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PubMed2007

Effects of loss of classical estrogen response element signaling on bone in male mice.

The role of estrogen signaling in the male skeleton via estrogen receptor (ER)-alpha is now well established. ERalpha can elicit responses through either classical estrogen response elements (ERE) pathways or nonclassical, non-ERE pathways. In the present study, we examined the effects of either the attenuation or loss of classical ERalpha signaling on the murine male skeleton. To accomplish this, we crossed male mice heterozygous for a knock-in mutation [nonclassical ERalpha knock-in (NERKI)], which abolishes the ERE-mediated pathway with female heterozygous ERalpha knockout mice (ERalpha+/-) and studied the F1 generation ERalpha+/+, ERalpha+/-, ERalpha+/NERKI, and ERalpha-/NERKI male progeny longitudinally using bone density and histomorphometry. The only ERalpha allele present in ERalpha-/NERKI mice is incapable of classical ERE-mediated signaling, whereas the heterozygous ERalpha+/NERKI mice have both one intact ERalpha and one NERKI allele. As compared with ERalpha+/+ littermates (n=10/genotype), male ERalpha+/NERKI and ERalpha-/NERKI mice displayed axial and appendicular skeletal osteopenia at 6, 12, 20, and 25 wk of age, as demonstrated by significant reductions in total bone mineral density (BMD) at representative sites (areal BMD by dual-energy x-ray absorptiometry at the lumbar vertebrae and femur and volumetric BMD by peripheral quantitative computed tomography at the tibia; P<0.05-0.001 vs. ERalpha+/+). The observed osteopenia in these mice was evident in both trabecular and cortical bone compartments. However, these decreases were more severe in mice lacking classical ERalpha signaling (ERalpha-/NERKI mice), compared with mice in which one wild-type ERalpha allele was present (ERalpha+/NERKI mice). Collectively, these data demonstrate that classical ERalpha signaling is crucial for the development of the murine male skeleton.

Animals↗

PubMed2004

Gene- and cell-type-specific effects of signal transduction cascades on metal-regulated gene transcription appear to be independent of changes in the phosphorylation of metal-response-element-binding transcription factor-1.

Post-translational modification of MTF-1 (metal-response-element-binding transcription factor-1) was suggested to play a role in its metalloregulatory functions. In the present study, pulse labelling and two-dimensional electrophoresis-Western blotting were used to demonstrate that, although MTF-1 is highly modified in vivo, its phosphorylation level does not rapidly change in response to metals, nor does its overall modification pattern. Recombinant MTF-1 was found to serve as an in vitro substrate for casein kinase II, c-Jun N-terminal kinase and protein kinase C, but inhibition of these kinases in vivo did not significantly change the modification pattern of MTF-1. Northern blotting revealed that inhibitors of casein kinase II and c-Jun N-terminal kinase severely attenuate the metal-induced transcription of the native chromatin-packaged metallothionein-I and zinc transporter-1 genes, whereas protein kinase C inhibitors exerted gene- and cell-type-specific effects on the metal regulation and basal expression of these two genes. A chromatin immunoprecipitation assay was used to demonstrate that none of these inhibitors prevent the metal-dependent recruitment of MTF-1 to the MT-I promoter. In brief, results of the present study suggest that protein kinases may not alter the phosphorylation state of MTF-1 during the rapid-response phase to metals, nor do they regulate the metal-dependent formation of a stable MTF-1-chromatin complex. Instead, protein kinases may exert their interdependent effects on metal-induced gene expression by acting on cofactors that interact with MTF-1.

Animals↗

PubMed1999

Characterization of an inverted repeat with a zero spacer (IR0)-type retinoic acid response element from the mouse nuclear orphan receptor TR2-11 gene.

An inverted repeat with zero nucleotides in the spacer (IR0, 5'-GGGTCA CGAACT-3') element was localized in the proximal promoter region of the mouse TR2-11 gene, and characterized as a functional retinoic acid response element (RARE). In gel mobility shift assays, heterodimers of retinoic acid receptor alpha (RARalpha) and retinoid X receptor beta (RXRbeta) bound specifically to this element. Neither receptor alone was able to bind to this element efficiently. The dissociation constant (Kd) with respect to RAR-RXR binding was estimated to be 8 nM. The biological activity of this IR0 element was assessed in a heterologous reporter system. The IR0-containing reporter was induced by RA in COS-1 cells in the presence of exogenously provided RARalpha and RXRbeta. In addition, the IR0 oligomers could be bound by nuclear extracts isolated from COS-1 cells harboring the expression vectors for RARalpha and RXRbeta, but not by extracts isolated from control COS-1 cells. RA responsiveness of this IR0 was further confirmed in P19 cells that expressed endogenous RARs and RXRs. Collectively, these data demonstrated, for the first time, the presence of a natural RARE of the IR0 type, and suggested a potential cross-talk between nuclear orphan receptor TR2-11 and RAR-RXR families.

5' Untranslated Regions↗

PubMed2000

Identification of functional estrogen response elements in the gene coding for the potent angiogenic factor vascular endothelial growth factor.

Vascular endothelial growth factor (VEGF) is a potent stimulator of angiogenesis and a prognostic factor for many tumors including those of endocrine-responsive tissues such as the breast and uterus. We and others have previously shown that VEGF is regulated by estradiol and tamoxifen in the uterus and by estradiol in human breast cancer cells, and pharmacological evidence has suggested that this regulation was mediated by transcriptional activation of the estrogen receptor (ER). This prompted us to investigate whether the VEGF gene contains sequences that bind the ER and confer hormonal inducibility to reporter constructs in the presence of the two ER subtypes. These studies identified two sequences homologous to the consensus estrogen response element, GGTCAnnnTGACC, which bind both ER-alpha and ER-beta. One of these elements is located in the 5'-untranslated region of the VEGF gene (GGGCAaagTGACT), and the other is located in the 3'-untranslated region (GAGCAcccTGCCC). Competition with excess unlabeled oligonucleotides indicates that these two elements bind both ERs specifically, mutations in either half-site of the two elements abolish receptor binding, and ER-alpha- and ER-beta-specific antibodies interact with complexes formed with the corresponding receptor subtypes. In cells containing either ER-alpha or ER-beta, the 3'-element behaves as a traditional enhancer that confers hormone inducibility to reporter constructs in an orientation-independent manner, and transcriptional activity is blocked by the pure antiestrogen ICI 182,780. The pattern of transcriptional activity of the element located in the 5'-flanking region is more complex. In the orientation found in the endogenous gene, this element is nonresponsive to ER-beta but confers estrogen-dependent inhibition of transcription with ER-alpha that is blunted by ICI 182,780. In the opposite orientation, the 5'-element confers hormone inducibility with either ER-alpha or -beta, and ICI 182,780 blocks activation by ER-alpha but not by ER-beta. These findings support the hypotheses that estrogens directly regulate VEGF transcription in target tissues and tumors, although such regulation appears likely to involve a complex interplay of cis- and trans-acting elements not previously observed for other hormone-responsive genes.

3' Untranslated Regions↗

PubMed2004

Anatomy of the estrogen response element.

Estrogens exert their regulatory potential on gene expression through different nuclear and non-nuclear mechanisms. A direct nuclear approach is the interaction of estrogen with specific target sequences of DNA, estrogen response elements (ERE) or units. EREs can be grouped into perfect and imperfect palindromic sequences with the imperfect sequences differing from the consensus sequence in one or more nucleotides and being less responsive to the activated estrogen-estrogen receptor (ER) complex. Differences in the ERE sequence and the ER subtype involved can substantially alter ER-ERE interaction. In addition, cross-talk between ERs and other nuclear transcription factors profoundly influences gene expression. Here, we focus on the recent advances in the understanding of the structure of EREs and how ERs are recruited to these. Identifying known target genes for estrogen action could help us to understand the potential risks and benefits of the administration of this steroid to humans.

Base Sequence↗

PubMed1997

Interplay of the E box, the cyclic AMP response element, and HTF4/HEB in transcriptional regulation of the neurospecific, neurotrophin-inducible vgf gene.

vgf is a neurotrophin response-specific, developmentally regulated gene that codes for a neurosecretory polypeptide. Its transcription in neuronal cells is selectively activated by the neurotrophins nerve growth factor (NGF), brain-derived neurotrophic factor, and neurotrophin 3, which induce survival and differentiation, and not by epidermal growth factor. We studied a short region of the rat vgf promoter which is essential for its regulated expression. A cyclic AMP response element (CRE) within this region is necessary for NGF induction of vgf transcription. Two sites upstream of CRE, an E box and a CCAAT sequence, bind nuclear protein complexes and are involved in transcriptional control. The E box has a dual role. It acts as an inhibitor in NIH 3T3 fibroblasts, together with a second E box located downstream, and as a stimulator in the NGF-responsive cell line PC12. By expression screening, we have isolated the cDNA for a basic helix-loop-helix transcription factor, a homolog of the HTF4/HEB E protein, that specifically binds the vgf promoter E box. The E protein was present in various cell lines, including PC12 cells, and was a component of a multiprotein nuclear complex that binds the promoter in vitro. The E box and CRE cooperate in binding to this complex, which may be an important determinant for neural cell-specific expression.

3T3 Cells↗

PubMed1999

Dual blockade of cyclic AMP response element- (CRE) and AP-1-directed transcription by CRE-transcription factor decoy oligonucleotide. gene-specific inhibition of tumor growth.

Alteration of gene transcription by inhibition of specific transcriptional regulatory proteins has important therapeutic potential. Synthetic double-stranded phosphorothioate oligonucleotides with high affinity for a target transcription factor can be introduced into cells as decoy cis-elements to bind the factors and alter gene expression. The CRE (cyclic AMP response element)-transcription factor complex is a pleiotropic activator that participates in the induction of a wide variety of cellular and viral genes. Because the CRE cis-element, TGACGTCA, is palindromic, a synthetic single-stranded oligonucleotide composed of the CRE sequence self-hybridizes to form a duplex/hairpin. Herein we report that the CRE-palindromic oligonucleotide can penetrate into cells, compete with CRE enhancers for binding transcription factors, and specifically interfere with CRE- and AP-1-directed transcription in vivo. These oligonucleotides restrained tumor cell proliferation, without affecting the growth of noncancerous cells. This decoy oligonucleotide approach offers great promise as a tool for defining cellular regulatory processes and treating cancer and other diseases.

Blotting, Western↗

PubMed1999

Androgen regulation of the cyclin-dependent kinase inhibitor p21 gene through an androgen response element in the proximal promoter.

Androgen is essential for the physiological maintenance of the integrity of prostatic epithelial cells, and castration causes the cells to undergo apoptosis. To study the molecular mechanism of androgen-dependent cell growth, we showed that androgen up-regulates the expression of the cyclin-dependent kinase inhibitor p21 (WAF1, CIP1, SDI1, CAP20) gene at both the mRNA and protein levels. Nuclear run-on assays demonstrated that androgen stimulates endogenous p21 gene expression at the transcriptional level. Transient transfection experiments showed that androgen can enhance the activity of a 2.4-kb promoter of the p21 gene linked to a luciferase reporter. These results suggested that a putative androgen response element (ARE), which mediates androgen response to enhance the p21 transcription, is included in the 2.4-kb promoter fragment. Deletion analysis of the promoter revealed a functional ARE (AGCACGCGAGGTTCC) located at -200 bp of the p21 gene proximal to the promoter region. Electrophoretic mobility shift assay further demonstrated that the androgen receptor specifically binds to this element. Wild-type ARE, but not mutant ARE, confers androgen responsiveness to a heterologous promoter. The up-regulation of p21 gene expression by androgen suggests that p21 may have an antiapoptotic function in prostatic epithelial cells. However, this hypothesis will need to be tested in future experiments.

Androgens↗

PubMed2006

Characterization of calcineurin-dependent response element binding protein and its involvement in copper-metallothionein gene expression in Neurospora.

In continuation of our recent observations indicating the presence of a lone calcineurin-dependent response element (CDRE) in the -3730bp upstream region of copper-induced metallothionein (CuMT) gene of Neurospora [K.S. Kumar, S. Dayananda, C. Subramanyam, Copper alone, but not oxidative stress, induces copper-metallothionein gene in Neurospora crassa, FEMS Microbiol. Lett. 242 (2005) 45-50], we isolated and characterized the CDRE-binding protein. The cloned upstream region of CuMT gene was used as the template to specifically amplify CDRE element, which was immobilized on CNBr-activated Sepharose 4B for use as the affinity matrix to purify the CDRE binding protein from nuclear extracts obtained from Neurospora cultures grown in presence of copper. Two-dimensional gel electrophoresis of the affinity purified protein revealed the presence of a single 17kDa protein, which was identified and characterized by MALDI-TOF. Peptide mass finger printing of tryptic digests and analysis of the 17kDa protein matched with the regulatory beta-subunit of calcineurin (Ca(2+)-calmodulin dependent protein phosphatase). Parallel identification of nuclear localization signals in this protein by in silico analysis suggests a putative role for calcineurin in the regulation of CuMT gene expression.

Amino Acid Sequence↗

PubMed2004

MINT, the Msx2 interacting nuclear matrix target, enhances Runx2-dependent activation of the osteocalcin fibroblast growth factor response element.

Msx2 promotes osteogenic lineage allocation from mesenchymal progenitors but inhibits terminal differentiation demarcated by osteocalcin (OC) gene expression. Msx2 inhibits OC expression by targeting the fibroblast growth factor responsive element (OCFRE), a 42-bp DNA domain in the OC gene bound by the Msx2 interacting nuclear target protein (MINT) and Runx2/Cbfa1. To better understand Msx2 regulation of the OCFRE, we have studied functional interactions between MINT and Runx2, a master regulator of osteoblast differentiation. In MC3T3E1 osteoblasts (with endogenous Runx2 and FGFR2), MINT augments transcription driven by the OCFRE that is further enhanced by FGF2 treatment. OCFRE regulation can be reconstituted in the naïve CV1 fibroblast cell background. In CV1 cells, MINT synergizes with Runx2 to enhance OCFRE activity in the presence of activated FGFR2. The RNA recognition motif domain of MINT (which binds the OCFRE) is required. Runx2 structural studies reveal that synergy with MINT uniquely requires Runx2 activation domain 3. In confocal immunofluorescence microscopy, MINT adopts a reticular nuclear matrix distribution that overlaps transcriptionally active osteoblast chromatin, extensively co-localizing with the phosphorylated RNA polymerase II meshwork. MINT only partially co-localizes with Runx2; however, co-localization is enhanced 2.5-fold by FGF2 stimulation. Msx2 abrogates Runx2-MINT OCFRE activation, and MINT-directed RNA interference reduces endogenous OC expression. In chromatin immunoprecipitation assays, Msx2 selectively inhibits Runx2 binding to OC chromatin. Thus, MINT enhances Runx2 activation of multiprotein complexes assembled by the OCFRE. Msx2 targets this complex as a mechanism of transcriptional inhibition. In osteoblasts, MINT may serve as a nuclear matrix platform that organizes and integrates osteogenic transcriptional responses.

Amino Acid Motifs↗

PubMed2001

Thyroid Hormone Stimulates Renin Gene Expression Through the Thyroid Hormone Response Element.

-We previously reported that thyroid hormone stimulates renin synthesis in vivo and in vitro. Here, we analyzed the 5'-flanking sequence of the human renin gene for promoter activity responsive to thyroid hormone using Calu-6 cells, which secrete renin endogenously and express thyroid hormone receptor-ss. The luciferase reporter gene was cloned together with 5'-flanking portions of the human renin gene of various lengths into the pGL3-Basic vector. Luciferase activity assays were performed using the Dual Luciferase Reporter Assay System. 3,3',5-Triiodo-L-thyronine stimulated the promoter activity of pGL3-Basic-1111/+12 and pGL3-Basic-1298/+12 by 2.3+/-0.1- and 1.7+/-0.1-fold, respectively. Shorter constructs (pGL3-Basic-144/+12, pGL3-Basic-226/+12, pGL3-Basic-452/+12, and pGL3-Basic-953/+12) were not stimulated by thyroid hormone. These results suggest that there is a possible thyroid hormone response element (5'-AGG TCA GGT CAc aat GTT CCT-3') between nucleotides -1111 and -953. In 3 constructs with site-directed mutations in this sequence, basal promoter activities were significantly increased, whereas promoter activation by thyroid hormone was abolished. Electrophoretic mobility shift assays showed that the -1111/-953 DNA fragment of the intact human renin gene was bound to nuclear proteins of Calu-6 cells; however, none of the 3 mutant probes were bound to any nuclear proteins. These results suggest that thyroid hormone stimulates the promoter activity of the human renin gene through thyroid hormone response element-dependent mechanisms in Calu-6 cells.

Journal Article↗

PubMed1996

A Purkinje cell protein-2 intronic thyroid hormone response element binds developmentally regulated thyroid hormone receptor-nuclear protein complexes.

Two thyroid hormone response elements (TREs), designated A1 TRE (-295/-268) and B1 TRE (+207/+227), have been identified within the Purkinje cell-expressed Pcp-2 gene. Previous studies have characterized the A1 TRE (Zou et al., 1994). This article analyzes the structural and functional characteristics of the intronic B1 TRE. The B1 sequence contains four overlapping TRE half-sites. The 3' DR4 motif, consisting of the second and forth half-sites, is responsible for the T3 induction observed with the B1 sequence. Gel-shift analysis reveals developmentally regulated complexes that are abundant in the fetus and at birth and then fall precipitously in the neonate bind to B1. The observed time-course of these complexes varies inversely with the rise in Pcp-2 expression, thus raising the possibility that the complexes may represent inhibitory factors. Supershift analysis indicates that endogenous TR alpha 1 is present in the fetal nuclear protein complexes that bind to B1. Competition analysis also indicates the second B1 TRE half-site is important in binding the TR alpha 1-TRAP complexes. These studies suggest that the B1 sequence may bind potential TR alpha 1-TRAP repressor complexes in the fetus, whereas in the neonate, these TRE sites may be involved in the activation of Pcp-2 by binding other TR-TRAP-activating complexes.

Animals↗

PubMed2003

Analysis of G/A polymorphism in the androgen response element I of the PSA gene and its interactions with the androgen receptor polymorphisms.

OBJECTIVES: To compare the in vitro functional activity of the prostate-specific antigen (PSA) androgen response element (ARE) I alleles alone or in combination with androgen receptor (AR) polymorphisms and to determine the association of ARE I alleles with serum PSA in men without clinical prostatic disease. Data are conflicting regarding the association of PSA promoter alleles with serum PSA in men. METHODS: In vitro functional analyses of ARE I and AR polymorphisms were conducted by luciferase reporter assays in LNCaP and PC-3 cells. Associations among serum PSA, ARE I, and AR genotypes were determined by genotyping 109 white and 71 African-American men determined to be free of clinical prostatic disease. RESULTS: We found no significant difference in the androgen responsiveness of the two alleles when cells were transfected with PSA promoter reporter constructs differing only in the ARE I single nucleotide polymorphism and treated with varying doses of androgen. The response to androgens of the ARE I alleles co-transfected with AR expression vectors of 9, 21, and 29 CAG repeat lengths were identical. No individual or combined effects of the ARE I genotype and the AR genotype on serum PSA were noted. CONCLUSIONS: Our data indicate that ARE I polymorphisms, alone or in combination with AR polymorphisms, have no functional effect on the activity of the PSA promoter in vitro and in vivo.

Adult↗

PubMed2003

A jasmonate-responsive element within the A. thaliana vsp1 promoter.

The vsp1 gene of Arabidopsis thaliana encodes a storage protein that accumulates in vegetative organs. Transgenic plants expressing a vsp1 promoter-gus (beta-glucuronidase) gene fusion were found to contain high GUS activity when challenged with jasmonate, a volatile plant hormone. The induction of vsp1-gus expression by jasmonate could be measured in tobacco mesophyll protoplasts, after transient expression. A number of deletions were operated in the vsp1 promoter in order to locate its jasmonate-responsive element. A 41 bp sequence taken approximately 150 bp upstream of the vsp1 TATA box could confer jasmonate responsiveness upon a short CaMV 35S promoter. Whereas the deletion of a CAAT box-like element within the 41 bp sequence did not affect promoter activity, mutation of a short palindromic motif completely abolished jasmonate responsiveness. This motif shows no homology with the jasmonate-responsive elements of other promoters.

Arabidopsis↗

PubMed2002

Cell type-specific replication of simian virus 40 conferred by hormone response elements in the late promoter.

The late genes of SV40 are not expressed at significant levels until after the onset of viral DNA replication. We previously identified two hormone response elements (HREs) in the late promoter that contribute to this delay. Mutants defective in these HREs overexpress late RNA at early, but not late, times after transfection of CV-1PD cells. Overexpression of nuclear receptors (NRs) that recognize these HREs leads to repression of the late promoter in a sequence-specific and titratable manner, resulting in a delay in late gene expression. These observations led to a model in which the late promoter is repressed at early times after infection by NRs, with this repression being relieved by titration of these repressors through simian virus 40 (SV40) genome replication to high copy number. Here, we tested this model in the context of the viral life cycle. SV40 genomes containing mutations in either or both HREs that significantly reduce NR binding without altering the coding of any proteins were constructed. Competition for replication between mutant and wild-type viruses in low-multiplicity coinfections indicated that the +1 HRE offered a significant selective advantage to the virus within a few cycles of infection in African green monkey kidney cell lines CV-1, CV-1P, TC-7, MA-134, and Vero but not in CV-1PD' cells. Interestingly, the +55 HRE offered a selective disadvantage in MA-134 cells but had no effect in CV-1, CV-1P, TC-7, Vero, and CV-1PD' cells. Thus, we conclude that these HREs are biologically important to the virus, but in a cell type-specific manner.

Animals↗

PubMed2003

Mutational analysis of the mouse aryl hydrocarbon receptor tyrosine residues necessary for recognition of dioxin response elements.

Tyrosine phosphorylation of the aryl hydrocarbon receptor (AhR), a member of the basic helix-loop-helix/PER-ARNT-SIM transcription factor family, has been shown to regulate its dioxin response elements (DRE) binding ability, although no specific residues have been directly demonstrated to be phosphorylated. Of the 23 tyrosines in the mouse AhR, 19 are conserved across all mammalian species sequenced thus far. The studies presented here were conducted to examine tyrosine residue(s) that are both likely candidates of phosphorylation and necessary for DNA binding and/or transcriptional activity of the AhR. Two-dimensional gel electrophoresis of phosphatase-treated AhR indicated that the receptor is phosphorylated on serine/threonine and tyrosine residues. Computational analysis predicted several highly conserved tyrosine residues to be phosphorylated. Both the N terminus (amino acids 1-399) and the C terminus (amino acids 399-805) of the mouse receptor synthesized in vitro using a rabbit reticulocyte lysate system are tyrosine phosphorylated as detected by antiphosphotyrosine antibodies. Furthermore, the N-terminal AhR bound DRE in a ligand-dependent manner similar to that by the full-length receptor, suggesting that phosphorylated tyrosines involved in DNA binding are likely located in the region between residues 1 and 399. Mouse AhR tyrosine (Y) residues were evaluated by phenylalanine (F) mutational analysis for both DNA binding (electrophoretic mobility shift assays; EMSAs) and ability to induce a DRE-driven reporter gene in transiently transfected AhR-deficient cells. Of the 12 tyrosine residues in the N-terminal AhR, only a tyrosine 9 mutant (AhRY9F) significantly decreased DRE binding as determined by EMSA. Similarly, only the AhRY9F mutant decreased the DRE-driven luciferase expression in AhR-deficient cells. Overall, these data strongly suggest that the putative posttranslational modification at, or mediated by, tyrosine 9, and not any other individual mouse AhR tyrosine residue, is necessary for AhR DRE binding and transcriptional activity.

Animals↗

PubMed2003

Identification of a novel Nrf2-regulated antioxidant response element (ARE) in the mouse NAD(P)H:quinone oxidoreductase 1 gene: reassessment of the ARE consensus sequence.

NQO1 [NAD(P)H:quinone oxidoreductase 1] has an integral role in cellular responses to oxidative stress. The expression of NQO1 is up-regulated in the mouse following challenge with electrophilic chemicals, in an Nrf2 (NF-E2 p45-related factor 2)-dependent fashion, but the molecular basis for this observation remains unexplained. Through characterization of the murine nqo1 5'-upstream region, we now show that Nrf2 regulates this gene directly via an ARE (antioxidant response element) that lies within a 24 bp region spanning nt -444 to -421. A comprehensive mutation study of this ARE revealed that it does not conform to the currently accepted ARE consensus sequence [(5'-TMAnnRTGAYnnnGCRwwww-3', with essential nucleotides shown in capitals); two cytosine residues (shown in bold in the following sequence) that have been designated 'n' previously because they were thought to be redundant (5'-gagTcA C aGTgAGt C ggCAaaatt-3') have now been found to be essential for enhancer activity; two guanines (also shown in bold) previously regarded as essential for ARE function (5'-gagTcACaGT g AGtCg g CAaaatt-3') have proven to be dispensable]. Examination of wild-type and nrf2 (-/-) mouse embryonic fibroblasts demonstrated that Nrf2 is essential for both constitutive expression of NQO1 and its induction by sulphoraphane. Electrophoretic mobility-shift and chromatin immunoprecipitation assays revealed that Nrf2 associates, in low amounts, with the nqo1 ARE under constitutive conditions, and following sulphoraphane challenge of cells, Nrf2 is recruited to the ARE in substantially greater quantities, as a heterodimer with the small Maf (musculoaponeurotic fibrosarcoma virus) protein, MafK. Also, MafK was found to bind the nqo1 ARE in an Nrf2-independent fashion, and may contribute to transcriptional repression of the oxidoreductase gene. These findings allow a model for transcriptional control of nqo1 through the ARE to be proposed. Furthermore, our results indicate that distinct AREs have differential sequence requirements, and a universally applicable consensus sequence cannot be derived.

5' Flanking Region↗

PubMed2000

Divergent and composite gonadotropin-releasing hormone-responsive elements in the rat luteinizing hormone subunit genes.

GnRH pulses regulate gonadotropin subunit gene transcription in a frequency-dependent, subunit-specific manner. The alpha-subunit gene is stimulated by constant GnRH and by rapid to intermediate pulse frequencies, while stimulation of LHbeta subunit gene transcription requires intermediate frequency pulses. We have defined the GnRH-responsive elements of the rat LH subunit gene promoters by deletion/mutation analysis and transfection studies in rat pituitary cells and two clonal gonadotrope cell lines. The alpha-subunit gene GnRH-responsive region lies between -411 and -375 bp. The region contains two Ets-domain protein binding sites, and mutating either site obliterates the response. DNA protein binding studies demonstrate the two sites are not equivalent, and that Ets-1 does not mediate this response. Studies of the LHbeta promoter reveal a major GnRH-responsive region between -456 and -342 bp. Within this region, two Sp1 binding sites contribute to the GnRH response, and the 3'Sp1 site is also critical for basal expression. The 5'Sp1 site partially overlaps a CArG box, and mutating the CArG element specifically eliminates the response to pulsatile GnRH. DNA containing this mutation cannot form intermediate mobility complexes with nuclear proteins, but retains Sp1 binding. Mutation of the 3'Sp1 site and either the 5'Sp1 or CArG element partially restores GnRH stimulation, suggesting a downstream element contributes to the full GnRH response. These studies demonstrate that unique composite elements and transcription factors are responsible for GnRH stimulation of the LH subunit genes and may contribute to their differential responses to GnRH pulses.

Animals↗