Recent reports have indicated the participation of tight junction (TJ) proteins in the regulation of gene expression and cell proliferation. Here, we have studied the role of zona occludens (ZO)-2, a TJ peripheral protein, in the regulation of cyclin D1 transcription. We found that ZO-2 down-regulates cyclin D1 transcription in a dose-dependent manner. To understand how ZO-2 represses cyclin D1 promoter activity, we used deletion analyses and found that ZO-2 negatively regulates cyclin D1 transcription via an E box and that it diminishes cell proliferation. Because ZO-2 does not associate directly with DNA, electrophoretic mobility shift assay and chromatin immunoprecipitation (ChIP) assay were used to identify the transcription factors mediating the ZO-2-repressive effect. c-Myc was found to bind the E box present in the cyclin D1 promoter, and the overexpression of c-Myc augmented the inhibition generated by ZO-2 transfection. The presence of ZO-2 and c-Myc in the same complex was further demonstrated by immunoprecipitation. ChIP and reporter gene assays using histone deacetylases (HDACs) inhibitors demonstrated that HDACs are necessary for ZO-2 repression and that HDAC1 is recruited to the E box. We conclude that ZO-2 down-regulates cyclin D1 transcription by interacting with the c-Myc/E box element and by recruiting HDAC1.

Histone deacetylases (HDACs) play a pivotal role in gene expression through their involvement in chromatin remodeling. The abnormal targeting or retention of HDACs to DNA regulatory regions is observed in many cancers, and hence HDAC inhibitors are being tested as promising anti-tumor agents. The results of previous kinetic studies, characterizing trichostatin A (TSA), as well as butyrate, as HDAC noncompetitive inhibitors, conflict with crystallographic and homology modeling data suggesting that TSA should act as a competitive inhibitor. Our results demonstrate that each of the HDAC inhibitors TSA and butyrate inhibits HDAC activity in a competitive fashion. Co-immunoprecipitation studies show that the inhibition of HDAC1 and HDAC2 activity by TSA does not disturb the extensive level of their association in the human breast cancer cell line MCF-7. Moreover, the inhibition of HDAC activity by TSA does not interfere with the interaction of HDAC1 and HDAC2 with Sin3A, a core component of the Sin3 complex. Thus, repressor complexes such as Sin3, appear to be stable in the presence of TSA. The association of HDAC2 with transcription factor Sp1 is also not affected by TSA.

Inflammation is essential for host defense but can cause tissue damage and organ failure if unchecked. How the inflammation is resolved remains elusive. Here we report that the transcription factor Miz1 was required for terminating lipopolysaccharide (LPS)-induced inflammation. Genetic disruption of the Miz1 POZ domain, which is essential for the transactivation or repression activity of Miz1, resulted in hyperinflammation, lung injury and greater mortality in LPS-treated mice but a lower bacterial load and mortality in mice with Pseudomonas aeruginosa pneumonia. Loss of the Miz1 POZ domain prolonged the expression of proinflammatory cytokines. After stimulation, Miz1 was phosphorylated at Ser178, which was required for recruitment of the histone deacetylase HDAC1 to repress transcription of the gene encoding C/EBP-δ, an amplifier of inflammation. Our data provide a long-sought mechanism underlying the resolution of LPS-induced inflammation.

The Forkhead box O (FoxO) transcription factors are activated, and necessary for the muscle atrophy, in several pathophysiological conditions, including muscle disuse and cancer cachexia. However, the mechanisms that lead to FoxO activation are not well defined. Recent data from our laboratory and others indicate that the activity of FoxO is repressed under basal conditions via reversible lysine acetylation, which becomes compromised during catabolic conditions. Therefore, we aimed to determine how histone deacetylase (HDAC) proteins contribute to activation of FoxO and induction of the muscle atrophy program. Through the use of various pharmacological inhibitors to block HDAC activity, we demonstrate that class I HDACs are key regulators of FoxO and the muscle-atrophy program during both nutrient deprivation and skeletal muscle disuse. Furthermore, we demonstrate, through the use of wild-type and dominant-negative HDAC1 expression plasmids, that HDAC1 is sufficient to activate FoxO and induce muscle fiber atrophy in vivo and is necessary for the atrophy of muscle fibers that is associated with muscle disuse. The ability of HDAC1 to cause muscle atrophy required its deacetylase activity and was linked to the induction of several atrophy genes by HDAC1, including atrogin-1, which required deacetylation of FoxO3a. Moreover, pharmacological inhibition of class I HDACs during muscle disuse, using MS-275, significantly attenuated both disuse muscle fiber atrophy and contractile dysfunction. Together, these data solidify the importance of class I HDACs in the muscle atrophy program and indicate that class I HDAC inhibitors are feasible countermeasures to impede muscle atrophy and weakness.

Hepatocellular carcinoma (HCC) is one of the most deadly cancers. Aberrant oncogenic activation of the Wnt/β-catenin signaling pathway contributes to hepatocellular carcinogenesis. Various epigenetic modifications of the Wnt antagonist secreted frizzled-related protein (SFRP) family have been implicated in regulating Wnt signaling. Here, we report that Hepatitis C virus (HCV) core protein downregulates SFRP1 expression when it is expressed in Huh7 and HepG2 cells. SFRP1 expression can be effectively restored by using either a DNA methylation inhibitor alone or in combination with a histone deacetylase inhibitor. DNA methylation analysis of the SFRP1 promoter revealed that cytosine-phosphate-guanine (CpG) islands close to the transcriptional start site (TSS) in the SFRP1 promoter were hypermethylated in core-expressing Huh7 cells, suggesting that HCV core protein may downregulate SFRP1 expression by inducing hypermethylation of the SFRP1 promoter. Chromatin immunoprecipitation revealed that HCV core protein markedly increased the expression level and binding of DNA methyltransferase-1 (Dnmt1) and histone deacetylase-1 (HDAC1) to the TSS of the SFRP1 promoter region, resulting in repression of acetyl-histone H3-binding capacity to SFRP1 promoter and the eventual epigenetic silencing of SFRP1 expression. Furthermore, the core protein-promoted cell proliferation, migration and invasiveness were effectively abrogated either by Dnmt1 knockdown or restoration of SFRP1 expression in hepatoma cells. Dnmt1 knockdown or SFRP1 overexpression also inhibited HCV core-induced epithelial-mesenchymal transition (EMT) and significantly decreased the expression levels of activated β-catenin and Wnt/β-catenin target genes, c-Myc and cyclin D1. We further showed that knockdown of Dnmt1 and restoration of SFRP1 inhibited core-induced in vivo tumor growth and aggressiveness in a xenograft HCC model. Taken together, our results strongly suggest that the HCV core-induced epigenetic silencing of SFRP1 may lead to the activation of the Wnt signaling pathway and thus contribute to HCC aggressiveness through induction of EMT.

Acetylation of the tumor suppressor gene p53 at the carboxy-terminal lysine (Lys) residues enhances its transcriptional activity associated with cell cycle arrest and apoptosis. Histone deacetylases (HDACs), a family of evolutionarily conserved enzymes, counterbalance the acetylation of lysine residues on histone and non-histone proteins. In this study, we demonstrate that green tea polyphenols (GTPs) and their major constituent, (-) epigallocatechin-3-gallate (EGCG), activate p53 through acetylation at the Lys373 and Lys382 residues by inhibiting class I HDACs in LNCaP human prostate cancer cells. Treatment of cells with GTPs (2.5-10 µg/ml) and EGCG (5-20 µM) resulted in dose- and time-dependent inhibition of class I HDACs (HDAC1, 2, 3 and 8), albeit at varying levels. Discontinuation of treatment with GTP/EGCG resulted in the loss of p53 acetylation at both the sites in these cells. GTP/EGCG treatment also resulted in increased expression of p21/waf1 and Bax at the protein and message levels in these cells. The increased GTP/EGCG-mediated p53 acetylation enhanced its binding on the promoters of p21/waf1 and Bax, which was associated with increased accumulation of cells in the G0/G1 phase of the cell cycle and induction of apoptosis. Our findings indicate that GTP/EGCG causes acetylation of p53 by inhibiting class I HDACs, a function that is likely to be part of the mechanisms that control the physiological activity of p53.

Transcription requires the progression of RNA polymerase II (RNAP II) through a permissive chromatin structure. Recent studies of Saccharomyces cerevisiae have demonstrated that the yeast Sin3 protein contributes to the restoration of the repressed chromatin structure at actively transcribed loci. Yet, the mechanisms underlying the restoration of the repressive chromatin structure at transcribed loci and its significance in gene expression have not been investigated in mammals. We report here the identification of a mammalian complex containing the corepressor Sin3B, the histone deacetylase HDAC1, Mrg15, and the PHD finger-containing Pf1 and show that this complex plays important roles in regulation of transcription. We demonstrate that this complex localizes at discrete loci approximately 1 kb downstream of the transcription start site of transcribed genes, and this localization requires both Pf1's and Mrg15's interaction with chromatin. Inactivation of this mammalian complex promotes increased RNAP II progression within transcribed regions and subsequent increased transcription. Our results define a novel mammalian complex that contributes to the regulation of transcription and point to divergent uses of the Sin3 protein homologues throughout evolution in the modulation of transcription.

mi-er1 (previously called er1) was first isolated from Xenopus laevis embryonic cells as a novel fibroblast growth factor-regulated immediate-early gene. Xmi-er1 was shown to encode a nuclear protein with an N-terminal acidic transcription activation domain. The human orthologue of mi-er1 (hmi-er1) displays 91% similarity to the Xenopus sequence at the amino acid level and was shown to be upregulated in breast carcinoma cell lines and tumors. Alternative splicing at the 3' end of hmi-er1 produces two major isoforms, hMI-ER1alpha and hMI-ER1beta, which contain distinct C-terminal domains. In this study, we investigated the role of hMI-ER1alpha and hMI-ER1beta in the regulation of transcription. Using fusion proteins of hMI-ER1alpha or hMI-ER1beta tethered to the GAL4 DNA binding domain, we show that both isoforms, when recruited to the G5tkCAT minimal promoter, function to repress transcription. We demonstrate that this repressor activity is due to interaction and recruitment of a trichostatin A-sensitive histone deacetylase 1 (HDAC1). Furthermore, deletion analysis revealed that recruitment of HDAC1 to hMI-ER1alpha and hMI-ER1beta occurs through their common ELM2 domain. The ELM2 domain was first described in the Caenorhabditis elegans Egl-27 protein and is present in a number of SANT domain-containing transcription factors. This is the first report of a function for the ELM2 domain, highlighting its role in the regulation of transcription.

Trichostatin A (TSA), a specific inhibitor of histone deacetylases (HDACs), induces acetylation of various non-histone proteins such as p53 and alpha-tubulin. We purified several acetylated proteins by the affinity to an anti-acetylated lysine (AcLys) antibody from cells treated with TSA and identified them by mass spectrometry. Here we report on acetylation of CFIm25, a component of mammalian cleavage factor Im (CF Im), and poly(A) polymerase (PAP), a polyadenylating enzyme for the pre-mRNA 3'-end. The residues acetylated in these proteins were mapped onto the regions required for interaction with each other. Whereas CBP acetylated these proteins, <em>HDAC1</em>, HDAC3, <em>HDAC1</em>0, SIRT1, and SIRT2 were involved in in vivo deacetylation. Acetylation of the CFIm25 occurred depending on the cleavage factor complex formation. Importantly, the interaction between PAP and CF Im complex was decreased by acetylation. We also demonstrated that acetylation of PAP inhibited the nuclear localization of PAP by inhibiting the binding to the importin alpha/beta complex. These results suggest that CBP and HDACs regulate the 3'-end processing machinery and modulate the localization of PAP through the acetylation and deacetylation cycle.

Retrotransposons are highly prevalent in mammalian genomes due to their ability to amplify in pluripotent cells or developing germ cells. Host mechanisms that silence retrotransposons in germ cells and pluripotent cells are important for limiting the accumulation of the repetitive elements in the genome during evolution. However, although silencing of selected individual retrotransposons can be relatively well-studied, many mammalian retrotransposons are seldom analysed and their silencing in germ cells, pluripotent cells or somatic cells remains poorly understood. Here we show, and experimentally verify, that cryptic repetitive element probes present in Illumina and Affymetrix gene expression microarray platforms can accurately and sensitively monitor repetitive element expression data. This computational approach to genome-wide retrotransposon expression has allowed us to identify the histone deacetylase Hdac1 as a component of the retrotransposon silencing machinery in mouse embryonic stem cells, and to determine the retrotransposon targets of Hdac1 in these cells. We also identify retrotransposons that are targets of other retrotransposon silencing mechanisms such as DNA methylation, Eset-mediated histone modification, and Ring1B/Eed-containing polycomb repressive complexes in mouse embryonic stem cells. Furthermore, our computational analysis of retrotransposon silencing suggests that multiple silencing mechanisms are independently targeted to retrotransposons in embryonic stem cells, that different genomic copies of the same retrotransposon can be differentially sensitive to these silencing mechanisms, and helps define retrotransposon sequence elements that are targeted by silencing machineries. Thus repeat annotation of gene expression microarray data suggests that a complex interplay between silencing mechanisms represses retrotransposon loci in germ cells and embryonic stem cells.

The class II transactivator (CIITA) is a master transcriptional regulator of major histocompatibility complex class II (MHC-II) promoters. CIITA does not bind DNA, but it interacts with the transcription factors RFX5, NF-Y, and CREB and associated chromatin-modifying enzymes to form an enhanceosome. This report examines the effects of histone deacetylases 1 and 2 (HDAC1/HDAC2) on MHC-II gene induction by gamma interferon (IFN-gamma) and CIITA. The results show that an inhibitor of HDACs, trichostatin A, enhances IFN-gamma-induced MHC-II expression, while HDAC1/HDAC2 inhibits IFN-gamma- and CIITA-induced MHC-II gene expression. mSin3A, a corepressor of HDAC1/HDAC2, is important for this inhibition, while NcoR, a corepressor of HDAC3, is not. The effect of this inhibition is directed at CIITA, since HDAC1/HDAC2 reduces transactivation by a GAL4-CIITA fusion protein. CIITA binds to overexpressed and endogenous HDAC1, suggesting that HDAC and CIITA may affect each other by direct or indirect association. Inhibition of HDAC activity dramatically increases the association of NF-YB and RFX5 with CIITA, the assembly of CIITA, NF-YB, and RFX5 enhanceosome, and the extent of H3 acetylation at the MHC-II promoter. These results suggest a model where HDAC1/HDAC2 affect the function of CIITA through a disruption of MHC-II enhanceosome and relevant coactivator-transcription factor association and provide evidence that CIITA may act as a molecular switch to modulate MHC-II transcription by coordinating the functions of both histone acetylases and HDACs.

During development and erythropoiesis, globin gene expression is finely modulated through an important network of transcription factors and chromatin modifying activities. In this report we provide in vivo evidence that endogenous Ikaros is recruited to the human beta-globin locus and targets the histone deacetylase HDAC1 and the chromatin remodeling protein Mi-2 to the human gamma-gene promoters, thereby contributing to gamma-globin gene silencing at the time of the gamma- to beta-globin gene transcriptional switch. We show for the first time that Ikaros interacts with GATA-1 and enhances the binding of the latter to different regulatory regions across the locus. Consistent with these results, we show that the combinatorial effect of Ikaros and GATA-1 impairs close proximity between the locus control region and the human gamma-globin genes. Since the absence of Ikaros also affects GATA-1 recruitment to GATA-2 promoter, we propose that the combinatorial effect of Ikaros and GATA-1 is not restricted to globin gene regulation.

Neural crest-derived pigment cell development has been used extensively to study cell fate specification, migration, proliferation, survival and differentiation. Many of the genes and regulatory mechanisms required for pigment cell development are conserved across vertebrates. The zebrafish mutant colgate (col)/histone deacetylase1 (hdac1) has reduced numbers, delayed differentiation and decreased migration of neural crest-derived melanophores and their precursors. In hdac1(col) mutants normal numbers of premigratory neural crest cells are induced. Later, while there is only a slight reduction in the number of neural crest cells in hdac1(col) mutants, there is a severe reduction in the number of mitfa-positive melanoblasts suggesting that hdac1 is required for melanoblast specification. Concomitantly, there is a significant increase in and prolonged expression of foxd3 in neural crest cells in hdac1(col) mutants. We found that partially reducing Foxd3 expression in hdac1(col) mutants rescues mitfa expression and the melanophore defects in hdac1(col) mutants. Furthermore, we demonstrate the ability of Foxd3 to physically interact at the mitfa promoter. Because mitfa is required for melanoblast specification and development, our results suggest that hdac1 is normally required to suppress neural crest foxd3 expression thus de-repressing mitfa resulting in melanogenesis by a subset of neural crest-derived cells.

Melanoma cells typically express wild-type p53, yet they are notoriously resistant to DNA-damaging agents. Here, we show that sodium butyrate (NaB), a histone deacetylase (HDAC) inhibitor, induced apoptosis in human melanoma cells in a dose- and time-dependent manner. Apoptosis was associated with HDAC1-dependent induction of Bax and acetylation of p53. Down-regulation of HDAC1 by an antisense vector sensitized the cells to NaB-induced apoptosis, whereas its overexpression conferred resistance to this agent. Increased HDAC1 levels and activity impaired NaB-mediated activation of Bax promoter and Bax protein levels. Finally, using p53-null melanoma cell line and RNA interference in cells expressing wild-type p53 protein, we show that Bax induction and NaB-mediated apoptosis is p53 dependent.

Epithelial-mesenchymal transition (EMT) has been implicated in embryonic development, fibrosis, and tumor metastasis. Histone deacetylases (HDACs) also play important roles in the control of various physiological and pathological events. However, whether HDACs are involved in the control of EMT in liver cells remains unidentified. Three structurally unrelated HDAC inhibitors completely suppress transforming growth factor-beta1 (TGF-beta1)-induced EMT in AML-12 murine hepatocytes and primary mouse hepatocytes. Expression of a dominant-negative mutant of HDAC1 but not HDAC2 or downregulation of HDAC1 but not HDAC2 by RNAi suppressed TGF-beta1-induced EMT. In addition, both HDAC inhibitor TSA and HDAC1 RNAi blocked cell migration. Overexpression of HDAC1 in invasive hepatocellular carcinoma (HCC) samples was detected. Further study showed that the mRNA levels of ZO-1 and E-cadherin were downregulated during TGF-beta1-induced EMT, and HDAC1 can downregulate the promoter activities of ZO-1 and E-cadherin.

CONCLUSIONS

our results demonstrate that HDAC1 is required for TGF-beta1-induced EMT and cell migration in hepatocytes. Its high expression levels in majority of invasive HCC samples suggest that, by promoting EMT, HDAC1 can be related with the invasiveness of HCC. The data also suggest that the repression of transcription of ZO-1 and E-cadherin by HDAC1 may be involved in TGF-beta1-induced EMT.

Zinc-dependent histone deacetylases (HDACs) are a family of hydrolases first identified as components of transcriptional repressor complexes, where they act by deacetylating lysine residues at the N-terminal extensions of core histones, thereby affecting transcription. To get more insight into the biological functions of the individual HDAC family members, we have used RNA interference in combination with microarray analysis in Drosophila S2 cells. Silencing of Drosophila HDAC1 (DHDAC1), but not of the other DHDAC family members, leads to increased histone acetylation. Silencing of either DHDAC1 or DHDAC3 leads to cell growth inhibition and deregulated transcription of both common and distinct groups of genes. Silencing DHDAC2 leads to increased tubulin acetylation levels but was not associated with a deregulation of gene expression. No growth of phenotype and no significant deregulation of gene expression was observed upon silencing of DHDAC4 and DHDACX. Loss of DHDAC1 or exposure of S2 cells to the small molecule HDAC inhibitor trichostatin both lead to a G(2) arrest and were associated with significantly overlapping gene expression signatures in which genes involved in nucleobase and lipid metabolism, DNA replication, cell cycle regulation, and signal transduction were over-represented. A large number of these genes were shown to also be deregulated upon loss of the co-repressor SIN3 (Pile, L. A., Spellman, P. T., Katzenberger, R. J., and Wassarman, D. A. (2003) J. Biol. Chem. 278, 37840-37848). We conclude the following. 1) DHDAC1 and -3 have distinct functions in the control of gene expression. 2) Under the tested conditions, DHDAC2, -4, and X have no detectable transcriptional functions in S2 cells. 3) The anti-proliferative and transcriptional effects of trichostatin are largely recapitulated by the loss of DHDAC1. 4) The deacetylase activity of DHDAC1 significantly contributes to the repressor function of SIN3.

Human T-cell lymphotropic virus type 1 (HTLV-1) is associated with adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). T-cell transformation is mainly due to the actions of the viral phosphoprotein Tax. Tax interacts with multiple transcriptional factors, aiding the transcription of many cellular genes. Here, we report that the cyclin-dependent kinase inhibitor p21/waf1 is overexpressed in all HTLV-1-infected cell lines tested as well as in ATL and HAM/TSP patient samples. Tax was found to be able to transactivate the endogenous p21/waf1 promoter, as detected by RNase protection, as well as activate a series of wild-type and 5'-deletion constructs linked to a luciferase reporter cassette. Wild-type but not a mutant form of Tax (M47) transactivated the p21/waf1 promoter in a p53-independent manner and utilized a minimal promoter that contained E2A and TATA box sequences. The p21/waf1 protein was reproducibly observed to be complexed with cyclin A/cdk2 and not with any other known G(1), S, or G(2)/M cyclins. Functionally, the association of p21/cyclin A/cdk2 decreased histone H1 phosphorylation in vitro, as observed in immunoprecipitations followed by kinase assays, and affected other substrates, such as the C terminus of Rb protein involved in c-Abl and histone deacetylase-1 (HDAC1) regulation. Interestingly, upon the use of a stress signal, such as gamma-irradiation, we found that the p21/cyclin A/cdk2 complex was able to block all known phosphorylation sites on the Rb molecule. Finally, using elutriated cell cycle fractions and a stress signal, we observed that the HTLV-1-infected T cells containing wild-type Tax, which had been in early or mid-G(1) phase prior to gamma-irradiation, arrested in G(1) and did not undergo apoptosis. This may be an important mechanism for an oncogenic virus such as HTLV-1 to stop the host at the G(1)/S boundary and to repair the damaged DNA upon injury, prior to S-phase entry.

For many years it has been noted that there is a correlation between acetylation of histones and an increase in transcriptional activity. One prediction, based on this correlation, is that hypomorphic or null mutations in histone deacetylase genes should lead to increased levels of histone acetylation and result in increased levels of transcription. It was therefore surprising when it was reported, in both yeast and fruit flies, that mutations that reduced or eliminated a histone deacetylase resulted in transcriptional silencing of genes subject to telomeric and heterochromatic position effect variegation (PEV). Here we report the first mutational analysis of a histone deacetylase in a multicellular eukaryote by examining six new mutations in HDAC1 of Drosophila melanogaster. We observed a suite of phenotypes accompanying the mutations consistent with the notion that HDAC1 acts as a global transcriptional regulator. However, in contrast to recent findings, here we report that specific missense mutations in the structural gene of HDAC1 suppress the silencing of genes subject to PEV. We propose that the missense mutations reported here are acting as antimorphic mutations that "poison" the deacetylase complex and propose a model that accounts for the various phenotypes associated with lesions in the deacetylase locus.

Activation of T cells leads to the induction of many cytokine genes that are required for appropriate immune responses, including IL-2, a key cytokine for T cell proliferation and homeostasis. The activating transcription factors such as nuclear factor of activated T cells, nuclear factor kappaB/Rel and activated protein-1 family members that regulate inducible IL-2 gene expression have been well documented. However, negative regulation of the IL-2 gene is less studied. Here we examine the role of zinc finger E-box-binding protein (ZEB) 1, a homeodomain/Zn finger transcription factor, as a repressor of IL-2 gene transcription. We show here that ZEB1 is expressed in non-stimulated and stimulated T cells and using chromatin immunoprecipitation assays we show that ZEB1 binds to the IL-2 promoter. Over-expression of ZEB1 can repress IL-2 promoter activity, as well as endogenous IL-2 mRNA production in EL-4 T cells, and this repression is dependent on the ZEB-binding site at -100. ZEB1 cooperates with the co-repressor C-terminal-binding protein (CtBP) 2 and with histone deacetylase 1 to repress the IL-2 promoter and this cooperation depends on the ZEB-binding site in the promoter as well as the Pro-X-Asp-Leu-Ser protein-protein interaction domain in CtBP2. Thus, ZEB1 may function to recruit a repressor complex to the IL-2 promoter.

We have developed previously a class of synthetic hybrid histone deacetylase (HDAC) inhibitors, which were built from hydroxamic acid of trichostatin A and pyridyl ring of MS-275. In this study we evaluated the antitumor effects of these novel hybrid synthetic HDAC inhibitors, SK-7041 and SK-7068, on human cancer cells. Both SK-7041 and SK-7068 effectively inhibited cellular HDAC activity at nanomolar concentrations and induced the time-dependent hyperacetylation of histones H3 and H4. These HDAC inhibitors preferentially inhibited the enzymatic activities of HDAC1 and HDAC2, as compared with the other HDAC isotypes, indicating that class I HDAC is the major target of SK-7041 and SK-7068. We found that these compounds exhibited potent antiproliferative activity against various human cancer cells in vitro. Growth inhibition effect of SK-7041 and SK-7068 was related with the induction of aberrant mitosis and apoptosis in human gastric cancer cells. Both compounds induced the accumulation of cells at mitosis after 6 h of treatment, which was demonstrated by accumulation of tetraploid cells, lack of G(2) cyclin/cyclin-dependent kinase inactivation, and higher mitotic index. After 12 h of treatment, apoptotic cells were increased through mitochondrial and caspase-mediated pathway. Finally, in vivo experiment showed that SK-7041 or SK-7068 was found to reduce the growth of implanted human tumors in nude mice. Therefore, based on isotype specificity and antitumor activity, SK-7041 and SK-7068 HDAC inhibitors are expected to be promising anticancer therapeutic agents and need additional clinical development.

Signal Transducer and Activator of Transcription 3 (STAT3) is a transcription factor that plays a crucial role in interleukin-6 (IL-6) signaling, mediating the acute-phase induction of the human Angiotensinogen (hAGT) gene in hepatocytes. We showed earlier that IL-6 induces acetylation of the STAT3 NH(2)-terminus by the recruitment of the p300 coactivator. We had also observed a physical interaction of STAT3 and Histone Deacetylase1 (HDAC1) in an IL-6-dependent manner that leads to transcriptional repression. In this study, we sought to elucidate the mechanism by which HDAC1 controls STAT3 transcriptional activity. Here, we mapped the interacting domains of both STAT3 and HDAC1 and found that the COOH-terminal domain of HDAC1 is necessary for IL-6-induced STAT3 transcriptional repression, whereas the NH(2)-terminal acetylation domain of STAT3 is required for HDAC1 binding. Interestingly, over expression of HDAC1 in HepG2 cells leads to significantly reduced amounts of nuclear STAT3 after IL-6 induction, whereas silencing of HDAC1 resulted in accumulation of total and acetylated STAT3 in the nucleus. We have found that HDAC1 knockdown also interferes with the responsiveness of the STAT3-dependent MCP1 target gene expression to IL-6, as confirmed by real-time RT-PCR analysis. Together, our study reveals the novel functional consequences of IL-6-induced STAT3-HDAC1 interaction on nucleocytoplasmic distribution of STAT3.

Histone deacetylases (HDACs) are known to play a central role in the regulation of several cellular properties interlinked with the development and progression of cancer. Recently, HDAC1 has been reported to be overexpressed in hepatocellular carcinoma (HCC), but its biological roles in hepatocarcinogenesis remain to be elucidated. In this study, we demonstrated overexpression of HDAC1 in a subset of human HCCs and liver cancer cell lines. HDAC1 inactivation resulted in regression of tumor cell growth and activation of caspase-independent autophagic cell death, via LC3B-II activation pathway in Hep3B cells. In cell cycle regulation, HDAC1 inactivation selectively induced both p21(WAF1/Cip1) and p27(Kip1) expressions, and simultaneously suppressed the expression of cyclin D1 and CDK2. Consequently, HDAC1 inactivation led to the hypophosphorylation of pRb in G1/S transition, and thereby inactivated E2F/DP1 transcription activity. In addition, we demonstrated that HDAC1 suppresses p21(WAF1/Cip1) transcriptional activity through Sp1-binding sites in the p21(WAF1/Cip1) promoter. Furthermore, sustained suppression of HDAC1 attenuated in vitro colony formation and in vivo tumor growth in a mouse xenograft model. Taken together, we suggest the aberrant regulation of HDAC1 in HCC and its epigenetic regulation of gene transcription of autophagy and cell cycle components. Overexpression of HDAC1 may play a pivotal role through the systemic regulation of mitotic effectors in the development of HCC, providing a particularly relevant potential target in cancer therapy.

The mammalian circadian clock is built on a molecular feedback loop in which the Period (PER) proteins, acting in a large, poorly understood complex, repress Clock-Bmal1, the transcription factor driving their expression. We found that mouse PER complexes include the histone methyltransferase HP1γ-Suv39h. PER proteins recruited HP1γ-Suv39h to the Per1 and Per2 promoters, and HP1γ-Suv39h proved important for circadian di- and trimethylation of histone H3 Lys9 (H3K9) at the Per1 promoter, feedback repression and clock function. HP1γ-Suv39h was recruited to the Per1 and Per2 promoters ~4 h after recruitment of HDAC1, a PER-associated protein previously implicated in clock function and H3K9 deacetylation at the Per1 promoter. PER complexes containing HDAC1 or HP1γ-Suv39h appeared to be physically separable. Circadian clock negative feedback by the PER complex thus involves dynamic, ordered recruitment of repressive chromatin modifiers to DNA-bound Clock-Bmal1.

HDAC1 and -2 are highly conserved enzymes and often coexist in the same coregulator complexes. Understanding the regulation of histone deacetylase activities is extremely important because these enzymes play key roles in epigenetic regulation in normal and cancer cells. We previously showed that HDAC1 is required for glucocorticoid receptor-mediated transcription activation and that its activity is regulated through acetylation by p300 during the induction cycle. Here, we showed that HDAC2 is also required for glucocorticoid receptor-mediated gene activation. HDAC2, however, is regulated through a different mechanism from that of HDAC1. HDAC2 is not acetylated by p300, although 5 of 6 acetylated lysine residues in HDAC1 are also present in HDAC2. More importantly, the activity of HDAC2 is inhibited by acetylated HDAC1. Additionally, we showed that acetylated HDAC1 can trans-regulate HDAC2 through heterodimerization. Thus, this study uncovered fundamental differences between HDAC1 and HDAC2. It also unveiled a new mechanism of collaborative regulation by HDAC1/2 containing coregulator complexes.