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At least 1,531 records · Page 85Linked to original sources
PubMed2005

Silencing of caspase-8 in murine hepatocellular carcinomas is mediated via methylation of an essential promoter element.

BACKGROUND & AIMS: Caspase-8 is the apical caspase essential for triggering Fas-induced apoptosis. In this study, we investigated caspase-8 expression in hepatocellular carcinomas (HCCs) using recently described HCC mouse models (c-myc and IgEGF transgenes). METHODS: HCCs were isolated from c-myc and IgEGF transgenic animals. Expression of caspase-8 was monitored by reverse-transcription polymerase chain reaction. The murine caspase-8 promoter was characterized by luciferase-reporter analysis and the analysis of promoter methylation was performed by bisulfite genomic sequencing. RESULTS: In HCCs investigated, we frequently found a lack of caspase-8 messenger RNA expression. Genomic deletions at the caspase-8 locus did not contribute to caspase-8 silencing. We examined tumor-derived promoter sequences and found significant hypermethylation at distinct CpG sites. In parallel, we characterized the murine caspase-8 promoter and identified a 30-bp promoter element that is indispensable for basal promoter activity. This minimal promoter element contained SP1 binding motifs that are colocalized with CpG sites and were methylated in tumor-derived promoter sequences. Electrophoretic mobility shift assay analysis showed that methylation of these SP1 sites is sufficient to prevent SP1 complex formation. To support our data, we mimicked the methylation pattern of a tumor-derived caspase-8 promoter in vitro using CpG methylase and found a strong reduction of promoter activity. CONCLUSIONS: We show that HCCs are correlated frequently with silencing of caspase-8 expression and provide data suggesting that caspase-8 silencing is a direct consequence of inhibiting SP1-dependent transactivation caused by CpG methylation at its essential binding sites in the promoter region. Our data support the hypothesis that inhibition of apoptosis triggers hepatocarcinogenesis.

Animals↗
PubMed1988

A conserved CATTCCT motif is required for skeletal muscle-specific activity of the cardiac troponin T gene promoter.

Transcription of the cardiac troponin T (cTNT) gene is restricted to cardiac and embryonic skeletal muscle tissue. A DNA segment containing 129 nucleotides upstream from the cTNT transcription initiation site (cTNT-129) directs expression of a heterologous marker gene in transfected embryonic skeletal muscle cells but is inactive in embryonic cardiac or fibroblast cells. By using chimeric promoter constructions, in which distal and proximal segments of cTNT-129 are fused to reciprocal segments of the herpes simplex virus thymidine kinase (HSV tk) gene promoter, the DNA segment responsible for this cell specificity can be localized to the cTNT distal promoter region, located between 50 and 129 nucleotides upstream of the transcription initiation site. The ability of the cTNT distal promoter region to confer skeletal muscle-specific activity upon a heterologous promoter is abolished when it is displaced 60 nucleotides upstream, indicating that its ability to direct skeletal muscle-specific transcription probably requires proximity to other components of the transcription initiation region. Two copies of the heptamer, CATTCCT ("muscle-CAT" or "M-CAT" motif), reside within the 80-nucleotide cTNT distal promoter region. A 3-nucleotide mutation in one of these copies inactivates the cTNT promoter in skeletal muscle cells. Therefore, the M-CAT motif is a distal promoter element required for expression of the cTNT promoter in embryonic skeletal muscle cells. Since the M-CAT motif is found in other contractile protein gene promoters, it may represent one example of a muscle-specific promoter element.

Animals↗
PubMed1991

Cell-free transcription directed by the 422 adipose P2 gene promoter: activation by the CCAAT/enhancer binding protein.

Previous investigations have shown that CCAAT/enhancer binding protein (C/EBP) can function as a trans-activator of the promoters of several adipocyte-specific genes--i.e., the 422 adipose P2 (422/aP2), stearoyl-CoA desaturase 1 (SCD1), and glucose transporter 4 (GLUT4) genes, in 3T3-L1 mouse preadipocytes. We now describe a cell-free system prepared from nuclei of 3T3-L1 cells that carries out transcription directed by these promoters. To measure transcript formation, we employed a polymerase chain reaction-assisted analysis. Nuclear extract from 3T3-L1 adipocytes that express C/EBP supports a higher rate of transcription of chimeric 422(aP2) promoter-chloramphenicol acetyltransferase (CAT) reporter gene constructs than nuclear extract from preadipocytes that lack C/EBP. A competitor oligonucleotide containing the C/EBP binding site sequence and antibodies raised against C/EBP inhibit transcription directed by the 422(aP2) promoter. The factor limiting transcription by nuclear extract from preadipocytes appears to be C/EBP, since recombinant C/EBP (rC/EBP) markedly activates transcription of the 422(aP2) promoter-CAT gene with preadipocyte extract but not with adipocyte extract. rC/EBP also activates cell-free transcription of SCD1 promoter-CAT and GLUT4 promoter-CAT chimeric genes. Point mutations within the C/EBP binding site in the 422(aP2) promoter markedly decrease transcription activated by rC/EBP. Consistent with activation by cAMP of the 422(aP2) promoter in intact preadipocytes, cAMP-dependent protein kinase activates transcription through this promoter with the cell-free system, this effect being independent of C/EBP. Thus, regulation of transcription directed by the 422(aP2) promoter in the cell-free system resembles that which occurs in intact 3T3-L1 cells.

Adipose Tissue↗
PubMed1998

In vivo genomic footprinting analysis reveals that the complex Bradyrhizobium japonicum fixRnifA promoter region is differently occupied by two distinct RNA polymerase holoenzymes.

The Bradyrhizobium japonicum fixRnifA operon is transcribed from two promoters: fixRp1, a -24/-12 promoter recognized by the sigma54-holoenzyme form of the RNA polymerase, and fixRp2, a -35/-10 promoter that is transcribed by a second, unidentified, form of RNA polymerase holoenzyme. The fixRp1 promoter is autoregulated during microaerobiosis by NifA, whereas fixRp2 is also activated, but by a different regulatory protein. The main transcription start sites for these promoters are just two nucleotides apart, such that the conserved -12 and -10 regions of fixRp1 and fixRp2, respectively, must overlap each other, whereas the -24 and -35 regions lie one DNA helical turn apart. Using in vivo genomic dimethyl sulfate and KMnO4 footprinting, we showed that the promoter region is differentially protected, depending upon which holoenzyme is bound. Mutagenesis analyses indicated that positions from -12 to -14 are critical for the activity of both promoters, whereas mutations at -10 and -11 affected mainly fixRp2 expression. When the sequence of the putative -35 region of fixRp2 was modified to match the putative consensus, expression from this promoter was increased 3-fold and the reactivity toward KMnO4, but not the transcriptional start site, moved two nucleotides further upstream, indicating that the altered promoter forms a different open complex. Additionally, we detected NifA-dependent methylation protection of two atypical NifA binding sites and protection of guanine -75. The latter residue is located in a region critical for fixRp2 promoter activation. The results present direct physical evidence of the complexity of the organization, regulation, and function of the fixRnifA promoter region.

Bacterial Proteins↗
PubMed1995

Two mutations in the promoter region of the human protein C gene both cause type I protein C deficiency by disruption of two HNF-3 binding sites.

Protein C is a vitamin K-dependent zymogen of a serine protease that inhibits blood coagulation by the proteolytic inactivation of factors Va and VIIIa. Individuals affected with protein C deficiency are at risk for thrombosis. Genetic analyses of affected individuals, to determine the cause of the protein C deficiency, revealed a large variety of mutations in the protein C gene, including several in the promoter region of this gene. Comparison of the region around two of these mutations, A-32-->G and T-27-->A, with transcription factor consensus sequences suggested the presence of two overlapping and inversely oriented HNF-3 binding sites. Direct evidence for the presence of the two HNF-3 binding sites in the protein C promoter was obtained using electrophoretic mobility shift assays and UV cross-linking experiments. These experiments revealed that HNF-3 can bind specifically to both putative HNF-3 sites in the wild-type protein C promoter. Due to the T-27-->A mutation, one binding site is completely lost, while the other site still binds HNF-3, but with strongly reduced affinity. As a consequence of the A-32-->G mutation, the protein C promoter loses all its HNF-3 binding capacity. Transient transfection experiments demonstrated that the binding of HNF-3 to the protein C promoter is of physiological significance. This followed from experiments in which the introduction of the A-32-->G or T-27-->A mutation resulted in a 4-5-fold reduced promoter activity in HepG2 cells. Furthermore, transactivation of the wild-type protein C promoter construct with HNF-3 showed a 4-5-fold increased promoter activity in HepG2 cells. In HeLa cells, significant wild-type promoter activity was only observed after transactivation with HNF-3. When a promoter construct containing the T-->A mutation at position -27 was used, the transactivation potential of HNF-3 was 2-fold reduced in HepG2 cells, whereas in HeLa cells no transactivation was observed. With the promoter construct containing the A-32-->G mutation, no transactivation by HNF-3 was found either in HepG2 or in HeLa cells.

Animals↗
PubMed1996

Identification of a bimodal regulatory element encompassing a canonical AP-1 binding site in the proximal promoter region of the human decorin gene.

We have previously described a tumor necrosis factor alpha (TNF-alpha) response element, located between residues -188 and -140 of the human decorin promoter, that mediates the inhibitory effect of TNF-alpha on decorin gene expression (Mauviel, A., Santra, M., Chen, Y.-Q., Uitto, J., and Iozzo, R. V. (1995) J. Biol. Chem. 270, 11692-11700). In this report, we demonstrate that interleukin 1 (IL-1), a pleiotropic cytokine that shares a wide variety of biological properties with TNF-alpha, uses the same cis element to up-regulate decorin gene expression. Specifically, IL-1 enhances the expression of the human decorin gene, and this effect is mediated by activation of the corresponding promoter, as shown in transient cell transfection experiments using decorin promoter-chloramphenicol acetyl transferase reporter gene constructs. Additional transfection experiments with various 5'-deletion promoter-chloramphenicol acetyltransferase constructs demonstrate that both the inhibitory effect of TNF-alpha and the stimulatory effect of IL-1 are mediated by a 48-base pair segment of the promoter, between residues -188 and -140. This region, which contains a canonical AP-1 binding site, TGAGTCA, allows an antagonistic effect of these two cytokines on the decorin promoter activity. When cloned upstream of the thymidine kinase promoter, this promoter fragment requires the AP-1 sequence to be responsive to IL-1. Supershift assays with various AP-1 antibodies identified c-Jun, Jun-B, and Fra-1 as components of the complex binding to the decorin promoter. Overexpression of c-jun, an oncogene encoding the c-Jun/AP-1 transcription factor, reduces the basal activity of both decorin and -188/-140 thymidine kinase promoter constructs. In contrast, blockage of c-jun expression with an antisense c-jun construct potentiates the stimulatory effect of IL-1 and reverses the response to TNF-alpha. These data indicate that the region between residues -188 and -140 of the human decorin promoter functions as a bimodal regulatory element and allows transcriptional repression by c-Jun/AP-1 complexes.

Binding Sites↗
PubMed1997

The keratinocyte-specific Epstein-Barr virus ED-L2 promoter is regulated by phorbol 12-myristate 13-acetate through two cis-regulatory elements containing E-box and Krüppel-like factor motifs.

We previously employed 782 base pairs of the Epstein-Barr virus ED-L2 early lytic cycle promoter in a transgenic mouse model to target cyclin D1 to the stratified squamous epithelium of the tongue and esophagus. This promoter is located 5' to the transcriptional start site of a short open reading frame BNLF-2A and is immediately 3' to the BNLF-1 (LMP-1 oncogene) open reading frame. We studied transcriptional regulation of the ED-L2 promoter by phorbol 12-myristate 13-acetate (PMA) as a means of understanding the tissue specificity of this promoter. The transcriptional activity of the ED-L2 promoter was stimulated 40-fold by PMA and could be blocked with the compound H7 through antagonism of protein kinase C. 5' deletion analysis of the 782-base pair promoter demonstrated that the sequences necessary for PMA-stimulated trans-activation were located in two separate cis-regulatory regions of the promoter: -187 to -164 and -144 to -114 base pairs from the transcription start site of BNLF-2A. Importantly, mutation of critical base pairs in each region was sufficient to abolish PMA-stimulated trans-activation in the native ED-L2 promoter. Region -187 to -164 contains a CACCTG (E-box) motif, and region -144 to -114 contains a CACACCC motif. Both of these motifs are necessary for trans-activation by PMA. These regions do not, however, demonstrate enhancer characteristics when tested in a heterologous minimal promoter system. Variations of the CACACCC motif are found in other keratinocyte-specific promoters, as well as in the DNA binding motifs of the Krüppel-like family of transcription factors. Electrophoretic mobility shift assays with specific competitors and factor-specific antibody supershift assays demonstrated that one complex binding the -187 to -164 region containing the CACCTG nucleotides has characteristics of the helix-loop-helix protein upstream stimulatory factor, whereas a factor binding the CACACCC motif may be a member of the Krüppel-like family. These experiments show how ubiquitous and tissue-specific transcription factors induced by PMA regulate the ED-L2 promoter in squamous epithelial cells.

Cell Lineage↗
PubMed1998

Mouse mu opioid receptor gene expression. A 34-base pair cis-acting element inhibits transcription of the mu opioid receptor gene from the distal promoter.

The 5'-flanking region of the mouse mu opioid receptor (MOR) gene has two promoters, referred to as distal and proximal, and the activities of each in the brain are quite different from each other. The 5'-distal promoter regulatory sequences (5'-DPRS), positioned between these two promoters, have strong inhibitory effects on the reporter gene expression driven by the MOR distal promoter. In our studies, detailed 3' deletion mapping of the 5'-DPRS narrowed down the negative cis-acting element to a 34-base pair (bp) segment (position -721 to -687). This 34-bp cis-acting element functions in both neuronal (NMB) and non-neuronal (CHO and RAW264.7) cultured cells. S1 nuclease protection assays indicated that this 34-bp cis-acting element suppresses distal promoter activity at the transcriptional level. Linker scanning mutagenesis demonstrated that nucleotides around position -721 and -689 in the 34-bp cis-acting element are essential for the regulation of distal promoter activity. Operational characterization of the 34-bp cis-acting element in the homologous MOR distal promoter and the heterologous SV40 promoter showed that its effects are position- and promoter-dependent while being orientation-independent in both promoters. Collectively, these data suggested that this 34-bp segment is a conditional transcriptional cis-acting element that blocks mouse MOR gene expression from the distal promoter.

Animals↗
PubMed2000

Identification of a novel cis element required for cell density-dependent down-regulation of insulin-like growth factor-2 P3 promoter activity in Caco2 cells.

The activity of the exogenous, full-length insulin-like growth factor-2 (IGF-2) P3 promoter is significantly up-regulated during the logarithmic growth phase but rapidly declines in confluent CaCo2 cells undergoing differentiation. Nuclear run-on assays confirmed cell density-dependent regulation of endogenous P3 promoter. To identify regulatory elements in the P3 promoter that may be required for regulating cell density-dependent transcriptional activity, we used the methods of promoter truncation, electrophoretic mobility shift assay, DNase footprinting, and mutation analysis. The relative activity of the full-length (-1229/+140) and truncated (-1090/+140) promoter was identical, being approximately 19, 27, 7, and 3% of pSV-luc activity on days 3, 5, 7, and 9 of cell culture, respectively. However, truncation to -1048 resulted in complete loss of cell density-dependent down-regulation of P3 promoter activity on days 7 and 9, suggesting the presence of regulatory elements between -1091 and -1048 sequence. Further stepwise truncation to -515 did not change promoter activity. Truncation to -138/+140 resulted in complete loss of promoter activity, suggesting that the core promoter was within the -515/-138 segment. A 14-base pair footprint (-1084/-1070) was identified by DNase footprinting within the distal -1091/-1048 segment. Electrophoretic mobility shift assay with wild type and mutant probes confirmed the presence of a novel 7-base pair (CGAGGGC) (-1084/-1078) cis element (P3-D); its mutation abolished binding. Functionality of P3-D cis element was confirmed by measuring the activity of core P3 promoter ligated to distal P3 segment containing either the mutant or wild type P3-D element. We have, therefore, identified a novel cis element, P3-D, that appears to play a critical role in regulating IGF-2 P3 promoter activity in a cell density/differentiation-dependent manner.

Base Sequence↗
PubMed2001

Aberrant expression of human mucin gene MUC5B in gastric carcinoma and cancer cells. Identification and regulation of a distal promoter.

In gastric cancer, altered expression of MUC1, MUC2, MUC5AC, and MUC6 mucin genes has already been described. We show in this report by the means of in situ hybridization, reverse transcriptase-polymerase chain reaction, and transfection assays that MUC5B is also abnormally expressed in gastric carcinomatous tissues and cell lines. We thus undertook to elucidate the molecular mechanisms that regulate the transcription of MUC5B in gastric cancer cells. To this end, high expressing (KATO-III) and low expressing (AGS) gastric cancer cell lines were chosen to study human mucin gene MUC5B expression and promoter activity. Sequencing of the promoter region revealed a distal TATA box located 1 kilobase upstream of the proximal TATA box. Functional activity of the promoter was addressed by using deletion mutants covering 2044 nucleotides upstream of the MUC5B transcription start site. We identified a distal promoter 10 times more active than the proximal promoter in KATO-III cells. In AGS cells, both promoters, much less active, showed the same range of activity. Binding assays allowed us to show that the transcription factor ATF-1 binds to a cis-element present in the distal promoter. Sp1, which binds to both promoters specifically transactivates the proximal promoter. Treatment of transfected cells with PMA, cholera toxin A subunit, and calcium ionophore showed that only PMA led to a substantial activation of the distal promoter. MUC5B 5'-flanking region having a high GC content, influence of methylation on the MUC5B expression was assessed. Our results indicate that repression of MUC5B expression visualized in AGS cells is due in part to the presence of numerous methylated cytosine residues throughout the 5'-flanking region. Altogether these results demonstrate that MUC5B expression in gastric cancer cells is governed by a highly active distal promoter that is up-regulated by protein kinase C and that repression is under the influence of methylation.

Adenocarcinoma↗
PubMed2001

p21-activated kinase-1 (PAK1) inhibition of the human scavenger receptor class B, type I promoter in macrophages is independent of PAK1 kinase activity, but requires the GTPase-binding domain.

Scavenger receptor class B, type I (SR-BI), is a high density lipoprotein receptor that mediates the flux of cholesterol between high density lipoprotein and cells. Recent evidence suggests that SR-BI plays a role in atherosclerosis and that inflammatory mediators down-regulate SR-BI in the macrophage. The purpose of this study was to evaluate the ability of lipopolysaccharide (LPS) to down-regulate the activity of the human SR-BI promoter in the macrophage and to delineate the mechanisms involved. Experiments with cultured cells and in vivo derived macrophages showed that LPS has a powerful suppressive effect on SR-BI expression both in vitro and in vivo. Transient transfection studies demonstrated that LPS represses SR-BI promoter activity in the macrophage cell line RAW 264.7. Cotransfection with either a constitutively active p21-activated protein kinase-1 (PAK1) construct (T423E) or a kinase-deficient PAK1 construct (K299R) resulted in repression of the SR-BI promoter, similar to LPS. These results demonstrate that PAK1-mediated down-regulation of the SR-BI promoter is independent of PAK1 kinase activity and suggest that PAK1 mediates the LPS-induced decrease in promoter activity. Cotransfection with constitutively active Cdc42 or Rac expression constructs also resulted in down-regulation of the promoter; whereas the dominant-negative Cdc42 and Rac constructs elevated basal promoter activity and blunted the LPS response. Cotransfection of PAK1 constructs containing mutations in both the kinase domain and the Cdc42/Rac-binding domain attenuated the PAK1-mediated down-regulation of the promoter, suggesting that Rac and Cdc42 are required for PAK1-mediated decreases in SR-BI promoter activity. 5'-Deletion analysis and gel shift data suggest that LPS inhibits binding of a novel transcription factor to a myeloid zing finger protein-1-like element (-476 to -456) in the human SR-BI promoter. These results demonstrate that the PAK1 pathway down-regulates the SR-BI promoter and suggest that activation of this pathway may play an important role in cholesterol trafficking in the vessel wall.

Animals↗
PubMed2003

Nucleotides from -16 to -12 determine specific promoter recognition by bacterial sigmaS-RNA polymerase.

The alternative sigma factor sigmaS, mainly active in stationary phase of growth, recognizes in vitro a -10 promoter sequence almost identical to the one for the main sigma factor, sigma70, thus raising the problem of how specific promoter recognition by sigmaS-RNA polymerase (EsigmaS) is achieved in vivo. We investigated the promoter features involved in selective recognition by EsigmaS at the strictly sigmaS-dependent aidB promoter. We show that the presence of a C nucleotide as first residue of the aidB -10 sequence (-12C), instead of the T nucleotide canonical for sigma70-dependent promoters, is the major determinant for selective recognition by EsigmaS. The presence of the -12C does not allow formation of an open complex fully proficient in transcription initiation by Esigma70. The role of -12C as specific determinant for promoter recognition by EsigmaS was confirmed by sequence analysis of known EsigmaS-dependent promoters as well as site-directed mutagenesis at the promoters of the csgB and sprE genes. We propose that EsigmaS, unlike Esigma70, can recognize both C and T as the first nucleotide in the -10 sequence. Additional promoter features such as the presence of a C nucleotide at position -13, contributing to open complex formation by EsigmaS, and a TG motif found at the unusual -16/-15 location, possibly contributing to initial binding to the promoter, also represent important factors for sigmaS-dependent transcription. We propose a new sequence, TG(N)0-2CCATA(c/a)T, as consensus -10 sequence for promoters exclusively recognized by EsigmaS.

Bacterial Proteins↗
PubMed2004

Identification of a novel NF-kappaB-binding site with regulation of the murine alpha2(I) collagen promoter.

Hepatic fibrosis is due to the increased synthesis and deposition of type I collagen. Acetaldehyde activates type I collagen promoters. Nuclear factor kappaB (NF-kappaB) was previously shown to inhibit expression of murine alpha(1)(I) and human alpha(2)(I) collagen promoters. The present study identifies binding of NF-kappaB, present in nuclear extracts of stellate cells, to a region between -553 and -537 of the murine alpha(2)(I) collagen promoter. The NF-kappaB (p65) expression vector inhibited promoter activity. Mutation of the promoter at the NF-kappaB-binding site increased basal promoter activity and abrogated the activating and inhibitory effects of transforming growth factor beta and tumor necrosis factor alpha, respectively, on promoter activity. Acetaldehyde increased IkappaB-alpha kinase activity and phosphorylated IkappaB-alpha, NF-kappaB nuclear protein, and its binding to the promoter. However, the activating effect of acetaldehyde was not affected by the mutation of the promoter. In conclusion, although acetaldehyde increases the binding of NF-kappaB to the murine alpha(2)(I) collagen promoter, this binding does not mediate the activating effect of acetaldehyde on promoter activity. The effects of acetaldehyde in increasing the translocation of NF-kappaB to the nucleus with increased DNA binding activity may be important in mediating the effects of acetaldehyde on other genes.

Acetaldehyde↗
PubMed2004

Differential regulation of human and mouse orphan nuclear receptor small heterodimer partner promoter by sterol regulatory element binding protein-1.

Small heterodimer partner (SHP; NR0B2) is an unusual orphan nuclear receptor that lacks a conventional DNA-binding domain and acts as a modulator of transcriptional activities of a number of nuclear receptors. Herein, we report that the human SHP promoter (hSHP) is activated by sterol regulatory element-binding protein-1 (SREBP-1), which regulates the expression of various genes involved in cholesterol and fatty acid synthesis. Overexpression of SREBP-1 activated the human but not mouse SHP promoter, although SREBP-2 had little effect on the SHP promoter in CV-1 cells. Serial deletion reporter assays revealed that SREBP-1-responsive region is located within the sequences from -243 to -120 bp in the hSHP promoter. DNase I footprinting, gel shift assays, and chromatin immunoprecipitation assays demonstrated that SREBP-1 binds directly to the hSHP promoter. Site-directed mutagenesis made it clear that the hSHP promoter activation by SREBP-1 is mostly mediated by the SRE1 (-186 to -195 bp) in the hSHP promoter, which is not conserved in the mouse SHP promoter. Moreover, adenovirus-mediated overexpression of SREBP-1c/ADD-1 induced SHP mRNA expression and repressed CYP7A1 expression in HepG2 cells. Finally, we found that a four-nucleotide deletion (-195CT-GAdel) in the hSHP promoter, which is reported to be associated with altered body weight and insulin secretion in human, coincides with the SRE1. This mutation strongly decreased both basal and SREBP-1 dependent activities of the hSHP promoter, because of the reduced binding of SREBP-1 to the mutated SRE1. Overall, our results demonstrate a differential regulation of human and mouse SHP promoters by SREBP-1. We propose a possible role of SREBP-1 in the species differential regulation of cholesterol and bile acid homeostasis via a novel mechanism of up-regulation of the hSHP gene expression.

Adenoviridae↗
PubMed2005

Identification of estrogen-responsive genes using a genome-wide analysis of promoter elements for transcription factor binding sites.

We developed a pipeline to identify novel genes regulated by the steroid hormone-dependent transcription factor, estrogen receptor, through a systematic analysis of upstream regions of all human and mouse genes. We built a data base of putative promoter regions for 23,077 human and 19,984 mouse transcripts from National Center for Biotechnology Information annotation and 8793 human and 6785 mouse promoters from the Data Base of Transcriptional Start Sites. We used this data base of putative promoters to identify potential targets of estrogen receptor by identifying estrogen response elements (EREs) in their promoters. Our program correctly identified EREs in genes known to be regulated by estrogen in addition to several new genes whose putative promoters contained EREs. We validated six genes (KIAA1243, NRIP1, MADH9, NME3, TPD52L, and ABCG2) to be estrogen-responsive in MCF7 cells using reverse transcription PCR. To allow for extensibility of our program in identifying targets of other transcription factors, we have built a Web interface to access our data base and programs. Our Web-based program for Promoter Analysis of Genome, PAGen@UIC, allows a user to identify putative target genes for vertebrate transcription factors through the analysis of their upstream sequences. The interface allows the user to search the human and mouse promoter data bases for potential target genes containing one or more listed transcription factor binding sites (TFBSs) in their upstream elements, using either regular expression-based consensus or position weight matrices. The data base can also be searched for promoters harboring user-defined TFBSs given as a consensus or a position weight matrix. Furthermore, the user can retrieve putative promoter sequences for any given gene together with identified TFBSs located on its promoter. Orthologous promoters are also analyzed to determine conserved elements.

Algorithms↗
PubMed2004

Dystrophin Dp71 expression is down-regulated during myogenesis: role of Sp1 and Sp3 on the Dp71 promoter activity.

Dp71 expression is present in myoblasts but declines during myogenesis to avoid interfering with the function of dystrophin, the predominant Duchenne muscular dystrophy gene product in differentiated muscle fibers. To elucidate the transcriptional regulatory mechanisms operating on the developmentally regulated expression of Dp71, we analyzed the Dp71 expression and promoter activity during myogenesis of the C2C12 cells. We demonstrated that the cellular content of Dp71 transcript and protein decrease in myotubes as a consequence of the negative regulation that the differentiation stimulus exerts on the Dp71 promoter. Promoter deletion analysis showed that the 224-bp 5'-flanking region, which contains several Sp-binding sites (Sp-A to Sp-D), is responsible for the Dp71 promoter basal activity in myoblasts as well as for down-regulation of the promoter in differentiated cells. Electrophoretic mobility shift and chromatin immunoprecipitation assays indicated that Sp1 and Sp3 transcription factors specifically bind to the Sp-binding sites in the minimal Dp71 promoter region. Site-directed mutagenesis assay revealed that Sp-A is the most important binding site for the proximal Dp71 promoter activity. Additionally, cotransfection of the promoter construct with Sp1- and Sp3-expressing vectors into Drosophila SL2 cells, which lack endogenous Sp family, confirmed that these proteins activate specifically the minimal Dp71 promoter. Endogenous Sp1 and Sp3 proteins were detected only in myoblasts and not in myotubes, which indicates that the lack of these factors causes down-regulation of the Dp71 promoter activity in differentiated cells. In corroboration, efficient promoter activity was restored in differentiated muscle cells by exogenous expression of Sp1 and Sp3.

Animals↗
PubMed2005

A novel role of sodium butyrate in the regulation of cancer-associated aromatase promoters I.3 and II by disrupting a transcriptional complex in breast adipose fibroblasts.

The aromatase gene encodes the key enzyme for estrogen formation. Aromatase enzyme inhibitors eliminate total body estrogen production and are highly effective therapeutics for postmenopausal breast cancer. A distal promoter (I.4) regulates low levels of aromatase expression in tumor-free breast adipose tissue. Two proximal promoters (I.3/II) strikingly induce in vivo aromatase expression in breast fibroblasts surrounding malignant cells. Treatment of breast fibroblasts with medium conditioned with malignant breast epithelial cells (MCM) or a surrogate hormonal mixture (dibutyryl (Bt2)cAMP plus phorbol diacetate (PDA)) induces promoters I.3/II. The mechanism of promoter-selective expression, however, is not clear. Here we reported that sodium butyrate profoundly decreased MCM- or Bt2cAMP + PDA-induced promoter I.3/II-specific aromatase mRNA. MCM, Bt2cAMP + PDA, or sodium butyrate regulated aromatase mRNA or activity only via promoters I.3/II but not promoters I.1 or I.4 in breast, ovarian, placental, and hepatic cells. Mechanistically, recruitment of phosphorylated ATF-2 by a CRE (-211/-199, promoter I.3/II) conferred inductions by MCM or Bt2cAMP + PDA. Chromatin immunoprecipitation-PCR and immunoprecipitation-immunoblotting assays indicated that MCM or Bt2cAMP + PDA stabilized a complex composed of phosphorylated ATF-2, C/EBPbeta, and cAMP-response element-binding protein (CREB)-binding protein in the common regulatory region of promoters I.3/II. Overall, histone acetylation patterns of promoters I.3/II did not correlate with sodium butyrate-dependent silencing of promoters I.3/II. Sodium butyrate, however, consistently disrupted the activating complex composed of phosphorylated ATF-2, C/EBPbeta, and CREB-binding protein. This was mediated, in part, by decreased ATF-2 phosphorylation. Together, these findings represent a novel mechanism of sodium butyrate action and provide evidence that aromatase activity can be ablated in a signaling pathway- and cell-specific fashion.

Activating Transcription Factor 2↗
PubMed2006

Regulation of rat hepatic L-pyruvate kinase promoter composition and activity by glucose, n-3 polyunsaturated fatty acids, and peroxisome proliferator-activated receptor-alpha agonist.

Carbohydrate regulatory element-binding protein (ChREBP), MAX-like factor X (MLX), and hepatic nuclear factor-4alpha (HNF-4alpha) are key transcription factors involved in the glucose-mediated induction of hepatic L-type pyruvate kinase (L-PK) gene transcription. n-3 polyunsaturated fatty acids (PUFA) and WY14643 (peroxisome proliferator-activated receptor alpha (PPARalpha) agonist) interfere with glucose-stimulated L-PK gene transcription in vivo and in rat primary hepatocytes. Feeding rats a diet containing n-3 PUFA or WY14643 suppressed hepatic mRNA(L-PK) but did not suppress hepatic ChREBP or HNF-4alpha nuclear abundance. Hepatic MLX nuclear abundance, however, was suppressed by n-3 PUFA but not WY14643. In rat primary hepatocytes, glucose-stimulated accumulation of mRNA(LPK) and L-PK promoter activity correlated with increased ChREBP nuclear abundance. This treatment also increased L-PK promoter occupancy by RNA polymerase II (RNA pol II), acetylated histone H3 (Ac-H3), and acetylated histone H4 (Ac-H4) but did not significantly impact L-PK promoter occupancy by ChREBP or HNF-4alpha. Inhibition of L-PK promoter activity by n-3 PUFA correlated with suppressed RNA pol II, Ac-H3, and Ac-H4 occupancy on the L-PK promoter. Although n-3 PUFA transiently suppressed ChREBP and MLX nuclear abundance, this treatment did not impact ChREBP-LPK promoter interaction. HNF4alpha-LPK promoter interaction was transiently suppressed by n-3 PUFA. Inhibition of L-PK promoter activity by WY14643 correlated with a transient decline in ChREBP nuclear abundance and decreased Ac-H4 interaction with the L-PK promoter. WY14643, however, had no impact on MLX nuclear abundance or HNF4alpha-LPK promoter interaction. Although overexpressed ChREBP or HNF-4alpha did not relieve n-3 PUFA suppression of L-PK gene expression, overexpressed MLX fully abrogated n-3 PUFA suppression of L-PK promoter activity and mRNA(L-PK) abundance. Overexpressed ChREBP, but not MLX, relieved the WY14643 inhibition of L-PK. In conclusion, n-3 PUFA and WY14643/PPARalpha target different transcription factors to control L-PK gene transcription. MLX, the heterodimer partner for ChREBP, has emerged as a novel target for n-3 PUFA regulation.

Animals↗