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PubMed1999

Transcriptional activity and regulatory protein binding of metal-responsive elements of the human metallothionein-IIA gene.

Multiple copies of a cis-acting DNA element, metal-responsive element (MRE) are required for heavy metal-induced transcriptional activation of mammalian metallothionein genes. To approach the regulatory mechanism mediated by these multiple elements, we studied the properties of seven MREs located upstream of the human metallothionein-IIA (hMT-IIA) gene in detail. Transfection assays of reporter gene constructs each containing one of these MREs as the promoter element revealed that only four MREs can mediate zinc response. With respect to the distribution of active MREs over the promoter region, the hMT-IIA gene is largely different from the mouse metallothionein-I gene, suggesting that MRE arrangement is not an important factor for metal regulation. Experiments using various model promoters showed that multiple MRE copies act highly synergistically, supporting the biological significance of the multiplicity. Only the four active MREs efficiently bound the purified transcription factor human MTF-1, and MRE mutants defective in binding this protein lost the ability to support zinc-induced reporter gene expression, strongly suggesting that the direct interaction between human MTF-1 and a set of the selected MREs plays the major role in heavy metal regulation. In protein/DNA binding reactions in vitro, the purified human MTF-1 was activated by zinc but not by other metallothionein-inducing heavy metals, supporting the idea that zinc is the direct modulator of human MTF-1.

DNA-Binding Proteins↗
PubMed2001

Involvement of cyclic AMP response element binding protein (CREB) and estrogen receptor phosphorylation in the synergistic activation of the estrogen receptor by estradiol and protein kinase activators.

Estrogen receptor (ER) and cAMP signaling pathways interact in a number of estrogen target tissues including mammary and uterine tissues. One aspect of this interaction is that estradiol and protein kinase A (PKA) activators can cooperate synergistically to activate ER-mediated transcription of both endogenous genes and reporter genes containing only estrogen response elements. The purpose of this study was to investigate the molecular mechanism of this interaction between signaling pathways. Site-directed mutagenesis of the potential PKA phosphorylation sites in the ER indicated that phosphorylation of these sites was not necessary for the observed transcriptional synergy. In transient transfection assays in two different cell lines using reporter constructs containing either cAMP response elements, estrogen response elements or both types of elements, with the addition or absence of cAMP response element binding protein (CREB) expression plasmid, we observed that only one of these cell lines exhibited estrogen/PKA transcriptional synergy. Experiments demonstrated that CREB itself was involved in the transcriptional synergy, and that transfection of CREB restored transcriptional synergy in the cell line in which it was lacking. A functional interaction between ER and CREB was also demonstrated using a mammalian cell protein interaction assay; a dominant negative mutant of CREB did not exhibit this interaction. Therefore, these data indicate that CREB protein is required for the transcriptional synergy between cAMP and estrogen signaling pathways. Furthermore, CREB cooperated with the ER on genes that did not contain cAMP response elements, but contained only estrogen response elements. We propose that activated CREB is recruited to estrogen responsive genes by an ER--coactivator complex containing proteins such as CREB binding protein (CBP) and that the interaction of CREB with ER may assist in stabilizing its interaction with CBP and in promoting estrogen-ER and PKA transcriptional synergy.

Animals↗
PubMed1990

Activation of the c-fos serum-response element by the activated c-Ha-ras protein in a manner independent of protein kinase C and cAMP-dependent protein kinase.

12-O-Tetradecanoylphorbol-13-acetate (TPA) activated the c-fos gene enhancer linked to the chloramphenicol acetyltransferase or luciferase reporter gene in the wild type PC-12 cells but not in the variant PC-12 cells that originated from the wild type cells. Transfection of the c-Ha-rasval12 complementary DNA (cDNA) or addition of dibutyryl cAMP to the wild type PC-12 cells as well as to the variant PC-12 cells activated the c-fos gene enhancer. Prolonged treatment of the wild type PC-12 cells with phorbol-12,13-dibutyrate caused down-regulation of protein kinase C. In these cells, TPA did not stimulate the c-fos gene enhancer any more, but transfection of the c-Ha-rasval12 cDNA still stimulated the c-fos gene enhancer to the same extent as induced in the control cells. Transfection of the c-Ha-rasval12 cDNA or addition of TPA to the wild type PC-12 cells stimulated the serum-response element but not the cAMP-response element. Dibutyryl cAMP stimulated both the serum-response element and the cAMP-response element in the wild type PC-12 cells. These results indicate that the c-Ha-rasval12 protein activates the serum-response element, but not the cAMP-response element in the c-fos gene enhancer, and that the signal pathway from the c-Ha-rasval12 protein to the c-fos serum-response element is independent of protein kinase C and cAMP-dependent protein kinase.

Blotting, Western↗
PubMed1996

The heme-responsive element of the mouse heme oxygenase-1 gene is an extended AP-1 binding site that resembles the recognition sequences for MAF and NF-E2 transcription factors.

Jun and Fos (AP-1) transcription factors were recently proposed to mediate induction of the mouse heme oxygenase-1 gene by different agents including heme and cadmium. In this report we show that the AP-1 binding sequence, TGAGTCA, is necessary but insufficient for gene activation in response to heme or cadmium. The minimal heme response element was identified as an extended AP-1 binding site, (T/C)GCTGAGTCA. In addition to the AP-1 heptad, this element also contains an interdigitated antioxidant response element, GCnnnGTCA. Specific antioxidant response elements from the NAD(P)H:quinone oxidoreductase-1 and the glutathione S-transferase Ya subunit genes were in fact responsive to heme but not all sequences that conform to the consensus antioxidant response element were activated by this agent. The heme response element resembles the consensus binding sites for the product of the maf oncogene and for the transcription factor NF-E2. The potential role of these and related transcription factors and the implication of the composite heme response element in heme oxygenase-1 gene regulation are discussed.

Animals↗
PubMed1997

Effects of multiple estrogen responsive elements, their spacing, and location on estrogen response of reporter genes.

Most highly estrogen-responsive genes possess multiple estrogen-responsive elements (EREs) that act synergistically to activate expression. Synergism between EREs appears to depend on structural features of the EREs and the promoter. To examine the activation process, we cloned single or multiple tandem copies of the consensus ERE into reporter plasmids. These plasmids contained either a chloramphenicol acetyl transferase reporter gene driven by a minimal promoter or a luciferase reporter gene driven by the Simian virus 40 (SV40) promoter. Using MCF-7 human breast cancer cells, we demonstrate that synergism among EREs depends on the number of EREs, their spacing, and the distance of the EREs from the promoter. The induction capacity of EREs falls off slowly with distance from the promoter. Remarkably, multiple EREs can induce effectively and synergize even when they are located more than 2000 nucleotides from the promoter. For EREs located immediately upstream of the promoter, both the distance separating the EREs and the distance to the promoter have to be optimal for synergy. Altering either distance changes the response from synergistic to additive. For distant EREs, presumed to interact by a looping mechanism at the promoter, the length of DNA between the EREs and the promoter is not critical. Synergy among closely spaced EREs that are far from the promoter only requires an optimal distance separating the ERE centers of symmetry. Interestingly, very widely separated EREs can also synergize, presumably also because of their ability to interact by looping. The estrogen response from single or multiple tandem copies of ERE half-palindromes near the SV40 promoter was also tested. The negligible induction capacity of a single half-site was not significantly increased in multiple sites. The biological role of half-EREs is not apparent in the system employed here.

Animals↗
PubMed1995

The rat quinone reductase antioxidant response element. Identification of the nucleotide sequence required for basal and inducible activity and detection of antioxidant response element-binding proteins in hepatoma and non-hepatoma cell lines.

The antioxidant response element (ARE) found in the 5'-flanking region of the rat quinone reductase gene has been further characterized by mutational and deletion analysis. The results indicate that the 31-base pair ARE, which contains a 13-base pair palindromic sequence, can be further separated into three regions, all three of which are required for elevated basal level gene expression. These three regions include the proximal and distal half-sites as well as a 3'-flanking region consisting of 4 adenine nucleotides. Neither the proximal nor the distal half-site alone mediates transcriptional activation by beta-naphthoflavone. However, when placed together the two half-sites restore responsiveness to the inducer. Interestingly, the presence of only 1 of the 4 adenine nucleotides in the 3'-flanking region of the proximal half-site is required for responsiveness to the inducer. Point mutations within the ARE indicate that several nucleotides in both the proximal and distal half-sites are required for basal level gene expression. Electrophoretic mobility shift analysis using the ARE as the probe indicates that enhancers found in the glutathione S-transferase Ya and P genes recognize a similar trans-acting factor(s) found in crude nuclear extracts from human Hep G2 cells. Further, this complex can be detected in nuclear extracts from rat liver and rat hepatoma cells but not in mouse Hepa 1c1c7 cells or in human HeLa cells. The ARE-nucleoprotein complex can also be detected in F9 cells which lack significant levels of Jun/Fos proteins. Although the rat ARE resembles the human quinone reductase ARE which contains a consensus TRE, the 2-nucleotide change in the core sequence (TGACTCA versus TGACTTG) eliminates the high affinity TRE motif in the rat ARE. The rat ARE forms a nucleoprotein complex in Hep G2 and other cells with different properties than AP-1.

Animals↗
PubMed2004

[Interaction of p53 with p53 response element like binding sequence at upstream of hepatitis B virus enhancer I].

BACKGROUND & OBJECTIVE: A p53 response element like binding sequence, 5'-TGCC(G)T-TGCCT-3' was found at upstream of hepatitis B virus (HBV) enhancer I from 1047 to 1059 nucleotides after analyzing the HBV genome by a computer program in our previous work. It indicated that the sequence could specifically bind P53 protein in vitro by electrophoretic mobility shift assay (EMSA) and electrophoretic mobility supershift assay (EMSSA). This study was designed to further investigate the interaction between P53 and p53 response element like binding sequence at upstream of HBV enhancer I. METHODS: HBV x gene enhancer and promoter which contains p53 response element like binding sequence TGCGT-TGCCT was in upstream of CAT enzyme in pX-CAT reporter plasmid. Firstly, we designed a point mutation in pX-CAT which change TGCGT TGCCT into TGTAT. TGTAT by polymerase chain reaction (PCR) in order to damage the p53 response element like sequence. After pX-CAT or mutation pX-CAT (mpX-CAT) transfected alone or cotransfected with pCMVp53 into HepG2 hepatoma cell, CAT activity was assayed to confirm the correlation of p53 protein with this DNA sequence. In addition, an antisense sequence corresponding to the p53 response element like sequence in HBV was reconstructed into the pZeoSV2 vector (alpha pZeoXP) and transfected into HepG2.2.15 cell line to block the binding of P53 with this sequence, the stable transfected HepG2.2.15 cell was observed about P53/P21 expression, cell cycle distribution and apoptosis rates. RESULTS: CAT enzyme of HepG2 had an higher expression in cotransfection with pCMVp53 and pX-CAT than alone pX-CAT (1.353 VS 0.738,P< 0.05), but it was lower in mpX-CAT(0.304) and pCMVp53 (0.402). Compared with the control group, P53/P21 expression and cell apoptosis rate decreased greatly in the stable transfected alpha pZeoXP HepG2.2.15 cell line (0.95% VS 7.84%), the cell number in S phase increased in the same cell line (16.37% VS 9.48%). CONCLUSION: Reporter gene expression of pX-CAT in intracellular could be promoted by P53, which further suggest that P53 could bind TGCC(G)T-TGCCT in upstream of HBV enhancer I and induce a prolonged P53 half life. Subsequently, P53 and p21 protein (downstream gene of P53) would have a higher expression and stronger activity.

Apoptosis↗
PubMed2002

Functionality versus strength -- has functional selection taken place in the case of the ecdysteroid receptor response element?.

Nuclear receptors are ligand-dependent transcription factors responsible for controlling differentiation, growth and development of higher eukaryotes. Three amino acids within the recognition alpha-helix of the DNA-binding domain of the nuclear receptors constitute the so-called "P-box" which determines response element specificity. In the ultraspiracle (Usp) protein, which together with EcR forms the heterodimeric ecdysone receptor, the P-box residues are E19, G20 and G23. Substitution of E19, the most characteristic amino acid for estrogen receptor-like P-boxes, with alanine showed that the mutation did not appreciably alter the affinity of the wild-type Usp DNA-binding domain (UspDBD(WT)) for a probe containing natural ecdysone response element (hsp27(wt)). Since in many cases E19 contacts a G/C base pair in position -4, which is absent in hsp27(wt), we analysed the interaction of UspDBD(WT), E19A and other P-box region mutants with the hsp27(wt) derivative which contains a G/C instead of an T/A base pair in position -4. UspDBD(WT) exhibited higher affinity for this element than for hsp27(wt). Moreover, a different interaction pattern of P-box region mutants was also observed. Thus we conclude that the E19 residue of UspDBD is not involved in any hsp27(wt) sequence-discerning contacts. However, substitution of the hsp27(wt) T/A base pair in position -4 with G/C generates target sequence with distinct functional characteristics and possibly with a new specificity. These results could serve as a basis for understanding the role of the presence of a T/A or G/C base-pair in the position -4 in the two types of ecdysone response elements found in nature.

Amino Acid Sequence↗
PubMed1997

DNA bending is essential for the site-specific recognition of DNA response elements by the DNA binding domain of the tumor suppressor protein p53.

We have used circular permutation assays to determine the extent and location of the DNA bend induced by the DNA binding domain of human wild type p53 (p53DBD) upon binding to several naturally occurring DNA response elements. We have found that p53DBD binding induces axial bending in all of the response elements investigated. In particular, response elements having a d(CATG) sequence at the junction of two consensus pentamers in each half-site favor highly bent complexes (bending angle is approximately 50 degrees ), whereas response elements having d(CTTG) bases at this position are less bent (bending angles from approximately 37 to approximately 25 degrees ). Quantitative electrophoretic mobility shift assays of different complexes show a direct correlation between the DNA bending angle and the binding affinity of the p53DBD with the response elements, i.e. the greater the stability of the complex, the more the DNA is bent by p53DBD binding. The study provides evidence that the energetics of DNA bending, as determined by the presence or absence of flexible sites in the response elements, may contribute significantly to the overall binding affinity of the p53DBD for different sequences. The results therefore suggest that both the structure and the stability of the p53-DNA complex may vary with different response elements. This variability may be correlated with variability in p53 function.

Base Sequence↗
PubMed1999

Arginine-aminoglycoside conjugates that bind to HIV transactivation responsive element RNA in vitro.

HIV gene expression is crucially dependent on binding of the viral Tat protein to the transactivation RNA response element. A number of synthetic Tat-transactivation responsive element interaction inhibitors of peptide/peptoid nature were described as potential antiviral drug prototypes. We present a new class of peptidomimetic inhibitors, conjugates of L-arginine with aminoglycosides. Using a gel-shift assay and affinity chromatography on an L-arginine column we found that these compounds bind specifically to the transactivation responsive element RNA in vitro with Kd values in the range of 20-400 nM, which is comparable to the Kd of native Tat bound to the transactivation responsive element (10-12 nM). Confocal microscopy studies demonstrated that fluorescein-labelled conjugate penetrates into live cells. High affinity to the transactivation responsive element, low toxicity, and relative simplicity of synthesis make these compounds attractive candidates for antiviral drug design.

Aminoglycosides↗
PubMed1991

Characteristics of the interaction of the ferritin repressor protein with the iron-responsive element.

The iron-responsive regulation of ferritin mRNA translation is mediated by the specific interaction of the ferritin repressor protein (FRP) with the iron-responsive element (IRE), a highly conserved 28-nucleotide sequence located in the 5' untranslated region of ferritin mRNAs. The IRE alone is necessary and sufficient to confer repression of translation by FRP upon a heterologous message, chloramphenicol acetyltransferase, in an in vitro translation system. The activity of FRP is sensitive to iron in vivo. Cytoplasmic extracts of rabbit kidney cells show reduction of FRP activity when grown in the presence of iron, as detected by RNA band shift assay. Using a nitrocellulose filter binding assay to examine the interaction of FRP with the IRE in more detail, we find that purified FRP has a single high-affinity binding site for the IRE with a Kd of 20-50 pM. Hemin pretreatment decreases the total amount of FRP which can bind to the IRE. This effect is dependent on hemin concentration. Interestingly, the FRP which remains active at a given hemin concentration binds to the IRE with the same high affinity as untreated FRP. A variety of hemin concentrations were examined for their effect on preformed FRP/IRE complexes. All hemin concentrations tested resulted in rapid complex breakdown. The final amount of complex breakdown corresponds to the concentration of hemin present in the reaction. The effect of hemin on FRP activity suggests that a specific hemin binding site exists on FRP.

Animals↗
PubMed1995

Composite structure of auxin response elements.

The auxin-responsive soybean GH3 gene promoter is composed of multiple auxin response elements (AuxREs), and each AuxRE contributes incrementally to the strong auxin inducibility to the promoter. Two independent AuxREs of 25 bp (D1) and 32 bp (D4) contain the sequence TGTCTC. Results presented here show that the TGTCTC element in D1 and D4 is required but not sufficient for auxin inducibility in carrot protoplast transient expression assays. Additional nucleotides upstream of TGTCTC are also required for auxin inducibility. These upstream sequences showed constitutive activity and no auxin inducibility when part or all of the TGTCTC element was mutated or deleted. In D1, the constitutive element overlaps the 5' portion of TGTCTC; in D4, the constitutive element is separated from TGTCTC. An 11-bp element in D1, CCTCGTGTCTC, conferred auxin inducibility to a minimal cauliflower mosaic virus 35S promoter in transgenic tobacco seedlings as well as in carrot protoplasts (i.e., transient expression assays). Both constitutive elements bound specifically to plant nuclear proteins, and the constitutive element in D1 bound to a recombinant soybean basic leucine zipper transcription factor with G-box specificity. To demonstrate further the composite nature of AuxREs and the ability of the TGTCTC element to confer auxin inducibility, we created a novel AuxRE by placing a yeast GAL4 DNA binding site adjacent to the TGTCTC element. Expression of a GAL4-c-Rel transactivator in the presence of this novel AuxRE resulted in auxin-inducible expression. Our results indicate that at least some AuxREs have a composite structure consisting of a constitutive element adjacent to a conserved TGTCTC element that confers auxin inducibility.

Animals↗
PubMed2001

New androgen response elements in the murine pem promoter mediate selective transactivation.

The Pem homeobox transcription factor is expressed under androgen control in the testis and epididymis. It is also transcribed in the ovary, muscle, and placenta. The mouse Pem gene promoter was cloned and sequenced. It was analyzed in transactivation tests using CV-1 and PC-3 cells expressing the AR and found to be strongly stimulated by androgens. EMSAs and mutational analysis of the Pem promoter allowed the identification of two functional androgen response elements named ARE-1 and ARE-2. They both differed from the consensus semipalindromic steroid response element and exhibited characteristics of direct repeats of the TGTTCT half-site. Unlike the steroid response element, both Pem androgen response elements were selectively responsive to androgen stimulation. Specific mutations in the left half-site of Pem ARE-1 and ARE-2, but not of the steroid response element, were still compatible with AR binding in the EMSA. In addition, Pem ARE-1, but not ARE-2 or the steroid response element, showed some flexibility with regard to spacing between half-sites. These results strongly suggest that the AR interacts differently with direct repeats than with inverted repeats, potentially leading to cis element-driven selective properties. Thus, the existence of several classes of DNA response elements might be an essential feature of differential androgen regulation.

Androgens↗
PubMed1997

5'-flanking sequences in thyroid hormone response element half-sites determine the requirement of retinoid X receptor for receptor-mediated gene expression.

Thyroid hormone receptors are ligand-inducible transcription factors that can potentially interact with thyroid hormone response elements as homodimers or heterodimers with the retinoid X receptor. It has generally been felt, however, that the heterodimer is responsible for induction of gene expression. We have demonstrated previously that the optimal thyroid hormone receptor binding sequence is not the consensus hexamer half-site AGGTCA but is an octamer, TAAGGTCA. Based upon these findings, we hypothesize that thyroid hormone response elements composed of optimal half-sites (TAAGGTCA) will bind thyroid hormone receptors readily and activate gene expression independently of the retinoid X receptor. In contrast, response elements composed of suboptimal half-sites (e.g. GCAGGTCA) will require the retinoid X receptor to facilitate thyroid hormone receptor-mediated gene expression. To test this hypothesis, we have reconstituted thyroid hormone receptor-mediated gene expression in yeast. Our studies confirm the hypothesis that the retinoid X receptor is required for gene expression from response elements composed of suboptimal half-sites, whereas thyroid hormone receptors are sufficient to activate gene expression maximally from response elements containing optimal half-sites. Furthermore, coexpression of steroid receptor coactivator-1 is required for ligand-dependent gene activation from single response elements. Surprisingly, however, coexpression of the retinoid X receptor decreases the steroid receptor coactivator-1-dependent thyroid hormone induction. Overall these data demonstrate that the architecture of the thyroid hormone response element dictates the nuclear receptor requirements for gene activation. The studies suggest that different coactivators may be required for gene activation depending upon the response element architecture and the nature of the bound thyroid hormone receptor complex (homo- versus heterodimer).

Animals↗
PubMed1992

v-Fps-responsiveness in the Egr-1 promoter is mediated by serum response elements.

Egr-1, a mitogen-responsive transcription factor, is rapidly induced by v-Fps in the absence of protein synthesis. Thus, Egr-1 is a primary response to the protein-tyrosine kinase activity of v-Fps. To determine the v-Fps-responsive elements in the Egr-1 promoter, deletion mutants of the Egr-1 promoter were used in transient expression assays. A v-Fps expression vector was contransfected into NIH 3T3 cells with chloramphenicol acetyl transferase (CAT) gene expression vectors under the control of the Egr-1 promoter or the Egr-1 promoter containing various deletions. Responsiveness to v-Fps was restricted to a region that contained repeated CC(A/T)6GG sequences, known as CArG boxes. CArG boxes form the core of serum response element (SREs). v-Fps-induced Egr-1 promoter activation was lost by sequential removal of four tandemly repeated SREs. This region, containing four SREs, was found to be sufficient for maximal Egr-1 induction by v-Fps when placed upstream from a heterologous promoter. Individual SREs from this region were able to respond to v-Fps, however, the activation of the individual SREs was lower than that observed for the clustered SREs. These data suggest that v-Fps-responsiveness in the Egr-1 promoter is mediated by SREs.

3T3 Cells↗
PubMed1992

An enhancer element responsive to ras and fms signaling pathways is composed of two distinct nuclear factor binding sites.

In order to precisely define the sequences that constitute the ras-responsive enhancers element present in the murine retrotransposon NVL3, point mutations were introduced into the previously defined minimal transcriptional enhancer DNA. Analyses of the effects of these point mutations in transient transfection experiments, in gel retention assays, and by methylation interference footprinting indicated that the enhancer element was composed of two binding sites for distinct nuclear factors. Both binding sites were required for activation of the enhancer by either ras or v-fms oncogenes, and the distinct nuclear factors were found in extracts from cells that contained either oncogene. UV cross-linking analysis revealed that the AP1-related binding site, TGACTCT, was recognized by a nuclear factor of apparent molecular size of 50 kilodaltons, that is probably c-jun. The other binding site, CAGGATAT, is very similar to sites recognized by the ets-family of transcription factors, and was recognized by the 120-kilodalton ras-responsive factor-1. Activation of the NVL3 element was reconstituted in an in vitro transcription assay. The ets-related binding site was necessary for this in vitro reconstitution of activity. Thus, the NVL3 enhancer is related to the previously described oncogene-responsive enhancer element present in polyoma virus and is also related to elements identified in several cellular genes known to be ras-responsive, including the transforming growth factor-beta 1 gene.

Base Sequence↗
PubMed1992

The basic region of AP-1 specifies glucocorticoid receptor activity at a composite response element.

Unrelated factors collaborate at composite response elements to confer novel patterns of transcriptional regulation. For example, AP-1 and glucocorticoid receptor bind and mutually affect their activities at a 25-bp composite element, plfG. We found that different members of the AP-1 factor family that behave similarly in the absence of receptor are strikingly distinct in its presence: They specify opposite (enhancement vs. repression) regulatory actions by the receptor. Four amino acids within the AP-1 DNA-binding domain were identified as crucial determinants of receptor regulatory activity at plfG. We conclude that interactions of factors from separate transcription factor families at composite response elements provide a mechanism by which a single factor can regulate both positively or negatively, and a potential resolution of the apparent functional redundancy within regulatory factor families.

Amino Acid Sequence↗
PubMed2000

Identification of Glucose-responsive and Insulin-responsive Elements in Promoter of Mouse ob Gene.

Glucose and insulin stimulate leptin gene expression in vitro and in vivo. To identify cis-elements that are responsible for the glucose and insulin effects, mouse 3T3-L1 adipocytes were transiently transfected with reporter constructs with serial deletions in mouse ob gene promoter. The cis-elements were identified with Gel mobility shift assays (GMSA), DNase I footprint assays and PCR mediated site-directed mutation assays. Transient transfections detected a negative cis-acting element, a glucose-responsive element (GLRE), and an insulin-responsive element (IRE) in the region from -1 719 bp to -1 452 bp of mouse ob gene. This region does not contain any known GLRE or IRE. GMSA identified a DNA binding protein which specifically binds a native probe prepared from mouse ob gene promoter (-1 719 bp/-1 452 bp), and the binding was repressed by glucose or insulin. DNase I footprint assays and PCR mediated site-directed mutations assays identified that the binding motif AGCAAAA, spanning -1 698 bp to -1 692 bp of the mouse ob gene promoter, was responsible for the effects of glucose and insulin on ob gene expression. These studies suggest that a negative cis-acting element is located between -1 719 bp and -1 452 bp of the mouse ob gene promoter, and glucose and insulin simulate mouse ob gene expression by repressing the binding of a transcription factor to this element. This element, AGCAAAA, spanning -1 698 bp to -1 692 bp is a novel GLRE and IRE.

Journal Article↗