated that there exist specific factors binding
to these DNA elements in the nuclear extracts of HeLa cells. Among them, Sp1 and
Sp3, and CAAT-box binding factor-A were identified to bind the GC-boxes and
CAAT-box, respectively. Expression of MyoD in HeLa cells stimulated the Arnt
promoter activity by binding to the E-box.

PMID: 9733792 [PubMed - indexed for MEDLINE]


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13: Nucleic Acids Res. 1998 Jun 15;26(12):3044-52. 

Functional and physical interactions between the estrogen receptor Sp1 and
nuclear aryl hydrocarbon receptor complexes.

Wang F, Hoivik D, Pollenz R, Safe S.

Department of Veterinary Physiology and Pharmacology, Texas A&M University,
College Station, TX 77843-4466, USA.

17beta-Estradiol (E2) induces cathepsin D gene expression in MCF-7 human breast
cancer cells and previous analyses of the proximal promoter region of this gene
identified two functional enhancer sequences; namely an
Sp1(N)23estrogen-responsive element (ERE) half-site (-199 to -165) and an
imperfect palindromic ERE (-119 to -107). A third region of the cathepsin D gene
promoter (CD/L, -145 to -119) was also E2 responsive in transient transfection
assays. A GC-rich sequence which contains two overlapping Sp1 binding sites
(-145 to -135) was responsible for ER-mediated transactivation and required
formation of an ER/Sp1 complex in which only the Sp1 protein bound DNA. E2
responsiveness of the CD/L sequence was also dependent on an adjacent
overlapping GCGTG motif corresponding to the dioxin-responsive element (DRE)
core binding sequence, which is the cognate response element for the
heterodimeric aryl hydrocarbon receptor (AhR)/AhR nuclear translocator (ARNT)
transcription factor complex. The results show that ER-mediated transactivation
of CD/L was associated with the Sp1(N)2-4DRE (core) motif and involved formation
of a multiprotein ER/Sp1-AhR/ARNT complex. These results illustrate a unique
example of an endogenous role for AhR/ARNT in the absence of added AhR agonist
and indicate that the cathepsin D gene proximal promoter region contains at
least three different functional motifs associated with ER-mediated
transactivation.

PMID: 9611253 [PubMed - indexed for MEDLINE]


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14: Mol Cell Biol. 1997 Jul;17(7):3497-507. 

Transactivation domains facilitate promoter occupancy for the dioxin-inducible
CYP1A1 gene in vivo.

Ko HP, Okino ST, Ma Q, Whitlock JP Jr.

Department of Molecular Pharmacology, Stanford University School of Medicine,
California 94306-5332, USA.

We have studied the transcriptional regulation of the dioxin-inducible mouse
CYP1A1 gene in its native chromosomal setting. We analyzed the ability of
aromatic hydrocarbon receptor (AhR) mutants and AhR chimeras to restore dioxin
responsiveness to the CYP1A1 gene in AhR-defective mouse hepatoma cells. Our
data reveal that transactivation domains in AhR's C-terminal half mediate
occupancy of the nuclear factor 1 site and TATA box for the CYP1A1 promoter in
vivo. Transactivation domains of VP16 and AhR nuclear translocator, but not Sp1,
can substitute for AhR's C-terminal half in facilitating protein binding at the
promoter. Our data also reveal an apparent linear relationship between promoter
occupancy and CYP1A1 gene expression in chromatin. These findings provide new
insights into the in vivo mechanism of transcriptional activation for an
interesting mammalian gene.

PMID: 9199285 [PubMed - indexed for MEDLINE]


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15: Arch Biochem Biophys. 1996 Sep 1;333(1):170-8. 

Differential regulation of mouse Ah receptor gene expression in cell lines of
different tissue origins.

FitzGerald CT, Fernandez-Salguero P, Gonzalez FJ, Nebert DW, Puga A.

Department of Environmental Health, University of Cincinnati Medical Center,
Ohio 45267-0056, USA.

The dioxin-binding Ah receptor (AHR) is a ligand-activated transcription factor
that regulates the expression of several drug-metabolizing enzymes and has been
implicated in immunosuppression, teratogenesis, cell-specific hyperplasia, and
certain types of malignancies and toxicities. In order to examine
tissue-specific regulation of the mouse Ah receptor gene (Ahr), we studied
chimeric deletion constructs, containing the Ahr 5' flanking region and the
firefly luciferase reporter gene (Luc). Transient transfection assays were
performed in five established mouse cell lines: Hepa-1c1c7 (derived from
hepatoma), JB6-C1 41-5a (epidermis), MLE-12 (lung epithelium), F9 (embryonal
carcinoma), and NIH/3T3 (fibroblasts). Treatment of the cell lines included:
dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin), retinoic acid (RA), cyclic
adenosine 3':5'-monophosphate (cAMP), or 12-O-tetradecanoylphorbol 13-acetate
(TPA). Expression levels of Luc varied widely from one untreated cell line to
another, this finding was also confirmed by measurements of AHR mRNA
steady-state levels. In all cell lines except F9 cells, maximal constitutive
expression was observed with constructs containing 78 bp of Ahr promoter
sequences, which include several putative binding sites for the transcription
factor Sp1. In contrast, in F9 cells, inclusion of sequences between -174 and
-78 resulted in a fourfold stimulation of constitutive expression, suggesting
that other transcription factors are important in Ahr gene expression in these
cells. In MLE-12 and 41-5a cells, expression was significantly decreased by
treatment with dioxin, RA, cAMP, or TPA. A similar inhibitory effect was
observed in cAMP-treated MLE-12 and F9 cells; this result was confirmed by
RT-PCR measurements of AHR mRNA steady-state levels. These results indicate that
both up- and down-regulation of the Ahr gene occur and exhibit tissue-and
cell-type specificity.

PMID: 8806768 [PubMed - indexed for MEDLINE]


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16: J Biol Chem. 1996 May 24;271(21):12310-6. 

Cooperative interaction between AhR.Arnt and Sp1 for the drug-inducible
expression of CYP1A1 gene.

Kobayashi A, Sogawa K, Fujii-Kuriyama Y.

Department of Chemistry, Faculty of Science, Tohoku University, Sendai, Japan.

Expression of CYP1A1 gene is regulated in a substrate-inducible manner through
at least two kinds of regulatory DNA elements in addition to the TATA sequence,
XRE (xenobiotic responsive element), and BTE (basic transcription element), a GC
box sequence. The trans-acting factor on the XRE is a heterodimer consisting of
arylhydrocarbon receptor (AhR) and AhR nuclear translocator (Arnt), while Sp1
acts as a regulatory factor on the BTE. We have investigated how these factors
interact with one another to induce expression of the CYP1A1 gene. Both in vivo
transfection assays using Drosophila Schneider line 2 (SL2) cells, which is
devoid of endogenous Sp1, AhR, and Arnt, and in vitro transcription assays using
baculovirus-expressed AhR, Arnt, and Sp1 proteins revealed that these factors
enhanced synergistically expression of the reporter genes driven by a model
CYP1A1 promoter, consisting of four repeated XRE sequences and a BTE sequence,
in agreement with previous observation (Yanagida, A., Sogawa, K., Yasumoto, K.,
and Fujii-Kuriyama, Y. (1990) Mol. Cell. Biol. 10, 1470-1475). We have proved by
coimmunoprecipitation assays and DNase I footprinting that both AhR and Arnt
interact with the zinc finger domain of Sp1 via their basic HLH/PAS domains.
When either the AhR.Arnt heterodimer of Sp1 was bound to its cognate DNA
element, DNA binding of the second factor was facilitated. Survey of DNA
sequences in the promoter region shows that the XRE and GC box elements are
commonly found in the genes whose expressions are induced by polycyclic aromatic
hydrocarbons, suggesting that the two regulatory DNA elements and their cognate
trans-acting factors constitute a common mechanism for induction of a group of
drug-metabolizing enzymes.

PMID: 8647831 [PubMed - indexed for MEDLINE]


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17: J Biol Chem. 1993 Oct 15;268(29):22203-9. 

Molecular characterization of the murine Ahr gene. Organization, promoter
analysis, and chromosomal assignment.

Schmidt JV, Carver LA, Bradfield CA.

Department of Pharmacology, Northwestern University Medical School, Chicago,
Illinois 60611.

The AH receptor is a ligand-activated transcription factor that mediates the
biological effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin. The AH receptor has
primary sequence homology to its dimerization partner the AH receptor nuclear
translocator, and to the Drosophila proteins Sim and Per. Characterization of
the gene encoding the murine AH receptor (Ahr gene) reveals that its structural
organization is also conserved with respect to the sim gene, since 6 of 11 Ahr
exons are spliced at homologous sites. Interestingly, little splicing homology
was observed between the Ahr and per genes. The promoter of the Ahr gene is
GC-rich and contains no TATA or CCAAT boxes; however, sequence analysis has
shown several binding sites for the transcription factor Sp1 (GC boxes).
Additionally we have identified a potential cAMP response element, AP-1 and E
box sites, and two elements demonstrated in other genes to confer
placenta-specific expression. Using a restriction fragment length polymorphism
in exon 7 and recombinant inbred mouse lines, the Ahr gene was found to be
concordant with the phenotypically defined Ahr locus, supporting the identity of
these two genetic elements.

PMID: 8408082 [PubMed - indexed for MEDLINE]


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18: Mol Cell Endocrinol. 1990 Feb 12;69(1):51-7. 

The binding of transformed aromatic hydrocarbon (Ah) receptor to its DNA
recognition site is not affected by metal depletion.

Denison MS, Deal RM.

Department of Biochemistry, Michigan State University, East Lansing 48823.

The biological effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin), a
potent environmental contaminant, are mediated by a soluble intracellular
protein, the aromatic hydrocarbon (Ah) receptor (AhR). TCDD:AhR complexes
activate gene transcription by binding to specific DNA sequences termed
dioxin-responsive elements adjacent to TCDD-responsive genes. Analogies between
the AhR and receptors for steroid hormones imply similarities in their mechanism
of action. The presence of chelatable, protein-bound metal(s), presumably zinc,
is required for DNA binding of several proteins, including steroid hormone
receptors and the transcription factor SP1. Utilizing gel retardation and
DNA-cellulose binding assays we have investigated the importance of metal in DNA
binding of transformed TCDD:AhR complexes. Here, we report that although
1,10-phenanthroline, a metal ion chelating agent, inhibited the DNA binding of
SP1 and transformed glucocorticoid receptor, no inhibition of transformed AhR
was observed. EDTA was similarly ineffective in inhibiting DNA binding of
transformed AhR. Our findings suggest that the AhR, although similar to steroid
receptors, appears not to require metals for binding to its specific DNA
recognition sequence.

PMID: 2157617 [PubMed - indexed for MEDLINE]


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