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At least 865 records · Page 48Linked to original sources
PubMed1994

Transcriptional activation of the alpha-1 acid glycoprotein gene by YY1 is mediated by its functional interaction with a negative transcription factor.

Regulation of alpha-1 acid glycoprotein (AGP) gene expression involves both positive and negative transcription factors. We have previously identified two dominant factors: positive and negative transcription factors, AGP/EBP and factor B, respectively, involved in the transcription of AGP and other acute-phase response genes. In this report, we present evidence showing that the transcription of the AGP gene is positively regulated by a transcription factor, YY1. The activation of AGP gene by YY1 is mediated by a negative element B in the AGP promoter region. YY1 can also activate the B motif linked to a heterologous promoter. However, YY1 does not bind directly to the B motif per se. Rather, our data suggest that the activation of AGP gene by YY1 may be mediated by its functional interaction with factor B, which recognizes the B motif.

Acute-Phase Reaction↗
PubMed1996

Species specificity of ribosomal gene transcription: a factor associated with human RNA polymerase I prevents transcription of mouse rDNA.

An intrinsic property of class I gene transcription by RNA polymerase I (Pol I) is the species specificity of the initiation reaction. Previous studies have demonstrated that species-specific rDNA promoter recognition is brought about by a TBP-TAF complex, termed TIF-IB in mouse and SL1 in man. We have compared the ability of affinity-purified TIF-IB and SL1 to direct transcription from the homologous rDNA template both in a reconstituted transcription system and in nuclear extracts prepared from mouse and human cells. We show that Pol I from both species and the individual transcription factors, with the exception of TIF-IB/SL1, are functionally interchangeable in the reconstituted transcription system containing purified proteins. In nuclear extracts, however, species-specific differences are obvious. Whereas SL1 reprograms a heterologous mouse extract to recognize the human promoter, TIF-IB fails to reprogram a human extract unless it is complemented with mouse Pol I. Crude human, but not mouse, Pol I exhibits species-specific differences that disappear after purification. We propose that in extracts and less purified fractions human Pol I exists as 'holoenzyme' containing associated protein(s) that prevent assembly of TIF-IB-directed initiation complexes at the murine rDNA promoter.

Animals↗
PubMed1996

Transcriptional regulation of the human cholecystokinin gene: composite action of upstream stimulatory factor, Sp1, and members of the CREB/ATF-AP-1 family of transcription factors.

We have examined cis-elements and trans-acting factors that regulate transcription of the human cholecystokinin (CCK) gene. Transient expression of CCK promoter deletion constructs in human SK-N-MC neuroblastoma cells depicted positive cis-elements between the positions -100 to -92, -84 to -74, and -58 to -37, 5' to the transcription initiation site. Correspondingly, DNase I protection analysis showed that transacting factors bound to elements within these regions. The sequences encompass a putative basic helix-loop-helix leucine zipper (bHLH-ZIP) element, an Sp1 element, and a combined cAMP- and TPA-responsive element (CRE/TRE) at positions -97 to -92, -39 to -34, and -80 to -73, respectively. Mobility and supershift assays demonstrated that upstream stimulatory factor (USF) and Sp1 bind to the former elements and competition experiments confirmed that CREB/ATF and AP-1 bind to the CRE/TRE element. Mutation of the bHLH-ZIP and CRE/TRE elements decreased the activity of the promoter by 65% and 42%, respectively. The activity of the promoter was increased six- and two-fold after stimulation with forskolin and TPA, respectively. Stimulation was eliminated after mutation of the CRE/TRE element. Co-transfection experiments with pRSV-c-jun, pSV-fos, and pRC-RSV-CREB constructs showed that jun, CREB, and AP-1 stimulate transcription. We conclude that USF, Sp1, and members of the CREB/ATF and AP-1 family of transcription factors are the major determinants of CCK gene transcription.

Base Sequence↗
PubMed2006

Pol I transcription and pre-rRNA processing are coordinated in a transcription-dependent manner in mammalian cells.

Pre-rRNA synthesis and processing are key steps in ribosome biogenesis. Although recent evidence in yeast suggests that these two processes are coupled, the nature of their association is unclear. In this report, we analyze the coordination between rDNA transcription and pre-rRNA processing in mammalian cells. We found that pol I transcription factor UBF interacts with pre-rRNA processing factors as analyzed by immunoprecipitations, and the association depends on active rRNA synthesis. In addition, injections of plasmids containing the human rDNA promoter and varying lengths of 18S rDNA into HeLa nuclei show that pol I transcription machinery can be recruited to rDNA promoters regardless of the product that is transcribed, whereas subgroups of pre-rRNA processing factors are recruited to plasmids only when specific pre-rRNA fragments are produced. Our observations suggest a model for sequential recruitment of pol I transcription factors and pre-rRNA processing factors to elongating pre-rRNA on an as-needed basis rather than corecruitment to sites of active transcription.

DNA, Ribosomal↗
PubMed2000

A yeast taf17 mutant requires the Swi6 transcriptional activator for viability and shows defects in cell cycle-regulated transcription.

In Saccharomyces cerevisiae, the Swi6 protein is a component of two transcription factors, SBF and MBF, that promote expression of a large group of genes in the late G1 phase of the cell cycle. Although SBF is required for cell viability, SWI6 is not an essential gene. We performed a synthetic lethal screen to identify genes required for viability in the absence of SWI6 and identified 10 complementation groups of swi6-dependent lethal mutants, designated SLM1 through SLM10. We were most interested in mutants showing a cell cycle arrest phenotype; both slm7-1 swi6Delta and slm8-1 swi6Delta double mutants accumulated as large, unbudded cells with increased 1N DNA content and showed a temperature-sensitive growth arrest in the presence of Swi6. Analysis of the transcript levels of cell cycle-regulated genes in slm7-1 SWI6 mutant strains at the permissive temperature revealed defects in regulation of a subset of cyclin-encoding genes. Complementation and allelism tests showed that SLM7 is allelic with the TAF17 gene, which encodes a histone-like component of the general transcription factor TFIID and the SAGA histone acetyltransferase complex. Sequencing showed that the slm7-1 allele of TAF17 is predicted to encode a version of Taf17 that is truncated within a highly conserved region. The cell cycle and transcriptional defects caused by taf17(slm7-1) are consistent with the role of TAF(II)s as modulators of transcriptional activation and may reflect a role for TAF17 in regulating activation by SBF and MBF.

Amino Acid Sequence↗
PubMed2003

Identification of a novel tissue-specific transcriptional activator FESTA as a protein that interacts with the transcription elongation factor S-II.

Transcription elongation factor S-II was originally purified as a specific stimulator of transcription by RNA polymerase II. Recent studies suggest that S-II participates in gene-specific transcriptional activation in vivo, despite the fact that it directly binds RNA polymerase II and does not recognize specific DNA sequences. In this study, under the hypothesis that S-II requires co-factors to regulate the expression of specific-genes in vivo, we searched for factors that directly interact with S-II using a yeast two-hybrid system, and isolated a novel nuclear protein, FESTA. FESTA is expressed specifically in kidney and spleen, supporting our notion that S-II participates in gene-specific regulation. Two mRNA isoforms of FESTA encoding proteins with different sizes were identified and named FESTA-S and FESTA-L. FESTA contains a serine-rich region and a C-terminal tail that are highly similar to those of the ELL-associated factor EAF1. Reporter gene assays indicated that both GAL4-FESTA-S and GAL4-FESTA-L fusion proteins have trans-activating ability. Furthermore, deletion of the C-terminal tail of FESTA dramatically reduced its trans-activating ability and abolished its interaction with S-II. This study is the first report of a transcriptional activator that directly interacts with S-II and contains a transcriptional activation domain that cooperates with S-II via direct interaction.

Amino Acid Sequence↗
PubMed1988

Heavy metal ions in transcription factors from HeLa cells: Sp1, but not octamer transcription factor requires zinc for DNA binding and for activator function.

Zinc is an important cofactor for many enzymes involved in nucleic acid metabolism such as DNA and RNA polymerases, reverse transcriptase and tRNA synthetases. We have developed an inducible in vitro transcription system using metal-depleted nuclear extracts to reveal the presence and functional relevance of heavy metal ions in transcription factors. Using protein-DNA binding assays (band shift and DNAase I footprint) we show that Sp1, a promoter-specific vertebrate transcription factor that binds to the "GC box" (Sequence in text), is reversibly inactivated by metal-depletion. Zinc is required for specific DNA binding in vitro and is also essential for Sp1 factor-directed transcription. In contrast, another factor from HeLa cells, the so-called octamer transcription factor (OTF) that binds to the sequence 5'-ATGCAAATNA, is not affected by metal-depletion and thus seems not to be a zinc metalloprotein.

Base Sequence↗
PubMed1993

The nucleolar transcription activator UBF relieves Ku antigen-mediated repression of mouse ribosomal gene transcription.

Previously we have shown that the RNA polymerase I (Pol I)-specific transcription factor UBF stimulates transcription by both facilitating transcription complex formation and by relieving repression exerted by a negative-acting factor which competes for binding of the murine factor TIF-IB to the ribosomal gene promoter (1). We have purified and functionally characterized this repressor protein from Ehrlich ascites cells. The final preparation contained two polypeptides with molecular masses of 75 and 90 kDa, respectively. Both polypeptides interact with the rDNA promoter as revealed by UV-crosslinking experiments. The specificity of binding to the ribosomal gene promoter was demonstrated in an electrophoretic mobility shift assay and by DNase footprinting. The biochemical properties of this negative-acting factor closely resemble those of the Ku antigen, a human nuclear DNA-binding heterodimer which is the target of autoantibodies in several autoimmune diseases. Anti-Ku antibodies precipitate the repressor activity and overcome transcription inhibition. The data demonstrate that regulation of Pol I gene transcription may involve an antirepression mechanism as already documented for Pol II genes and suggest that Ku protein may be causally involved in repressor-mediated down regulation of rRNA synthesis.

Animals↗
PubMed1998

Correct usage of multiple transcription initiation sites and C/EBP-dependent transcription activation of the rat XDH/XO TATA-less promoter requires downstream elements located in the coding region of the gene.

In the present study, we have shown that a downstream element located in the coding region of the TATA-less rat xanthine dehydrogenase/oxidase (XDH/XO) gene (-7 to +42) plays an important role in transcription initiation and C/EBP transcriptional activation. Previous work from our laboratory has shown that the promoter is organized with multiple initiator elements (Inr 1, 2, 3 and 4) which are important for transcription initiation. Additionally, we had identified two C/EBP binding sites upstream of this promoter. Deletional and mutational studies revealed that C/EBP binding was not essential for the basal level of transcriptional initation. However when XO-luciferase constructs include downstream sequence extending to +42 there is development of C/EBP sensitivity as well as a shift in the initiator usage. In the absence of the downstream element, primer extension analyses reveals Inr 3 and 4 to be the major start sites but in the presence of this additional sequence the usage is shifted to Inr 1 and 2. This shift in Inr usage more closely resembles that seen in intact macrophages or liver cells. Gel mobility shift assays indicate the presence of several binding factors located in this downstream region, one of which has been identified as YY-1. We postulate that YY-1 allows DNA bending which permits the upstream C/EBP elements to exhibit a transcriptional activation which is not seen when the downstream element is absent. This study presents a potential model for regulation of the XDH/XO promoter.

Animals↗
PubMed2004

Phosphorylation of rat mitochondrial transcription termination factor (mTERF) is required for transcription termination but not for binding to DNA.

Despite the crucial importance of mitochondrial transcription, knowledge of its regulation is poor. Therefore, characterization of mammalian mitochondrial transcription termination factor (mTERF) functionality and regulation is of fundamental biological interest in order to understand the regulation of mitochondrial transcription. Here we report that mTERF is the first protein having a role in mammalian mitochondrial gene expression that appears to be controlled by phosphorylation. Recombinant mature rat mTERF protein has specific DNA-binding capacity for the sequence required for transcription termination. Furthermore, unlike recombinant human mTERF, the rat protein bound to its mitochondrial DNA binding site promotes the termination of transcription initiated with heterologous RNA polymerase. Interestingly, mTERF is a phosphoprotein with four phosphate groups, and while the DNA-binding activity of mTERF is unaffected by the phosphorylation/dephosphorylation state, only the phosphorylated form of the protein is active for termination activity. Moreover, natural human mTERF is also a phosphoprotein and its termination activity is inhibited by dephosphorylation. These data suggest that mTERF functioning in vivo is regulated by phosphorylation.

Amino Acid Sequence↗
PubMed1997

The transcriptional activators of the PHO regulon, Pho4p and Pho2p, interact directly with each other and with components of the basal transcription machinery in Saccharomyces cerevisiae.

The transcriptional regulators Pho4p and Pho2p are involved in transcription of several genes in the PHO regulon of Saccharomyces cerevisiae. Genetic evidence with temperature-sensitive pho4 and pho2 mutants suggested that Pho4p and Pho2p interact with each other. Immunoprecipitation experiments showed that Pho4p and Pho2p form a complex on a 36-bp sequence bearing an upstream activation site (UAS) and protein binding assays indicated that these proteins interact directly. DNA-binding experiments with crude extracts prepared from yeast strains expressing T7-PHO4, encoding Pho4p tagged with the T7 epitope, indicated that Pho2p interacts with T7-Pho4p and enhances the binding affinity of T7-Pho4p to the UAS. Protein binding experiments also showed that both Pho4p and Pho2p could bind with the general transcription factors, TBP, TFIIB, and TFIIEbeta, suggesting that the Pho4p-Pho2p complex bound to the UAS activates transcription of the PHO genes by direct interaction with the general transcription factors.

Basal Metabolism↗
PubMed1998

Hematopoietic transcription factor GATA-2 activates transcription from HIV-1 long terminal repeat.

OBJECTIVES: To study the role of the hematopoietic transcription factor GATA-2 in long terminal repeat (LTR)-directed transcriptional activation of HIV-1 in hematopoietic progenitor cells, and to investigate possible GATA-2 binding sites in HIV-1 LTR. DESIGN AND METHODS: Wild-type HIV-1 LTR, or mutants, ligated to a luciferase reporter gene with or without a GATA-2 expression vector, were transfected into COS cells, and standardized luciferase activity was examined. The binding activity of GATA-2 to these sites was examined by electrophoretic mobility shift assay. These wild-type or mutant reporter genes were also transfected into the murine hematopoietic progenitor cells, BAF3, in which GATA-2 was the predominantly expressed transcription factor of the GATA family, to assay LTR-directed transcription in intact hematopoietic machinery. Using a Tat expression plasmid for cotransfection, the influence of Tat protein on GATA-2-induced transactivation was determined. RESULTS: In COS cells, LTR-dependent transactivation was highly enhanced by the coexpression of GATA-2. Experiments with mutant LTR suggested the presence of multiple GATA-2 binding sites, of which the major sites were identified. Cotransfection of Tat with GATA-2 indicated that GATA-2 and Tat synergistically enhanced the transcriptional activity. Transfection experiments in BAF3 cells showed that the disruption of these GATA sites diminished LTR-driven activity to 40% of the wild-type. CONCLUSIONS: GATA-2 may be a key host cell regulator of HIV-1 expression in hematopoietic stem cells. Manipulating this transactivation may represent a valuable approach to controlling virus production in infected hematopoietic progenitors. To elucidate the possible interaction between GATA-2 and Tat protein in vivo might give new insights to the mechanism of impaired hematopoiesis in AIDS patients.

Animals↗
PubMed1995

Transcriptional activation and repression, two properties of the lymphoid-specific transcription factor Oct-2a.

The lymphoid-specific transcription factor Oct-2a contains two transcriptional activation domains which are located within the N-terminal and C-terminal regions. To study their differential activation properties, we linked the isolated effector domains to the GAL4 DNA-binding domain. We have shown that both activating regions of Oct-2a, isolated from their natural context, can activate transcription as promoter factors. In contrast to the C-terminus, activation by the N-terminal domain is dependent on a yet unidentified factor(s) binding to the simian virus 40 enhancer. The results obtained by duplication of activation domains or their mixed combination suggest that the domains are functionally independent. However, activation from a remote position could only be achieved with the C-terminus of Oct-2a in B cells. In lymphoid cells, higher activation levels were observed, suggesting that distinct B-cell-specific cofactors in concert with the effector domains of Oct-2a might be involved in mediating transcription from proximal and remote positions. Furthermore, we identified a repression domain at the N-terminus of Oct-2a. When transferred to a potent activator, transcriptional stimulation was inhibited efficiently. These results underscore the modular structure of Oct-2a with separable domains for activation and repression and suggest that Oct-2a might have complex regulatory functions in vivo.

B-Lymphocytes↗
PubMed2006

Induction of immunoglobulin heavy-chain transcription through the transcription factor Bright requires TFII-I.

Bright/ARID3a/Dril1, a member of the ARID family of transcription factors, is expressed in a highly regulated fashion in B lymphocytes, where it enhances immunoglobulin transcription three- to sixfold. Recent publications from our lab indicated that functional, but not kinase-inactive, Bruton's tyrosine kinase (Btk) is critical for Bright activity in an in vitro model system, yet Bright itself is not appreciably tyrosine phosphorylated. These data suggested that a third protein, and Btk substrate, must contribute to Bright-enhanced immunoglobulin transcription. The ubiquitously expressed transcription factor TFII-I was identified as a substrate for Btk several years ago. In this work, we show that TFII-I directly interacts with human Bright through amino acids in Bright's protein interaction domain and that specific tyrosine residues of TFII-I are essential for Bright-induced activity of an immunoglobulin reporter gene. Moreover, inhibition of TFII-I function in a B-cell line resulted in decreased heavy-chain transcript levels. These data suggest that Bright functions as a three-component protein complex in the immunoglobulin locus and tie together previous data indicating important roles for Btk and TFII-I in B lymphocytes.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed1991

The ubiquitous transcription factor Oct-1 and the liver-specific factor HNF-1 are both required to activate transcription of a hepatitis B virus promoter.

The liver-specific transcription factor HNF-1 activates transcription of several mammalian hepatocyte-specific genes. The hepatitis B virus preS1 promoter shows hepatocyte specificity, which has been ascribed to binding of HNF-1 to a cognate DNA sequence upstream of the TATA box. We show here that there is an adjacent site that binds the ubiquitous transcription factor Oct-1. Both the Oct-1 and HNF-1 sites are necessary for liver-specific transcription of the preS1 promoter, but neither site alone activates transcription. The Oct-1 site is also necessary for activation of the preS1 promoter in HeLa cells, expressing transfected HNF-1. Our results show that while Oct-1 is not restricted to hepatocytes, it nevertheless can play a critical role in the expression of a liver-specific gene.

Base Sequence↗
PubMed1994

Microphthalmia-associated transcription factor as a regulator for melanocyte-specific transcription of the human tyrosinase gene.

Tyrosinase is a rate-limiting enzyme in melanin biosynthesis and is specifically expressed in differentiated melanocytes. We have identified the enhancer element in the 5'-flanking region of the human tyrosinase gene that is responsible for its pigment cell-specific transcription and have termed it tyrosinase distal element (TDE) (positions -1861 to -1842). Transient expression assays showed that TDE confers efficient expression of a firefly luciferase reporter gene linked to the tyrosinase gene promoter in MeWo pigmented melanoma cells but not in HeLa cells, which do not express tyrosinase. TDE was specifically bound by nuclear proteins of MeWo and HeLa cells, the binding properties of which were indistinguishable in gel mobility shift assays. TDE contains the CATGTG motif in its center, and mutation analysis indicates that the CA dinucleotides of this motif are crucial for protein binding and pigment cell-specific enhancer function. The CATGTG motif is consistent with the consensus sequence recognized by a large family of transcription factors with a basic helix-loop-helix structure, which prompted us to examine the possible involvement of a ubiquitous transcription factor, USF, and a novel factor, microphthalmia-associated transcription factor (MITF), recently cloned as the human homolog of the mouse microphthalmia (mi) gene product. The mi phenotype is associated with a mutant mi locus and characterized by small eyes and loss of melanin pigments. Both USF and MITF are predicted to contain a basic helix-loop-helix structure and a leucine zipper structure. We provide evidence that USF binds to TDE, whereas we were unable to detect the DNA-binding activity of MITF. Transient coexpression assays showed that MITF specifically transactivates the promoter activity of the tyrosinase gene through the CATGTG motif of TDE but not the promoter of the ubiquitously expressed heme oxygenase gene, while USF is able to activate both promoters. These results indicate that MITF is a cell-type-specific factor that is capable of activating transcription of the tyrosinase gene.

Base Sequence↗
PubMed1994

The yeast and mammalian Ras pathways control transcription of heat shock genes independently of heat shock transcription factor.

Yeast strains in which the Ras-cyclic AMP (cAMP) pathway is constitutively active are sensitive to heat shock, whereas mutants in which the activity of this pathway is low are hyperresistant to heat shock. To determine the molecular basis for these differences, we examined the transcriptional induction of heat shock genes in various yeast strains. Activation of heat shock genes was attenuated in the strains in which the Ras-cAMP pathway is constitutively active. In contrast, in a strain deficient in cAMP production, several heat shock genes were induced by removal of cAMP from the medium. These results indicate that the Ras-cAMP pathway affects the induction of heat shock genes. In all of the mutants, heat shock transcription factor expression and activity were identical to those in wild-type cells. The response to heat shock in Ha-ras-transformed rat fibroblasts was also studied. While no induction of Hsp68 was observed in Ha-ras-transformed cells, proper regulation of heat shock transcription factor was found. Therefore, in mammals, as in Saccharomyces cerevisiae, the Ras pathway controls the transcription of heat shock genes via a mechanism not involving the heat shock transcription factor.

3T3 Cells↗
PubMed1999

Coactivator PC4 mediates AP-2 transcriptional activity and suppresses ras-induced transformation dependent on AP-2 transcriptional interference.

ras oncogene-transformed PA-1 human teratocarcinoma cells have abundant AP-2 mRNA but, paradoxically, little AP-2 transcriptional activity. We have previously shown that overexpression of AP-2 in nontumorigenic variants of PA-1 cells results in inhibition of AP-2 activity and induction of tumorigenicity similar to that caused by ras transformation of PA-1 cells. Evidence indicated the existence of a novel mechanism of inhibition of AP-2 activity involving sequestering of transcriptional coactivators. In this study, we found that PC4 is a positive coactivator of AP-2 and can restore AP-2 activity in ras-transformed PA-1 cells. Relative to vector-transfected ras cell lines, ras cell lines stably transfected with and expressing the PC4 cDNA have a diminished growth rate and exhibit a loss of anchorage-independent growth, and they are unable to induce the formation of tumors in nude mice. These data suggest that a transcriptional coactivator, like a tumor suppressor, can have a growth-suppressive effect on cells. Our experiments are the first to show that ras oncogenes and oncogenic transcription factors can induce transformation through effects on the transcription machinery rather than through specific programs of gene expression.

Animals↗