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At least 253 records · Page 14Linked to original sources
PubMed1991

Transcriptional and post-transcriptional regulation of 72-kDa gelatinase/type IV collagenase by transforming growth factor-beta 1 in human fibroblasts. Comparisons with collagenase and tissue inhibitor of matrix metalloproteinase gene expression.

The temporal aspects and mechanisms of the regulation of the matrix metalloproteinase (MMP) 72-kDa gelatinase/type IV collagenase (MMP-2) by transforming growth factor-beta 1 (TGF-beta 1) were investigated in early passage human gingival fibroblasts and compared with the regulation of the genes for collagenase (MMP-1) and TIMP, the tissue inhibitor of MMPs. Northern hybridization analyses revealed that 1.0 ng/ml TGF-beta 1 increased the abundance of MMP-2 mRNA/cell approximately 1.5-fold at 24 h, an increase similar to that observed in the level of [35S]methionine pulse-labeled MMP-2 at 24 h (1.9-fold). At 48 and 72 h, the increase in MMP-2 mRNA abundance remained elevated by 1.5-2.2-fold on a per cell basis whereas TIMP mRNA levels were elevated by up to 3.3-fold. In contrast, the relative levels of collagenase mRNA were reduced by 66-75%. The changes in the MMP-2, collagenase, and TIMP mRNA concentrations in response to TGF-beta 1 were blocked by cycloheximide indicating that protein synthesis was required to mediate the effects of TGF-beta 1 on these mRNA levels. TGF-beta 1 was also found to increase the half-life of the MMP-2 mRNA from approximately 46 to approximately 150 h but did not alter the stability of TIMP mRNA (t1/2 approximately 60 h). Nuclear run-off transcription assays revealed that MMP-2 gene transcription was increased approximately 5-fold 7 h following TGF-beta 1-treatment but returned to control levels by 24 h. In comparison, increased TIMP gene transcription was only detectable after 24 h whereas collagenase gene transcription, although low in control cells, was undetectable at 24 h. Gene transcription, mRNA levels, and message stability of the genes for the extracellular matrix proteins type I collagen and fibronectin were also increased by TGF-beta 1. Thus, the similarity in the control of MMP-2, alpha 1 (I) procollagen, and fibronectin expression at the transcriptional and post-transcriptional levels indicates that these genes may share regulatory elements. In comparison, TGF-beta 1 reduced the level of collagenase mRNA and increased the level of TIMP mRNA as a result of altered transcriptional activities, through pathways that required protein synthesis, and without changes in mRNA stability.

Blotting, Northern↗
PubMed1983

Mutations of the beta subunit of RNA polymerase alter both transcription pausing and transcription termination in the trp operon leader region in vitro.

RNA polymerase was purified from rifampicin-resistant mutants of Escherichia coli which exhibit altered transcription termination at the trp operon attenuator in vivo. These mutant polymerases were used to investigate transcription pausing at the trp leader pause site and transcription termination at the trp attenuator. The mutant polymerases examined in vitro mimic their in vivo termination responses; i.e. RNA polymerase isolated from a mutant which displays high transcriptional read-through of the trp operon in vivo allows greater transcriptional read-through in vitro, while RNA polymerase prepared from a mutant which has reduced read-through in vivo exhibits greater termination of transcription in vitro. The observed differences are not due to the presence of--or response to--alternate secondary structures in the trp leader transcript since deletion of the DNA segment corresponding to some of these alternate structures does not affect termination efficiency. The mutant polymerases also have comparable effects on the kinetics of transcription pausing at the trp leader pause site; the termination-deficient polymerase exhibits diminished pausing while the termination-proficient polymerase displays enhanced pausing. We suggest that this correlation reflects polymerase recognition of similar features of RNA secondary structures in the pause and termination events. In addition, since single mutational changes in RNA polymerase affect two activities, pausing and termination, it is likely that a single site or region of the polymerase is involved in both events.

Base Sequence↗
PubMed1993

Human CD3-CD16+ natural killer cells express the hGATA-3 T cell transcription factor and an unrearranged 2.3-kb TcR delta transcript.

In this study we analyzed the T cell receptor(TcR) delta transcripts expressed by CD3-CD16+ cells and we investigated whether these cells expressed the hGATA-3 T cell transcription factor and the recombination-activating gene (RAG)-1. Multiple TcR delta transcripts deriving from an unrearranged TcR delta gene were detected in both polyclonal and clonal CD3-CD16+ natural killer(NK) cell lines. Two unrearranged TcR delta transcripts had a size similar to that of the functional TcR delta mRNA (2.3 and 1.3 kb) found in TcR gamma/delta+ T lymphocytes. Sequence analysis of nine different 2.3-kb cDNA clones obtained from NK-derived polyA+ RNA confirmed that they corresponded to an unrearranged TcR delta gene. These cDNA were 2343 bp long and their transcription initiation site was located 814 bp upstream from the J delta 1 segment. The sequence located upstream of the J delta 1 segment corresponded to the previously reported germ-line sequence. The J delta 1 segment was correctly spliced to C delta; in addition the four C delta exons were found to be already assembled. Two polyadenylation sites were present in the fourth C delta exon. However, only that located at the 3' end appeared to be utilized in the 2.3-kb cDNA. The expression of hGATA-3, a T cell-specific factor known to be involved in the regulation of the transcription of TcR delta locus, was analyzed by Northern blot, in cultured NK cell population and clones (but not in freshly derived cell populations). All NK clones and cell lines studied were found to express hGATA-3-specific mRNA, suggesting that hGATA-3 may be involved in the regulation of the unrearranged TcR delta gene expression in NK cells. Finally, no transcription of the RAG-1 gene could be detected in all NK cell lines or clones analyzed.

Base Sequence↗
PubMed1995

The age-related decline in the induction of IL-2 transcription is correlated to changes in the transcription factor NFAT.

The effect of aging on the induction of IL-2 transcription by concanavalin A was studied in spleen lymphocytes isolated from young (4-6 months) and old (24 months) male Fischer 344 rats. The induction of IL-2 mRNA levels and IL-2 transcription were 40 to 50% lower for spleen lymphocytes isolated from old rats compared to spleen lymphocytes isolated from young rats. Because the transcription factor NFAT (nuclear factor activating IL-2 transcription) plays a major role in the regulation of IL-2 transcription, the DNA binding activity of NFAT in nuclear extracts of concanavalin A-stimulated lymphocytes was measured using a gel shift assay. The ability of nuclear extracts isolated from either total spleen lymphocytes or T cells to bind the NFAT oligonucleotide decreased 50 to 60% with age. Therefore, the age-related decrease in the expression of IL-2 appears to occur at the level of transcription and involves the transcription factor NFAT.

Aging↗
PubMed1998

The activities of acidic and glutamine-rich transcriptional activation domains in plant cells: design of modular transcription factors for high-level expression.

The aim of this work was to design strong transcriptional activators that can be used to regulate plant gene expression. The contribution of different components in a transcription factor and target gene system was assayed by measuring transcriptional activation. Each component was optimised to achieve maximal reporter gene expression in transient protoplast transformation assays. The DNA-binding domain of the yeast transcriptional activator GAL4 was studied in the context of fusion proteins with activation domains of the herpes simplex virus protein VP16 or the tomato Myb-like activator THM18. Multimerisation of the activation domain and insertion of a homopolymeric glutamine stretch was used to increase transcription factor potency. Evidence is presented that these modifications can result in even more active transcription factors when they are combined. Finally, it was demonstrated using competition experiments that transcription factors with acidic activation domains can mutually suppress their activation potentials when expressed at high levels.

Base Sequence↗
PubMed1998

The transcription of the intercellular adhesion molecule-1 is regulated by Ets transcription factors.

The Ets family of transcription factors comprises several members which are involved to regulate gene transcription. Although several consensus binding sites for Ets proteins can be found in a wide series of promoter, only a limited number of them are indeed activated by these transcription factors. The human intercellular adhesion molecule-1 (ICAM-1) plays a crucial role in immune responses by enabling the binding of effector cells to various target cell types. ICAM-1 is constitutively expressed at different levels in the absence of stimuli in different cell types, and its expression is upregulated by several proinflammatory cytokines. We have here examined the transcriptional regulation of human ICAM-1 expression by Ets proteins, and more particularly by ERM, a member of this family of transcription factors. Transient transfection assays revealed that Ets-2 and ERM significantly activate the transcription of ICAM-1 promoter, whereas the less-related Ets family member, Spi-1/Pu.1, failed to do so. Transfection of a series of ICAM-1 promoter deletion mutants together with ERM expression plasmids have shown that an Ets responsive element is located within the first 176 bp upstream from the translational start site. Electrophoretic mobility shift assays and DNase I footprinting analysis have enabled us to identify two Ets binding sites at positions -158 and -138 from the ATG, respectively. Site directed mutagenesis of these elements has shown that the distal site is the major element required for the ERM-mediated activation of the ICAM-1 promoter. We can thus conclude that expression of ICAM-1 can be regulated by Ets transcription factors.

Animals↗
PubMed1990

Engrailed, a homeodomain protein, can repress in vitro transcription by competition with the TATA box-binding protein transcription factor IID.

Engrailed (En) is a homeodomain protein that binds to a consensus sequence (NP) and plays an important role during Drosophila development. Purified En, which is produced in Escherichia coli, binds not only to this consensus sequence but also to the TATA box of the Drosophila Hsp70 promoter and of other eukaryotic promoters. Interestingly, En represses transcription of these promoters in an in vitro-reconstituted mammalian transcription system and footprint analyses show that En competes with the TATA box-binding protein transcription factor IID for binding to the TATA box. In contrast, a stable template-committed complex formed by preincubation of transcription factor IID with the promoter is not disrupted by addition of En, and in this case transcription is not repressed. These in vitro studies suggest a transcriptional repression mechanism, involving competition between En and transcription factor IID for TATA box binding, that may be involved in En-mediated repression in vivo.

Animals↗
PubMed1996

Overexpression of the transcription factor UBF1 is sufficient to increase ribosomal DNA transcription in neonatal cardiomyocytes: implications for cardiac hypertrophy.

The accelerated protein accumulation characteristic of cardiomyocyte hypertrophy results from increased cellular protein synthetic capacity (elevated ribosome content). The rate limiting step in ribosome accumulation is transcription of the rRNA genes. During neonatal cardiomyocyte hypertrophy induced by norepinephrine or spontaneous contraction, changes in the expression of a ribosomal DNA transcription factor, UBF, correlated with increased rates of ribosome biogenesis. We hypothesized that elevated expression of UBF was part of the mechanism by which these hypertrophic stimuli effected increases in the rate of transcription from the rDNA promoter. In this study, we have examined directly the effect of overexpressing UBF on rDNA transcription in neonatal cardiomyocytes in culture. In control experiments, a novel reporter construct for rDNA transcription (pSMECAT) showed similar increases in activity in response to hypertrophic stimuli (10(-4) M phenylephrine, 10(-7) M endothelin, and spontaneous contraction) as did the endogenous rRNA genes. When contraction-arrested cardiomyocytes were cotransfected with pSMECAT and increasing amounts of a UBF1 expression vector; a dose-dependent (3-5 fold) increase in rDNA transcription was observed. Western blot analysis confirmed that the overexpressed, FLAG-tagged UBF accumulated in the cardiomyocyte nuclei. The observation that overexpression of UBF1 is sufficient to increase rDNA transcription in neonatal cardiomyocytes provides evidence in support of the hypothesis that the regulation of UBF is a key component of the increased ribosome biogenesis and protein accumulation associated with cardiomyocyte hypertrophy.

Animals↗
PubMed2000

Regulation of transcription of the human presenilin-1 gene by ets transcription factors and the p53 protooncogene.

The expression of the human presenilin-1 cellular gene is suppressed by the p53 protooncogene. The rapid kinetic of the down-regulation has suggested that it may result from a primary mechanism. We show here that p53 also suppresses the transcription of a presenilin-1 promoter-chloramphenicol acetyltransferase reporter synthetic gene in transient infection assays in neuroblastoma (SK-N-SH) and hepatoma (HepG2) cell lines. Only a minimum promoter including sequences from -35 to + 6 from the transcription initiation is sufficient to confer down-regulation. We have previously defined a crucial DNA element controlling 90% of the expression of the gene within the same short area, and the identification of the transcription factors involved should also provide insights into the regulation of PS1 by p53. This region contains an Ets transcription factor binding motif, and a 2-base pair alteration within the core sequence (GGAA to TTAA) of the Ets consensus also reduced transcription by more than 90%. We now show that Ets1 and Ets2 indeed transactivate a PS1 promoter-chloramphenicol acetyltransferase reporter including the (-35 to +6) fragment. Furthermore, in vitro translated Ets2 binds specifically to the -10 Ets motif in electrophoretic mobility shift assays. Therefore, Ets1/2 factors bind specifically to the -10 Ets element and activate PS1 transcription. We also show that the coactivator p300 enhances the activation by Ets1 and Ets2 as well as the repression by p53. p300 is known to interact with p53 as well as with Ets1 and Ets2. We show that p53 does not bind directly to the PS1 promoter. Hence the repression of PS1 transcription by p53 is likely to be mediated through protein-protein interactions.

Amino Acid Motifs↗
PubMed1996

Functional domains of transcription factor hGABP beta1/E4TF1-53 required for nuclear localization and transcription activation.

Transcription factor E4TF1 is the human homolog of GABP and has been renamed hGABP (human GABP). hGABP is composed of two types of subunits; hGABP beta1/E4TF1-53 and the ets-related protein hGABP alpha/E4TF1-60. Both bind together to form an (alpha)2(beta1)2 heterotetrameric complex on DNA and activate transcription at specific promoters in vitro. Tetramer formation depends on two regions of hGABP beta1; the N-terminal region containing the Notch/ankyrin-type repeats is necessary for binding to hGABP alpha and the C-terminal region is necessary for homodimerization. In this report, we constructed various deletion mutants of hGABP beta1 in order to delimit the functional regions required for nuclear localization and transcription activity. We found that hGABP beta1 localization in the nucleus is dependent on a region located between amino acids 243 and 330 and that the presence of hGABP beta1 influences the efficiency of hGABP alpha transport into the nucleus. Next, we demonstrated that the hGABP complex composed of alpha and beta1 subunits activates transcription from the adenovirus early 4 promoter in vivo. This transcription activation needs the C-terminal region of hGABP beta1 and is consistent with results obtained with the in vitro assay. Furthermore, site-directed mutagenesis analysis of the C-terminal region reveals that the alpha-helix structure and the leucine residues are important for formation of a heterotetrameric complex with hGABP alpha in vitro and for transcription activation in vivo. These results suggest that hGABP beta1 stimulates transcription as part of a heterotetrameric complex with hGABP alpha in vivo.

Animals↗
PubMed1998

New core promoter element in RNA polymerase II-dependent transcription: sequence-specific DNA binding by transcription factor IIB.

A sequence element located immediately upstream of the TATA element, and having the consensus sequence 5'-G/C-G/C-G/A-C-G-C-C-3', affects the ability of transcription factor IIB to enter transcription complexes and support transcription initiation. The sequence element is recognized directly by the transcription factor IIB. Recognition involves alpha-helices 4' and 5' of IIB, which comprise a helix-turn-helix DNA-binding motif. These observations establish that transcription initiation involves a fourth core promoter element, the IIB recognition element (BRE), in addition to the TATA element, the initiator element, and the downstream promoter element, and involves a second sequence-specific general transcription factor, IIB, in addition to transcription factor IID.

Amino Acid Sequence↗
PubMed1995

A 10-amino-acid sequence in the N-terminal A/B domain of thyroid hormone receptor alpha is essential for transcriptional activation and interaction with the general transcription factor TFIIB.

The effects of the thyroid hormone (3,5,3'-triiodo-L-thyronine [T3]) on gene transcription are mediated by nuclear T3 receptors (T3Rs). alpha- and beta-isoform T3Rs (T3R alpha and -beta) are expressed from different genes and are members of a superfamily of ligand-dependent transcription factors that also includes the receptors for steroid hormones, vitamin D, and retinoids. Although T3 activates transcription by mediating a conformational change in the C-terminal approximately 220-amino-acid ligand-binding domain (LBD), the fundamental mechanisms of T3R-mediated transcriptional activation remain to be determined. We found that deletion of the 50-amino-acid N-terminal A/B domain of chicken T3R alpha (cT3R alpha) decreases T3-dependent stimulation of genes regulated by native thyroid hormone response elements about 10- to 20-fold. The requirement of the A/B region for transcriptional activation was mapped to amino acids 21 to 30, which contain a cluster of five basic amino acids. The A/B region of cT3R alpha is not required for T3 binding or for DNA binding of the receptor as a heterodimer with retinoid X receptor. In vitro binding studies indicate that the N-terminal region of cT3R alpha interacts efficiently with TFIIB and that this interaction requires amino acids 21 to 30 of the A/B region. In contrast, the LBD interacts poorly with TFIIB. The region of TFIIB primarily involved in the binding of cT3R alpha includes an amphipathic alpha helix contained within residues 178 to 201. Analysis using a fusion protein containing the DNA-binding domain of GAL4 and the entire A/B region of cT3R alpha suggests that this region does not contain an intrinsic activation domain. These and other studies indicate that cT3R alpha mediates at least some of its effects through TFIIB in vivo and that the N-terminal region of DNA-bound cT3R alpha acts to recruit and/or stabilize the binding of TFIIB to the transcription complex. T3 stimulation could then result from ligand-mediated changes in the LBD which may lead to the interaction of other factors with cT3R alpha, TFIIB, and/or other components involved in the initiation of transcription.

Amino Acid Sequence↗
PubMed1998

Regulation of transcription by hypoxia requires a multiprotein complex that includes hypoxia-inducible factor 1, an adjacent transcription factor, and p300/CREB binding protein.

Molecular adaptation to hypoxia depends on the binding of hypoxia-inducible factor 1 (HIF-1) to cognate response elements in oxygen-regulated genes. In addition, adjacent sequences are required for hypoxia-inducible transcription. To investigate the mechanism of interaction between these cis-acting sequences, the multiprotein complex binding to the lactate dehydrogenase A (LDH-A) promoter was characterized. The involvement of HIF-1, CREB-1/ATF-1, and p300/CREB binding protein (CBP) was demonstrated by techniques documenting in vitro binding, in combination with transient transfections that test the in vivo functional importance of each protein. In both the LDH-A promoter and the erythropoietin 3' enhancer, formation of multiprotein complexes was analyzed by using biotinylated probes encompassing functionally critical cis-acting sequences. Strong binding of p300/CBP required interactions with multiple DNA binding proteins. Thus, the necessity of transcription factor binding sites adjacent to a HIF-1 site for hypoxically inducible transcription may be due to the requirement of p300 to interact with multiple transcription factors for high-affinity binding and activation of transcription. Since it has been found to interact with a wide range of transcription factors, p300 is likely to play a similar role in other genes, mediating interactions between DNA binding proteins, thereby activating stimulus-specific and tissue-specific gene transcription.

Activating Transcription Factor 1↗
PubMed1999

Transcriptional repression of Stat6-dependent interleukin-4-induced genes by BCL-6: specific regulation of iepsilon transcription and immunoglobulin E switching.

The BCL-6 proto-oncogene encodes a POZ/zinc-finger transcription factor that is expressed in B cells and a subset of CD4(+) T cells within germinal centers. Recent evidence suggests that BCL-6 can act as a sequence-specific repressor of transcription, but the target genes for this activity have not yet been identified. The binding site for BCL-6 shares striking homology to the sites that are the target sequence for the interleukin-4 (IL-4)-induced Stat6 (signal transducers and activators of transcription) signaling molecule. Electrophoretic mobility shift assays demonstrate that BCL-6 can bind, with different affinities, to several DNA elements recognized by Stat6. Expression of BCL-6 can repress the IL-4-dependent induction of immunoglobulin (Ig) germ line epsilon transcripts, but does not repress the IL-4 induction of CD23 transcripts. Consistent with the role of BCL-6 in modulating transcription from the germ line epsilon promoter, BCL-6(-/-) mice display an increased ability to class switch to IgE in response to IL-4 in vitro. These animals also exhibit a multiorgan inflammatory disease characterized by the presence of a large number of IgE(+) B cells. The apparent dysregulation of IgE production is abolished in BCL-6(-/-) Stat6(-/-) mice, indicating that BCL-6 regulation of Ig class switching is dependent upon Stat6 signaling. Thus, BCL-6 can modulate the transcription of selective Stat6-dependent IL-4 responses, including IgE class switching in B cells.

Animals↗
PubMed1988

An RNA polymerase II transcription factor inactivated in poliovirus-infected cells copurifies with transcription factor TFIID.

Inhibition of host cell RNA polymerase II-mediated transcription by poliovirus infection was studied in vitro. Whole-cell extracts prepared from poliovirus-infected HeLa cells at 3 h postinfection were shown to be deficient in a factor required for specific transcription from the adenovirus major late promoter. Three lines of evidence suggest that transcription factor TFIID is deficient in poliovirus-infected cells. First, the activity required to specifically restore transcription in poliovirus-infected cell extracts was shown to copurify with TFIID through three chromatographic steps. Second, transcription reactions reconstituted with phosphocellulose-derived chromatographic fractions revealed a fourfold decrease in the specific activity of the TFIID-containing fraction prepared from poliovirus-infected cells compared with that of the same fraction prepared from mock-infected cells. Finally, TFIID and the activity required to specifically restore transcription in virus-infected cell extracts were shown to have the same kinetics of heat inactivation. Together, these results suggest that inactivation of TFIID is an early event in the inhibition of host cell RNA polymerase II transcription by poliovirus.

Cell Transformation, Viral↗
PubMed1993

Negative transcriptional regulation of human interleukin 2 (IL-2) gene by glucocorticoids through interference with nuclear transcription factors AP-1 and NF-AT.

IL-2 gene transcription is affected by several nuclear proteins. We asked whether dexamethasone (Dex) and cyclosporin A (CsA) inhibit IL-2 gene transcription by interfering with the activity of nuclear proteins that bind to the IL-2 promoter. Nuclear extracts from primary human T lymphocytes were analyzed by electrophoretic DNA mobility shift assays. Both Dex and CsA inhibited the binding of transcription factors AP-1 and NF-AT, but not of NF-kB and OCT-1/OAF, to their corresponding sites on the IL-2 gene promoter. To correlate changes in nuclear factor binding in vitro with transcriptional activity in vivo and define the structural requirements for IL-2 promoter repression, we used transient DNA transfections. Jurkat cells were transfected with plasmids containing either the intact IL-2 promoter or its AP-1, NF-AT, and NF-kB motifs. Dex inhibited the IL-2 promoter and the AP-1, but not the NF-AT and NF-kB plasmids. In contrast, CsA inhibited the IL-2 promoter and the NF-AT, but not the AP-1 and NF-kB plasmids. These results suggest that in human T lymphocytes both Dex and CsA inhibited IL-2 gene transcription through interference with transcription factors AP-1 and NF-AT. We propose that, while maximum inhibition may involve interaction with both transcription factors, AP-1 is the primary target of Dex.

Cyclosporine↗
PubMed2001

Cooperative effects of STAT5 (signal transducer and activator of transcription 5) and C/EBPbeta (CCAAT/enhancer-binding protein-beta) on beta-casein gene transcription are mediated by the glucocorticoid receptor.

Beta-casein gene transcription is controlled primarily by a composite response element (CoRE) that integrates signaling from the lactogenic hormones, PRL, insulin, and hydrocortisone, in mammary epithelial cells. This CoRE contains binding sites for STAT5 (signal transducer and activator of transcription 5) and C/EBPbeta (CCAAT/enhancer-binding protein-beta) and several half-sites for glucocorticoid receptor (GR). To examine how interactions among these three transcription factors might regulate beta-casein gene transcription, a COS cell reconstitution system was employed. Cooperative transactivation was observed when all three factors were expressed, but unexpectedly was not seen between STAT5 and C/EBPbeta in the absence of full-length, transcriptionally active GR. Cooperativity required the amino-terminal transactivation domain of C/EBPbeta, and neither C/EBPalpha nor C/EBPdelta was able to substitute for C/EBPbeta when cotransfected with STAT5 and GR. Different GR determinants were needed for transcriptional cooperation between STAT5 and GR as compared with those required for all three transcription factors. These studies provide some new insights into the mechanisms responsible for high level, tissue-specific expression conferred by the beta-casein CoRE.

Animals↗
PubMed2000

Regulation of tyrosine hydroxylase gene transcription by the cAMP-signaling pathway: involvement of multiple transcription factors.

The conversion of L-tyrosine to 3,4-dihydroxy-L-phenylalanine by tyrosine hydroxylase (TH) is the first and rate-limiting step in biosynthesis of catecholamine neurotransmitters. TH gene expression is regulated in a cell type-specific and cAMP-dependent manner. Evidence from this laboratory and others indicates that the cAMP response element (CRE), residing at -45 to -38 bp upstream of the transcription initiation site, is essential for both basal and cAMP-inducible transcription of the TH gene. To understand the control mechanisms of TH gene transcription in greater detail, we sought to identify and characterize the transcription factors involved in recognition and activation of the CRE of the TH gene. Remarkably, electrophoretic mobility shift assay and antibody supershift experiments indicated that all three major CRE-binding protein factors, i.e. CREB, ATF1, and CREM, may participate in forming specific DNA/protein complexes with the CRE of the TH gene. To address the transcriptional activation function of individual factors, we replaced the TH CRE with a GAL4-binding site and cotransfected this modified TH promoter-reporter gene with an effector plasmid that encodes GAL4-fused transcription factor. Our results indicate that CREB but not ATF1 can support basal promoter activity while both can robustly induce the promoter activity in response to co-expression of the catalytic subunit of cAMP-dependent protein kinase (PKA). We further show that the coactivator CBP up-regulates PKA-mediated activation of the TH promoter and, if tethered to the TH promoter by a GAL4-fusion, can robustly transactivate the TH promoter even in the absence of PKA. Collectively, our results suggest that multiple CRE-binding factors interact with the CRE and regulate, in conjunction with the coactivator CBP, the transcriptional activity of the TH gene.

Activating Transcription Factor 1↗