Human SWI-SNF component BRG1 represses transcription of the c-fos gene.
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Yeast and mammalian SWI-SNF complexes regulate transcription through active modification of chromatin structure. Human SW-13 adenocarcinoma cells lack BRG1 protein, a component of SWI-SNF that has a DNA-dependent ATPase activity essential for SWI-SNF function. Expression of BRG1 in SW-13 cells potentiated transcriptional activation by the glucocorticoid receptor, which is known to require SWI-SNF function. BRG1 also specifically repressed transcription from a transfected c-fos promoter and correspondingly blocked transcriptional activation of the endogenous c-fos gene. Mutation of lysine residue 798 in the DNA-dependent ATPase domain of BRG1 significantly reduced its ability to repress c-fos transcription. Repression by BRG1 required the cyclic AMP response element of the c-fos promoter but not nearby binding sites for Sp1, YY1, or TFII-I. Using human C33A cervical carcinoma cells, which lack BRG1 and also express a nonfunctional Rb protein, transcriptional repression by BRG1 was weak unless wild-type Rb was also supplied. Interestingly, Rb-dependent repression by BRG1 was found to take place through a pathway that is independent of transcription factor E2F. (+info)
Human Cdc34 and Rad6B ubiquitin-conjugating enzymes target repressors of cyclic AMP-induced transcription for proteolysis.
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Ubiquitin-mediated proteolysis controls diverse physiological processes in eukaryotes. However, few in vivo targets of the mammalian Cdc34 and Rad6 ubiquitin-conjugating enzymes are known. A yeast-based genetic assay to identify proteins that interact with human Cdc34 resulted in three cDNAs encoding bZIP DNA binding motifs. Two of these interactants are repressors of cyclic AMP (cAMP)-induced transcription: hICERIIgamma, a product of the CREM gene, and hATF5, a novel ATF homolog. Transfection assays with mammalian cells demonstrate both hCdc34- and hRad6B-dependent ubiquitin-mediated proteolysis of hICERIIgamma and hATF5. This degradation requires an active ubiquitin-conjugating enzyme and results in abrogation of ICERIIgamma- and ATF5-mediated repression of cAMP-induced transcription. Consistent with these results, the endogenous ICER protein is elevated in cells which are null for murine Rad6B (mHR6B-/-) or transfected with dominant negative and antisense constructs of human CDC34. Based on the requirement for CREM/ICER and Rad6B proteins in spermatogenesis, we determined expression of Cdc34, Rad6B, CREM/ICER isoforms, and the Skp1-Cullin-F-box ubiquitin protein ligase subunits Cul-1 and Cul-2, which are associated with Cdc34 activity during murine testicular development. Cdc34, Rad6B, and the Cullin proteins are expressed in a developmentally regulated manner, with distinctly different patterns for Cdc34 and the Cullin proteins in germ cells. The Cdc34 and Rad6B proteins are significantly elevated in meiotic and postmeiotic haploid germ cells when chromatin modifications occur. Thus, the stability of specific mammalian transcription factors is the result of complex targeting by multiple ubiquitin-conjugating enzymes and may have an impact on cAMP-inducible gene regulation during both meiotic and mitotic cell cycles. (+info)
Identification of an erythroid active element in the transferrin receptor gene.
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Hemoglobin synthesis consumes most of the iron that is taken up by cells from plasma transferrin, and this process requires very high expression of transferrin receptors (TfR) at the membranes of erythroid cells. Studies in our and other laboratories indicate that a dramatic increase in TfR levels during erythroid differentiation occurs at the transcriptional level. In this study, we investigated the transcriptional regulation of the TfR in terms of its promoter activity and DNA-protein binding in murine erythroleukemia cells. Reporter gene assays revealed that the TfR promoter activity was stimulated 6-8-fold in murine erythroleukemia cells induced to differentiate into hemoglobin-synthesizing cells by either Me(2)SO or N,N'-hexamethylene-bis-acetamide. A minimal region (-118 to +14) was required for the differentiation-induced promoter activity. Mutation of either an Ets-binding site or an activator protein-1/cyclic AMP-response element-like motif within this region, but not disruption of the adjacent GC-rich/specificity protein-1 sequence, inhibited the inducible promoter activity. Electrophoresis mobility shift assays suggest that the cyclic AMP-response element-binding proteins/activating transcription factor-like factors and Ets-like factors bind constitutively to this bipartite element. Upon induction of differentiation, a shift in the pattern of the cyclic AMP-response element-binding protein/activating transcription factor-like binding factors was observed. Our data indicate that the TfR gene promoter contains an erythroid active element that stimulates the receptor gene transcription upon induction of hemoglobin synthesis. (+info)
Aca1 and Aca2, ATF/CREB activators in Saccharomyces cerevisiae, are important for carbon source utilization but not the response to stress.
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In Saccharomyces cerevisiae, the family of ATF/CREB transcriptional regulators consists of a repressor, Acr1 (Sko1), and two activators, Aca1 and Aca2. The AP-1 factor Gen4 does not activate transcription through ATF/CREB sites in vivo even though it binds these sites in vitro. Unlike ATF/CREB activators in other species, Aca1- and Aca2-dependent transcription is not affected by protein kinase A or by stress, and Aca1 and Aca2 are not required for Hog1-dependent salt induction of transcription through an optimal ATF/CREB site. Aca2 is important for a variety of biological functions including growth on nonoptimal carbon sources, and Aca2-dependent activation is modestly regulated by carbon source. Strains lacking Aca1 are phenotypically normal, but overexpression of Aca1 suppresses some defects associated with the loss of Aca2, indicating a functional overlap between Aca1 and Aca2. Acr1 represses transcription both by recruiting the Cyc8-Tup1 corepressor and by directly competing with Aca1 and Aca2 for target sites. Acr1 does not fully account for osmotic regulation through ATF/CREB sites, and a novel Hog1-dependent activator(s) that is not a bZIP protein is required for ATF/CREB site activation in response to high salt. In addition, Acr1 does not affect a number of phenotypes that arise from loss of Aca2. Thus, members of the S. cerevisiae ATF/CREB family have overlapping, but distinct, biological functions and target genes. (+info)
Protein kinase A and mitogen-activated protein kinase pathways antagonistically regulate fission yeast fbp1 transcription by employing different modes of action at two upstream activation sites.
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A significant challenge to our understanding of eukaryotic transcriptional regulation is to determine how multiple signal transduction pathways converge on a single promoter to regulate transcription in divergent fashions. To study this, we have investigated the transcriptional regulation of the Schizosaccharomyces pombe fbp1 gene that is repressed by a cyclic AMP (cAMP)-dependent protein kinase A (PKA) pathway and is activated by a stress-activated mitogen-activated protein kinase (MAPK) pathway. In this study, we identified and characterized two cis-acting elements in the fbp1 promoter required for activation of fbp1 transcription. Upstream activation site 1 (UAS1), located approximately 900 bp from the transcriptional start site, resembles a cAMP response element (CRE) that is the binding site for the atf1-pcr1 heterodimeric transcriptional activator. Binding of this activator to UAS1 is positively regulated by the MAPK pathway and negatively regulated by PKA. UAS2, located approximately 250 bp from the transcriptional start site, resembles a Saccharomyces cerevisiae stress response element. UAS2 is bound by transcriptional activators and repressors regulated by both the PKA and MAPK pathways, although atf1 itself is not present in these complexes. Transcriptional regulation of fbp1 promoter constructs containing only UAS1 or UAS2 confirms that the PKA and MAPK regulation is targeted to both sites. We conclude that the PKA and MAPK signal transduction pathways regulate fbp1 transcription at UAS1 and UAS2, but that the antagonistic interactions between these pathways involve different mechanisms at each site. (+info)
Cartilage degradation and invasion by rheumatoid synovial fibroblasts is inhibited by gene transfer of a cell surface-targeted plasmin inhibitor.
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OBJECTIVE: Joint destruction in rheumatoid arthritis (RA) is a result of degradation and invasion of the articular cartilage by the pannus tissue. The present study was undertaken to examine the role of the plasminogen activation system in cartilage degradation and invasion by synovial fibroblasts and investigate a novel gene therapeutic approach using a cell surface-targeted plasmin inhibitor (ATF.BPTI). METHODS: Adenoviral vectors were used for gene transfer. The effects of ATF.BPTI gene transfer on RA synovial fibroblast-dependent cartilage degradation were studied in vitro, and cartilage invasion was studied in vivo in the SCID mouse coimplantation model. RESULTS: The results indicate that cartilage matrix degradation by rheumatoid synovial fibroblasts is plasmin mediated and depends on urokinase-type plasminogen activator for activation. Targeting plasmin inhibition to the cell surface of the fibroblasts by gene transfer of a cell surface-binding plasmin inhibitor resulted in a significant reduction of cartilage matrix degradation in vitro and of cartilage invasion in vivo. Compared with uninfected rheumatoid synovial fibroblasts, the mean +/-SEM cartilage degradation in vitro was reduced to 87.9+/-0.9% after LacZ gene transfer versus a reduction to 24.0+/-1.6% after ATF.BPTI gene transfer (P<0.0001). The mean +/- SEM in vivo cartilage invasion score was 3.1+/-0.4 in the control-transduced fibroblasts and 1.8+/-0.4 in the ATF.BPTI-transduced fibroblasts (P<0.05). CONCLUSION: These results indicate a role of the plasminogen activation system in synovial fibroblast-dependent cartilage degradation and invasion in RA, and demonstrate an effective way to inhibit this by gene transfer of a cell surface-targeted plasmin inhibitor. (+info)
Construction and in vitro characterization of attenuated feline immunodeficiency virus long terminal repeat mutant viruses.
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AP-1- and ATF-binding sites are cis-acting transcriptional elements within the U3 domain of the feline immunodeficiency virus (FIV) long terminal repeat (LTR) that serve as targets for cellular activation pathways and may regulate virus replication. We report that FIV LTR mutant proviruses encoding U3 deletions of the ATF-binding sequence exhibited restricted virus expression and replication in both feline lymphocytes and macrophages. In contrast, deletion of the AP-1 site had negligible effects on virus expression and replication. FIV LTR mutant proviruses encoding deletions of both the AP-1 and ATF sites or a 72-bp deletion encompassing the AP-1 site, duplicated C/EBP sites, and ATF sites were severely restricted for virus expression. These results demonstrate that deletion of either the ATF-binding site or multiple cis-acting transcriptional elements attenuates FIV. These attenuated FIV mutants provide opportunities to characterize the role of cis-acting elements in virus replication in vivo and to test LTR mutants as attenuated virus vaccines. (+info)
CREB/ATF-dependent repression of cyclin a by human T-cell leukemia virus type 1 Tax protein.
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Expression of the human T-cell leukemia virus type 1 (HTLV-1) oncoprotein Tax is correlated with cellular transformation contributing to the development of adult T-cell leukemia. Tax has been shown to modulate the activities of several cellular promoters. Existing evidence suggests that Tax need not directly bind to DNA to accomplish these effects but rather that it can act through binding to cellular factors, including members of the CREB/ATF family. Exact mechanisms of HTLV-1 transformation of cells have yet to be fully defined, but the process is likely to include both activation of cellular-growth-promoting factors and repression of cellular tumor-suppressing functions. While transcriptional activation has been well studied, transcriptional repression by Tax, reported recently from several studies, remains less well understood. Here, we show that Tax represses the TATA-less cyclin A promoter. Repression of the cyclin A promoter was seen in both ts13 adherent cells and Jurkat T lymphocytes. Two other TATA-less promoters, cyclin D3 and DNA polymerase alpha, were also found to be repressed by Tax. Interestingly, all three promoters share a common feature of at least one conserved upstream CREB/ATF binding site. In electrophoretic mobility shift assays, we observed that Tax altered the formation of a complex(es) at the cyclin A promoter-derived ATF site. Functionally, we correlated removal of the CREB/ATF site from the promoter with loss of repression by Tax. Furthermore, since a Tax mutant protein which binds CREB repressed the cyclin A promoter while another mutant protein which does not bind CREB did not, we propose that this Tax repression occurs through protein-protein contact with CREB/ATF. (+info)