Showing papers on "Psychological repression published in 2001"

Repression Domains of Class II ERF Transcriptional Repressors Share an Essential Motif for Active Repression

Abstract: We reported previously that three ERF transcription factors, tobacco ERF3 (NtERF3) and Arabidopsis AtERF3 and AtERF4, which are categorized as class II ERFs, are active repressors of transcription. To clarify the roles of these repressors in transcriptional regulation in plants, we attempted to identify the functional domains of the ERF repressor that mediates the repression of transcription. Analysis of the results of a series of deletions revealed that the C-terminal 35 amino acids of NtERF3 are sufficient to confer the capacity for repression of transcription on a heterologous DNA binding domain. This repression domain suppressed the intermolecular activities of other transcriptional activators. In addition, fusion of this repression domain to the VP16 activation domain completely inhibited the transactivation function of VP16. Comparison of amino acid sequences of class II ERF repressors revealed the conservation of the sequence motif L / F DLN L / F (x)P. This motif was essential for repression because mutations within the motif eliminated the capacity for repression. We designated this motif the ERF-associated amphiphilic repression (EAR) motif, and we identified this motif in a number of zinc-finger proteins from wheat, Arabidopsis, and petunia plants. These zinc finger proteins functioned as repressors, and their repression domains were identified as regions that contained an EAR motif.

TGF-β-induced repression of CBFA1 by Smad3 decreases cbfa1 and osteocalcin expression and inhibits osteoblast differentiation

Abstract: Transforming growth factor-β (TGF-β), a secreted factor present at high levels in bone, inhibits osteoblast differentiation in culture; yet, the mechanism of this inhibition remains unclear. We studied the effects of TGF-β and its effectors, the Smads, on the expression and function of the osteoblast transcription factor CBFA1. TGF-β inhibited the expression of the cbfa1 and osteocalcin genes, whose expression is controlled by CBFA1 in osteoblast-like cell lines. This inhibition was mediated by Smad3, which interacts physically with CBFA1 and represses its transcriptional activity at the CBFA1-binding OSE2 promoter sequence. The repression of CBFA1 function by Smad3 contrasts with previous observations that Smads function as transcription activators. This repression occurred in mesenchymal but not epithelial cells, and depended on the promoter sequence. Smad3-mediated repression of CBFA1 provides a central regulatory mechanism for the inhibition of osteoblast differentiation by TGF-β, since it inhibits both cbfa1 transcription and transcriptional activation of osteoblast differentiation genes by CBFA1. Altering Smad3 signaling influenced osteoblast differentiation in the presence or absence of TGF-β, implicating Smad3/TGF-β-mediated repression in autocrine regulation of osteoblast differentiation.

Polypyrimidine Tract Binding Protein Antagonizes Exon Definition

Abstract: PTB appears to be a global repressor of weak or regulated exons. We propose here that PTB multimerization sequesters these exons to prevent exon definition. This is likely critical not only to prevent inclusion of pseudo-exons but also to set up cell-type-specific exon definition. What remains unclear about PTB can probably be broken down into two basic questions. First, what is the precise mechanism of repression? Second, how is this mechanism circumvented? Most of the research to resolve the first question has focused primarily on identifying instances of PTB repression but has done little to understand how that repression is achieved. Recently, both in vivo and in vitro assays for PTB repression have been developed (8, 65); thus, a detailed structure-function analysis can be done. Information from this approach may address mechanistic questions such as if PTB multimerization is required for repression or if there are PTB cofactors. Understanding how this repression is lifted will probably be a more complicated issue. Overwhelming PTB may occur by numerous mechanisms, such as strengthening weak splice sites via activators such as TIA-1 (18), causing the enhancement of inclusion via a tissue-specific expression of antagonizing RNA-binding proteins, or simply by modulating the expression of a PTB cofactor.

TGF-beta inhibits muscle differentiation through functional repression of myogenic transcription factors by Smad3.

Abstract: Transforming growth factor-beta (TGF-beta) is a potent inhibitor of skeletal muscle differentiation, but the molecular mechanism and signaling events that lead to this inhibition are poorly characterized. Here we show that the TGF-beta intracellular effector Smad3, but not Smad2, mediates the inhibition of myogenic differentiation in MyoD-expressing C3H10T1/2 cells and C2C12 myoblasts by repressing the activity of the MyoD family of transcriptional factors. The Smad3-mediated repression was directed at the E-box sequence motif within muscle gene enhancers and the bHLH region of MyoD, the domain required for its association with E-protein partners such as E12 and E47. The repression could be overcome by supplying an excess of E12, and covalent tethering of E47 to MyoD rendered the E-box-dependent transcriptional activity refractory to the effects of Smad3 and TGF-beta. Smad3 physically interacted with the HLH domain of MyoD, and this interaction correlated with the ability of Smad3 to interfere with MyoD/E protein heterodimerization and binding of MyoD complexes to oligomerized E-box sites. Together, these results reveal a model for how TGF-beta, through Smad3-mediated transcriptional repression, inhibits myogenic differentiation.

Methyl CpG-binding proteins and transcriptional repression.

Abstract: Since its discovery, methylation of DNA in mammalian cells has been correlated with transcriptional repression and with specialized chromatin structures Recently, considerable progress has been reported in the identification of protein factors with a highly conserved DNA interaction surface, termed the methyl CpG-binding domain or MBD A subset has been biochemically linked to histone deacetylases, suggesting a molecular mechanism for the functional properties of methylated DNA Despite several obvious attractions, the connection between MBD proteins and histone deacetylases fails to explain all the existing data In fact, the biochemistry and DNA-binding properties of most MBD family members have not been adequately described and considerable evidence exists for alternative mechanisms in the repression of methylated loci Null mutations have been generated in mice for several MBD family members, the phenotypes of the mutant animals raise important questions regarding the functions of the MBD family Here, I review the biochemistry, DNA-binding properties, and genetics of the MBD proteins that are linked to transcriptional repression, namely, MeCP2, MBD1, MBD2, and MBD3 Several models to account for the functional properties of methylated DNA are presented

Defective repression of c-myc in breast cancer cells: A loss at the core of the transforming growth factor β growth arrest program

Abstract: Loss of growth inhibitory responses to the cytokine transforming growth factor β (TGF-β) in cancer cells may result from mutational inactivation of TGF-β receptors or their signal transducers, the Smad transcription factors. In breast cancer, however, loss of TGF-β growth inhibition often occurs without a loss of these signaling components. A genome-wide analysis of rapid TGF-β gene responses in MCF-10A human mammary epithelial cells and MDA-MB-231 breast cancer cells shows that c-myc repression, a response that is key to the TGF-β program of cell cycle arrest, is selectively lost in the cancer cell line. Transformation of MCF-10A cells with c-Ha-ras and c-erbB2 oncogenes also led to a selective loss of c-myc repression and cell cycle arrest response. TGF-β stimulation of epithelial cells rapidly induces the formation of a Smad complex that specifically recognizes a TGF-β inhibitory element in the c-myc promoter. Formation of this complex is deficient in the oncogenically transformed breast cells. These results suggest that a Smad complex that specifically mediates c-myc repression is a target of oncogenic signals in breast cancer.

Transcriptional repression: the long and the short of it

Abstract: Gene-specific repression of transcription plays a central role in gene regulation. This is true for the spatial control of gene activity in development, during which boundaries of gene expression are often determined by the spatially restricted localization or activity of transcriptional repressors (Mannervik et al. 1999). It is also true for the control of gene expression by extracellular signals, in which genes are often maintained in an off state by repressor proteins until signal transduction alleviates the repression (e.g., Roose and Clevers 1999). One of the most useful ways of categorizing repressors is according to whether they mediate long-range or short-range repression (Gray and Levine 1996b). In longrange repression, a repressor makes a promoter resistant to the influence of all enhancers, even if those enhancers are located thousands of base pairs from the repressor binding site. This kind of repression is often referred to as silencing because an entire chromosomal locus is inactivated. In contrast, short-range repressors function in a less general manner. Rather than interfering with all transcription at a locus, they block the function of nearby DNA-bound activators while not interfering with more distantly bound activators. In this review, we discuss examples of both long-range and short-range repression, showing that long-range repression may often involve the assembly of a multiprotein complex termed a repressosome that is analogous in many ways to the enhanceosomes known to mediate activation. Furthermore, we discuss how both long-range and short-range repression may involve the recruitment of histone deacetylases to the template and discuss models that may allow these enzymes to mediate both types of repression. Finally, we consider the possibility that interactions between repressors and the basal machinery as well as between repressors and activators play roles in long-range and short-range repression.

Amino acid-induced translation of TOP mRNAs is fully dependent on phosphatidylinositol 3-kinase-mediated signaling, is partially inhibited by rapamycin, and is independent of S6K1 and rpS6 phosphorylation.

Abstract: Vertebrate TOP mRNAs contain an oligopyrimidine tract at their 5' termini (5'TOP) and encode components of the translational machinery. Previously it has been shown that they are subject to selective translational repression upon growth arrest and that their translational behavior correlates with the activity of S6K1. We now show that the translation of TOP mRNAs is rapidly repressed by amino acid withdrawal and that this nutritional control depends strictly on the integrity of the 5'TOP motif. However, neither phosphorylation of ribosomal protein (rp) S6 nor activation of S6K1 per se is sufficient to relieve the translational repression of TOP mRNAs in amino acid-starved cells. Likewise, inhibition of S6K1 activity and rpS6 phosphorylation by overexpression of dominant-negative S6K1 mutants failed to suppress the translational activation of TOP mRNAs in amino acid-refed cells. Furthermore, TOP mRNAs were translationally regulated by amino acid sufficiency in embryonic stem cells lacking both alleles of the S6K1 gene. Inhibition of mTOR by rapamycin led to fast and complete repression of S6K1, as judged by rpS6 phosphorylation, but to only partial and delayed repression of translational activation of TOP mRNAs. In contrast, interference in the phosphatidylinositol 3-kinase (PI3-kinase)-mediated pathway by chemical or genetic manipulations blocked rapidly and completely the translational activation of TOP mRNAs. It appears, therefore, that translational regulation of TOP mRNAs, at least by amino acids, (i) is fully dependent on PI3-kinase, (ii) is partially sensitive to rapamycin, and (iii) requires neither S6K1 activity nor rpS6 phosphorylation.

Repression of transcription of the p27(Kip1) cyclin-dependent kinase inhibitor gene by c-Myc.

Abstract: Upon engagement of the B Cell Receptor (BCR) of WEHI 231 immature B cells, a drop in c-Myc expression is followed by activation of the cyclin-dependent kinase inhibitor (CKI) p27(Kip1), which induces growth arrest and apoptosis. Here, we report inverse patterns of p27 and c-Myc protein expression follow BCR engagement. We present evidence demonstrating, for the first time, that the p27(Kip1) gene is a target of transcriptional repression by c-Myc. Specifically, the changes in p27 protein levels correlated with changes in p27 mRNA levels, and gene transcription. Induction of p27 promoter activity followed BCR engagement of WEHI 231 cells, and this induction could be repressed upon co-transfection of a c-Myc expression vector. Inhibition of the TATA-less p27 promoter by c-Myc was also observed in Jurkat T cells, vascular smooth muscle, and Hs578T breast cancer cells, extending the observation beyond immune cells. Consistent with a putative Inr element CCAGACC (where +1 is underlined) at the start site of transcription in the p27 promoter, deletion of Myc homology box II reduced the extent of repression. Furthermore, enhanced repression was observed upon transfection of the c-Myc 'super-repressor', with mutation of Phe115 to Leu. The sequences mediating transcriptional activity and c-Myc repression were mapped to bp -20 to +20 of the p27 gene. Finally, binding of Max was shown to facilitate c-Myc binding and repression of p27 promoter activity. Overall, these studies identify the p27 CKI gene as a new target whereby c-Myc can control cell proliferation, survival and neoplastic transformation.

Catabolite repression mediated by the CcpA protein in Bacillus subtilis: novel modes of regulation revealed by whole-genome analyses.

Abstract: Previous studies have shown that the CcpA protein of Bacillus subtilis is a major transcription factor mediating catabolite repression. We report here whole-transcriptome analyses that characterize CcpA-dependent, glucose-dependent gene expression and correlate the results with full-genome computer analyses of DNA binding (CRE) sites for CcpA. The data obtained using traditional approaches show good agreement with those obtained using the transcriptome approach. About 10% of all genes in B. subtilis are regulated > 3x by glucose, with repressed genes outnumbering activated genes three to one. Eighty per cent of these genes depend on CcpA for regulation. Classical approaches have provided only evidence for CcpA-mediated, glucose-dependent activation or repression. We show here that CcpA also mediates glucose-independent activation or repression, and that glucose may alter either the direction or the intensity of either effect. Computer analyses revealed the presence of CRE sites in most operons subject to CcpA-mediated glucose repression, but not in those subject to glucose activation, suggesting that either secondary transcription factors regulate the latter genes or activation by CcpA involves a dissimilar binding site. Operons encoding the constituents of ABC-type transporters that are subject to CcpA-mediated glucose regulation show two distinct patterns: either all genes in the operon are regulated in parallel (the minor class) or the gene encoding the extracytoplasmic solute-binding receptor is preferentially regulated (the major class). Genes subject to CcpA-independent catabolite repression are primarily concerned with sporulation. Several transcription factors were identified that are themselves regulated by CcpA at the transcriptional level. Representative data with functionally characterized genes are presented to illustrate the novel findings. The comprehensive transcriptome data are available on our website: www.biology.uesd.edu/~MSAIER/regulation/ and also on http://www.blackwell-science.com/ products/journals/suppmat/MMI/MMI2328/MMI2328sm.htm

Id1 regulation of cellular senescence through transcriptional repression of p16/Ink4a

Abstract: The Id family of helix–loop–helix (HLH) transcriptional regulatory proteins does not possess a basic DNA-binding domain and functions as a negative regulator of basic HLH transcription factors. Id proteins coordinate cell growth and differentiation pathways within mammalian cells and have been shown to regulate G1-S cell-cycle transitions. Although much recent data has implicated Id1 in playing a critical role in modulating cellular senescence, no direct genetic evidence has been reported to substantiate such work. Here we show that Id1-null primary mouse embryo fibroblasts undergo premature senescence despite normal growth profiles at early passage. These cells possess increased expression of the tumor-suppressor protein p16/Ink4a but not p19/ARF, and have decreased cyclin-dependent kinase (cdk) 2 and cdk4 kinase activity. We also show that Id1 is able to directly inhibit p16/Ink4a but not p19/ARF promoter activity via its HLH domain, and that Id1inhibits transcriptional activation at E-boxes within the p16/Ink4a promoter. Our data provide, to our knowledge, the first genetic evidence for a role for Id1 as an inhibitor of cellular senescence and suggest that Id1 functions to delay cellular senescence through repression of p16/Ink4a. Because epigenetic and genetic abrogation of p16/Ink4a function has been implicated in the evolution of several human malignancies, we propose that transcriptional regulation of p16/Ink4a may also provide a mechanism for the dysregulation of normal cellular growth controls during the evolution of human malignancies.

BRCA1 mediates ligand-independent transcriptional repression of the estrogen receptor

Abstract: Mutational inactivation of BRCA1 confers a cumulative lifetime risk of breast and ovarian cancers. However, the underlying basis for the tissue-restricted tumor-suppressive properties of BRCA1 remains poorly defined. Here we show that BRCA1 mediates ligand-independent transcriptional repression of the estrogen receptor α (ERα), a principal determinant of the growth, differentiation, and normal functional status of breasts and ovaries. In Brca1-null mouse embryo fibroblasts and BRCA1-deficient human ovarian cancer cells, ERα exhibited ligand-independent transcriptional activity that was not observed in Brca1-proficient cells. Ectopic expression in Brca1-deficient cells of wild-type BRCA1, but not clinically validated BRCA1 missense mutants, restored ligand-independent repression of ERα in a manner dependent upon apparent histone deacetylase activity. In estrogen-dependent human breast cancer cells, chromatin immunoprecipitation analysis revealed the association of BRCA1 with ERα at endogenous estrogen-response elements before, but not after estrogen stimulation. Collectively, these results reveal BRCA1 to be a ligand-reversible barrier to transcriptional activation by unliganded promoter-bound ERα and suggest a possible mechanism by which functional inactivation of BRCA1 could promote tumorigenesis through inappropriate hormonal regulation of mammary and ovarian epithelial cell proliferation.

HERP, a Novel Heterodimer Partner of HES/E(spl) in Notch Signaling

Abstract: HERP1 and -2 are members of a new basic helix-loop-helix (bHLH) protein family closely related to HES/E(spl), the only previously known Notch effector. Like that of HES, HERP mRNA expression is directly up-regulated by Notch ligand binding without de novo protein synthesis. HES and HERP are individually expressed in certain cells, but they are also coexpressed within single cells after Notch stimulation. Here, we show that HERP has intrinsic transcriptional repression activity. Transcriptional repression by HES/E(spl) entails the recruitment of the corepressor TLE/Groucho via a conserved WRPW motif, whereas unexpectedly the corresponding—but modified—tetrapeptide motif in HERP confers marginal repression. Rather, HERP uses its bHLH domain to recruit the mSin3 complex containing histone deacetylase HDAC1 and an additional corepressor, N-CoR, to mediate repression. HES and HERP homodimers bind similar DNA sequences, but with distinct sequence preferences, and they repress transcription from specific DNA binding sites. Importantly, HES and HERP associate with each other in solution and form a stable HES-HERP heterodimer upon DNA binding. HES-HERP heterodimers have both a greater DNA binding activity and a stronger repression activity than do the respective homodimers. Thus, Notch signaling relies on cooperation between HES and HERP, two transcriptional repressors with distinctive repression mechanisms which, either as homo- or as heterodimers, regulate target gene expression.

The hexokinase 2 protein regulates the expression of the GLK1, HXK1 and HXK2 genes of Saccharomyces cerevisiae.

Abstract: The key glycolytic HXK2 gene, coding for the enzyme hexokinase 2 (Hxk2p), is expressed when cells of the yeast Saccharomyces cerevisiae are grown on a fermentable medium using glucose, fructose or mannose as a carbon source. After shifting the cells to a non-fermentable carbon source, the HXK2 gene is repressed and the HXK1 and GLK1 genes are rapidly de-repressed, producing the enzymes hexokinase 1 (Hxk1p) and glucokinase (Glk1p) respectively. Because the in vivo functions of the Hxk1p and Glk1p enzymes have remained a mystery so far, we have investigated this glucose-induced regulatory process. Here we demonstrate the involvement of Hxk2p in the glucose-induced repression of the HXK1 and GLK1 genes and the glucose-induced expression of the HXK2 gene. We have also demonstrated the involvement of Hxk1p as a negative factor in the expression of the GLK1 and HXK2 genes. Further experimental evidence, using mutant cells expressing a truncated version of Hxk2p unable to enter the nucleus, shows that nuclear localization of Hxk2p is necessary for glucose-induced repression signalling of the HXK1 and GLK1 genes and for glucose-induced expression of the HXK2 gene. Gel mobility-shift analysis shows that Hxk2p-mediated regulation is exerted through ERA (ethanol repression autoregulation)-like regulatory sequences present in the HXK1 and GLK1 promoters and in two downstream repressing sequences of the HXK2 gene. These findings reveal a novel mechanism of gene regulation whereby the product of a glycolytic gene, normally resident in the cytosol, interacts directly with nuclear proteins to regulate the transcription of the HXK1 and GLK1 genes and to autoregulate its own transcription.

When the SWI/SNF complex remodels...the cell cycle.

Abstract: Mammalian cells contain several chromatin-remodeling complexes associated with the Brm and Brg1 helicase-like proteins. These complexes likely represent the functional homologs of the SWI/SNF and RSC complexes found in Saccharomyces cerevisiae. The mammalian chromatin-remodeling complexes are involved in both activation and repression of a variety of genes. Several lines of evidence also indicate that they play a specific role in the regulation of cell growth. Brm is down-regulated by ras signaling and its forced re-expression suppresses transformation by this oncogene. Besides, the Brg1 gene is silenced or mutated in several tumors cell lines and a Brg1-associated complex was recently found to co-purify with BRCA1, involved in breast and ovarian cancers. Finally, the gene encoding SNF5/Ini1, a subunit common to all mammalian SWI/SNF complexes, is inactivated in rhabdoid sarcomas, a very aggressive form of pediatric cancer. The current review will address observations made upon inactivation of Brm, Brg1 and SNF5/Ini1 by homologous recombination in the mouse, as well as the possible implication of these factors in the regulation of the Retinoblastoma pRb-mediated repression of the transcription factor E2F.

Translational repression determines a neuronal potential in Drosophila asymmetric cell division

Abstract: Asymmetric cell division is a fundamental strategy for generating cellular diversity during animal development1. Daughter cells manifest asymmetry in their differential gene expression. Transcriptional regulation of this process has been the focus of many studies, whereas cell-type-specific ‘translational’ regulation has been considered to have a more minor role. During sensory organ development in Drosophila, Notch signalling directs the asymmetry between neuronal and non-neuronal lineages2, and a zinc-finger transcriptional repressor Tramtrack69 (TTK69) acts downstream of Notch as a determinant of non-neuronal identity3,4. Here we show that repression of TTK69 protein expression in the neuronal lineage occurs translationally rather than transcriptionally. This translational repression is achieved by a direct interaction between cis-acting sequences in the 3′ untranslated region of ttk69 messenger RNA and its trans-acting repressor, the RNA-binding protein Musashi (MSI)5. Although msi can act downstream of Notch, Notch signalling does not affect MSI expression. Thus, Notch signalling is likely to regulate MSI activity rather than its expression. Our results define cell-type-specific translational control of ttk69 by MSI as a downstream event of Notch signalling in asymmetric cell division.

Methyl-CpG-binding protein 2 represses LINE-1 expression and retrotransposition but not Alu transcription

Abstract: In order to explore the defense mechanism by which retrotransposons are repressed, we assessed the ability of methyl-CpG-binding protein 2, MeCP2, to influence LINE-1 (L1) and Alu transcription and, furthermore, L1 retrotransposition. In transient transfection assays, targeting of the transcriptional-repression domain (TRD) of MeCP2 (via a linked Gal4 DNA-binding domain) to the transcriptional start site of L1 promoter-driven reporter constructs efficiently repressed transcription. The Gal4-linked TRD of the related methyl-CpG-binding protein MBD1 also repressed transcription but not that of MBD2. Furthermore, full-length MeCP2 effectively repressed transcription of a HpaII-methylated L1 reporter. Secondly, we used a genetic assay employing a full-length neo-marked L1 reporter construct to study L1 retrotransposition. We found the Gal4-linked TRD of MeCP2 to repress effectively L1 retrotransposition when targeted to the retrotransposition reporter. Retrotransposition was also reduced in response to in vitro HpaII methylation of the reporter and was further decreased by co-expressed full-length MeCP2. In striking contrast expression of the Gal4-linked TRD of MeCP2 had no inhibiting effect on transcription of an AluSx reporter tagged with a 7S-upstream sequence. Furthermore, full-length MeCP2 abrogated the methylation-induced repression of this reporter. Our results indicate that MeCP2 serves a role in repression of L1 expression and retrotransposition but has no inhibiting effect on Alu transcription.

A Conserved α-Helical Motif Mediates the Interaction of Sp1-Like Transcriptional Repressors with the Corepressor mSin3A

Abstract: Sp1-like proteins are defined by three highly homologous C(2)H(2) zinc finger motifs that bind GC-rich sequences found in the promoters of a large number of genes essential for mammalian cell homeostasis. Here we report that TIEG2, a transforming growth factor beta-inducible Sp1-like protein with antiproliferative functions, represses transcription through recruitment of the mSin3A-histone deacetylase complex. The interaction of TIEG2 with mSin3A is mediated by an alpha-helical repression motif (alpha-HRM) located within the repression domain (R1) of TIEG2. This alpha-HRM specifically associates with the second paired amphipathic helix (PAH2) domain of mSin3A. Mutations in the TIEG2 alpha-HRM domain that disrupt its helical structure abolish its ability to both bind mSin3A and repress transcription. Interestingly, the alpha-HRM is conserved in both the TIEG (TIEG1 and TIEG2) and BTEB (BTEB1, BTEB3, and BTEB4) subfamilies of Sp1-like proteins. The alpha-HRM from these proteins also mediates direct interaction with mSin3A and represses transcription. Surprisingly, we found that the alpha-HRM of the Sp1-like proteins characterized here exhibits structural and functional resemblance to the Sin3A-interacting domain previously described for the basic helix-loop-helix protein Mad1. Thus, our study defines a mechanism of transcriptional repression via the interactions of the alpha-HRM with the Sin3-histone deacetylase complex that is utilized by at least five Sp1-like transcriptional factors. More importantly, we demonstrate that a helical repression motif which mediates Sin3 interaction is not an exclusive structural and functional characteristic of the Mad1 subfamily but rather has a wider functional impact on transcriptional repression than previously demonstrated.

A cytosolic NAD-dependent deacetylase, Hst2p, can modulate nucleolar and telomeric silencing in yeast

Abstract: In budding yeast, the silent information regulator Sir2p is a nuclear NAD-dependent deacetylase that is essential for both telomeric and rDNA silencing. All eukaryotic species examined to date have multiple homologues of Sir two (HSTs), which share a highly conserved globular core domain. Here we report that yeast Hst2p and a mammalian Hst2p homologue, hSirT2p, are cytoplasmic in yeast and human cells, in contrast to yHst1p and ySir2p which are exclusively nuclear. Although yHst2p cannot restore silencing in a sir2 deletion, overexpression of yHst2p influences nuclear silencing events in a SIR2 strain, derepressing subtelomeric silencing while increasing repression in the rDNA. In contrast, a form of ySir2p carrying a point mutation in the conserved core domain disrupts both telomeric position effect (TPE) and rDNA repression at low expression levels. This argues that non-nuclear yHst2p can compete for a substrate or ligand specifically required for telomeric, and not rDNA repression.

Factor recruitment and TIF2/GRIP1 corepressor activity at a collagenase-3 response element that mediates regulation by phorbol esters and hormones.

Abstract: To investigate determinants of specific transcriptional regulation, we measured factor occupancy and function at a response element, col3A, associated with the collagenase-3 gene in human U2OS osteosarcoma cells; col3A confers activation by phorbol esters, and repression by glucocorticoid and thyroid hormones. The subunit composition and activity of AP-1, which binds col3A, paralleled the intracellular level of cFos, which is modulated by phorbol esters and glucocorticoids. In contrast, a similar AP-1 site at the collagenase-1 gene, not inducible in U2OS cells, was not bound by AP-1. The glucocorticoid receptor (GR) associated with col3A through protein–protein interactions with AP-1, regardless of AP-1 subunit composition, and repressed transcription. TIF2/GRIP1, reportedly a coactivator for GR and the thyroid hormone receptor (TR), was recruited to col3A and potentiated GR-mediated repression in the presence of a GR agonist but not antagonist. GRIP1 mutants deficient in GR binding and coactivator functions were also defective for corepression, and a GRIP1 fragment containing the GR-interacting region functioned as a dominant-negative for repression. In contrast, repression by TR was unaffected by GRIP1. Thus, the composition of regulatory complexes, and the biological activities of the bound factors, are dynamic and dependent on cell and response element contexts. Cofactors such as GRIP1 probably contain distinct surfaces for activation and repression that function in a context-dependent manner.

p53 Down-Regulates CHK1 through p21 and the Retinoblastoma Protein

Abstract: Both fission yeast and mammalian cells require the function of the checkpoint kinase CHK1 for G2 arrest after DNA damage. The tumor suppressor p53, a well-studied stress response factor, has also been shown to play a role in DNA damage G2 arrest, although in a manner that is probably independent of CHK1. p53, however, can be phosphorylated and regulated by both CHK1 as well as another checkpoint kinase, hCds1 (also called CHK2). It was therefore of interest to determine whether reciprocally, p53 affects either CHK1 or CHK2. We found that induction of p53 either by diverse stress signals or ectopically using a tetracycline-regulated promoter causes a marked reduction in CHK1 protein levels. CHK1 downregulation by p53 occurs as a result of reduced CHK1 RNA accumulation, indicating that repression occurs at the level of transcription. Repression of CHK1 by p53 requires p21, since p21 alone is sufficient for this to occur and cells lacking p21 cannot downregulate CHK1. Interestingly, pRB is also required for CHK1 downregulation, suggesting the possible involvement of E2F-dependent transcription in the regulation of CHK1. Our results identify a new repression target of p53 and suggest that p53 and CHK1 play interdependent and complementary roles in regulating both the arrest and resumption of G2 after DNA damage.

Temporal recruitment of the mSin3A-histone deacetylase corepressor complex to the ETS domain transcription factor Elk-1

Abstract: The transcriptional status of eukaryotic genes is determined by a balance between activation and repression mechanisms. The nuclear hormone receptors represent classical examples of transcription factors that can regulate this balance by recruiting corepressor and coactivator complexes in a ligand-dependent manner. Here, we demonstrate that the equilibrium between activation and repression via a single transcription factor, Elk-1, is altered following activation of the Erk mitogen-activated protein kinase cascade. In addition to its C-terminal transcriptional activation domain, Elk-1 contains an N-terminal transcriptional repression domain that can recruit the mSin3A-histone deacetylase 1 corepressor complex. Recruitment of this corepressor is enhanced in response to activation of the Erk pathway in vivo, and this recruitment correlates kinetically with the shutoff of one of its target promoters, c-fos. Elk-1 therefore undergoes temporal activator-repressor switching and contributes to both the activation and repression of target genes following growth factor stimulation.

Post‐transcriptional regulation through the HO 3′‐UTR by Mpt5, a yeast homolog of Pumilio and FBF

Abstract: Drosophila Pumilio (Pum) and Caenorhabditis elegans FBF bind to the 3′-untranslated region (3′-UTR) of their target mRNAs and repress translation. Pum and FBF are members of a large and evolutionarily conserved protein family, the Puf family, found in Drosophila, C.elegans, humans and yeasts. Budding yeast, Saccharomyces cerevisiae, has five proteins with conserved Puf motifs: Mpt5/Uth4, Ygl014w, Yll013c, Jsn1 and Ypr042c. Here we report that Mpt5 negatively regulates expression of the HO gene. Loss of MPT5 increased expression of reporter genes integrated into the ho locus, whereas overexpression of MPT5 decreased expression. Repression required the 3′-UTR of HO, which contains a tetranucleotide, UUGU, also found in the binding sites of Pum and FBF. Mutation of UUGU to UACU in the HO 3′-UTR abolished Mpt5-mediated repression. Studies using a three-hybrid assay for RNA binding indicate that Mpt5 binds to the 3′-UTR of HO mRNA containing a UUGU sequence but not a UACU sequence. These observations suggest that the yeast Puf homolog, Mpt5, negatively regulates HO expression post- transcriptionally.