Several recent reports have identified TET1 as the main enzyme modulating DNA methylation and gene transcription via hydroxylation of 5-methylcytosine. However, little is known about the protein network that controls TET1 activity. By using a new proximity ligation in situ assay, we identified MeCP2, HDAC1/6/7, EZH2, mSin3A, PCNA, and LSD1 as TET1-interacting proteins. We also discerned that TET1/PCNA acts as a demethylator of the cyclical methylation/demethylation process, the perturbation of which promotes the aberrant methylation hallmarks frequently observed in cancer cells.

Epigenetic mechanisms play important roles in brain development, orchestrating proliferation, differentiation, and morphogenesis. Lysine-Specific Demethylase 1 (LSD1 also known as KDM1A and AOF2) is a histone modifier involved in transcriptional repression, forming a stable core complex with the corepressors corepressor of REST (CoREST) and histone deacetylases (HDAC1/2). Importantly, in the mammalian CNS, neuronal LSD1-8a, an alternative splicing isoform of LSD1 including the mini-exon E8a, sets alongside LSD1 and is capable of enhancing neurite growth and morphogenesis. Here, we describe that the morphogenic properties of neuronal LSD1-8a require switching off repressive activity and this negative modulation is mediated in vivo by phosphorylation of the Thr369b residue coded by exon E8a. Three-dimensional crystal structure analysis using a phospho-mimetic mutant (Thr369bAsp), indicate that phosphorylation affects the residues surrounding the exon E8a-coded amino acids, causing a local conformational change. We suggest that phosphorylation, without affecting demethylase activity, causes in neurons CoREST and HDAC1/2 corepressors detachment from LSD1-8a and impairs neuronal LSD1-8a repressive activity. In neurons, Thr369b phosphorylation is required for morphogenic activity, converting neuronal LSD1-8a in a dominant-negative isoform, challenging LSD1-mediated transcriptional repression on target genes.

The Drosophila fruitless (fru) gene encodes a set of putative transcription factors that promote male sexual behavior by controlling the development of sexually dimorphic neuronal circuitry. However, the mechanism whereby fru establishes the sexual fate of neurons remains enigmatic. Here, we show that Fru forms a complex with the transcriptional cofactor Bonus (Bon), which, in turn, recruits either of two chromatin regulators, Histone deacetylase 1 (HDAC1), which masculinizes individual sexually dimorphic neurons, or Heterochromatin protein 1a (HP1a), which demasculinizes them. Manipulations of HDAC1 or HP1a expression change the proportion of male-typical neurons and female-typical neurons rather than producing neurons with intersexual characteristics, indicating that on a single neuron level, this sexual switch operates in an all-or-none manner.

Two of the most common signalling pathways in breast cancer are the ER (oestrogen receptor) ligand activation pathway and the E-cadherin snai1 slug EMT (epithelial-mesenchymal transition) pathway. Although these pathways have been thought to interact indirectly, the present study is the first to observe direct interactions between these pathways that involves the regulation of slug expression. Specifically we report that ligand-activated ERalpha suppressed slug expression directly by repression of transcription and that knockdown of ERalpha with RNA interference increased slug expression. More specifically, slug expression was down-regulated in ERalpha-negative MDA-MB-468 cells transfected with ERalpha after treatment with E2 (17beta-oestradiol). The down-regulation of slug in the ERalpha-positive MCF-7 cell line was mediated by direct repression of slug transcription by the formation of a co-repressor complex involving ligand-activated ERalpha protein, HDAC1 (histone deacetylase 1) and N-CoR (nuclear receptor co-repressor). This finding was confirmed by sequential ChIP (chromatin immunoprecipitation) studies. In the MCF-7 cell line, slug expression normally was low. In addition, knockdown of ERalpha with RNA interference in this cell line increased slug expression. This effect could be partially reversed by treatment of the cells with E2. The efficacy of the effect of ERalpha on slug repression was dependent on the overall level of ERalpha. These observations confirmed that slug was an E2-responsive gene.

Epigenetic modifications play an important role during carcinogenesis. The main goal of this study was to examine expression levels of two critical enzymes, DNA methyltransferase-1 (DNMT1) and histone deacetylase-1 (HDAC1), by immunohistochemistry (IHC) in human pancreatic cancer and precancerous lesions: 20 foci containing normal ductal epithelial cells without an inflammatory back-ground (DE), 30 containing ductal epithelial cells with an inflammatory background (DEI), 48 of pancreatic intraepithelial neoplasia-1A (PanIN-1A), 103 of PanIN-1B, 99 of PanIN-2, 30 of PanIN-3, 18 of intraductal papillary mucinous neoplasm A (IPMA), 10 of IPMB, 20 of IPMC, and 54 of pancreatic ductal adenocarcinoma (PDAC). The expression levels of both DNMT1 and HDAC1 increased from normal to precancerous lesions to pancreatic cancer, in a malignancy-dependent manner. Correlations between expression levels and clinicopathological features of the 54 PDAC patients were also analyzed. The expression of DNMT1 significantly correlated with nerve infiltration, degree of tumor differentiation and TNM staging (p<0.05), while that of HDAC1 correlated with proliferative activity, degree of tumor differentiation and TNM staging (p<0.05). Patients with higher expression of DNMT1 and/or HDAC1 had an overall lower survival than those with lower expression (p<0.05). Higher expression of DNMT1 and HDAC1 correlated with advanced stages of the disease and reflect the malignancy of pancreatic carcinoma. They may become new prognostic markers and potential therapeutic targets for pancreatic cancer.

Recent studies on enzymes and reader proteins for histone crotonylation support a function of histone crotonylation in transcription. However, the enzyme(s) responsible for histone decrotonylation (HDCR) remains poorly defined. Moreover, it remains to be determined if histone crotonylation is physiologically significant and functionally distinct from or redundant to histone acetylation. Here we present evidence that class I histone deacetylases (HDACs) rather than sirtuin family deacetylases (SIRTs) are the major histone decrotonylases, and that histone crotonylation is as dynamic as histone acetylation in mammalian cells. Notably, we have generated novel HDAC1 and HDAC3 mutants with impaired HDAC but intact HDCR activity. Using these mutants we demonstrate that selective HDCR in mammalian cells correlates with a broad transcriptional repression and diminished promoter association of crotonylation but not acetylation reader proteins. Furthermore, we show that histone crotonylation is enriched in and required for self-renewal of mouse embryonic stem cells.

Histone deacetylase inhibitors (HDACIs) interfere with the epigenetic process of histone acetylation and are known to have analgesic properties in models of chronic inflammatory pain. The aim of this study was to determine whether these compounds could also affect neuropathic pain. Different class I HDACIs were delivered intrathecally into rat spinal cord in models of traumatic nerve injury and antiretroviral drug-induced peripheral neuropathy (stavudine, d4T). Mechanical and thermal hypersensitivity was attenuated by 40% to 50% as a result of HDACI treatment, but only if started before any insult. The drugs globally increased histone acetylation in the spinal cord, but appeared to have no measurable effects in relevant dorsal root ganglia in this treatment paradigm, suggesting that any potential mechanism should be sought in the central nervous system. Microarray analysis of dorsal cord RNA revealed the signature of the specific compound used (MS-275) and suggested that its main effect was mediated through HDAC1. Taken together, these data support a role for histone acetylation in the emergence of neuropathic pain.

Cytokine-induced beta-cell apoptosis is important to the etiology of type-1 diabetes. Although previous reports have shown that general inhibitors of histone deacetylase (HDAC) activity, such as suberoylanilide hydroxamic acid and trichostatin A, can partially prevent beta-cell death, they do not fully restore beta-cell function. To understand HDAC isoform selectivity in beta cells, we measured the cellular effects of 11 structurally diverse HDAC inhibitors on cytokine-induced apoptosis in the rat INS-1E cell line. All 11 compounds restored ATP levels and reduced nitrite secretion. However, caspase-3 activity was reduced only by MS-275 and CI-994, both of which target HDAC1, 2, and 3. Importantly, both MS-275 and genetic knockdown of Hdac3 alone were sufficient to restore glucose-stimulated insulin secretion in the presence of cytokines. These results suggest that HDAC3-selective inhibitors may be effective in preventing cytokine-induced beta-cell apoptosis.

OBJECTIVE

Histone deacetylases (HDACs) are promising pharmacological targets in cancer and autoimmune diseases. All 11 classical HDACs (HDAC1-11) are found in the pancreatic beta cell, and HDAC inhibitors (HDACi) protect beta cells from inflammatory insults. We investigated which HDACs mediate inflammatory beta cell damage and how the islet content of these HDACs is regulated in recent-onset type 1 diabetes.

METHODS

The rat beta cell line INS-1 and dispersed primary islets from rats, either wild type or HDAC1-3 deficient, were exposed to cytokines and HDACi. Molecular mechanisms were investigated using real-time PCR, chromatin immunoprecipitation and ELISA assays. Pancreases from healthy children and children with type 1 diabetes were assessed using immunohistochemistry and immunofluorescence.

RESULTS

Screening of 19 compounds with different HDAC selectivity revealed that inhibitors of HDAC1, -2 and -3 rescued INS-1 cells from inflammatory damage. Small hairpin RNAs against HDAC1 and -3, but not HDAC2, reduced pro-inflammatory cytokine-induced beta cell apoptosis in INS-1 and primary rat islets. The protective properties of specific HDAC knock-down correlated with attenuated cytokine-induced iNos expression but not with altered expression of the pro-inflammatory mediators Il1α, Il1β, Tnfα or Cxcl2. HDAC3 knock-down reduced nuclear factor κB binding to the iNos promoter and HDAC1 knock-down restored insulin secretion. In pancreatic sections from children with type 1 diabetes of recent onset, HDAC1 was upregulated in beta cells whereas HDAC2 and -3 were downregulated in comparison with five paediatric controls.

CONCLUSIONS

These data demonstrate non-redundant functions of islet class I HDACs and suggest that targeting HDAC1 and HDAC3 would provide optimal protection of beta cell mass and function in clinical islet transplantation and recent-onset type 1 diabetic patients.

BACKGROUND

Hepatic insulin resistance is a major characteristic of type 2 diabetes mellitus. LncRNA MEG3 has been shown to correlate to hepatic glucose production; however, the underlying mechanism remains unclear. This study aims to investigate the role of MEG3 in hepatic insulin resistance.

METHODS

High-fat diet mice, ob/ob mice and mice primary hepatocytes were used in this study. Expression of MEG3, FoxO1, G6pc and Pepck were determined by real-time PCR. FoxO1, G6pc, Pepck, HDAC1 and HDAC3 protein levels were analyzed by western blotting. Hepatic gluconeogenesis, glycogen accumulation, triglyceride and glycogen contents were measured by corresponding assay or kit, and body weight was monitored after an overnight fast.

RESULTS

Gene expression of MEG3 was upregulated in high-fat diet and ob/ob mice and increased by palmitate, oleate or linoleate. MEG3 overexpression significantly increased FoxO1, G6pc, Pepck mRNA expressions and hepatic gluconeogenesis and suppressed insulin-stimulated glycogen synthesis in primary hepatocytes, whereas palmitate-induced increase of FoxO1, G6pc and Pepck protein expressions could be reversed by MEG3 interference. In addition, high fat enhanced expression of lncRNA MEG3 in hepatocytes through histone acetylation. Furthermore, MEG3 interference could reverse the up-regulation of triglyceride as well as impaired glucose tolerance and down-regulation of glucogen content in high-fat diet mice or ob/ob mice.

CONCLUSIONS

Upregulation of lncRNA MEG3 enhances hepatic insulin resistance via increasing foxO1expression, suggesting that MEG3 may be a potential target and therapeutic strategy for diabetes.

Histone deacetylases (HDACs) play a crucial role in tumorigenesis. Over-expression of HDACs has been reported in lung cancer. The mechanism of highly expressed HDAC1 in lung cancer has yet not been determined. In the present study, we showed that miR-449a/b regulates HDAC1 by directly binding with the 3' untranslated region of the HDAC1. The expression of miR-449a/b was down-regulated and the expression of HDAC1 was up-regulated in primary lung cancer. The down expression of miR-449a/b might be one mechanism for over-expression of HDAC1 in lung cancer. miR-449a/b inhibited cell growth and anchorage-independent growth. Furthermore, co-treatment with miR-449a and HDAC inhibitors had a significant growth reduction compared with HDAC inhibitor mono-treatment. These results suggest that miR-449a/b may have a tumor suppressor function and might be a potential therapeutic candidate in patients with primary lung cancer.

HIPK2 has been implicated in restraining tumor progression by more than one mechanism, involving both its catalytic and transcriptional co-repressor functions. Starting from the finding that HIPK2 knockdown by RNA-interference (HIPK2i) induced significant up-regulation of HIF-1alpha mRNA and of its target VEGF in tumor cells, we evaluated the role of HIPK2 in transcriptional regulation of HIF-1alpha. We found that HIPK2 overexpression downmodulated both HIF-1alpha reporter activity and mRNA levels and showed that HIPK2 was bound in vivo to the HIF-1alpha promoter likely in a multiprotein co-repressor complex with histone deacetylase 1 (HDAC1). Thus, the HIF-1alpha promoter was strongly acetylated following HIPK2 knockdown. The HIF-1alpha-dependent VEGF transcription was evaluated by co-transfection of a dominant negative (DN) construct of HIF-1alpha that inhibited VEGF reporter activity induced by HIPK2 knockdown. HIF-1alpha and VEGF up-regulation in HIPK2i cells correlated with increased vascularity of tumor xenografts in vivo and tube formation in HUVEC in vitro. These findings provide the first evidence of HIPK2-mediated transcriptional regulation of HIF-1alpha that might play a critical role in VEGF expression.

Histone acetylation plays an important role in chromatin remodeling and gene expression. The molecular mechanisms involved in cell-specific expression of endothelial nitric-oxide synthase (eNOS) are not fully understood. In this study we investigated whether histone deacetylation was involved in repression of eNOS expression in non-endothelial cells. Induction of eNOS expression by histone deacetylase (HDAC) inhibitors trichostatin A (TSA) and sodium butyrate was observed in all four different types of non-endothelial cells examined. Chromatin immunoprecipitation assays showed that the induction of eNOS expression by TSA was accompanied by a remarkable increase of acetylation of histone H3 associated with the eNOS 5'-flanking region in the non-endothelial cells. Moreover, DNA methylation-mediated repression of eNOS promoter activity was partially reversed by TSA treatment, and combined treatment of TSA and 5-aza-2'-deoxycytidine (AzadC) synergistically induced eNOS expression in non-endothelial cells. The proximal Sp1 site is critical for basal activity of eNOS promoter. The induction of eNOS by inhibition of HDACs in non-endothelial cells, however, appeared not mediated by the changes in Sp1 DNA binding activity. We further showed that Sp1 bound to the endogenous eNOS promoter and associated with HDAC1 in non-endothelial HeLa cells. Combined TSA and AzadC treatment increased Sp1 binding to the endogenous eNOS promoter but decreased the association between HDAC1 and Sp1 in HeLa cells. Our data suggest that HDAC1 plays a critical role in eNOS repression, and the proximal Sp1 site may serve a key target for HDCA1-mediated eNOS repression in non-endothelial cells.

The NF-kappaB component RelB is essential for dendritic cell (DC) differentiation and maturation. The vitamin D receptor (VDR) is a nuclear receptor that mediates inhibition of DC maturation and transcriptional repression of relB after engagement of its ligand, 1alpha,25-dihydroxyvitamin D(3), or related analogs (D(3) analogs). Ligand-dependent relB suppression was abolished by a histone deacetylase (HDAC) inhibitor. Constitutive association of VDR with the relB promoter was demonstrated in DCs by chromatin immunoprecipitation. Promoter binding by VDR was enhanced by ligand and reduced by LPS. Association of HDAC3 and HDAC1 with the relB VDR-binding site was observed, but only HDAC3 was reciprocally modulated by D(3) analog and LPS. Overexpression of HDAC3 caused relB promoter suppression, increased sensitivity to D(3) analog, and resistance to LPS. Depletion of HDAC3 attenuated relB suppression by D(3) analog. In vivo, D(3) analog resulted in reduced RelB in DCs from VDR WT mice but not VDR knockout mice. Other NF-lation of RelB and c-Rel in control animals. We conclude that vitamin D-regulated relB transcription in DCs is controlled by chromatin remodeling by means of recruitment of complexes including HDAC3.

Sphingosine kinase (there are two isoforms, SK1 and SK2) catalyses the formation of sphingosine 1-phosphate (S1P), a bioactive lipid that can be released from cells to activate a family of G protein-coupled receptors, termed S1P1-5. In addition, S1P can bind to intracellular target proteins, such as HDAC1/2, to induce cell responses. There is increasing evidence of a role for S1P receptors (e.g. S1P4) and SK1 in cancer, where high expression of these proteins in ER negative breast cancer patient tumours is linked with poor prognosis. Indeed, evidence will be presented here to demonstrate that S1P4 is functionally linked with SK1 and the oncogene HER2 (ErbB2) to regulate mitogen-activated protein kinase pathways and growth of breast cancer cells. Although much emphasis is placed on SK1 in terms of involvement in oncogenesis, evidence will also be presented for a role of SK2 in both T-cell and B-cell acute lymphoblastic leukemia. In patient T-ALL lymphoblasts and T-ALL cell lines, we have demonstrated that SK2 inhibitors promote T-ALL cell death via autophagy and induce suppression of c-myc and PI3K/AKT pathways. We will also present evidence demonstrating that certain SK inhibitors promote oxidative stress and protein turnover via proteasomal degradative pathways linked with induction of p53-and p21-induced growth arrest. In addition, the SK1 inhibitor, PF-543 exacerbates disease progression in an experimental autoimmune encephalomyelitis mouse model indicating that SK1 functions in an anti-inflammatory manner. Indeed, sphingosine, which accumulates upon inhibition of SK1 activity, and sphingosine-like compounds promote activation of the inflammasome, which is linked with multiple sclerosis, to stimulate formation of the pro-inflammatory mediator, IL-1β. Such compounds could be exploited to produce antagonists that diminish exaggerated inflammation in disease. The therapeutic potential of modifying the SK-S1P receptor pathway in cancer and inflammation will therefore, be reviewed.

The polycomb group (PcG) proteins are known to be involved in maintaining the silenced state of several developmentally regulated genes. Enhancer of zeste homolog 2 (Ezh2), a member of this large protein family, has also been shown to be deregulated in different tumor types and its role, both as a potential primary effector and as a mediator of tumorigenesis, has become a subject of increased interest. We observed that Ezh2 binds to pRb2/p130, a member of the retinoblastoma family; as such, we were led to consider the possible ability of Ezh2 to modulate cell cycle progression. Both Ezh2 and pRb2/p130 repress gene expression by recruiting histone deacetylase (HDAC1), which decreases DNA accessibility for activating transcription factors. Additionally, we observed that Ezh2 interacts with the C-terminal region of pRb2/p130, essential for interaction with HDAC1. We show that Ezh2 is able to reverse pRb2/p130-HDAC1-mediated repression of the cyclin A promoter. This indicates a functional role of this complex in regulating cyclin A expression, known to be crucial in mediating cell cycle advancement. We also detected a significant decrease in the retention of HDAC1 activity associated with pRb2/p130 when Ezh2 was overexpressed. Finally, electromobility shift assays (EMSA) demonstrated that overexpression of Ezh2 caused the abrogation of the pRb2/p130-HDAC1 complex on the cyclin A promoter. These data, taken together, suggest that Ezh2 competes with HDAC1 in binding to pRb2/p130, disrupting their occupancy on the cyclin A promoter. In this study, we propose a new mechanism for the functional inactivation of pRb2/p130 that ultimately contributes to cell cycle progression and malignant transformation.

Neonatal exposure to diethylstilbestrol (DES) causes permanent alterations in female reproductive tract gene expression, infertility, and uterine cancer in mice. To determine whether epigenetic mechanisms could explain these phenotypes, we first tested whether DES altered uterine expression of chromatin-modifying proteins. DES treatment significantly reduced expression of methylcytosine dioxygenase TET oncogene family, member 1 (TET1) on postnatal day 5; this decrease was correlated with a subtle decrease in DNA 5-hydroxymethylcytosine in adults. There were also significant reductions in histone methyltransferase enhancer of zeste homolog 2 (EZH2), histone lysine acetyltransferase 2A (KAT2A), and histone deacetylases HDAC1, HDAC2, and HDAC3. Uterine chromatin immunoprecipitation was used to analyze the locus-specific association of modified histones with 2 genes, lactoferrin (Ltf) and sine oculis homeobox 1 (Six1), which are permanently upregulated in adults after neonatal DES treatment. Three histone modifications associated with active transcription, histone H3 lysine 9 acetylation (H3K9ac), H3 lysine 4 trimethylation (H3K4me3), and H4 lysine 5 acetylation (H4K5ac) were enriched at specific Ltf promoter regions after DES treatment, but this enrichment was not maintained in adults. H3K9ac, H4K5ac, and H3K4me3 were enriched at Six1 exon 1 immediately after neonatal DES treatment. As adults, DES-treated mice had greater differences in H4K5ac and H3K4me3 occupancy at Six1 exon 1 and new differences in these histone marks at an upstream region. These findings indicate that neonatal DES exposure temporarily alters expression of multiple chromatin-modifying proteins and persistently alters epigenetic marks in the adult uterus at the Six1 locus, suggesting a mechanism for developmental exposures leading to altered reproductive function and increased cancer risk.

The intestinal epithelium possesses a remarkable self-renewal ability, which is mediated by actively proliferating Lgr5+ stem cells. Bone morphogenetic protein (BMP) signalling represents one major counterforce that limits the hyperproliferation of intestinal epithelium, but the exact mechanism remains elusive. Here we demonstrate that epithelial BMP signalling plays an indispensable role in restricting Lgr5+ stem cell expansion to maintain intestinal homeostasis and prevent premalignant hyperproliferation on damage. Mechanistically, BMP inhibits stemness of Lgr5+ stem cells through Smad-mediated transcriptional repression of a large number of stem cell signature genes, including Lgr5, and this effect is independent of Wnt/β-catenin signalling. Smad1/Smad4 recruits histone deacetylase HDAC1 to the promoters to repress transcription, and knockout of Smad4 abolishes the negative effects of BMP on stem cells. Our findings therefore demonstrate that epithelial BMP constrains the Lgr5+ stem cell self-renewal via Smad-mediated repression of stem cell signature genes to ensure proper homeostatic renewal of intestinal epithelium.

Earlier we identified a survival role for NF-kappaB in ventricular myocytes, however, the underlying mechanism was undefined. In this report we provide new mechanistic evidence that the hypoxia-inducible death factor BNIP3 is transcriptionally silenced by NF-kappaB through a mechanism that involves the cooperative actions of HDAC1. Activation of the NF-kappaB signaling pathway in ventricular myocytes suppressed basal and hypoxia-inducible BNIP3 gene activity. Basal Bnip3 gene expression was increased in cells derived from p65(-/-) deficient mice. The histone deacetylase (HDAC) inhibitor Trichostatin A (TSA 10 nM) suppressed the inhibitory actions of NF-kappaB on Bnip3 gene transcription. Basal and hypoxia- induced Bnip3 transcription was repressed by wild type but not a catalytically inactive mutant of HDAC1. Immunoprecipitation assays verified interaction of HDAC1 with wild type p65 NF-kappaB and mutations of p65 defective for transactivation in ventricular myocytes. Deletion analysis revealed canonical NF-kappaB elements within the Bnip3 promoter to be important for repression of Bnip3 gene expression by HDAC1. Further, the ability of HDAC1 to repress Bnip3 gene transcription was lost in cells derived from p65(-/-) deficient mice but was restored by repletion of p65 NF-kappaB into p65(-/-) cells. Mutations of p65 NF-kappaB defective for DNA binding but not for transactivation abrogated the inhibitory actions of HDAC1 on the Bnip3 gene transcription. Together, our findings provide new mechanistic insight into the cytoprotective actions conferred by NF-kappaB that extend to the active transcriptional repression of the death factor Bnip3 through a mechanism that is mutually dependent on HDAC-1.

PLZF, the promyelocytic leukaemia zinc-finger protein, is a transcriptional repressor essential to development. In some acute leukaemias, a chromosomal translocation fusing the PLZF gene to that encoding the retinoic acid receptor RARalpha gives rise to a fusion protein, PLZF-RARalpha, thought to be responsible for constitutive repression of differentiation-associated genes in these cells. Repression by both PLZF and PLZF-RARalpha is sensitive to the histone deacetylase inhibitor TSA, and PLZF was previously shown to interact physically with HDAC1, a class I histone deacetylase. We here asked whether class II histone deacetylases, known to be generally involved in differentiation processes, participate in the repression mediated by PLZF and PLZF-RARalpha, and found that PLZF interacts with HDAC4 in both GST-pull-down and co-immunoprecipitation assays. Furthermore, HDAC4 is indeed involved in PLZF and PLZF-RARalpha-mediated repression, since an enzymatically dead mutant of HDAC4 released the repression, as did an siRNA that blocks HDAC4 expression. Taken together, our data indicate that recruitment of HDAC4 is necessary for PLZF-mediated repression in both normal and leukaemic cells.

The multi-zinc finger proteins of the Sal family regulate organogenesis. Genetic evidence from Drosophila has shown that spalt (sal) can alter gene expression in a cell autonomous fashion, but Sal proteins have never been directly analyzed for their ability to activate or repress transcription. In this report, we show that a member of the Sal family, mouse Sall1, is a potent transcriptional repressor. When fused to a heterologous DNA-binding domain, Sall1 represses transcription of a luciferase reporter by over 100-fold. Expression of the N terminus alone is sufficient for dose-responsive repression that, as shown by deletion analysis, requires the extreme N-terminal amino acids of the protein. The N terminus of Sall1 can repress at both short and long range relative to the promoter, and treatment with the histone deacetylase (HDAC) inhibitor, trichostatin A, alleviates repression by 3-fold. The same regions of the protein that are required for repression physically interact with components of chromatin remodeling complexes, HDAC1, HDAC2, RbAp46/48, MTA-1, and MTA-2. Finally, we demonstrate that Sall1 is localized to discrete nuclear foci and this localization depends on the N-terminal repression domain. Together, these results suggest that the N terminus of mouse Sall1 can recruit HDAC complexes to mediate transcriptional repression.

Infected-cell protein 0 encoded by bovine herpesvirus 1 (BHV-1) (bICP0) is necessary for efficient productive infection, in large part, because it activates all 3 classes of BHV-1 genes (U. V. Wirth, C. Fraefel, B. Vogt, C. Vlcek, V. Paces, and M. Schwyzer, J. Virol. 66:2763-2772, 1992). Although bICP0 is believed to be a functional homologue of herpes simplex virus type 1-encoded ICP0, the only well-conserved domain between the proteins is a zinc ring finger located near the amino terminus of both proteins. Our previous studies demonstrated that bICP0 is toxic to transfected cells but does not appear to directly induce apoptosis (Inman, M., Y. Zhang, V. Geiser, and C. Jones, J. Gen. Virol. 82:483-492, 2001). C-terminal sequences in the last 320 amino acids of bICP0 mediate subcellular localization. Mutagenesis of the zinc ring finger within bICP0 revealed that this domain was important for transcriptional activation. In this study, we demonstrate that bICP0 interacts with histone deacetylase 1 (HDAC1), which results in activation of a simple promoter containing four consensus Myc-Max binding sites. The interaction between bICP0 and HDAC1 correlated with inhibition of Mad-dependent transcriptional repression. In resting CV-1 cells, bICP0 relieved HDAC1-mediated transcriptional repression. The zinc ring finger was required for relieving HDAC1-induced repression but not for interacting with HDAC1. In fetal bovine lung cells but not in a human epithelial cell line, bICP0 expression correlated with reduced steady-state levels of HDAC1 in crude cytoplasmic extracts. We hypothesize that the ability of bICP0 to overcome HDAC1-induced repression plays a role in promoting productive infection in highly differentiated cell types.

The MAPK pathway mediates TLR signaling during innate immune responses. We discovered previously that MKP-1 is acetylated, enhancing its interaction with its MAPK substrates and deactivating TLR signaling. As HDACs modulate inflammation by deacetylating histone and nonhistone proteins, we hypothesized that HDACs may regulate LPS-induced inflammation by deacetylating MKP-1. We found that mouse macrophages expressed a subset of HDAC isoforms (HDAC1, HDAC2, and HDAC3), which all interacted with MKP-1. Genetic silencing or pharmacologic inhibition of HDAC1, -2, and -3 increased MKP-1 acetylation in cells. Furthermore, knockdown or pharmacologic inhibition of HDAC1, -2, and -3 decreased LPS-induced phosphorylation of the MAPK member p38. Also, pharmacologic inhibition of HDAC did not decrease MAPK signaling in MKP-1 null cells. Finally, inhibition of HDAC1, -2, and -3 decreased LPS-induced expression of TNF-α, IL-1β, iNOS (NOS2), and nitrite synthesis. Taken together, our results show that HDAC1, -2, and -3 deacetylate MKP-1 and that this post-translational modification increases MAPK signaling and innate immune signaling. Thus, HDAC1, -2, and -3 isoforms are potential therapeutic targets in inflammatory diseases.

The effect of trichostatin A (TSA), histone deacetylase inhibitor, on cell growth and the mechanism of growth modulation was examined in 8 gastric and 3 oral carcinoma cell lines which included 9-cis-retinoic acid resistant (MKN-7 and Ho-1-N-1) and IFN-beta resistant cell lines (MKN-7, -28 and -45). TSA inhibited growth in all cell lines examined. Apoptotic cell death was confirmed by apoptotic ladder formation and induction of a cleaved form (85 kDa) of poly (ADP-ribose) polymerase (PARP) induction. TSA enhanced the protein expression of p21(WAF1), CREB-binding protein, cyclinE, cyclin A, Bak and Bax, while it reduced the expression of E2F-1, E2F-4, HDAC1, p53 and hyperphosphorylated form of Rb. Furthermore, TSA induced morphological changes, such as elongation of cytoplasm and cell-to-cell detachment, in gastric and oral carcinoma cell lines. These results suggest that TSA may inhibit cell growth and induce apoptosis of gastric and oral carcinoma cells through modulation of the expression of cell cycle regulators and apoptosis-regulating proteins.