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PubMed2006

Polycomb complexes repress developmental regulators in murine embryonic stem cells.

The mechanisms by which embryonic stem (ES) cells self-renew while maintaining the ability to differentiate into virtually all adult cell types are not well understood. Polycomb group (PcG) proteins are transcriptional repressors that help to maintain cellular identity during metazoan development by epigenetic modification of chromatin structure. PcG proteins have essential roles in early embryonic development and have been implicated in ES cell pluripotency, but few of their target genes are known in mammals. Here we show that PcG proteins directly repress a large cohort of developmental regulators in murine ES cells, the expression of which would otherwise promote differentiation. Using genome-wide location analysis in murine ES cells, we found that the Polycomb repressive complexes PRC1 and PRC2 co-occupied 512 genes, many of which encode transcription factors with important roles in development. All of the co-occupied genes contained modified nucleosomes (trimethylated Lys 27 on histone H3). Consistent with a causal role in gene silencing in ES cells, PcG target genes were de-repressed in cells deficient for the PRC2 component Eed, and were preferentially activated on induction of differentiation. Our results indicate that dynamic repression of developmental pathways by Polycomb complexes may be required for maintaining ES cell pluripotency and plasticity during embryonic development.

Animals↗
PubMed2006

Repression of p53 activity by Smyd2-mediated methylation.

Specific sites of lysine methylation on histones correlate with either activation or repression of transcription. The tumour suppressor p53 (refs 4-7) is one of only a few non-histone proteins known to be regulated by lysine methylation. Here we report a lysine methyltransferase, Smyd2, that methylates a previously unidentified site, Lys 370, in p53. This methylation site, in contrast to the known site Lys 372, is repressing to p53-mediated transcriptional regulation. Smyd2 helps to maintain low concentrations of promoter-associated p53. We show that reducing Smyd2 concentrations by short interfering RNA enhances p53-mediated apoptosis. We find that Set9-mediated methylation of Lys 372 inhibits Smyd2-mediated methylation of Lys 370, providing regulatory cross-talk between post-translational modifications. In addition, we show that the inhibitory effect of Lys 372 methylation on Lys 370 methylation is caused, in part, by blocking the interaction between p53 and Smyd2. Thus, similar to histones, p53 is subject to both activating and repressing lysine methylation. Our results also predict that Smyd2 may function as a putative oncogene by methylating p53 and repressing its tumour suppressive function.

Animals↗
PubMed2006

Clustered DNA motifs mark X chromosomes for repression by a dosage compensation complex.

Gene expression in metazoans is regulated not only at the level of individual genes but also in a coordinated manner across large chromosomal domains (for example centromeres, telomeres and imprinted gene clusters) and along entire chromosomes (for example X-chromosome dosage compensation). The primary DNA sequence usually specifies the regulation of individual genes, but the nature of cis-acting information that controls genes over large regions has been elusive: higher-order DNA structure, specific histone modifications, subnuclear compartmentalization and primary DNA sequence are possibilities. One paradigm of chromosome-wide gene regulation is Caenorhabditis elegans dosage compensation in which a large dosage compensation complex (DCC) is targeted to both X chromosomes of hermaphrodites to repress transcript levels by half. This essential process equalizes X-linked gene expression between the sexes (XO males and XX hermaphrodites). Here we report the discovery and dissection of cis-acting sites that mark nematode X chromosomes as targets for gene repression by the DCC. These rex (recruitment element on X) sites are widely dispersed along X and reside in promoters, exons and intergenic regions. rex sites share at least two distinct motifs that act in combination to recruit the DCC. Mutating these motifs severely reduces or abolishes DCC binding in vivo, demonstrating the importance of primary DNA sequence in chromosome-wide regulation. Unexpectedly, the motifs are not enriched on X, but altering motif numbers within rex sites demonstrates that motif co-occurrence in unusually high densities is essential for optimal DCC recruitment. Thus, X-specific repression is established through sequences not specific to X. The distribution of common motifs provides the foundation for repression along an entire chromosome.

Animals↗
PubMed2004

Imprinting along the Kcnq1 domain on mouse chromosome 7 involves repressive histone methylation and recruitment of Polycomb group complexes.

Imprinted genes are clustered in domains, and their allelic repression is mediated by imprinting control regions. These imprinting control regions are marked by DNA methylation, which is essential to maintain imprinting in the embryo. To explore how imprinting is regulated in placenta, we studied the Kcnq1 domain on mouse distal chromosome 7. This large domain is controlled by an intronic imprinting control region and comprises multiple genes that are imprinted in placenta, without the involvement of promoter DNA methylation. We found that the paternal repression along the domain involves acquisition of trimethylation at Lys27 and dimethylation at Lys9 of histone H3. Eed-Ezh2 Polycomb complexes are recruited to the paternal chromosome and potentially regulate its repressive histone methylation. Studies on embryonic stem cells and early embryos support our proposal that chromatin repression is established early in development and is maintained in the placenta. In the embryo, however, imprinting is stably maintained only at genes that have promoter DNA methylation. These data underscore the importance of histone methylation in placental imprinting and identify mechanistic similarities with X-chromosome inactivation in extraembryonic tissues, suggesting that the two epigenetic mechanisms are evolutionarily linked.

Animals↗
PubMed2006

Reconstitution of chromatin transcription with purified components reveals a chromatin-specific repressive activity of p300.

Here we describe an in vitro chromatin transcription system in which chromatin assembly and transcription are carried out with purified and defined factors. With basal (also known as general) transcription factors and sequence-specific DNA-binding activators, we observed chromatin-specific, activation domain-dependent transcription. We then examined the biochemical function of purified p300 in the absence of the endogenous factor and other related activities and found, unexpectedly, that p300 has a chromatin-specific, transcriptional repression activity that can be relieved by the addition of acetyl-CoA. This p300-mediated repression is reversible, requires the p300 bromodomain but not the acetyltransferase region, and does not involve the formation of a stable, nuclease-resistant nucleoprotein complex. Hence, the mechanism of transcriptional repression by p300 is distinct from that of histone H1, PARP-1 or Sir2. These findings reveal a novel chromatin-specific repressive function of p300.

Acetyl Coenzyme A↗
PubMed2004

The SINE-encoded mouse B2 RNA represses mRNA transcription in response to heat shock.

Cells respond to changes in environmental conditions via orchestrated modifications in gene expression. For example, in response to heat shock, cells execute a program of gene-specific transcriptional activation and repression. Although the activation of genes upon heat shock has been widely studied, the mechanism of mRNA transcriptional repression upon heat shock is unexplained. Here we show that during the heat shock response in mouse cells, a small noncoding RNA polymerase III transcript, B2 RNA, associates with RNA polymerase II and represses transcription of specific mRNA genes. These studies define a unique transcriptional regulatory mechanism involving an RNA regulator and reveal how mRNA transcription is repressed upon heat shock. Moreover, we identify a function for B2 RNA, which is transcribed from short interspersed elements that are abundant in the mouse genome and historically considered to be 'junk DNA.'

Animals↗
PubMed2005

CtBP represses p300-mediated transcriptional activation by direct association with its bromodomain.

Histone acetyltransferase coactivators bind to acetylated histones through their bromodomains and catalyze the acetylation of histone H3 and H4 tails for transcriptional activation. C-terminal binding protein (CtBP) serves as a transcriptional corepressor by recruiting histone deacetylases. However, the precise mechanism by which CtBP represses transcription has not been determined. In this study we found that CtBP1 directly associates with p300 by binding to the PXDLS motif in the bromodomain of p300. Moreover, CtBP1 blocks the accessibility of p300 to histones in an NADH-sensitive manner and thus represses p300-mediated histone acetylation and transcriptional activation. In addition, an NADH-nonresponsive, monomeric mutant, CtBP1 (G183V), was found to strongly repress p300-mediated transcriptional activation. Thus, the dissociation of NADH from CtBP1 leads to the repression of p300-driven general transcriptional activity by CtBP1. These results suggest a novel mechanism whereby CtBP1 serves as an energy-sensing repressor of histone acetyltransferase(s) and thus affects general transcription.

Acetyltransferases↗
PubMed2005

Positive and negative regulation of EAAT2 by NF-kappaB: a role for N-myc in TNFalpha-controlled repression.

The glutamate transporter gene, EAAT2/GLT-1, is induced by epidermal growth factor (EGF) and downregulated by tumor necrosis factor alpha (TNFalpha). While TNFalpha is generally recognized as a positive regulator of NF-kappaB-dependent gene expression, its ability to control transcriptional repression is not well characterized. Additionally, the regulation of NF-kappaB by EGF is poorly understood. Herein, we demonstrate that both TNFalpha-mediated repression and EGF-mediated activation of EAAT2 expression require NF-kappaB. We show that EGF activates NF-kappaB independently of signaling to IkappaB. Furthermore, TNFalpha can abrogate IKKbeta- and p65-mediated activation of EAAT2. Our results suggest that NF-kappaB can intrinsically activate EAAT2 and that TNFalpha mediates repression through a distinct pathway also requiring NF-kappaB. Consistently, we find that N-myc is recruited to the EAAT2 promoter with TNFalpha and that N-myc-binding sites are required for TNFalpha-mediated repression. Moreover, N-myc overexpression inhibits both basal and p65-induced activation of EAAT2. Our data highlight the remarkable specificity of NF-kappaB activity to regulate gene expression in response to diverse cellular signals and have implications for glutamate homeostasis and neurodegenerative disease.

Amino Acid Transport System X-AG↗
PubMed2005

FOG-1 recruits the NuRD repressor complex to mediate transcriptional repression by GATA-1.

Transcription factor GATA-1 and its cofactor FOG-1 coordinate erythroid cell maturation by activating erythroid-specific genes and repressing genes associated with the undifferentiated state. Here we show that FOG-1 binds to the NuRD corepressor complex in vitro and in vivo. The interaction is mediated by a small conserved domain at the extreme N-terminus of FOG-1 that is necessary and sufficient for NuRD binding. This domain defines a novel repression module found in diverse transcriptional repressors. NuRD is present at GATA-1/FOG-1-repressed genes in erythroid cells in vivo. Point mutations near the N-terminus of FOG-1 that abrogate NuRD binding block gene repression by FOG-1. Finally, the ability of GATA-1 to repress transcription was impaired in erythroid cells expressing mutant forms of FOG-1 that are defective for NuRD binding. Together, these studies show that FOG-1 and likely other FOG-like proteins are corepressors that link GATA factors to histone deacetylation and nucleosome remodeling.

Amino Acid Motifs↗
PubMed2005

Repression of Runx2 function by TGF-beta through recruitment of class II histone deacetylases by Smad3.

Transforming growth factor-beta (TGF-beta) inhibits osteoblast differentiation through inhibition of the function of Runx2 (Cbfa1) by Smad3. The mechanism through which TGF-beta/Smad3 inhibits Runx2 function has not been characterized. We show that TGF-beta induces histone deacetylation, primarily of histone H4, at the osteocalcin promoter, which is repressed by TGF-beta, and that histone deacetylation is required for repression of Runx2 by TGF-beta. This repression occurs through the action of the class IIa histone deacetylases (HDAC)4 and 5, which are recruited through interaction with Smad3 to the Smad3/Runx2 complex at the Runx2-binding DNA sequence. Accordingly, HDAC4 or 5 is required for efficient TGF-beta-mediated inhibition of Runx2 function and is involved in osteoblast differentiation. Our results indicate that class IIa HDACs act as corepressors for TGF-beta/Smad3-mediated transcriptional repression of Runx2 function in differentiating osteoblasts and are cell-intrinsic regulators of osteoblast differentiation.

Animals↗
PubMed2006

Regulation of MBD1-mediated transcriptional repression by SUMO and PIAS proteins.

In mammalian cells, DNA methylation is associated with heritable and stable gene repression, mediated in part by methyl-CpG-binding domain (MBD) proteins that recruit corepressors to modify chromatin. MBD1 protein, a member of the MBD family, forms a complex with SETDB1 histone methylase to silence transcription at target promoters by methylation of lysine 9 of histone H3. How MBD1-mediated transcriptional repression is regulated is currently unknown. Here we show that MBD1 is a target for sumoylation by PIAS1 (Protein Inhibitors of Activated STAT 1) and PIAS3 E3 SUMO (small ubiquitin-like modifier)-ligases, at two conserved lysine residues within the C-terminus of MBD1. Although sumoylated MBD1 binds to methylated DNA, it does not incorporate into a complex with SETDB1 and does not efficiently repress transcription of a target gene, p53BP2, in HeLa cells. Our data suggest that transcriptional silencing by MBD1 is regulated by a PIAS-mediated conjugation of SUMO1, which antagonizes the formation of a repressive complex with SETDB1.

Amino Acid Sequence↗
PubMed2006

Repression of ADH1 and ADH3 during zinc deficiency by Zap1-induced intergenic RNA transcripts.

The transcriptional activator Zap1 induces target gene expression in response to zinc deficiency. We demonstrate that during zinc starvation, Zap1 is required for the repression of ADH1 expression. ADH1 encodes the major zinc-dependent alcohol dehydrogenase that is utilized during fermentation. During zinc starvation, Zap1 binds upstream of the activator Rap1 and induces an intergenic RNA transcript, ZRR1. ZRR1 expression leads to the transient displacement of Rap1 from the ADH1 promoter resulting in ADH1 repression. Using a microarray-based approach, we screened for additional genes repressed by Zap1 intergenic transcripts. We found that ADH3, the major mitochondrial alcohol dehydrogenase, is regulated in a manner similar to ADH1. Thus, during zinc deficiency, Zap1 mediates the repression of two of the most abundant zinc-requiring enzymes.

Alcohol Dehydrogenase↗
PubMed2004

The Snf1 kinase controls glucose repression in yeast by modulating interactions between the Mig1 repressor and the Cyc8-Tup1 co-repressor.

Among lower eukaryotes, glucose repression is a conserved, widely spread mechanism regulating carbon catabolism. The yeast Snf1 kinase, the Mig1 DNA-binding repressor and the Mig1-interacting co-repressor complex Cyc8(Ssn6)-Tup1 are central components of this pathway. Previous experiments suggested that cytoplasmic translocation of Mig1, upon its phosphorylation by Snf1 in the nucleus, is the key regulatory step for releasing glucose repression. In this report we re-evaluate this model. We establish the coordinated repressive action of Mig1 and Cyc8-Tup1 on GAL1 transcription, but we find that Cyc8-Tup1 is not tethered by Mig1 to the promoter DNA. We demonstrate that both negative regulators occupy GAL1 continuously under either repression or activation conditions, although the majority of the Mig1 is redistributed to the cytoplasm upon activation. We show that Snf1-dependent phosphorylation of Mig1 abolishes interaction with Cyc8-Tup1, and we propose that regulation of this interaction, not the Mig1 cytoplasmic localization, is the molecular switch that controls transcriptional repression/de-repression.

DNA-Binding Proteins↗
PubMed2005

Epigenetic changes in the repression and induction of asparagine synthetase in human leukemic cell lines.

In common with certain other lymphoid neoplasms, cells of the human lymphocytic leukemia lines 1873 and 1929 are asparagine (ASN) auxotrophs. Asparagine synthetase (ASY), which is a housekeeping gene, is repressed and the promoting region of the gene is highly methylated. We now demonstrate in these cells multiple levels in control of the expression of this gene, in a system of cocultivation with macrophages and other cell types. In this system, mediated by cell-to-cell contact, ASY becomes expressed by the leukemic cells and they become prototrophic. Demethylation of ASY occurs; it follows expression and is permanent over multiple cell generations, but the cells return to auxotrophy with rapid repression of ASY on removal from cell contact. With ASY expression, the associated histone H3 at lysine position 9 (H3K9) becomes acetylated and H3K4, methylated. In contrast to other systems, H3K9 methylation does not characterize the repressed state. The changes leading from repression to induction of ASY and demethylation parallel the physiological changes specific to functional maturation of normal lymphoid precursors. The lability of expression of ASY has potential significance in determining the sensitivity of leukemic cells to L-asparaginase.

Acetylation↗
PubMed1997

Mapping of adenovirus 5 E1A domains responsible for suppression of neu-mediated transformation via transcriptional repression of neu.

Overexpression of neu (also known as c-erbB-2 or HER-2) commonly occurs in human cancer and is also known to enchance tumor metastasis and chemoresistance. Our earlier reports showed that the adenovirus 5 E1A can suppresss the neu-mediated transformation by repression of neu. Thus, E1A has the potential to be used as a therapeutic agent against the neu-overexpressing human cancers. However, a serious concern to this approach is that E1A is also capable of immortalizing primary culture cells and can co-operate with ras or E1B oncogenes to transform them. The E1A CR2 domain (amino acid residues 120 to 140) necessary for binding to RB is believed to be required for this oncogenic function. Here, we report that deletion of CR2 region did not affect E1A's capability to repress neu. Interestingly, deletion of the amino acid residues 4 to 25 or 40 to 80 completely disrupted E1A-mediated neu repression. By deleting the amino acid residues from 81 to 185, we have successfully generated a mini-E1A mutant that was sufficient to inhibit neu promoter activity and suppress neu-mediated transformation. The mini-E1A mutant does not contain the CR2 domain that is crucial for RB binding and immortalization, and hence, may serve as a more selective tumor suppressor, and a safer therapeutic agent. It may also be a useful tool to further investigate the molecular mechanism(s) of neu overexpression and E1A-mediated transcriptional repression in cancer cells.

3T3 Cells↗
PubMed1997

Inhibition of the DNA-binding and transcriptional repression activity of the Wilms' tumor gene product, WT1, by cAMP-dependent protein kinase-mediated phosphorylation of Ser-365 and Ser-393 in the zinc finger domain.

The Wilms' tumor suppressor gene, WT1, encodes a transcription factor in the zinc finger family, which binds to GC-rich sequences and functions as a transcriptional activator or repressor. The WT1 protein plays a crucial role in urogenital development in mammals and its function is thought to be conserved during vertebrate evolution. Although accumulating evidence suggests that WT1 regulates a subset of genes including growth factor and growth factor receptor genes, little is known about regulators or signal cascades that could modulate the function of WT1. In this study, we show that the WT1 protein expressed exogenously in fibroblasts was phosphorylated in vivo, and that treatment with forskolin, which activates the cAMP-dependent protein kinase (PKA) in vivo, induced phosphorylation of additional sites in WT1. We identified the forskolin-induced phosphorylation sites as Ser-365 and Ser-393, which lie in the zinc finger domain in zinc fingers 2 and 3, respectively. PKA phosphorylated WT1 at Ser-365 and Ser-393 in vitro, as well as at additional sites, and this phosphorylation abolished the DNA-binding activity of WT1 in vitro. Using WT1 mutants in which Ser-365 and Ser-393 were mutated to Ala individually and in combination, we showed that phosphorylation of these sites was critical for inhibition of DNA binding in vivo. Thus, coexpression of the PKA catalytic subunit with wild type WT1 reduced the level of WT1 DNA-binding activity detected in nuclear extracts, and decreased transcriptional repression activity in vivo. In contrast to wild type WT1, all of the phosphorylation site mutants retained significant DNA-binding activity and repression activity in the presence of PKA. Analysis of the mutants showed that phosphorylation of Ser-365 and Ser-395 had additive inhibitory effects on WT1 DNA-binding in vivo and that phosphorylation at both sites was required for neutralization of repression activity. Therefore, we conclude that PKA modulates the activity of WT1 in vivo through phosphorylation of Ser-365 and Ser-393, which inhibits DNA binding. This in turn results in a decrease in WT1 transcriptional repression. Our findings provide the first evidence that the function of WT1 can be modulated by its phosphorylation in vivo.

Animals↗
PubMed1999

PAX3 and PAX7 exhibit conserved cis-acting transcription repression domains and utilize a common gain of function mechanism in alveolar rhabdomyosarcoma.

The t(2;13) and t(1;13) translocations of alveolar rhabdomyosarcoma (ARMS) result in chimeric PAX3-FKHR or PAX7-FKHR transcription factors, respectively. In each chimera, a PAX DNA-binding domain is fused to the C-terminal FKHR transactivation domain. Previously we demonstrated that PAX3-FKHR is more potent than PAX3 because the FKHR transactivation domain is resistant to repression mediated by the PAX3 N-terminus. Here we test the hypothesis that the cis-acting repression domain is a conserved feature of PAX3 and PAX7 and that PAX7-FKHR gains function similarly. Using PAX-specific DNA-binding sites, we found that PAX7 was virtually inactive, while PAX7-FKHR exhibited activity 600-fold above background and was comparable to PAX3-FKHR. Deletion analysis showed that the transactivation domains of PAX7 and PAX7-FKHR are each more potent than either full-length protein, and resistance to cis-repression is responsible for the PAX7-FKHR gain of function. Further deletion mapping and domain swapping experiments with PAX3 and PAX7 showed that their transactivation domains exhibit subtle dose-dependent differences in potency, likely due to regions of structural divergence; while their repression domains are structurally and functionally conserved. Thus, the data support the hypothesis and demonstrate that PAX3 and PAX7 utilize a common gain of function mechanism in ARMS.

3T3 Cells↗
PubMed2000

HTLV-1 tax oncoprotein represses the p53-mediated trans-activation function through coactivator CBP sequestration.

The human T-cell leukemia virus type 1 (HTLV-1) Tax oncoprotein repressed the transcriptional activity of wild-type p53 through its N-terminal trans-activation domain. Although Tax did not directly bind to p53, this repression required the activation of CREB pathway by Tax. In contrast to a recent report by Pise-Masison et al. (1998a, b) we found that the phosphorylation of p53 on Ser 15 is not a major cause of the Tax-mediated inactivation of p53. However, Tax with a mutation in the coactivator CBP-binding site (K88A), which activates NF-kappaB but not the CREB pathway, could not repress the p53 trans-activation function. Moreover, Tax inhibited p53 binding to CBP in vitro and inhibited synergistic activation of transcription by CBP and p53. Thus, Tax is likely to compete with p53 in binding with CBP, thereby repressing its trans-activation function.

Binding, Competitive↗