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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Hox genes encode homeodomain-containing proteins that are presumed to control spatial patterning during murine embryogenesis through their actions as transcriptional regulatory proteins. In this study, we have investigated the transcriptional function of a prototypic member of this family, HoxA7. We demonstrate that HoxA7 function as a potent transcriptional repressor and that its action as such requires several domains, including both activator and repressor regions. The repressor regions are contained within the homeodomain and a C-terminal acidic region, both of which are well conserved among members of the Hox family. Accordingly, we show that two other members of this family also function as repressors, although they vary in their relative repressor potency. Finally, we explore the novel observation that the homeodomain of HoxA7 functions as a transcriptional repressor domain. We show that the homeodomain compared with two other DNA-binding domains, is unique in its ability to function as a repressor domain and that repression requires conserved residues, in helix III. We further show that residues in the N-terminal arm of the homeodomain contribute to the differential repressor actions of various Hox proteins. These findings demonstrate that the transcriptional function of HoxA7 and possibility of Hox proteins in general is determined by their unique combination of conserved and nonconserved regions as well as through the complex actions of their homeodomains.
Mol Cell Biol 1996 Jun
PMID:Repression by HoxA7 is mediated by the homeodomain and the modulatory action of its N-terminal-arm residues. 864 75

Mad1 is a basic helix-loop-helix-leucine zipper protein that is induced upon differentiation of a number of distinct cell types. Mad1 dimerizes with Max and recognizes the same DNA sequences as do Myc:Max dimers. However, Mad1 and Myc appear to have opposing functions. Myc:Max heterodimers activate transcription while Mad:Max heterodimers repress transcription from the same promoter. In addition Mad1 has been shown to block the oncogenic activity of Myc. Here we show that ectopic expression of Mad1 inhibits the proliferative response of 3T3 cells to signaling through the colony-stimulating factor-1 (CSF-1) receptor. The ability of over-expressed Myc and cyclin D1 to complement the mutant CSF-1 receptor Y809F (containing a Y-to-F mutation at position 809) is also inhibited by Mad1. Cell cycle analysis of proliferating 3T3 cells transfected with Mad1 demonstrates a significant decrease in the fraction of cells in the S and G2/M phases and a concomitant increase in the fraction of G1 phase cells, indicating that Mad1 negatively influences cell cycle progression from the G1 to the S phase. Mutations in Mad1 which inhibit its activity as a transcription repressor also result in loss of Mad1 cell cycle inhibitory activity. Thus, the ability of Mad1 to inhibit cell cycle progression is tightly coupled to its function as a transcriptional repressor.
Mol Cell Biol 1996 Jun
PMID:Inhibition of cell proliferation by the Mad1 transcriptional repressor. 864 88

We have isolated and analyzed human CTCF cDNA clones and show here that the ubiquitously expressed 11-zinc-finger factor CTCF is an exceptionally highly conserved protein displaying 93% identity between avian and human amino acid sequences. It binds specifically to regulatory sequences in the promoter-proximal regions of chicken, mouse, and human c-myc oncogenes. CTCF contains two transcription repressor domains transferable to a heterologous DNA binding domain. One CTCF binding site, conserved in mouse and human c-myc genes, is found immediately downstream of the major P2 promoter at a sequence which maps precisely within the region of RNA polymerase II pausing and release. Gel shift assays of nuclear extracts from mouse and human cells show that CTCF is the predominant factor binding to this sequence. Mutational analysis of the P2-proximal CTCF binding site and transient-cotransfection experiments demonstrate that CTCF is a transcriptional repressor of the human c-myc gene. Although there is 100% sequence identity in the DNA binding domains of the avian and human CTCF proteins, the regulatory sequences recognized by CTCF in chicken and human c-myc promoters are clearly diverged. Mutating the contact nucleotides confirms that CTCF binding to the human c-myc P2 promoter requires a number of unique contact DNA bases that are absent in the chicken c-myc CTCF binding site. Moreover, proteolytic-protection assays indicate that several more CTCF Zn fingers are involved in contacting the human CTCF binding site than the chicken site. Gel shift assays utilizing successively deleted Zn finger domains indicate that CTCF Zn fingers 2 to 7 are involved in binding to the chicken c-myc promoter, while fingers 3 to 11 mediate CTCF binding to the human promoter. This flexibility in Zn finger usage reveals CTCF to be a unique "multivalent" transcriptional factor and provides the first feasible explanation of how certain homologous genes (i.e., c-myc) of different vertebrate species are regulated by the same factor and maintain similar expression patterns despite significant promoter sequence divergence.
Mol Cell Biol 1996 Jun
PMID:An exceptionally conserved transcriptional repressor, CTCF, employs different combinations of zinc fingers to bind diverged promoter sequences of avian and mammalian c-myc oncogenes. 864 89

Transcriptional activation in eukaryotic organisms normally requires combinatorial interactions of multiple transcription factors. In most cases, the precise role played by each transcription factor is not known. The upstream activating sequence (UAS) elements of glycolytic enzyme genes in Saccharomyces cerevisiae are excellent model systems for the study of combinatorial interactions. The yeast protein known as Rap1p acts as both a transcriptional repressor and an activator, depending on sequence context. Rap1p-binding sites are found adjacent to Gcr1p-binding sites in the UAS elements of glycolytic enzyme genes. These UAS elements constitute some of the strongest activating sequences known in S. cerevisiae. In this study, we have investigated the relationship between Rap1p- and Gcr1p-binding sites and the proteins that bind them. In vivo DNA-binding studies with rap1ts mutant strains demonstrated that the inability of Rap1p to bind at its site resulted in the inability of Gcr1p to bind at adjacent binding sites. Synthetic oligonucleotides, modeled on the UAS element of PYK1, in which the relative positions of the Rap1p- and Gcr1p-binding sites were varied prepared and tested for their ability to function as UAS elements. The ability of the oligonucleotides to function as UAS elements was dependent not only on the presence of both binding sites but also on the relative distance between the binding sites. In vivo DNA-binding studies showed that the ability of Rap1p bind its site was independent of Gcr1p but that the ability of Gcr1p to bind its site was dependent on the presence of an appropriately spaced and bound Rap1p-binding site. In vitro binding studies showed Rap1p-enhanced binding of Gcr1p on oligonucleotides modeled after the native PYK1 UAS element but not when the Rap1p- and Gcr1p-binding sites were displaced by 5 nucleotides. This work demonstrates that the role of the Rap1p in the activation of glycolytic enzyme genes is to bind in their UAS elements and to facilitate the binding of Gcr1p at adjacent binding sites.
Mol Cell Biol 1996 Jun
PMID:Activation mechanism of the multifunctional transcription factor repressor-activator protein 1 (Rap1p). 864 29

p107 is a member of the pocket family of proteins that includes the retinoblastoma tumor suppressor. Overexpression of p107 arrests cells in G1, suggesting that it is important for cell cycle control. This growth suppression is mediated at least in part through the interaction of p107 with a member of the E2F family of cell cycle transcription factors, and this interaction can be disrupted by oncoproteins from DNA tumor viruses such as adenovirus E1a that bind p107. Not only does the binding of p107 to E2F inactivate E2F, but also we show that when p107 is tethered to the promoter through binding to E2F it functions as a general transcriptional repressor. This general repressor activity was also evident when p107 was fused to the DNA binding domain of Gal4 so that it could be directly targeted to the promoter in an E2F-independent fashion. Using p107 mutants, we compared the regions of the protein required for transcriptional repression and cell growth suppression. We found that the pocket domain is sufficient for inactivation of E2F, general repressor activity, and most of the growth suppressor activity. Binding of conserved region 1 from Ela to p107 blocked interaction with E2F, but it did not affect general repressor activity, demonstrating that binding and inactivation of E2F and general repressor activity are distinguishable properties of p107. Within the pocket, two conserved domains, A and B, were sufficient for growth suppression and transcriptional repressor activity. Surprisingly, we found that these two domains were fully functional when they were coexpressed as separate proteins, and we present results suggesting that the domains may interact at the promoter to form an active pocket.
Mol Cell Biol 1996 Jul
PMID:Transcriptional repression and growth suppression by the p107 pocket protein. 866 77

The SIN3 gene in Saccharomyces cerevisiae encodes a negative regulator of transcription of a large number of genes. Mouse homologs of SIN3 have been identified through screens for proteins interacting with the mammalian Mad1 protein, a transcriptional repressor. We find that yeast Sin3 (ySin3) interacts with Madl and that, as for mouse Sin3, the N terminus of Mad1 interacts with the PAH2 domain of ySin3. Although Mad1 (a basic helix-loop-helix leucine zipper [bHLH-Zip) protein) forms a heterodimer with the Max bHLH-Zip protein, LexA-Mad1 and VP16-Max do not activate transcription of a reporter gene in a two-hybrid assay. This failure in activation is due to direct repression by ySin3, as LexA-Mad1 and VP16-Max are able to activate the two-hybrid reporter in a sin3 mutant. This inhibition of activation by LexA-Mad1 and VP16-Max requires the PAH2 domain of ySin3 and the N-terminal interaction region of Mad1. These data demonstrate that ySin3 functions as a transcriptional repressor by being brought to promoters by interacting with proteins bound to DNA.
Mol Cell Biol 1996 Aug
PMID:SIN3-dependent transcriptional repression by interaction with the Mad1 DNA-binding protein. 875 21

The retinoblastoma protein (Rb) is a tumor suppressor that regulates progression from the G1 phase to the S phase of the cell cycle. Previously, we found that Rb is a transcriptional repressor that is selectively targeted to promoters through an interaction with the E2F family of cell cycle transcription factors--when Rb is tethered to a promoter through E2F, it not only blocks E2F activity, it also binds surrounding transcription factors, preventing their interaction with the basal transcription complex, thus resulting in a dominant inhibitory effect on transcription of cell cycle genes. Here we examine the repressor motif of Rb. The two domains in the Rb pocket, A and B, which are conserved across species and in the Rb-related proteins p107 and p130, are both required for repressor activity. The nonconserved spacer separating A and B is not required. Although neither A nor B alone had any repressor activity, surprisingly, repressor activity was observed when the domains were coexpressed on separate proteins. Transfection assays suggest that one domain can recruit the other to the promoter to form a repressor motif that can both interact with E2F and have a dominant inhibitory effect on transcription. Using coimmunoprecipitation and in vitro binding assays, we show that A and B interact directly and that mutations which disrupt this interaction inhibit repressor activity. The Rb pocket was originally defined as the binding site for oncoproteins from DNA tumor viruses such as adenovirus E1a. We present evidence that E1a interacts with a site formed by the interaction of A and B and that this interaction with A and B induces or stabilizes the A-B interaction.
Mol Cell Biol 1996 Sep
PMID:Domains A and B in the Rb pocket interact to form a transcriptional repressor motif. 875 45

Glycogenosis type II is a recessively inherited disorder caused by mutations in the acid maltase (GAA) gene. Clinically, three different phenotypes are recognized: Infantile, juvenile and adult forms. A majority of compound heterozygous adult-onset patients carry a t-13g mutation in intron 1 associated with splicing out the first coding exon (exon 2). We have studied the mechanism of this mutation in a model system with wild-type and mutant minigenes expressed in a GAA deficient cell line. We have demonstrated that the mutation does not prevent normal splicing; low levels of correctly spliced mRNA are generated with the mutant construct. The data explain why the mutation is restricted to a milder, adult-onset phenotype. We also demonstrate that splicing out of exon 2 occurs with the wild-type construct, and thus represents alternative splicing which takes place in normal cells. Three splice variants (SV1, SV2 and SV3) are made with both the mutant and the wild-type constructs. Furthermore, as shown by RNAse protection assay, these mRNA variants are less abundant with the mutant construct. Thus, a major effect of the mutation appears to be a low splicing efficiency, since the total amount of all the transcripts generated from the mutant construct is reduced compared with the wild type. The removal of approximately 90% of the intron 1 (2.6 kb) sequence resulted in a dramatic increase in the levels of correctly spliced mRNA, indicating that the intron may contain a powerful transcriptional repressor.
Hum Mol Genet 1996 Jul
PMID:A model of mRNA splicing in adult lysosomal storage disease (glycogenosis type II). 881 37

Transcription of the Escherichia coli dnaA gene, encoding DnaA protein required for initiation of chromosomal DNA replication at oriC in E. coli, starts from two promoters, 1P and 2P. Gel-shift and DNase I-protection assays revealed that IciA protein, an inhibitor of initiation of in vitro E. coli chromosomal DNA replication at oriC, bound to two sites in the dnaA promoter region. One site is located upstream of promoter 1P, and the second is located downstream of promoter 2P. Whereas IciA protein did not affect transcription from the promoter 2P, transcription from the promoter 1P was specifically enhanced by IciA protein in vivo and in vitro. DnaA protein bound to the DnaA box between the two promoters 1P and 2P, acts as an transcriptional repressor. Under this condition, IciA protein counteracted the repressive effect of DnaA protein on the promoter 1P. These findings suggest that IciA protein may regulate the initiation of chromosomal DNA replication at oriC by controlling expression of the dnaA gene, as well as by inhibiting the initiation of chromosomal DNA replication at oriC.
Mol Microbiol 1996 Jan
PMID:Transcriptional activation of the dnaA gene encoding the initiator for oriC replication by IciA protein, an inhibitor of in vitro oriC replication in Escherichia coli. 882 83

Recent studies in our laboratory indicated that the upstream stimulatory factor (USF) in the sea urchin embryo of Lytechinus acts as a transcriptional repressor for the aboral ectoderm-specific expression of the LpS1 genes. Disruption of the extracellular matrix (ECM) arrests development prior to gastrulation and inactivates the LpS1 genes. We wanted to determine whether the inactivation of the LpS1 genes by ECM disruption may be due to an increase in USF expression. In the course of the investigation, a second L, variegatus USF cDNA clone (LvUSF2) was isolated and sequenced. The deduced amino acid sequence of LvUSF2 is nearly identical to LvUSF1 except at the amino end, where they are sharply divergent. Like LvUSF1, LvUSF2 has a USF-specific, a basic/hefixloop-helix, and a leucine zipper domain. Genomic DNA blots indicated that the two cDNA clones are derived from one gene, which suggested that the Lytechinus USF1 and USF2 mRNAs, of approximately 6.0 and 4.0 kb, respectively, are the result of differential RNA splicing. ECM disruption in Lytechinus embryos caused a relative drop in USF RNA accumulation levels to approximately 60% of control embryos, while LpS1 RNA accumulation levels dropped to less than 5%. USF protein levels and DNA binding activities in ECM-disrupted embryos also dropped to approximately 60% to that of control embryos. A mutation at the USF binding site in an LpS1 promoter-chloramphenicol acetyl transferase (CAT) fusion DNA construct did not cause a relative increase in CAT activity in ECM disrupted embryos. These results suggest that the induced drop in LpS1 gene expression by ECM disruption is not due to an increase in the repressive activity of USF.
Mol Reprod Dev 1996 Sep
PMID:Two distinct forms of USF in the Lytechinus sea urchin embryo do not play a role in LpS1 gene inactivation upon disruption of the extracellular matrix. 887 63


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