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PubMed2006

RUNX1 associates with histone deacetylases and SUV39H1 to repress transcription.

RUNX1 (AML1) is a gene that is frequently disrupted by chromosomal translocations in acute leukemia. Like its Drosophila homolog Runt, RUNX1 both activates and represses transcription. Both Runt and RUNX1 are required for gene silencing during development and a central domain of RUNX1, termed repression domain 2 (RD2), was defined as being required for transcriptional repression and for the silencing of CD4 during T-cell maturation in thymic organ cultures. Although transcriptional co-repressors are known to contact other repression domains in RUNX1, the factors that bind to RD2 had not been defined. Therefore, we tested whether RD2 contacts histone-modifying enzymes that may mediate both repression and gene silencing. We found that RD2 contacts SUV39H1, a histone methyltransferase, via two motifs and that endogenous Suv39h1 associates with a Runx1-regulated repression element in murine erythroleukemia cells. In addition, one of these SUV39H1-binding motifs is also sufficient for binding to histone deacetylases 1 and 3, and both of these domains are required for full RUNX1-mediated transcriptional repression. The association between RUNX1, histone deacetylases and SUV39H1 provides a molecular mechanism for repression and possibly gene silencing mediated by RUNX1.

Animals↗
PubMed1998

Repression of basal transcription by vitamin D receptor: evidence for interaction of unliganded vitamin D receptor with two receptor interaction domains in RIP13delta1.

Repression of basal transcription of a 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) responsive 25-hydroxyvitamin D3-24-hydroxylase (CYP24) promoter construct as observed in kidney cells in the absence of ligand and this repression was dependent on a functional vitamin D response element (VDRE). Basal repression was also seen with a construct where a consensus DR-3-type VDRE was fused to the thymidine kinase promoter. Expression of a dominant negative vitamin D receptor (VDR) isoform that strongly bound to the VDRE motif in the CYP24 promoter ablated basal repression. This VDR isoform lacked sequence in the hinge- and ligand-binding domains implicating one or both of these domains in basal repression. It is well known that thyroid hormone and retinoic acid receptors silence basal transcription of target genes in the absence of ligands and this repressor function can be mediated by the nuclear receptor corepressor N-CoR. Two variants of N-CoR have been described, RIP13a and RIP13delta1. N-CoR and the variants contain two receptor interaction domains, ID-I and ID-II, which are identical except region ID-II in RIP13delta1 has an internal deletion. We have used the mammalian two hybrid system to investigate whether VDR, in the absence of ligand 1,25-(OH)2D3, can interact with these domains. The data showed that unliganded VDR does not interact with either ID-I or ID-II from RIP13a and RIP13delta1, but does interact strongly with a composite domain of ID-I and ID-II from RIP13delta1 (but not from RIP13a) and this strong interaction is abrogated in the presence of ligand. This finding implicates RIP13delta1 in VDR-dependent basal repression of the promoter constructs under investigation. However, over-expression of RIP13delta1 in kidney cell lines did not alter basal expression of the CYP24 promoter construct. It is concluded that either the level of endogenous RIP13delta1 in these kidney cells permits maximal repression or that repression occurs by a mechanism that is independent of RIP13delta1. Alternatively, repression may be dependent on RIP13delta1 but requires an additional cofactor that is limiting in these cells.

Animals↗
PubMed2005

Expression, purification and partial characterization of the Krüppel-associated box (KRAB) from the human ZNF2 protein.

The Krüppel-associated box (KRAB) domain is a potent transcription repression bipartite domain, shared by over 400 zinc finger proteins in humans, involved in the regulation of many functions. KRAB domains are both physically and functionally bipartite (A and B modules). The lone KRAB-A and composite KRAB-AB domains from the human ZNF2 protein were over-expressed as recombinant proteins in E. coli, isolated and purified to homogeneity to investigate their structure to function relationship.

Amino Acid Sequence↗
PubMed1998

The yeast protein complex containing cdc68 and pob3 mediates core-promoter repression through the cdc68 N-terminal domain.

Transcription of nuclear genes usually involves trans-activators, whereas repression is exerted by chromatin. For several genes the transcription mediated by trans-activators and the repression mediated by chromatin depend on the CP complex, a recently described abundant yeast nuclear complex of the Pob3 and Cdc68/Spt16 proteins. We report that the N-terminal third of the Saccharomyces cerevisiae Cdc68 protein is dispensable for gene activation but necessary for the maintenance of chromatin repression. The absence of this 300-residue N-terminal domain also decreases the need for the Swi/Snf chromatin-remodeling complex in transcription and confers an Spt- effect characteristic of chromatin alterations. The repression domain, and indeed the entire Cdc68 protein, is highly conserved, as shown by the sequence of the Cdc68 functional homolog from the yeast Kluyveromyces lactis and by database searches. The repression-defective (truncated) form of Cdc68 is stable but less active at high temperatures, whereas the known point-mutant form of Cdc68, encoded by three independent mutant alleles, alters the N-terminal repression domain and destabilizes the mutant protein.

Adenosine Triphosphatases↗
PubMed2002

POZ domain transcription factor, FBI-1, represses transcription of ADH5/FDH by interacting with the zinc finger and interfering with DNA binding activity of Sp1.

The POZ domain is a protein-protein interaction motif that is found in many transcription factors, which are important for development, oncogenesis, apoptosis, and transcription repression. We cloned the POZ domain transcription factor, FBI-1, that recognizes the cis-element (bp -38 to -22) located just upstream of the core Sp1 binding sites (bp -22 to +22) of the ADH5/FDH minimal promoter (bp -38 to +61) in vitro and in vivo, as revealed by electrophoretic mobility shift assay and chromatin immunoprecipitation assay. The ADH5/FDH minimal promoter is potently repressed by the FBI-1. Glutathione S-transferase fusion protein pull-down showed that the POZ domains of FBI-1, Plzf, and Bcl-6 directly interact with the zinc finger DNA binding domain of Sp1. DNase I footprinting assays showed that the interaction prevents binding of Sp1 to the GC boxes of the ADH5/FDH promoter. Gal4-POZ domain fusions targeted proximal to the GC boxes repress transcription of the Gal4 upstream activator sequence-Sp1-adenovirus major late promoter. Our data suggest that POZ domain represses transcription by interacting with Sp1 zinc fingers and by interfering with the DNA binding activity of Sp1.

Aldehyde Oxidoreductases↗
PubMed1990

The yeast heat shock transcription factor contains a transcriptional activation domain whose activity is repressed under nonshock conditions.

Transcription of heat shock genes is induced by exposure of cells to elevated temperatures or other stress conditions. In yeast, it is thought that induction of transcription is mediated by conversion of a DNA-bound transcriptionally inactive form of the heat shock transcription factor (HSTF) to a DNA-bound transcriptionally active form. We have identified domains in HSTF involved in transcriptional activation and in repression of transcriptional activation at non-shock temperatures. We present evidence that a temperature-regulated transcriptional activation domain exists in HSTF and that this domain is essential for survival of yeast cells at heat shock temperatures. We propose a model for temperature-regulated transcriptional activation by a derepression mechanism.

Amino Acid Sequence↗
PubMed2001

Biological activity of mammalian transcriptional repressors.

Research on the regulation of transcription in mammals has focused in recent years mainly on the mechanism of transcriptional activation. However, transcriptional repression mediated by repressor proteins is a common regulatory mechanism in mammals and might play an important role in many biological processes. To understand the molecular mechanism of transcriptional repression, the activity of eight mammalian repressors or repressor domains was investigated using a set of model promoters in combination with two different transcriptional detection methods. The repressors studied were: REST, the thyroid hormone receptors alpha and beta, the zinc finger protein NK10 containing a 'krüppel-associated box' (KRAB), repressor domains derived from the proteins Egr-1, Oct2A and Dr1 and the repressor/activator protein YY1. Here we show that the repressor domains of REST, Egr-1, the thyroid hormone receptors alpha< and beta and NK10 were transferable to a heterologous DNA-binding domain and repressed transcription from proximal and distal positions. Moreover, these repressor domains also blocked the activity of a strong viral enhancer in a 'remote position'. Thus, these domains are 'general' transcriptional repressor domains. The 'krüppel-associated box' was the most powerful repressor domain tested. In contrast, the repressor domains derived from Oct2A and Dr1 were inactive when fused to a heterologous DNA-binding domain. The repressor domain of YY1 exhibited transcriptional repression activity only in one of the transcriptional assay systems. The recruitment of histone deacetylases to the proximity of the basal transcriptional apparatus was recently discussed as a mechanism for some mammalian transcriptional repressor proteins. Here we show here that histone deacetylase 2, targeted to the reporter gene via DNA-protein interaction, functions as a transcriptional repressor protein regardless of the location of its binding site within the transcription unit.

Animals↗
PubMed2000

PML and the oncogenic nuclear domains in regulating transcriptional repression.

Promyelocytic leukemia protein (PML) is a tumor suppressor involved in the t(15;17) translocation that causes acute promyelocytic leukemia (APL). PML is located at multiple nuclear domains known as PML oncogenic domains (PODs), whose structures are dynamically regulated and disrupted in t(15;17) APL cells. PML is involved in several important cellular processes; however, its exact function is unclear. Recently, a POD-associated protein was found to be transcriptional repressor, suggesting a new role for PODs in regulating transcriptional repression.

Animals↗
PubMed1997

Functional domains of the human orphan receptor ARP-1/COUP-TFII involved in active repression and transrepression.

The orphan receptor ARP-1/COUP-TFII, a member of the chicken ovalbumin upstream promoter transcription factor (COUP-TF) subfamily of nuclear receptors, strongly represses transcriptional activity of numerous genes, including several apolipoprotein-encoding genes. Recently it has been demonstrated that the mechanism by which COUP-TFs reduce transcriptional activity involves active repression and transrepression. To map the domains of ARP-1/COUP-TFII required for repressor activity, a detailed deletion analysis of the protein was performed. Chimeric proteins in which various segments of the ARP-1/COUP-TFII carboxy terminus were fused to the GAL4 DNA binding domain were used to characterize its active repression domain. The smallest segment confering active repressor activity to a heterologous DNA binding domain was found to comprise residues 210 to 414. This domain encompasses the region of ARP-1/COUP-TFII corresponding to helices 3 to 12 in the recently published crystal structure of other members of the nuclear receptor superfamily. It includes the AF-2 AD core domain formed by helix 12 but not the hinge region, which is essential for interaction with a corepressor in the case of the thyroid hormone and retinoic acid receptor. Attachment of the nuclear localization signal from the simian virus 40 large T antigen (Flu tag) to the amino terminus of ARP-1/COUP-TFII abolished its ability to bind to DNA without affecting its repressor activity. By using a series of Flu-tagged mutants, the domains required for transrepressor activity of the protein were mapped. They include the DNA binding domain and the segment spanning residues 193 to 399. Transcriptional activity induced by liver-enriched transactivators such as hepatocyte nuclear factor 3 (HNF-3), C/EBP, or HNF-4 was repressed by ARP-1/COUP-TFII independent of the presence of its cognate binding site, while basal transcription or transcriptional activity induced by ATF or Sp1 was not perturbed by the protein. In conclusion, our results demonstrate that the domains of ARP-1/COUP-TFII required for active repression and transrepression do not coincide. Moreover, they strongly suggest that transrepression is the predominant mechanism underlying repressor activity of ARP-1/COUP-TFII. This mechanism most likely involves interaction of the protein with one or several transcriptional coactivator proteins which are employed by various liver-enriched transactivators but not by ubiquitous factors such as Sp1 or ATF.

Apolipoproteins B↗
PubMed1994

The oncogenic transcription factor IRF-2 possesses a transcriptional repression and a latent activation domain.

IRF-1 and IRF-2 are two structurally related transcription factors originally identified as regulators of the type I interferon (IFN) system. IRF-1 functions as an activator whereas IRF-2 binds to the same cis-elements and can repress IRF-1 action. More recently these two factors have been shown to act in a mutually antagonistic manner to regulate cell growth; overexpression of the repressor IRF-2 leads to cell transformation, whereas concomitant overexpression of IRF-1 leads to reversion. Previous studies have identified DNA-binding domains in IRF-1 and IRF-2 and an activation domain in IRF-1. In the present study we show that IRF-2 also possesses a transcriptional repression domain in its carboxyl terminal region. We further observe that a LexA-IRF2 fusion can inhibit the function of an activator positioned nearby in the promoter. Thus, repression by IRF-2 may involve both competition with IRF-1 for binding to the promoter as well as the 'silencing' of nearby activators. Furthermore, we demonstrate the presence of a latent activation domain in the central region of IRF-2 and speculate that IRF-2 may contribute to gene activation under certain conditions.

Animals↗
PubMed2003

MCAF mediates MBD1-dependent transcriptional repression.

DNA methylation is involved in a variety of genome functions, including gene control and chromatin dynamics. MBD1 is a transcriptional regulator through the cooperation of a methyl-CpG binding domain, cysteine-rich CXXC domains, and a transcriptional repression domain. A yeast two-hybrid screen was performed to investigate the role of MBD1 in methylation-based transcriptional repression. We report a mediator, MBD1-containing chromatin-associated factor (MCAF), that interacts with the transcriptional repression domain of MBD1. MCAF harbors two conserved domains that allow it to interact with MBD1 and enhancer-like transactivator Sp1. MCAF possesses a coactivator-like activity, and it seems to facilitate Sp1-mediated transcription. In contrast, the MBD1-MCAF complex blocks transcription through affecting Sp1 on methylated promoter regions. These data provide a mechanistic basis for direct inhibition of gene expression via methylation-dependent and histone deacetylation-resistant processes.

Binding Sites↗
PubMed2005

[RIP140 and hormone signaling].

Nuclear hormone receptors belong to a superfamily of ligand-activated transcription factors which regulate fundamental physiological processes. Their activity is controlled by a large number of coregulatory proteins which are, in most cases, recruited by nuclear receptors in the presence of ligand. RIP140 (receptor interacting protein of 140 kDa) was one of the first transcription cofactors to be identified almost ten years ago. This molecule is an atypical cofactor which interacts with agonist-liganded nuclear receptors but negatively regulates their transactivation potential. RIP140 exhibits nine leucine-rich motifs (LxxLL) which mediate the specific docking on the nuclear receptor ligand-binding domain. Transcription repression exerted by this cofactor implicates different mechanisms. Not only it involves a competition with coactivators such as those belonging to the p160 family, but also relies on active intrinsic repression through at least four different domains which allow recruitement of downstream repressors such as histone deacetylases (HDACs) or C-terminal binding proteins (CtBPs). The biological role of RIP140 has been investigated by disrupting the gene in mice. The lack of RIP140 expression in ovaries prevents follicle rupture and ovulation, rising to female infertility. In addition, this cofactor is also required for the control of fat storage and utilization through the regulation of genes involved in thermogenesis. Finally, RIP140 could play a role in the hormonal control of cancer cell proliferation by negatively regulating the activity of estrogen and retinoic acid receptors which are key actors in cancer growth. Interestingly, both estrogens and retinoic acid regulate RIP140 gene expression, revealing an increased level of complexity. In conclusion, RIP140 is an atypical transcription inhibitor which, by repressing nuclear hormone receptor activity, plays fundamental physiopathological roles.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Hyperacetylated chromatin domains: lessons from heterochromatin.

A small but growing number of loci that exhibit covalent histone modifications, such as hyperacetylation, over broad regions of 10 kb or more have been characterized. These hyperacetylated domains occur exclusively at loci containing highly expressed, tissue-specific genes, and the available evidence suggests that they are involved in the activation of these genes. Although to date little is known concerning the formation or function of these domains, rather more is known concerning repressive, heterochromatic domains, and the example provided by heterochromatin may be instructive in considering mechanisms of active domain formation.

Acetylation↗
PubMed1992

Regions controlling hyperphosphorylation and conformation of the retinoblastoma gene product are independent of domains required for transcriptional repression.

Cell cycle-dependent phosphorylation appears to control the function of the retinoblastoma gene product, p110RB1. We have previously shown that both the N-terminal portion and the portion corresponding to exon 23 are important in the hyperphosphorylation of p110RB1. To gain further insight into the domains involved in these phosphorylations, we have created a series of mutations in the murine cDNA and investigated their effects on phosphorylation, protein conformation and transcription of genes controlled by an E2 element. Three series of p110RB1 constructs were made: (1) mutations and/or deletions in the cluster of potential p34cdc2 consensus sequences of exon 23, (2) deletions for the entire region C-terminal to the large T antigen (TAg)-binding domains or (3) mutations in eight potential p34cdc2 sites, including four in exon 23. Following transfections into COS cells, phosphorylation, TAg binding and protein conformation were evaluated. Constructs were also tested for their ability to repress transcription from the adenovirus early promoter. All mutant proteins bound to SV40 TAg. Mutation of Ser-781, Ser-787 and Ser-788 had no detectable effect on the phosphorylation pattern of p110RB1, producing both the fast-migrating, hypophosphorylated and slower migrating, hyperphosphorylated bands on SDS-PAGE gels. However, mutation or deletion of Ser-800 and Ser-804 resulted in proteins which failed to show the characteristic shift in molecular weight associated with the hyperphosphorylated form of p110RB1. A protein mutated only at Pro-805, the residue adjacent to Ser-804, behaved similarly to mutants with both Ser-800 and Ser-804 altered; mutation of Pro-801 had no effect on the mobility of the hyperphosphorylated species. While differences were seen in the ability of these proteins to be hyperphosphorylated, the wild-type function of repression of adenovirus early promoter activity was retained. These results are consistent with the large shift in apparent molecular weight of p110RB1 upon hyperphosphorylation being due to phosphorylation of Ser-804. Phosphorylation of this p34cdc2 consensus sequence and the subsequent conformational change in p110RB1 detected in denaturing gels is not necessary, however, for the phosphorylation-dependent modulation of p110RB1-TAg interaction.

Amino Acid Sequence↗
PubMed1996

Multiple domains participate in distance-independent LAZ3/BCL6-mediated transcriptional repression.

The LAZ3/BCL6 gene implicated in diffuse large cell lymphomas encodes a transcriptional repressor containing Krüppel-fike zinc fingers. It harbours at its N-terminus a conserved protein/protein interaction motif, the BTB/POZ domain, which is also an autonomous transcriptional repression domain. We demonstrate here using several GAL4-LAZ3/BCL6 chimeras that the BTB/POZ domain plays an important but not exclusive role as its deletion gives rise to a GAL4 chimera that mediates significant, albeit reduced, transcriptional repression. Moreover, the repressive effect mediated either by LAZ3/BCL6 or by the isolated domains occurs with unaltered efficiency even at long distance (1.6 Kbp), ruling out steric hindrance mechanisms. Finally, though the absence of a TATA box appears to weaken this activity, it is largely promoter independent. Taken together, our results demonstrate that multiple domains participate in the promoter and distance-independent LAZ3/BCL6-mediated transcriptional repression.

Animals↗
PubMed1997

Characterization of receptor interaction and transcriptional repression by the corepressor SMRT.

SMRT (silencing mediator of retinoic acid and thyroid hormone receptor) and N-CoR (nuclear receptor corepressor) are two related transcriptional corepressors that contain separable domains capable of interacting with unliganded nuclear receptors and repressing basal transcription. To decipher the mechanisms of receptor interaction and transcriptional repression by SMRT/N-CoR, we have characterized protein-protein interacting surfaces between SMRT and nuclear receptors and defined transcriptional repression domains of both SMRT and N-CoR. Deletional analysis reveals two individual nuclear receptor domains necessary for stable association with SMRT and a C-terminal helix essential for corepressor dissociation. Coordinately, two SMRT domains are found to interact independently with the receptors. Functional analysis reveals that SMRT contains two distinct repression domains, and the corresponding regions in N-CoR also repress basal transcription. Both repression domains in SMRT and N-CoR interact weakly with mSin3A, which in turn associates with a histone deacetylase HDAC1 in a mammalian two-hybrid assay. Far-Western analysis demonstrates a direct protein-protein interaction between two N-CoR repression domains with mSin3A. Finally we demonstrate that overexpression of full-length SMRT further represses basal transcription from natural promoters. Together, these results support a role of SMRT/N-CoR in corepression through the utilization of multiple mechanisms for receptor interactions and transcriptional repression.

Amino Acid Sequence↗
PubMed2004

Phosphorylation of MRF4 transactivation domain by p38 mediates repression of specific myogenic genes.

Skeletal myogenesis is associated with the activation of four muscle regulatory factors (MRFs): Myf5, MyoD, Myogenin and MRF4. Here we report that p38 mitogen-activated protein kinase represses the transcriptional activity of MRF4 (involved in late stages of myogenesis), resulting in downregulation of specific muscle genes. MRF4 is phosphorylated in vitro and in vivo by p38 on two serines (Ser31 and Ser42) located in the N-terminal transactivation domain, resulting in reduced MRF4-mediated transcriptional activity. In contrast, nonphosphorylatable MRF4 mutants display increased transcriptional activity and are able to advance both myoblast fusion and differentiation. We also show that expression of desmin and alpha-actin, but not muscle creatin kinase, decreased at late stages of muscle differentiation, correlating with the induction of MRF4 and p38 activation. Accordingly, inhibition of p38 during late myogenesis results in the upregulation of both desmin and alpha-actin. We propose that repression of MRF4 activity by p38 phosphorylation may represent a new mechanism for the silencing of specific muscle genes at the terminal stages of muscle differentiation.

Actins↗
PubMed1996

Genetic evidence for interdomain regulation of the phenol-responsive final sigma54-dependent activator DmpR.

The final sigma54-dependent DmpR activator regulates transcription of the dmp operon that encodes the enzymes for catabolism of (methyl)phenols. DmpR is expressed constitutively, but its transcriptional promoting activity is controlled positively in direct response to the presence of aromatic pathway substrates (effectors). DmpR has a distinct domain structure with the amino-terminal A-domain controlling the specificity of activation of the regulator by aromatic effectors (signal reception), a central C-domain mediating an ATPase activity essential for transcriptional activation, and a carboxyl-terminal D-domain involved in DNA binding. Deletion of the A-domain has been shown previously to result in an effector-independent transcriptional activator with constitutive ATPase activity. These results, in conjunction with the location of mutations within the A- and C-domains which exhibit an effector-independent (semiconstitutive) property, have led to a working model in which the A-domain serves to mask the ATPase and transcriptional promoting activity of the C-domain in the absence of effectors. To investigate the mechanism by which the A-domain exerts its repressive effect, we developed a genetic system to select positively for intramolecular second site revertants of DmpR. The results demonstrate (i) that mutations within the A-domain can suppress the semiconstitutive activity of C-domain located mutations and vice versa; (ii) that the C-domain located mutations do not influence the intrinsic ATPase and transcriptional promoting property of the C-domain in the absence of the A-domain; and (iii) that semiconstitutive mutations of the A- and C-domain have an additive effect. Taken together these results support a model in which the A-domain represses the function(s) of the C-domain by direct interactions between residues of the two domains.

Adenosine Triphosphatases↗