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At least 1,369 records · Page 76Linked to original sources
PubMed2003

Regulation of proglucagon transcription by activated transcription factor (ATF) 3 and a novel isoform, ATF3b, through the cAMP-response element/ATF site of the proglucagon gene promoter.

Glucagon, the second major glucose-regulated hormone in the control of glucose homeostasis, functions as a counter-regulator to insulin and is specifically produced by the pancreatic alpha cells. Its excessive biosynthesis and secretion is associated with diabetes mellitus. The expression of the proglucagon gene has been demonstrated to be regulated by a cAMP-dependent pathway through cAMP-response element-binding protein (CREB) and possibly other transcription factors bound to its cAMP-response element (CRE)/activated transcription factor (ATF) site. Elsewhere we have shown that ATF3, a member of the ATF/CREB subfamily of the basic leucine zipper domain proteins, is expressed predominantly in the alpha cells of the pancreatic islets. In our attempts to further dissect the role of ATF3 proteins in alpha cells, we have identified and characterized a novel alternatively spliced form, ATF3b, and have compared the specific binding ability of ATF3 and ATF3b on the CRE/ATF motif of the proglucagon promoter. Our findings indicate the existence of a novel mechanism by which the transcription of the proglucagon gene is regulated in response to cAMP signals, in addition to CREB and in relation to glucose fluctuations in pancreatic alpha cells.

3T3 Cells↗
PubMed2003

Coupling of the transcriptional regulation of glutathione biosynthesis to the availability of glutathione and methionine via the Met4 and Yap1 transcription factors.

Depletion of the cellular pool of glutathione is detrimental to eukaryotic cells and in Saccharomyces cerevisiae leads to sensitivity to oxidants and xenobiotics and an eventual cell cycle arrest. Here, we show that the Yap1 and Met4 transcription factors regulate the expression of gamma-glutamylcysteine synthetase (GSH1), encoding the rate-limiting enzyme in glutathione biosynthesis to prevent the damaging effects of glutathione depletion. Transcriptional profiling of a gsh1 mutant indicates that glutathione depletion leads to a general activation of Yap1 target genes, but the expression of Met4-regulated genes remains unaltered. Glutathione depletion appears to result in Yap1 activation via oxidation of thioredoxins, which normally act to down-regulate the Yap1-mediated response. The requirement for Met4 in regulating GSH1 expression is lost in the absence of the centromere-binding protein Cbf1. In contrast, the Yap1-mediated effect is unaffected, indicating that Met4 acts via Cbf1 to regulate the Yap1-mediated induction of GSH1 expression in response to glutathione depletion. Furthermore, yeast cells exposed to the xenobiotic 1-chloro-2,4-dintrobenzene are rapidly depleted of glutathione, accumulate oxidized thioredoxins, and elicit the Yap1/Met4-dependent transcriptional response of GSH1. The addition of methionine, which promotes Met4 ubiquitination and inactivation, specifically represses GSH1 expression after 1-chloro-2,4-dintrobenzene exposure but does not affect Yap1 activation. These results indicate that the Yap1-dependent activation of GSH1 expression in response to glutathione depletion is regulated by the sulfur status of the cell through a specific Met4-dependent mechanism.

Basic Helix-Loop-Helix Leucine Zipper Transcriptio↗
PubMed1992

Transcription factor IID mutants defective for interaction with transcription factor IIA.

Transcription factor IID (TFIID) recognizes the TATA element of promoters transcribed by RNA polymerase II (RNAPII) and serves as the base for subsequent association by other general transcription factors and RNAPII. The carboxyl-terminal domain of TFIID is highly conserved and contains an imperfect repetition of a 60-amino acid sequence. These repeats are separated by a region rich in basic amino acids. Mutagenesis of the lysines in this region resulted in a conditioned phenotype in vivo, and the mutant proteins were defective for interactions with transcription factor IIA in vitro. Binding of TFIID to DNA was unaffected. These results suggest that the basic domain of TFIID is important for protein-protein interactions.

Amino Acid Sequence↗
PubMed1999

Human transcription factor hTAF(II)150 (CIF150) is involved in transcriptional regulation of cell cycle progression.

Here we present evidence that CIF150 (hTAF(II)150), the human homolog of Drosophila TAF(II)150, plays an important and selective role in establishing gene expression patterns necessary for progression through the cell cycle. Gel filtration experiments demonstrate that CIF150 (hTAF(II)150) seems to be less tightly associated with human transcription factor IID than hTAF(II)130 is associated with hTAF(II)250. The transient functional knockout of CIF150 (hTAF(II)150) protein led to cell cycle arrest at the G(2)/M transition in mammalian cell lines. PCR display analysis with the RNA derived from CIF150-depleted cells indicated that CIF150 (hTAF(II)150) is required for the transcription of only a subset of RNA polymerase II genes. CIF150 (hTAF(II)150) directly stimulated cyclin B1 and cyclin A transcription in cotransfection assays and in vitro assays, suggesting that the expression of these genes is dependent on CIF150 (hTAF(II)150) function. We defined a CIF150 (hTAF(II)150) consensus binding site and demonstrated that a CIF150-responsive cis element is present in the cyclin B1 core promoter. These results suggest that one function of CIF150 (hTAF(II)150) is to select specific RNA polymerase II core promoter elements involved in cell cycle progression.

Cell Cycle↗
PubMed2001

The novel transcription factor e(y)2 interacts with TAF(II)40 and potentiates transcription activation on chromatin templates.

Weak hypomorph mutations in the enhancer of yellow genes, e(y)1 and e(y)2, of Drosophila melanogaster were discovered during the search for genes involved in the organization of interaction between enhancers and promoters. Previously, the e(y)1 gene was cloned and found to encode TAF(II)40 protein. Here we cloned the e(y)2 gene and demonstrated that it encoded a new ubiquitous evolutionarily conserved transcription factor. The e(y)2 gene is located at 10C3 (36.67) region and is expressed at all stages of Drosophila development. It encodes a 101-amino-acid protein, e(y)2. Vertebrates, insects, protozoa, and plants have proteins which demonstrate a high degree of homology to e(y)2. The e(y)2 protein is localized exclusively to the nuclei and is associated with numerous sites along the entire length of the salivary gland polytene chromosomes. Both genetic and biochemical experiments demonstrate an interaction between e(y)2 and TAF(II)40, while immunoprecipitation studies demonstrate that the major complex, including both proteins, appears to be distinct from TFIID. Furthermore, we provide genetic evidence suggesting that the carboxy terminus of dTAF(II)40 is important for mediating this interaction. Finally, using an in vitro transcription system, we demonstrate that recombinant e(y)2 is able to enhance transactivation by GAL4-VP16 on chromatin but not on naked DNA templates, suggesting that this novel protein is involved in the regulation of transcription.

Amino Acid Sequence↗
PubMed1998

Testicular leukemia inhibitory factor (LIF) and LIF receptor mediate phosphorylation of signal transducers and activators of transcription (STAT)-3 and STAT-1 and induce c-fos transcription and activator protein-1 activation in rat Sertoli but not germ cells.

Increasing amounts of evidence suggest noninflammatory roles for growth factor and cytokines in development and differentiation. Leukemia inhibitory factor (LIF) belongs to a gp130 pleiotropic family of growth factors that has recently been shown to enhance the survival of rat testicular gonocytes and Sertoli cells. In this study, we show the expression of gp130 and LIF messenger RNAs (mRNAs) in the somatic (the Sertoli and Leydig cells) and specific germ cells (spermatogonia, pachytene, round, and elongated spermatids) of rodent testis, suggestive of cell-specific LIF-mediated functions. LIF receptor mRNA was demonstrated in rat somatic cells, rat elongating spermatids, and all of the mouse germ cells. In addition, we characterized the effects of LIF on the signal transducers and activators of transcription (STAT)-3 and STAT-1, c-fos gene expression, and activator protein-1 regulation in primary rat Sertoli cells. Electrophoretic mobility shift assay and Western blot analysis demonstrated that LIF translocates STAT-3 (and to a lesser extent STAT-1) transcription factor(s) to the nucleus within 2 min of exposure in a tyrosine but not serine/threonine phosphorylation-dependent pathway. Quantitative solution hybridization analysis revealed a transient increase in c-fos mRNA levels by 20-fold following 30-45 min of LIF treatment, an effect that was inhibited by the tyrosine, as well as serine/threonine kinase inhibitors, genistein, and H7. Subsequently, LIF treatment of the Sertoli cells increased nuclear activator protein-1 binding proteins at 2 h after its addition, an effect that was also sensitive to genistein and H7 pretreatments. In contrast, LIF treatment of primary rat germ cells did not alter c-fos mRNA levels. Species specificity in the expression of LIF receptor as well as ligand binding may play a role in LIF signaling in these germ cells. Thus, using a primary Sertoli cell model, we demonstrated that the testicular LIF signaling pathway is contingent on the phosphorylation of latent transcription factors. Our data are consistent with LIF-mediated signaling events involving both somatic and germ cells during spermatogenesis.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1999

Transcription factor GATA-4 enhances Müllerian inhibiting substance gene transcription through a direct interaction with the nuclear receptor SF-1.

Secretion of Müllerian-inhibiting substance (MIS) by Sertoli cells of the fetal testis and subsequent regression of the Müllerian ducts in the male embryo is a crucial event that contributes to proper sex differentiation. The zinc finger transcription factor GATA-4 and nuclear receptor SF-1 are early markers of Sertoli cells that have been shown to regulate MIS transcription. The fact that the GATA and SF-1 binding sites are adjacent to one another in the MIS promoter raised the possibility that both factors might transcriptionally cooperate to regulate MIS expression. Indeed, coexpression of both factors resulted in a strong synergistic activation of the MIS promoter. GATA-4/SF-1 synergism was the result of a direct protein-protein interaction mediated through the zinc finger region of GATA-4. Remarkably, synergy between GATA-4 and SF-1 on a variety of different SF-1 targets did not absolutely require GATA binding to DNA. Moreover, synergy with SF-1 was also observed with other GATA family members. Thus, these data not only provide a clearer understanding of the molecular mechanisms that control the sex-specific expression of the MIS gene but also reveal a potentially novel mechanism for the regulation of SF-1-dependent genes in tissues where SF-1 and GATA factors are coexpressed.

Animals↗
PubMedDate not supplied

The pro-oncoprotein EWS (Ewing's Sarcoma protein) interacts with the Brn-3a POU transcription factor and inhibits its ability to activate transcription.

The Brn-3a POU family transcription factor is able to induce the expression of a number of neuronally-expressed genes as well as to enhance neuronal differentiation and inhibit apoptosis. Many of these effects are mediated by the C-terminal POU domain of Brn-3a which acts both as a DNA binding domain and a transcriptional activation domain. To identify the mechanisms by which this domain acts, we carried out a yeast two hybrid assay to identify proteins which interact with it. We show that both full length Brn-3a and the isolated POU domain interact with the EWS transcription factor and its oncogenic derivative EWS-Fli1. Moreover, EWS can block Brn-3a-mediated activation of the Bcl-x promoter whereas this effect is lost in EWS-Fli1. The significance of this novel interaction is discussed in terms of the manner in which Brn-3a regulates its target promoters and the mechanism of oncogenic transformation by EWS-Fli1.

Cell Line↗
PubMed1994

The macromolecular state of the transcription factor E2F and glucocorticoid regulation of c-myc transcription.

Glucocorticoids inhibit transcription of the proto-oncogene c-myc in lymphoid cells of thymic origin. To determine if this effect is associated with changes in the properties of the transcription factor E2F, extracts were prepared from control and glucocorticoid-treated P1798 murine T lymphoma cells, and the macromolecular state of E2F was assessed by gel-mobility shift. Control extracts exhibit two predominant gel-mobility shift entities of which one corresponds to "free" E2F. A second entity, complex C, has properties similar to those described for the complex containing E2F, p107, cyclin A, and Cdk2. Complex C disappears after addition of dexamethasone and is replaced by complex D. The mobility of this complex and its sensitivity to SV40 T antigen suggest that complex D corresponds to an E2F-p105Rb-1 complex. Extracts from control and glucocorticoid-treated cells yield identical DNase I protection patterns on the c-myc P2 promoter. Furthermore, such extracts transcribe the c-myc P2 promoter in vitro with equal activity. The relative abundance of the E2F complexes was measured after addition of dexamethasone. Complex C disappears as cells withdraw from S phase, and complex D appears at this time. The genes encoding thymidine kinase (Tk-1) and p34cdc2 (cdc2) are regulated with kinetics similar to those observed for changes in the macromolecular state of E2F. However, regulation of c-myc expression occurs long before any change in E2F. The macromolecular state of E2F may regulate expression of genes at the G1/S boundary. However, the data are not consistent with the hypothesis that association of E2F with tumor suppressor gene products such as p107 or p105Rb-1 is relevant to glucocorticoid regulation of c-myc transcription.

Animals↗
PubMed1998

Transcriptional regulation of lysosomal acid lipase in differentiating monocytes is mediated by transcription factors Sp1 and AP-2.

Human lysosomal acid lipase (LAL) is a hydrolase required for the cleavage of cholesteryl esters and triglycerides derived from plasma lipoproteins. It is shown here that during monocyte to macrophage differentiation, the expression of LAL-mRNA is induced. This induction is dependent on protein kinase C activity and protein synthesis. The cell type-specific increase in LAL expression is further investigated in the THP-1 cell line with respect to transcriptional regulation. The human monocytic leukemia cell line THP-1 differentiates into macrophage-like cells when treated with phorbol esters. In order to determine the cis-acting elements necessary for both basal and phorbol 12-myristate-13 acetate (PMA)-enhanced promoter activity, we performed deletion analysis and reporter gene assays. A PMA responsive element has been identified between -182 bp and -107 bp upstream of the major transcription start site. Gel mobility shift assays demonstrated that binding of Sp1 and AP-2 to the LAL promoter is increased by PMA in THP-1 cells. Co-transfections with expression plasmids for Sp1 and AP-2 further emphasized the important role of these transcription factors in both basal and PMA-enhanced LAL expression. Our data suggest that differentiation dependent increase of lysosomal acid lipase (LAL) expression in THP-1 cells is mediated by a concerted action of Sp1 and AP-2.

Cell Differentiation↗
PubMed1997

Transcriptional and post-transcriptional control of lysyl oxidase expression in vascular smooth muscle cells: effects of TGF-beta 1 and serum deprivation.

Transforming growth factor-beta 1 (TGF-beta 1) markedly reduced cell proliferation and elevated steady state lysyl oxidase (LO) mRNA 3-fold in neonatal rat aorta smooth muscle cells cultured in medium containing 10% fetal bovine serum. The increase in LO mRNA was prevented by the presence of cycloheximide, indicative of controlling events at the level of protein synthesis. The basal level of mRNA in cells proliferating in 10% fetal bovine serum in the absence of TGF-beta 1 was enhanced 7-fold upon decreasing growth by shifting to medium containing 0.5% serum. Changes in LO activity paralleled those in LO mRNA. Nuclear run-on assays revealed that the stimulation of expression in 0.5% serum involved increased gene transcription whereas that caused by TGF-beta 1 was mostly post-transcriptional in origin. LO mRNA was quite labile (t1/2 approximately 3 h) in 10% serum but was markedly stabilized (t1/2 > 12 h) by the presence of TGF-beta 1 in the 10% serum medium. LO mRNA was also considerably more stable under retarded growth conditions (0.5% serum) in the absence of TGF-beta 1. LO promoter activity in luciferase reporter constructs transfected into these cells was low and not significantly affected by the addition of TGF-beta 1 to the 10% serum medium but was markedly elevated by shifting from 10 to 0.5% serum in the absence of TGF-beta 1. Thus, LO expression is inversely correlated with cell proliferation, and is subject to control at transcriptional and post-transcriptional levels. TGF-beta 1 enhances LO expression in these cells by dramatically stabilizing LO mRNA.

Animals↗
PubMed1990

Transcriptional and post-transcriptional regulation of tyrosine hydroxylase gene by protein kinase C.

The role played by protein kinase C (PKC) in TH gene regulation was investigated at transcriptional and post-transcriptional levels using PC12 cells. The cells were treated with the phorbol ester TPA, which not only activates PKC but also causes down-regulation. PKC levels were monitored by [3H]PDBU binding assay and by using an anti-PKC antibody that detected intact PKC (79 kd) as well as its catalytic and regulatory domains. The [3H]PDBU binding to the membrane-associated PKC increased within 15-30 min of TPA treatment; thereafter total cellular [3H]PDBU binding decreased to a minimum of 20% of the control at 8 h. The rate of decrease in binding was greater than the decrease in the intensity of the staining of PKC holo enzyme visualized by anti-PKC antibody. TH mRNA levels, measured over the same time period, rose within 15 min of TPA treatment to peak at 4 h and subsequently declined below control level, paralleling the depletion of PKC. If cells depleted of PKC were reincubated in the normal medium, a recovery in PKC level was seen and, in parallel, TH mRNA levels increased to above control level. Furthermore, if down-regulation of PKC was prevented by incubating the cells with the protease inhibitor leupeptin, a decrease beyond control level in TH mRNA was not observed. TPA rapidly induced TH gene transcription; a maximal increase of two-fold was observed at 15 min, but the transcriptional rate then declined although it did not decrease beyond control values after 8 and 24 h of TPA treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaloids↗
PubMed1990

Transcriptional and post-transcriptional regulation of TcR, CD4 and CD8 gene expression during activation of normal human T lymphocytes.

We previously showed that the turnover rates of the messengers coding for the T cell receptor (TcR) alpha, beta and gamma, CD4 and CD8 molecules composing the multireceptor complex vary in normal human mature T lymphocytes according to their state of activation. Activation by soluble anti-CD3 which does not induce proliferation, promotes a weak up-modulation of the corresponding five mRNAs. In contrast, activation signals such as anti-CD3 + PMA, which lead to lymphokine mRNA expression and T cell proliferation, promote a decrease of the TcR, CD4 and CD8 mRNA levels within 4 h post-activation, followed by their gradual re-expression. Here we show that the down-modulation of these mRNAs results from early regulation controls at transcriptional and post-transcriptional levels, i.e. through a concomitant inhibition of transcription and destabilization of the mRNA. Moreover, later re-expression of the mRNA results from recovery of transcription and marked increase of the mRNA stability. Finally, down-modulation is specific for TcR, CD4 and CD8 mRNAs, all submitted to similar regulation processes. These results strongly suggest a direct correlation between down-modulation of the multireceptor complex mRNAs, and lymphokine mRNA expression, and cellular proliferation.

Antibodies, Monoclonal↗
PubMed1995

Transcriptional studies on yeast SEC genes provide no evidence for regulation at the transcriptional level.

A number of proteins have been identified as components of the secretory pathway of Saccharomyces cerevisiae (SEC gene products). However, very little is known about the expression of these components and their regulation at the transcriptional level. In this study yeast cells were exposed to conditions that changed the secretory activity of the cells. The conditions analysed include the different stages of the cell cycle, overexpression of secretory proteins, and block of secretion and endocytosis. The effect of these conditions on the transcriptional expression levels of a number of SEC genes (SAR1, SEC1, SEC14, SEC17, SEC18, SEC23, SEC62, YPT1) was analysed. In summary, no major changes in transcriptional expression levels could be detected. From these results we conclude that the components of the secretory pathway are expressed constitutively and that no general regulation of transcription exists, that could adjust the expression level of the SEC genes to the secretory activity of the cells.

Cell Cycle↗
PubMed1999

Thrombin activates transcription factors sp1, NF-kappaB, and CREB: importance of the use of phosphatase inhibitors during nuclear protein extraction for the assessment of transcription factor DNA-binding activities.

Thrombin, a serine protease, is an important effector of many cellular processes and has been shown to up-regulate the expression of several genes. The mechanisms underlying thrombin-mediated regulation of gene transcription remain poorly understood. The original aim of this work was to study the effects of thrombin on the activation of transcription factors, Sp1, NF-kappaB, and CREB by means of electrophoretic mobility-shift assays (EMSA). However, an inconsistent pattern of results was observed. We raised the possibility that some EMSA results may have been erroneous by the fact that during the nuclear protein extraction and EMSA procedure, transcription factors are dephosphorylated by cellular phosphatases and hence their DNA-binding capacities are modified. Therefore, we have altered the original nuclear extraction protocol by including a mixture of phosphatase inhibitors during protein extraction and subsequent EMSA steps. We show here that this simple measure led to significant changes in both basal and thrombin-induced levels of activation of Sp1 and CREB, but not of NF-kappaB. In light of the data presented here, it would be important to reexamine the conclusions of many reports in which EMSA was used to assess the basal and agonist-induced levels of transcription factor DNA-binding activities.

Cells, Cultured↗
PubMed1993

Cyclic AMP-induced transcriptional repression of the insulin-responsive glucose transporter (GLUT4) gene: identification of a promoter region required for down-regulation of transcription.

The mechanism(s) by which cyclic AMP represses transcription of the GLUT4 gene was investigated. 3T3-L1 preadipocytes were stably transfected with a series of 5' deletion mutants of the mouse GLUT4 gene promoter fused to the bacterial CAT gene and then were induced to differentiate into adipocytes. A method based on reverse transcription/polymerase chain reaction (PCR) amplification was developed and optimized to quantitate expression of CAT mRNA transcripts. Treatment with 8-bromo-cAMP down-regulated the level of CAT mRNA in adipocytes transfected with the -7000/CAT, -785/CAT and -469/CAT constructs, but not the -78/CAT construct. Thus, the regulatory element(s) which mediates transcriptional repression by cAMP resides in the proximal promoter of the GLUT4 gene between positions -469 and -78. Since down-regulation of GLUT4 mRNA is unaffected by inhibitors of protein synthesis, cAMP (and insulin) may activate phosphorylation or dephosphorylation of an existing transcription factor that interacts with the GLUT4 proximal promoter.

3T3 Cells↗
PubMed1996

Requirement for phosphorylation of RNA polymerase II C-terminal domain in transcription is both transcript length and promoter dependent.

RNA polymerase II exists in both phosphorylated and nonphosphorylated forms. The interconversion between these two forms is suggested to be the molecular switch that regulates the transition from transcription initiation to elongation. Here, the ATP analogue H8 is used as a CTD kinase inhibitor to study the role of CTD phosphorylation in transcription with a HeLa nuclear extract in vitro. The results demonstrate that (i) CTD phosphorylation is not required for transcription of the promoter proximal region from TATA containing promoters, (ii) the primary role of CTD phosphorylation is to promote productive elongation, and (iii) the contribution of CTD phosphorylation to transcription differs among TATA containing promoters as well as between TATA less and TATA containing promoters.

Animals↗
PubMed1999

Recognition of target DNA and transcription activation by the CO-sensing transcriptional activator CooA.

CooA from Rhodospirillum rubrum is a heme-based CO-sensing transcriptional activator, in which CO acts as a physiological effector. In this study, we examined the mechanism of site-specific recognition and transcriptional activation by CooA by elucidating the transcriptional activator activity of the mutant CooA proteins and the chimeric proteins derived from CRP and CooA and the promoter activity of the mutant promoters. Site-directed mutagenesis has revealed that Arg(177), Gln(178), and Ser(181) on the recognition helix of the helix-turn-helix motif in CooA are responsible for the site-specific recognition. The side chains of these amino acid residues at positions 177, 178, and 181 are believed to be hydrogen bonding to the G:A, T:A, and C:G pairs at positions 2/15, 3/14, and 4/13 in the CooA-dependent promoters to recognize the DNA site for CooA. The properties of the CRP/CooA chimeric proteins constructed in this work suggest that CooA activates transcription by a similar mechanism to that of CRP at Class II CRP-dependent promoters.

Amino Acid Sequence↗