Corepressor mSin3A Research Articles

Correction for Knight et al., "Epstein-Barr Virus Nuclear Antigen 3C Recruits Histone Deacetylase Activity and Associates with the Corepressors mSin3A and NCoR in Human B-Cell Lines".

Volume 77, no. 7, p. 4261–4272, 2003, https://jvi.asm.org/content/77/7/4261.abstract. Page 4267, Fig. 7: The Western blots show expression of two proteins of interest, prothymosin (ProTα) and EBNA3C (E3C). The bottom image represents Ponceau S staining (labeled “Protein”) that seeks to confirm that total protein loading was roughly equal in each lane. A mistake was made in the rightmost “Protein” image. Only eight lanes are displayed rather than the expected nine. Given the age of the article, we have been unable to locate the original images.

Cdk5 controls IL-2 gene expression via repression of the mSin3a-HDAC complex (IRM6P.719)

Abstract Cyclin-dependent kinase 5 (Cdk5) is a unique member of a family of serine/threonine cyclin-dependent protein kinases. We previously demonstrated disruption of Cdk5 gene expression in mice impairs T-cell function and ameliorates T-cell-mediated neuroinflammation. Here, we show Cdk5 modulates gene expression during T-cell activation by impairing the repression of gene transcription by histone deacetylase 1 (HDAC1). This effect is mediated through a direct phosphorylation of the mSin3a protein by Cdk5, which prevents the accumulation of the mSin3a protein and the formation of a functional mSin3a/HDAC1 complex during T cell activation. Disruption of Cdk5 activity in T-cells enhances HDAC activity and binding of the HDAC1/mSin3a complex to the IL-2 promoter, ultimately leading to suppression of IL-2 expression. These data point to essential roles for Cdk5 in regulating gene expression in T-cells and transcriptional regulation by the co-repressor mSin3a.

Immediate effects of a single exercise bout on protein O-GlcNAcylation and chromatin regulation of cardiac hypertrophy

Cardiac hypertrophy induced by pathological stimuli is regulated by a complex formed by the repressor element 1-silencing transcription factor (REST) and its corepressor mSin3A. We previously reported that hypertrophic signaling is blunted by O-linked attachment of β-N-acetylglucosamine (O-GlcNAc) to proteins. Regular exercise induces a physiological hypertrophic phenotype in the heart that is associated with decreased O-GlcNAc levels, but a link between O-GlcNAc, the REST complex, and initiation of exercise-induced cardiac hypertrophy is not known. Therefore, mice underwent a single 15- or 30-min bout of moderate- to high-intensity treadmill running, and hearts were harvested immediately and compared with sedentary controls. Cytosolic O-GlcNAc was lower (P < 0.05) following 15 min exercise with no differences in nuclear levels (P > 0.05). There were no differences in cytosolic or nuclear O-GlcNAc levels in hearts after 30 min exercise (P > 0.05). Cellular compartment levels of O-GlcNAc transferase (OGT, the enzyme that removes the O-GlcNAc moiety from proteins), REST, mSin3A, and histone deacetylases (HDACs) 1, 2, 3, 4, and 5 were not changed with exercise. Immunoprecipitation revealed O-GlcNAcylation of OGT and HDACs 1, 2, 4, and 5 that was not changed with acute exercise; however, exercised hearts did exhibit lower interactions between OGT and REST (P < 0.05) but not between OGT and mSin3A. These data suggest that hypertrophic signaling in the heart may be initiated by as little as 15 min of exercise via intracellular changes in protein O-GlcNAcylation distribution and reduced interactions between OGT and the REST chromatin repressor.

The Rett Syndrome Protein MeCP2 Regulates Synaptic Scaling

Synaptic scaling is a form of homeostatic synaptic plasticity characterized by cell-wide changes in synaptic strength in response to changes in overall levels of neuronal activity. Here we report that bicuculline-induced increase in neuronal activity leads to a decrease in mEPSC amplitude and a decrease in expression of the AMPA receptor subunit GluR2 in rat hippocampal cultures. Bicuculline treatment also leads to an increase in the levels of the transcriptional repressor MeCP2, which binds to the GluR2 promoter along with the corepressors HDAC1 and mSin3A. Downregulation of MeCP2 by shRNA expression or genetic deletion blocks the bicuculline-induced decrease in GluR2 expression and mEPSC amplitude. These observations indicate that MeCP2 mediates activity-dependent synaptic scaling, and suggest that the pathophysiology of Rett syndrome, which is caused by mutations in MeCP2, may involve defects in activity-dependent regulation of synaptic currents.

Epigenetics and nutrition – nature versus nurture

It is becoming increasingly apparent that the in utero environment in which a fetus grows and develops may have long-term effects on subsequent health and survival. The abnormal intrauterine milieu of intrauterine growth retardation (IUGR) permanently alters gene expression and function of pancreatic beta-cells, leading to the development of diabetes in adulthood. Beta-cell function is markedly impaired in IUGR offspring in very early life. This is accompanied by a reduction in beta-cell proliferation and neogenesis. Expression of the pancreatic and duodenal homeobox 1 (Pdx1) transcription factor is permanently reduced in IUGR and epigenetic modifications are responsible for this decrease. Pdx1 encodes a homeobox transcription factor critically important for beta-cell function and development. Expression of Pdx1 is permanently reduced in IUGR beta-cells and epigenetic modifications are responsible for this. The fetal IUGR state is characterized by loss of upstream stimulatory factor 1 (USF1) binding at the proximal promoter of Pdx1, with deacetylation of histones H3 and H4 due to recruitment of histone deacetylase 1 (HDAC1) and the co-repressor mSin3A. After birth, histone 3 lysine 4 (H3K4) is demethylated and histone 3 lysine 9 (H3K9) is methylated. During the neonatal period, the reduction in Pdx1 expression and these epigenetic changes can be reversed by HDAC inhibition. Finally, once diabetes occurs, DNA methylation of the cytosine-phosphate-guanine (CpG) island in the proximal promoter ensues, resulting in permanent silencing of the Pdx1 locus.

P53 directly suppresses BNIP3 expression to protect against hypoxia-induced cell death

Hypoxia stabilizes the tumour suppressor p53, allowing it to function primarily as a transrepressor; however, the function of p53 during hypoxia remains unclear. In this study, we showed that p53 suppressed BNIP3 expression by directly binding to the p53-response element motif and recruiting corepressor mSin3a to the BNIP3 promoter. The DNA-binding site of p53 must remain intact for the protein to suppress the BNIP3 promoter. In addition, taking advantage of zebrafish as an in vivo model, we confirmed that zebrafish nip3a, a homologous gene of mammalian BNIP3, was indeed induced by hypoxia and p53 mutation/knockdown enhanced nip3a expression under hypoxia resulted in cell death enhancement in p53 mutant embryos. Furthermore, p53 protected against hypoxia-induced cell death mediated by p53 suppression of BNIP3 as illustrated by p53 knockdown/loss assays in both human cell lines and zebrafish model, which is in contrast to the traditional pro-apoptotic role of p53. Our results suggest a novel function of p53 in hypoxia-induced cell death, leading to the development of new treatments for ischaemic heart disease and cerebral stroke.

Cigarette Smoke Exposure Alters mSin3a and Mi-2α/β Expression; implications in the control of pro-inflammatory gene transcription and glucocorticoid function

BackgroundThe key co-repressor complex components HDAC-2, Mi-2α/β and mSin3a are all critical to the regulation of gene transcription. HDAC-2 function is impaired by oxidative stress in a PI3Kδ dependant manner which may be involved in the chronic glucocorticoid insensitive inflammation in the lungs of COPD patients. However, the impact of cigarette smoke exposure on the expression of mSin3a and Mi2α/β and their role in glucocorticoid responsiveness is unknown.MethodsWild type, PI3Kγ knock-out (PI3Kγ-/-) and PI3K kinase dead knock-in (PI3KδD910/A910) transgenic mice were exposed to cigarette smoke for 3 days and the expression levels of the co-repressor complex components HDAC-2, mSin3a, Mi-2α and Mi-2β and HDAC-2 activity in the lungs were assessed.ResultsCigarette smoke exposure impaired glucocorticoid function and reduced HDAC-2 activity which was protected in the PI3KδD910/A910 mice. Both mSin3a and Mi-2α protein expression was reduced in smoke-exposed mice. Budesonide alone protected mSin3a protein expression with no additional effect seen with abrogation of PI3Kγ/δ activity, however Mi-2α, but not Mi-2β, expression was protected in both PI3KδD910/A910 and PI3Kγ-/- budesonide-treated smoke-exposed mice. The restoration of glucocorticoid function coincided with the protection of both HDAC activity and mSin3a and Mi-2α protein expression.ConclusionsCigarette smoke exposure induced glucocorticoid insensitivity and alters co-repressor activity and expression which is prevented by blockade of PI3K signaling with glucocorticoid treatment. Inhibition of PI3Kδ signalling in combination with glucocorticoid treatment may therefore provide a therapeutic strategy for restoring oxidant-induced glucocortiocid unresponsiveness.

Abstract 4359: Folylpolyglutamate synthetase expression is transcriptionally regulated by chromatin remodeling and recruitment of a multiprotein corepressor complex associated to non-random fusions in ALL

Abstract Non-random translocations in acute lymphoblastic leukemia (ALL) are known to alter gene transcription and molecular signaling pathways. Understanding the effects of these “altered pathways” on drug metabolism will provide the biological rationale to design combination strategies for ALL using proven antileukemic drugs, such as methotrexate (MTX), and novel targeted agents. The TEL-AML1 gene fusion is the most common translocation in childhood ALL (25%) and the E2A-PBX1 occurs in ∼5% of B-precursor (Bp) ALL. Both T-ALL and B-p ALL expressing TEL-AML1 and E2A-PBX1 fusions were shown to accumulate lower level of long-chain MTX-PGs when compared to other Bp- ALL phenotypes. Folylpolyglutamate synthetase (FPGS) expression, responsible for the synthesis of MTX-PG, is both lineage-specific and proliferation dependent in ALL cells. Using real-time qRT-PCR, we determined that primary cells and cell lines expressing TEL-AML1 and E2A-PBX1 exhibit significantly decreased FPGS mRNA expression. Using an FPGS-luciferase reporter gene assay, we determined that both TEL-AML1 and E2A-PBX1 fusions lead to down regulation of the FPGS promoter activity. Co-immunoprecipitation and ChIP assays demonstrated that TEL-AML1 decreased FPGS transcription by recruiting co-repressors (mSin3A, Rb) and HDAC1 to the native chromatin structure of the FPGS promoter region. These data suggest that TEL-AML1 associates with mSin3A and HDAC1 to repress FPGS transcription, leading to lower intracellular MTX-PGs accumulation. We also investigated the cell cycle dependence of FPGS expression and determined whether TEL-AML1 and E2A-PBX1 influence its expression. When CCRF-CEM (T-ALL) and NALM6 (Bp-ALL) cells were synchronized to G1 and S-phase, we found that their relative level of FPGS mRNA expression was respectively 1.4 and 2.9-fold higher in G1-phase than in S-phase, indicating FPGS mRNA expression is upregulated in G1-phase prior to progress into S-phase. Using ChIP assays, we demonstrated that NFYB, Sp1, E2F and Rb proteins interact with the native chromatin structure of the FPGS promoter region, suggesting they may associate with non-random fusions to regulate FPGS expression during cell cycle progression. Consistent with this observation, REH cells expressing TEL-AML1, exhibited a greater percentage of cells arrested in G0/G1 compared to NALM6 control cells (51% vs. 40%). These data suggest that TEL-AML1, and possibly other fusions, alter FPGS mRNA levels by determining the cell cycle dependence of FPGS gene expression. Our data demonstrate for the first time the molecular mechanism leading to lower MTX-PGs accumulation mediated by lower FPGS expression in TEL-AML1 lymphoblasts, and suggest that recruitment of this multiprotein corepressor complex may also regulate FPGS expression during cell cycle progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4359.

Metallothionein-1 Isoforms and Vimentin Are Direct PU.1 Downstream Target Genes in Leukemia Cells

PU.1 is a key transcription factor for hematopoiesis and plays important roles in various hematological malignancies. To clarify the molecular function of PU.1, we initially tried to identify bona fide target genes regulated by PU.1. Dual microarrays were employed for this study to compare PU.1-knockdown K562 cells (K562PU.1KD) stably expressing PU.1 short inhibitory RNAs versus control cells and PU.1-overexpressing K562 cells (K562PU.1OE) versus control cells. In these analyses, we found that several genes, including metallothionein (MT)-1 isoforms (MT-1G and MT-1A) and vimentin (VIM), were markedly induced while Jun dimerization protein (JDP) 2 was suppressed in K562PU.1KD cells. Furthermore, the mRNA expressions of the MT-1 and VIM genes were inversely correlated and the mRNA expression of JDP2 was positively correlated with PU.1 mRNA expression in 43 primary acute myeloid leukemia specimens (MT-1G: R = -0.50, p < 0.001; MT-1A: R = -0.58, p < 0.0005; VIM: R = -0.39, p < 0.01; and JDP2: R = 0.30, p < 0.05). Next, we analyzed the regulation of the MT-1 and VIM genes. We observed increased associations of acetylated histones H3 and H4 with the promoters of these genes in K562PU.1KD cells. Sequence analyses of the regions approximately 1 kb upstream from the transcription start sites of these genes revealed numerous CpG sites, which are potential targets for DNA methylation. Chromatin immunoprecipitation assays revealed that methyl CpG-binding protein 2 (MeCP2) and PU.1 bound to the CpG-rich regions in the MT-1 and VIM promoters. Bisulfite sequencing analyses of the PU.1-bound regions of these promoters revealed that the proportions of methylated CpG sites were tightly related to the PU.1 expression levels.

Kaposi's Sarcoma-Associated Herpesvirus RTA Promotes Degradation of the Hey1 Repressor Protein through the Ubiquitin Proteasome Pathway

The Kaposi's sarcoma-associated herpesvirus (KSHV) replication and transcription activator (RTA) protein regulates the latent-lytic switch by transactivating a variety of KSHV lytic and cellular promoters. RTA is a novel E3 ubiquitin ligase that targets a number of transcriptional repressor proteins for degradation by the ubiquitin proteasome pathway. Herein, we show that RTA interacts with the cellular transcriptional repressor protein Hey1. We demonstrate that Hey1 is a target for RTA-mediated ubiquitination and is subsequently degraded by the proteasome. Moreover, a Cys-plus-His-rich region within RTA is important for RTA-mediated degradation of Hey1. We confirm that Hey1 represses the RTA promoter and, furthermore, show that Hey1 binds to the RTA promoter. An interaction was observed between Hey1 and the corepressor mSin3A, and this interaction was abolished in the presence of RTA. Additionally, mSin3A associated with the RTA promoter in nonreactivated, but not reactivated, BCBL1 cells. Small interfering RNA knockdown of Hey1 in HEK 293T cells latently infected with the recombinant virus rKSHV.219 led to increased levels of RTA expression upon reactivation but was insufficient to induce complete lytic reactivation. These results suggest that other additional transcriptional repressors are also important in maintenance of KSHV latency. Taken together, our results suggest that Hey1 has a contributory role in the maintenance of KSHV latency and that disruption of the Hey1 repressosome by RTA-targeted degradation may be one step in the mechanism to regulate lytic reactivation.

Decreasing Intracellular Superoxide Corrects Defective Ischemia-induced New Vessel Formation in Diabetic Mice

Tissue ischemia promotes vasculogenesis through chemokine-induced recruitment of bone marrow-derived endothelial progenitor cells (EPCs). Diabetes significantly impairs this process. Because hyperglycemia increases reactive oxygen species in a number of cell types, and because many of the defects responsible for impaired vasculogenesis involve HIF1-regulated genes, we hypothesized that HIF1 function is impaired in diabetes because of reactive oxygen species-induced modification of HIF1alpha by the glyoxalase 1 (GLO1) substrate methylglyoxal. Decreasing superoxide in diabetic mice by either transgenic expression of manganese superoxide dismutase or by administration of an superoxide dismutase mimetic corrected post-ischemic defects in neovascularization, oxygen delivery, and chemokine expression, and normalized tissue survival. In hypoxic fibroblasts cultured in high glucose, overexpression of GLO1 prevented reduced expression of both the EPC mobilizing chemokine stromal cell-derived factor-1 (SDF-1) and of vascular epidermal growth factor, which modulates growth and differentiation of recruited EPCs. In hypoxic EPCs cultured in high glucose, overexpression of GLO1 prevented reduced expression of both the SDF-1 receptor CXCR4, and endothelial nitric-oxide synthase, an enzyme essential for EPC mobilization. HIF1alpha modification by methylglyoxal reduced heterodimer formation and HIF1alpha binding to all relevant promoters. These results provide a basis for the rational design of new therapeutics to normalize impaired ischemia-induced vasculogenesis in patients with diabetes.

Chromatin-Bound p53 Anchors Activated Smads and the mSin3A Corepressor To Confer Transforming Growth Factor β-Mediated Transcription Repression

In hepatic cells, Smad and SnoN proteins converge with p53 to repress transcription of AFP, an oncodevelopmental tumor marker aberrantly reactivated in hepatoma cells. Using p53- and SnoN-depleted hepatoma cell clones, we define a mechanism for repression mediated by this novel transcriptional partnership. We find that p53 anchors activated Smads and the corepressor mSin3A to the AFP distal promoter. Sequential chromatin immunoprecipitation analyses and molecular modeling indicate that p53 and Smad proteins simultaneously occupy overlapping p53 and Smad regulatory elements to establish repression of AFP transcription. In addition to its well-known function in antagonizing transforming growth factor beta (TGF-beta) responses, we find that SnoN actively participates in AFP repression by positively regulating mSin3A protein levels. We propose that activation of TGF-beta signaling restores a dynamic interplay between p53 and TGF-beta effectors that cooperate to effectively target mSin3A to tumor marker AFP and reestablish transcription repression.

Ebp1-mediated inhibition of cell growth requires serine 363 phosphorylation

Ebp1 is an ErbB3 binding phosphoprotein with pleiotropic effects. Overexpression of Ebp1 represses transcription of E2F1 responsive cell cycle regulated genes and inhibits cell growth. However, the effect of phosphorylation on Ebp1-mediated transcriptional repression and cell growth inhibition is currently unknown. In this study, we show that serine 363 (S363) of Ebp1 is phosphorylated in vivo. Although total Ebp1 is located in the nucleus, organelles and the cytoplasm, Ebp1 phosphorylated at S363 (Ebp1 pS363) is localized exclusively to the nucleus. Mutation of S363 to alanine did not change the subcellular localization of Ebp1. However, the S363A mutation significantly decreased the ability of Ebp1 to repress transcription and abrogated its ability to inhibit cell growth. We have previously shown that Ebp1 can bind the E2F1 promoter in vitro and in vivo as part of a protein complex and that Ebp1-transcriptional repression is mediated via its interaction with the co-repressors HDAC2 and mSin3a present in this complex. Although Ebp1 S363A interacted with an E2F1 promoter element, it did not bind HDAC2 and mSin3a. These results indicate the importance of S363 phosphorylation in the function of Ebp1.

Regulation of C/EBPδ‐dependent transactivation by histone deacetylases in intestinal epithelial cells

The C/EBPdelta transcription factor is involved in the positive regulation of the intestinal epithelial cell acute phase response. C/EBPdelta regulation by histone deacetylases (HDACs) during the course of inflammation remains to be determined. Our aim was to examine the effect of HDACs on C/EBPdelta-dependent regulation of haptoglobin, an acute phase protein induced in intestinal epithelial cells in response to pro-inflammatory cytokines. HDAC1, HDAC3, and HDAC4 were expressed in intestinal epithelial cells, as determined by Western blot. GST pull-down assays showed specific HDAC1 interactions with the transcriptional activation and the b-ZIP C/EBPdelta domains, while the co-repressor mSin3A interacts with the C-terminal domain. Immunoprecipitation assays confirmed the interaction between HDAC1 and the N-terminal C/EBPdelta amino acid 36-164 domain. HDAC1 overexpression decreased C/EBPdelta transcriptional activity of the haptoglobin promoter, as assessed by transient transfection and luciferase assays. Chromatin immunoprecipitation analysis showed a displacement of HDAC1 from the haptoglobin promoter in response to inflammatory stimuli and an increased acetylation of histone H3 and H4. HDAC1 silencing by shRNA expression increased both basal and IL-1beta-induced haptoglobin mRNA levels in epithelial intestinal cells. Our results suggest that interactions between C/EBPs and HDAC1 negatively regulate C/EBPdelta-dependent haptoglobin expression in intestinal epithelial cells.

High Glucose Increases Angiopoietin-2 Transcription in Microvascular Endothelial Cells through Methylglyoxal Modification of mSin3A

Methylglyoxal is a highly reactive dicarbonyl degradation product formed from triose phosphates during glycolysis. Methylglyoxal forms stable adducts primarily with arginine residues of intracellular proteins. The biologic role of this covalent modification in regulating cell function is not known. Here we report that in mouse kidney endothelial cells, high glucose causes increased methylglyoxal modification of the corepressor mSin3A. Methylglyoxal modification of mSin3A results in increased recruitment of O-GlcNAc-transferase, with consequent increased modification of Sp3 by O-linked N-acetylglucosamine. This modification of Sp3 causes decreased binding to a glucose-responsive GC-box in the angiopoietin-2 (Ang-2) promoter, resulting in increased Ang-2 expression. Increased Ang-2 expression induced by high glucose increased expression of intracellular adhesion molecule 1 and vascular cell adhesion molecule 1 in cells and in kidneys from diabetic mice and sensitized microvascular endothelial cells to the proinflammatory effects of tumor necrosis factor alpha. This novel mechanism for regulating gene expression may play a role in the pathobiology of diabetic vascular disease.

Glucose Mediates the Translocation of NeuroD1 by O-Linked Glycosylation

O-Linked GlcNAc modification of nuclear and cytosolic proteins has been shown to regulate the function of many cellular proteins. Increased O-linked glycosylation, observed under chronic hyperglycemia conditions, has been implicated in the pathogenesis of diabetes. However, the exact role of O-GlcNAc modification in regulating glucose homeostasis remains to be established. We report here that the subcellular localization of the pancreatic beta cell-specific transcription factor NeuroD1 is regulated by O-linked glycosylation in the mouse insulinoma cell line MIN6. Under low glucose conditions, NeuroD1 is mainly in the cytosol. However, treatment of MIN6 cells with high glucose results in O-linked GlcNAc modification of NeuroD1 and its subsequent translocation into the nucleus. Consistent with these data, treatment of MIN6 cells with O-(2-acetamido-2-deoxy-d-glucopyranosylidene)-amino N-phenylcarbamate, an inhibitor of O-GlcNAcase, causes Neuro-D1 localization to the nucleus and induction of insulin gene expression even on low glucose. Furthermore, we demonstrate that NeuroD1 interacts with the O-GlcNAc transferase, OGT only at high concentrations of glucose and depletion of OGT by using small interfering RNA oligos interferes with the nuclear localization of NeuroD1 on high glucose. On low glucose NeuroD1 interacts with the O-GlcNAcase and becomes deglycosylated, which is likely to be important for export of Neuro-D1 into cytosol in the presence of low glucose. In summary, the presented data suggest that glucose regulates the subcellular localization of NeuroD1 in pancreatic beta cells via O-linked GlcNAc modification of NeuroD1 by OGT.

Suppression of Viral Gene Expression in Bovine Leukemia Virus-Associated B-Cell Malignancy: Interplay of Epigenetic Modifications Leading to Chromatin with a Repressive Histone Code

Ovine leukemia/lymphoma resulting from bovine leukemia virus infection of sheep offers a large animal model for studying mechanisms underlying leukemogenesis. Silencing of viral information including Tax, the major contributor to the oncogenic potential of the virus, is critical if not mandatory for tumor progression. In this study, we have identified epigenetic mechanisms that govern the complete suppression of viral expression, using a lymphoma-derived B-cell clone carrying a silent provirus. Silencing was not relieved by injection of the malignant B cells into sheep. However, exogenous expression of Tax or treatment with either the DNA methyltransferase inhibitor 5'azacytidine or the histone deacetylase (HDAC) inhibitor trichostatin A rescued viral expression, as demonstrated by in vivo infectivity trials. Comparing silent and reactivated provirus, we found mechanistic connections between chromatin conformation and tumor-associated transcriptional repression. Silencing is associated with DNA methylation and decreased accessibility of promoter sequences. HDAC1 and the transcriptional corepressor mSin3A are associated with the inactive but not the reactivated promoter. Silencing correlates with a repressed chromatin structure marked by histone H3 and H4 hypoacetylation, a loss of methylation at H3 lysine 4, and an increase of H3 lysine 9 methylation. These observations point to the critical role of epigenetic mechanisms in tumor-specific virus/oncogene silencing, a potential strategy to evade immune response and favor the propagation of the transformed cell.

Repression of p53-mediated Transcription by Adenovirus E1B 55-kDa Does Not Require Corepressor mSin3A and Histone Deacetylases

The Ad E1B 55-kDa protein (E1B) is a potent transcriptional repressor. In vitro biochemical studies revealed that direct p53-E1B interaction is essential for E1B to block p53-activated transcription and a corepressor may be involved. To understand how E1B represses p53-mediated transcription in vivo, we expressed E1B in several tumor cell lines that express wild type p53. Here we show that E1B strongly suppresses the expression of p53 target genes such as p21 and Puma-alpha in normal growth conditions or after cells were treated with p53-activating chemotherapeutic agents, suggesting that E1B-mediated gene repression is dominant and cannot be reversed via p53 activation. Interestingly, we found that E1B binds to corepressor mSin3A. Mutagenesis analysis indicated that the sequence motif "LHLLA" near the NH(2) terminus of E1B is responsible for mSin3A binding, and this motif is conserved among E1B proteins from different Ad serotypes. The conserved paired amphipathic helix domain 1 of mSin3A is critical for mSin3A-E1B interaction. Surprisingly, E1B mutants that cannot bind to mSin3A can still repress p53 target genes, indicating that it is not the corepressor required for E1B-mediated gene repression. In support of this notion, repression of p53 target genes by E1B is insensitive to HDAC inhibitor trichostatin A. We further show that both the NH(2)- and COOH-terminal domains of E1B are required for the repression function. Therefore, E1B employs a unique repression mechanism to block p53-mediated transcription.

Wnt-mediated Down-regulation of Sp1 Target Genes by a Transcriptional Repressor Sp5

Wnt/beta-catenin signaling regulates many processes during vertebrate development. To study transcriptional targets of canonical Wnt signaling, we used the conditional Cre/loxP system in mouse to ectopically activate beta-catenin during central nervous system development. We show that the activation of Wnt/beta-catenin signaling in the embryonic mouse telencephalon results in the up-regulation of Sp5 gene, which encodes a member of the Sp1 transcription factor family. A proximal promoter of Sp5 gene is highly evolutionarily conserved and contains five TCF/LEF binding sites that mediate direct regulation of Sp5 expression by canonical Wnt signaling. We provide evidence that Sp5 works as a transcriptional repressor and has three independent repressor domains, called R1, R2, and R3, respectively. Furthermore, we show that the repression activity of R1 domain is mediated through direct interaction with a transcriptional corepressor mSin3a. Finally, our data strongly suggest that Sp5 has the same DNA binding specificity as Sp1 and represses Sp1 target genes such as p21. We conclude that Sp5 transcription factor mediates the downstream responses to Wnt/beta-catenin signaling by directly repressing Sp1 target genes.

Recruitment of the SWI/SNF protein Brg1 by a multiprotein complex effects transcriptional repression in murine erythroid progenitors

SWI/SNF complexes are involved in both activation and repression of transcription. While one of two homologous ATPases, Brg1 [Brm (Brahma)-related gene 1] or Brm, is required for their chromatin remodelling function, less is known about how these complexes are recruited to DNA. We recently established that a DNA-binding complex containing TAL1/SCL, E47, GATA-1, LMO2 and Ldb1 stimulates P4.2 (protein 4.2) transcription in erythroid progenitors via two E box-GATA elements in the gene's proximal promoter. We show here that the SWI/SNF protein Brg1 is also associated with this complex and that both the E box and GATA DNA-binding sites in these elements are required for Brg1 recruitment. Further, Brg1 occupancy of the P4.2 promoter decreased with terminal erythroid differentiation in association with increased P4.2 transcription, while enforced expression of Brg1 in murine erythroleukaemia cells reduced P4.2 gene expression. Overexpression of Brg1 was associated with increased occupancy of the P4.2 promoter by the nuclear co-repressor mSin3A and HDAC2 (histone deacetylase 2) and with reduced histone H3 and H4 acetylation. Finally, a specific HDAC inhibitor attenuated Brg1-directed repression of P4.2 promoter activity in transfected cells. These results provide insight into the mechanism by which SWI/SNF proteins are recruited to promoters and suggest that transcription of P4.2, and most likely other genes, is actively repressed until the terminal differentiation of erythroid progenitors.