BACKGROUND

Chronic obstructive pulmonary disease (COPD) is characterized by chronic airway inflammation that is greater in patients with advanced disease. We asked whether there is a link between the severity of disease and the reduction in histone deacetylase (HDAC) activity in the peripheral lung tissue of patients with COPD of varying severity. HDAC is a key molecule in the repression of production of proinflammatory cytokines in alveolar macrophages.

METHODS

HDAC activity and histone acetyltransferase (HAT) activity were determined in nuclear extracts of specimens of surgically resected lung tissue from nonsmokers without COPD, patients with COPD of varying severity, and patients with pneumonia or cystic fibrosis. Alveolar macrophages from nonsmokers, smokers, and patients with COPD and bronchial-biopsy specimens from nonsmokers, healthy smokers, patients with COPD, and those with mild asthma were also examined. Total RNA extracted from lung tissue and macrophages was used for quantitative reverse-transcriptase-polymerase-chain-reaction assay of HDAC1 through HDAC8 and interleukin-8. Expression of HDAC2 protein was quantified with the use of Western blotting. Histone-4 acetylation at the interleukin-8 promoter was evaluated with the use of a chromatin immunoprecipitation assay.

RESULTS

Specimens of lung tissue obtained from patients with increasing clinical stages of COPD had graded reductions in HDAC activity and increases in interleukin-8 messenger RNA (mRNA) and histone-4 acetylation at the interleukin-8 promoter. The mRNA expression of HDAC2, HDAC5, and HDAC8 and expression of the HDAC2 protein were also lower in patients with increasing severity of disease. HDAC activity was decreased in patients with COPD, as compared with normal subjects, in both the macrophages and biopsy specimens, with no changes in HAT activity, whereas HAT activity was increased in biopsy specimens obtained from patients with asthma. Neither HAT activity nor HDAC activity was changed in lung tissue from patients with cystic fibrosis or pneumonia.

CONCLUSIONS

Patients with COPD have a progressive reduction in total HDAC activity that reflects the severity of the disease.

Regulation of NF-kappaB transactivation function is controlled at several levels, including interactions with coactivator proteins. Here we show that the transactivation function of NF-kappaB is also regulated through interaction of the p65 (RelA) subunit with histone deacetylase (HDAC) corepressor proteins. Our results show that inhibition of HDAC activity with trichostatin A (TSA) results in an increase in both basal and induced expression of an integrated NF-kappaB-dependent reporter gene. Chromatin immunoprecipitation (ChIP) assays show that TSA treatment causes hyperacetylation of the wild-type integrated NF-kappaB-dependent reporter but not of a mutant version in which the NF-kappaB binding sites were mutated. Expression of HDAC1 and HDAC2 repressed tumor necrosis factor (TNF)-induced NF-kappaB-dependent gene expression. Consistent with this, we show that HDAC1 and HDAC2 target NF-kappaB through a direct association of HDAC1 with the Rel homology domain of p65. HDAC2 does not interact with NF-kappaB directly but can regulate NF-kappaB activity through its association with HDAC1. Finally, we show that inhibition of HDAC activity with TSA causes an increase in both basal and TNF-induced expression of the NF-kappaB-regulated interleukin-8 (IL-8) gene. Similar to the wild-type integrated NF-kappaB-dependent reporter, ChIP assays showed that TSA treatment resulted in hyperacetylation of the IL-8 promoter. These data indicate that the transactivation function of NF-kappaB is regulated in part through its association with HDAC corepressor proteins. Moreover, it suggests that the association of NF-kappaB with the HDAC1 and HDAC2 corepressor proteins functions to repress expression of NF-kappaB-regulated genes as well as to control the induced level of expression of these genes.

Mammalian DNA is methylated at many CpG dinucleotides. The biological consequences of methylation are mediated by a family of methyl-CpG binding proteins. The best characterized family member is MeCP2, a transcriptional repressor that recruits histone deacetylases. Our report concerns MBD2, which can bind methylated DNA in vivo and in vitro and has been reported to actively demethylate DNA (ref. 8). As DNA methylation causes gene silencing, the MBD2 demethylase is a candidate transcriptional activator. Using specific antibodies, however, we find here that MBD2 in HeLa cells is associated with histone deacetylase (HDAC) in the MeCP1 repressor complex. An affinity-purified HDAC1 corepressor complex also contains MBD2, suggesting that MeCP1 corresponds to a fraction of this complex. Exogenous MBD2 represses transcription in a transient assay, and repression can be relieved by the deacetylase inhibitor trichostatin A (TSA; ref. 12). In our hands, MBD2 does not demethylate DNA. Our data suggest that HeLa cells, which lack the known methylation-dependent repressor MeCP2, use an alternative pathway involving MBD2 to silence methylated genes.

Members of the Mad family of bHLH-Zip proteins heterodimerize with Max to repress transcription in a sequence-specific manner. Transcriptional repression by Mad:Max heterodimers is mediated by ternary complex formation with either of the corepressors mSin3A or mSin3B. We report here that mSin3A is an in vivo component of large, heterogeneous multiprotein complexes and is tightly and specifically associated with at least seven polypeptides. Two of the mSin3A-associated proteins, p50 and p55, are highly related to the histone deacetylase HDAC1. The mSin3A immunocomplexes possess histone deacetylase activity that is sensitive to the specific deacetylase inhibitor trapoxin. mSin3A-targeted repression of a reporter gene is reduced by trapoxin treatment, suggesting that histone deacetylation mediates transcriptional repression through Mad-Max-mSin3A multimeric complexes.

Long interspersed nuclear elements-1 (LINE-1 or L1s) are abundant retrotransposons that comprise approximately 20% of mammalian genomes. Active L1 retrotransposons can impact the genome in a variety of ways, creating insertions, deletions, new splice sites or gene expression fine-tuning. We have shown previously that L1 retrotransposons are capable of mobilization in neuronal progenitor cells from rodents and humans and evidence of massive L1 insertions was observed in adult brain tissues but not in other somatic tissues. In addition, L1 mobility in the adult hippocampus can be influenced by the environment. The neuronal specificity of somatic L1 retrotransposition in neural progenitors is partially due to the transition of a Sox2/HDAC1 repressor complex to a Wnt-mediated T-cell factor/lymphoid enhancer factor (TCF/LEF) transcriptional activator. The transcriptional switch accompanies chromatin remodelling during neuronal differentiation, allowing a transient stimulation of L1 transcription. The activity of L1 retrotransposons during brain development can have an impact on gene expression and neuronal function, thereby increasing brain-specific genetic mosaicism. Further understanding of the molecular mechanisms that regulate L1 expression should provide new insights into the role of L1 retrotransposition during brain development. Here we show that L1 neuronal transcription and retrotransposition in rodents are increased in the absence of methyl-CpG-binding protein 2 (MeCP2), a protein involved in global DNA methylation and human neurodevelopmental diseases. Using neuronal progenitor cells derived from human induced pluripotent stem cells and human tissues, we revealed that patients with Rett syndrome (RTT), carrying MeCP2 mutations, have increased susceptibility for L1 retrotransposition. Our data demonstrate that L1 retrotransposition can be controlled in a tissue-specific manner and that disease-related genetic mutations can influence the frequency of neuronal L1 retrotransposition. Our findings add a new level of complexity to the molecular events that can lead to neurological disorders.

The human HDAC (histone deacetylase) family, a well-validated anticancer target, plays a key role in the control of gene expression through regulation of transcription. While HDACs can be subdivided into three main classes, the class I, class II and class III HDACs (sirtuins), it is presently unclear whether inhibiting multiple HDACs using pan-HDAC inhibitors, or targeting specific isoforms that show aberrant levels in tumours, will prove more effective as an anticancer strategy in the clinic. To address the above issues, we have tested a number of clinically relevant HDACis (HDAC inhibitors) against a panel of rhHDAC (recombinant human HDAC) isoforms. Eight rhHDACs were expressed using a baculoviral system, and a Fluor de Lystrade mark (Biomol International) HDAC assay was optimized for each purified isoform. The potency and selectivity of ten HDACs on class I isoforms (rhHDAC1, rhHDAC2, rhHDAC3 and rhHDAC8) and class II HDAC isoforms (rhHDAC4, rhHDAC6, rhHDAC7 and rhHDAC9) was determined. MS-275 was HDAC1-selective, MGCD0103 was HDAC1- and HDAC2-selective, apicidin was HDAC2- and HDAC3-selective and valproic acid was a specific inhibitor of class I HDACs. The hydroxamic acid-derived compounds (trichostatin A, NVP-LAQ824, panobinostat, ITF2357, vorinostat and belinostat) were potent pan-HDAC inhibitors. The growth-inhibitory effect of the HDACis on HeLa cells showed that both pan-HDAC and class-I-specific inhibitors inhibited cell growth. The results also showed that both pan-HDAC and class-I-specific inhibitor treatment resulted in increased acetylation of histones, but only pan-HDAC inhibitor treatment resulted in increased tubulin acetylation, which is in agreement with their activity towards the HDAC6 isoform.

BACKGROUND

Persistent infection in resting CD4+ T cells prevents eradication of HIV-1. Since the chromatin remodeling enzyme histone deacetylase 1 (HDAC1) maintains latency of integrated HIV, we tested the ability of the HDAC inhibitor valproic acid to deplete persistent, latent infection in resting CD4+ T cells.

METHODS

We did a proof-of-concept study in four volunteers infected with HIV and on highly-active antiretroviral therapy (HAART). After intensifying the effect of HAART with subcutaneous enfuvirtide 90 mug twice daily for 4-6 weeks to prevent the spread of HIV, we added oral valproic acid 500-750 mg twice daily to their treatment regimen for 3 months. We quantified latent infection of resting CD4+ T cells before and after augmented treatment by limiting-dilution culture of resting CD4+ T cells after ex-vivo activation.

RESULTS

The frequency of resting cell infection was stable before addition of enfuvirtide and valproic acid, but declined thereafter. This decline was significant in three of four patients (mean reduction 75%, range 68% to >84%). Patients had slight reactions to enfuvirtide at the injection site, but otherwise tolerated treatment well.

CONCLUSIONS

Combination therapy with an HDAC inhibitor and intensified HAART safely accelerates clearance of HIV from resting CD4+ T cells in vivo, suggesting a new and practical approach to eliminate HIV infection in this persistent reservoir. This finding, though not definitive, suggests that new approaches will allow the cure of HIV in the future.

Gene expression is in part controlled by chromatin remodeling factors and the acetylation state of nucleosomal histones. The latter process is regulated by histone acetyltransferases and histone deacetylases (HDACs). Previously, three human and five yeast HDAC enzymes had been identified. These can be categorized into two classes: the first class represented by yeast Rpd3-like proteins and the second by yeast Hda1-like proteins. Human HDAC1, HDAC2, and HDAC3 proteins are members of the first class, whereas no class II human HDAC proteins had been identified. The amino acid sequence of Hda1p was used to search the GenBank/expressed sequence tag databases to identify partial sequences from three putative class II human HDAC proteins. The corresponding full-length cDNAs were cloned and defined as HDAC4, HDAC5, and HDAC6. These proteins possess certain features present in the conserved catalytic domains of class I human HDACs, but also contain additional sequence domains. Interestingly, HDAC6 contains an internal duplication of two catalytic domains, which appear to function independently of each other. These class II HDAC proteins have differential mRNA expression in human tissues and possess in vitro HDAC activity that is inhibited by trichostatin A. Coimmunoprecipitation experiments indicate that these HDAC proteins are not components of the previously identified HDAC1 and HDAC2 NRD and mSin3A complexes. However, HDAC4 and HDAC5 associate with HDAC3 in vivo. This finding suggests that the human class II HDAC enzymes may function in cellular processes distinct from those of HDAC1 and HDAC2.

Oligodendrocyte development is regulated by the interaction of repressors and activators in a complex transcriptional network. We found that two histone-modifying enzymes, HDAC1 and HDAC2, were required for oligodendrocyte formation. Genetic deletion of both Hdac1 and Hdac2 in oligodendrocyte lineage cells resulted in stabilization and nuclear translocation of beta-catenin, which negatively regulates oligodendrocyte development by repressing Olig2 expression. We further identified the oligodendrocyte-restricted transcription factor TCF7L2/TCF4 as a bipartite co-effector of beta-catenin for regulating oligodendrocyte differentiation. Targeted disruption of Tcf7l2 in mice led to severe defects in oligodendrocyte maturation, whereas expression of its dominant-repressive form promoted precocious oligodendrocyte specification in developing chick neural tube. Transcriptional co-repressors HDAC1 and HDAC2 compete with beta-catenin for TCF7L2 interaction to regulate downstream genes involved in oligodendrocyte differentiation. Thus, crosstalk between HDAC1/2 and the canonical Wnt signaling pathway mediated by TCF7L2 serves as a regulatory mechanism for oligodendrocyte differentiation.

Cells latently infected with HIV represent a currently insurmountable barrier to viral eradication in infected patients. Using the J-Lat human T-cell model of HIV latency, we have investigated the role of host factor binding to the kappaB enhancer elements of the HIV long terminal repeat (LTR) in the maintenance of viral latency. We show that NF-kappaB p50-HDAC1 complexes constitutively bind the latent HIV LTR and induce histone deacetylation and repressive changes in chromatin structure of the HIV LTR, changes that impair recruitment of RNA polymerase II and transcriptional initiation. Knockdown of p50 expression with specific small hairpin RNAs reduces HDAC1 binding to the latent HIV LTR and induces RNA polymerase II recruitment. Similarly, inhibition of histone deacetylase (HDAC) activity with trichostatin A promotes binding of RNA polymerase II to the latent HIV LTR. This bound polymerase complex, however, remains non-processive, generating only short viral transcripts. Synthesis of full-length viral transcripts can be rescued under these conditions by expression of Tat. The combination of HDAC inhibitors and Tat merits consideration as a new strategy for purging latent HIV proviruses from their cellular reservoirs.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized clinically by cognitive impairments that progress to dementia and death. The earliest symptoms of AD present as a relatively pure deficit in memory retrieval. Therefore, drug treatments that intervene in the early stages of AD by rescuing memory deficits could be promising therapies to slow, or even reverse progression of the disease. In this study, we tested the potential of systemic histone deacetylase inhibitor (HDACi) treatment to rescue cognitive deficits in a mouse model of AD. APPswe/PS1dE9 mice showed pronounced contextual memory impairments beginning at 6 months of age. Chronic HDACi injections (2-3 weeks) did not alter contextual memory formation in normal mice, but had profound effects in transgenic animals. Injections of sodium valproate, sodium butyrate, or vorinostat (suberoylanilide hydroxamic acid; Zolinza) completely restored contextual memory in these mutant mice. Further behavioral testing of the HDACi-treated transgenic mice showed that the newly consolidated memories were stably maintained over a 2-week period. Measurement of the HDAC isoform selectivity profile of sodium valproate, sodium butyrate, and vorinostat revealed the common inhibition of class I HDACs (HDAC1, 2, 3, 8) with little effect on the class IIa HDAC family members (HDAC4, 5, 7, 9) and inhibition of HDAC6 only by vorinostat. These preclinical results indicate that targeted inhibition of class I HDAC isoforms is a promising avenue for treating the cognitive deficits associated with early stage AD.

Nanog and Oct4 are essential transcription factors that regulate self-renewal and pluripotency of ES cells. However, the mechanisms by which Nanog and Oct4 modulate ES cell fate remain unknown. Through characterization of endogenous Nanog and Oct4 protein complexes in mouse ES cells, we found that these transcription factors interact with each other and associate with proteins from multiple repression complexes, including the NuRD, Sin3A and Pml complexes. In addition, Nanog, Oct4 and repressor proteins co-occupy Nanog-target genes in mouse ES cells, suggesting that Nanog and Oct4 together may communicate with distinct repression complexes to control gene transcription. To our surprise, of the various core components in the NuRD complex with which Nanog and Oct4 interact, Mta1 was preferred, whereas Mbd3 and Rbbp7 were either absent or present at sub-stoichiometric levels. We named this unique Hdac1/2- and Mta1/2-containing complex NODE (for Nanog and Oct4 associated deacetylase). Interestingly, NODE contained histone deacetylase (HDAC) activity that seemed to be comparable to NuRD, and retained its association with Nanog and Oct4 in Mbd3(-/-) ES cells. In contrast to Mbd3 loss-of-function, knockdown of NODE subunits led to increased expression of developmentally regulated genes and ES-cell differentiation. Our data collectively suggest that Nanog and Oct4 associate with unique repressor complexes on their target genes to control ES cell fate.

The acetylation state of histones can influence transcription. Acetylation, carried out by acetyltransferases such as CBP/p300 and P/CAF, is commonly associated with transcriptional stimulation, whereas deacetylation, mediated by the three known human deacetylases HDAC1, 2 and 3, causes transcriptional repression. The known human deacetylases represent a single family and are homologues of the yeast RPD3 deacetylase. Here we identify and characterize HDAC4, a representative of a new human histone deacetylase family, which is homologous to the yeast HDA1 deacetylase. We show that HDAC4, unlike other deacetylases, shuttles between the nucleus and the cytoplasm in a process involving active nuclear export. In the nucleus, HDAC4 associates with the myocyte enhancer factor MEF2A. Binding of HDAC4 to MEF2A results in the repression of MEF2A transcriptional activation, a function that requires the deacetylase domain of HDAC4. These results identify MEF2A as a nuclear target for HDAC4-mediated repression and suggests that compartmentalization may be a novel mechanism for controlling the nuclear activity of this new family of deacetylases.

An important event in gene expression is the covalent modification of histone proteins. We have found that the mammalian transcriptional repressor Sin3 (mSin3) exists in a complex with histone deacetylases HDAC1 and HDAC2. Consistent with the observation that mSin3-mediated repression of transcription involves the modification of histone polypeptides, we found that the mSin3-containing complex includes polypeptides that tether the mSin3 complex to core histone proteins. In addition, two novel mSin3-associated polypeptides, SAP18 and SAP30, were identified. We isolated a cDNA encoding human SAP18 and found that SAP18 is a component of an mSin3-containing complex in vivo. Moreover, we demonstrate a direct interaction between SAP18 and mSin3. SAP18 represses transcription in vivo when tethered to the promoter, consistent with the ability of SAP18 to interact with mSin3.

Intrauterine growth retardation (IUGR) has been linked to the onset of diseases in adulthood, including type 2 diabetes, and has been proposed to result from altered gene regulation patterns due to epigenetic modifications of developmental genes. To determine whether epigenetic modifications may play a role in the development of adult diabetes following IUGR, we used a rodent model of IUGR that expresses lower levels of Pdx1, a pancreatic and duodenal homeobox 1 transcription factor critical for beta cell function and development, which develops diabetes in adulthood. We found that expression of Pdx1 was permanently reduced in IUGR beta cells and underwent epigenetic modifications throughout development. The fetal IUGR state was characterized by loss of USF-1 binding at the proximal promoter of Pdx1, recruitment of the histone deacetylase 1 (HDAC1) and the corepressor Sin3A, and deacetylation of histones H3 and H4. Following birth, histone 3 lysine 4 (H3K4) was demethylated and histone 3 lysine 9 (H3K9) was methylated. During the neonatal period, these epigenetic changes and the reduction in Pdx1 expression could be reversed by HDAC inhibition. After the onset of diabetes in adulthood, the CpG island in the proximal promoter was methylated, resulting in permanent silencing of the Pdx1 locus. These results provide insight into the development of type 2 diabetes following IUGR and we believe they are the first to describe the ontogeny of chromatin remodeling in vivo from the fetus to the onset of disease in adulthood.

The three-way connection between DNA methylation, gene activity and chromatin structure has been known for almost two decades. Nevertheless, the molecular link between methyl groups on the DNA and the positioning of nucleosomes to form an inactive chromatin configuration was missing. This review discusses recent experimental data that may, for the first time, shed light on this molecular link. MeCP2, which is a known methylcytosine-binding protein, has been shown to possess a transcriptional repressor domain (TRD) that binds the corepressor mSin3A. This corepressor protein constitutes the core of a multiprotein complex that includes histone deacetylases (HDAC1 and HDAC2). Transfection and injection experiments with methylated constructs have revealed that the silenced state of a methylated gene, which is associated with a deacetylated nucleosomal structure, could be relieved by the deacetylase inhibitor, trichostatin A. Thus, methylation plays a pivotal role in establishing and maintaining an inactive state of a gene by rendering the chromatin structure inaccessible to the transcription machinery.

Gene transcription is critically influenced by chromatin structure and the modification status of histone tails. Methylation of lysine residues in histone tails is dynamically regulated by the opposing activities of histone methyltransferases and histone demethylases. Here we show that JARID1C/SMCX, a JmjC-domain-containing protein implicated in X-linked mental retardation and epilepsy, possesses H3K4 tri-demethylase activity and functions as a transcriptional repressor. An SMCX complex isolated from HeLa cells contains additional chromatin modifiers (the histone deacetylases HDAC1 and HDAC2, and the histone H3K9 methyltransferase G9a) and the transcriptional repressor REST, suggesting a direct role for SMCX in chromatin dynamics and REST-mediated repression. Chromatin immunoprecipitation reveals that SMCX and REST co-occupy the neuron-restrictive silencing elements in the promoters of a subset of REST target genes. RNA-interference-mediated depletion of SMCX derepresses several of these targets and simultaneously increases H3K4 trimethylation at the sodium channel type 2A (SCN2A) and synapsin I (SYN1) promoters. We propose that loss of SMCX activity impairs REST-mediated neuronal gene regulation, thereby contributing to SMCX-associated X-linked mental retardation.

Inhibitors of histone deacetylases (HDACs) induce growth arrest, differentiation, and apoptosis of colon cancer cell lines in vitro and have demonstrated anti-cancer efficacy in clinical trials. Whereas a role for HDAC1 and -2 in mediating components of the HDAC inhibitor response has been reported, the role of HDAC3 is unknown. Here we demonstrate increased protein expression of HDAC3 in human colon tumors and in duodenal adenomas from Apc1638(N/+) mice. HDAC3 was also maximally expressed in proliferating crypt cells in normal intestine. Silencing of HDAC3 expression in colon cancer cell lines resulted in growth inhibition, a decrease in cell survival, and increased apoptosis. Similar effects were observed for HDAC2 and, to a lesser extent, for HDAC1. HDAC3 silencing also selectively induced expression of alkaline phosphatase, a marker of colon cell maturation. Concurrent with its effect on cell growth, overexpression of HDAC3 and other Class I HDACs inhibited basal and butyrate-induced p21 transcription in a Sp1/Sp3-dependent manner, whereas silencing of HDAC3 stimulated p21 promoter activity and expression. However, the magnitude of the effects elicited by silencing of individual Class I HDACs was significantly less than that induced by HDAC inhibitors. These findings identify HDAC3 as a gene deregulated in human colon cancer and as a novel regulator of colon cell maturation and p21 expression. These findings also demonstrate that multiple Class I HDACs are involved in repressing p21 and suggest that the growth-inhibitory and apoptotic effects induced by HDAC inhibitors are probably mediated through the inhibition of multiple HDACs.

Understanding the cell type-specific molecular mechanisms by which distinct signaling pathways combinatorially control proliferation during organogenesis is a central issue in development and disease. Here, we report that the bicoid-related transcription factor Pitx2 is rapidly induced by the Wnt/Dvl/beta-catenin pathway and is required for effective cell-type-specific proliferation by directly activating specific growth-regulating genes. Regulated exchange of HDAC1/beta-catenin converts Pitx2 from repressor to activator, analogous to control of TCF/LEF1. Pitx2 then serves as a competence factor required for the temporally ordered and growth factor-dependent recruitment of a series of specific coactivator complexes that prove necessary for Cyclin D2 gene induction. The molecular strategy underlying interactions between the Wnt and growth factor-dependent signaling pathways in cardiac outflow tract and pituitary proliferation is likely to be prototypic of cell-specific proliferation strategies in other tissues.

The tumor suppressor p53 is stabilized and activated in response to cellular stress through post-translational modifications including acetylation. p300/CBP-mediated acetylation of p53 is negatively regulated by MDM2. Here we show that MDM2 can promote p53 deacetylation by recruiting a complex containing HDAC1. The HDAC1 complex binds MDM2 in a p53-independent manner and deacetylates p53 at all known acetylated lysines in vivo. Ectopic expression of a dominant-negative HDAC1 mutant restores p53 acetylation in the presence of MDM2, whereas wild-type HDAC1 and MDM2 deacetylate p53 synergistically. Fibroblasts overexpressing a dominant negative HDAC1 mutant display enhanced DNA damage-induced p53 acetylation, increased levels of p53 and a more pronounced induction of p21 and MDM2. These results indicate that acetylation promotes p53 stability and function. As the acetylated p53 lysine residues overlap with those that are ubiquitylated, our results suggest that one major function of p53 acetylation is to promote p53 stability by preventing MDM2-dependent ubiquitylation, while recruitment of HDAC1 by MDM2 promotes p53 degradation by removing these acetyl groups.

Histone deacetylase (HDAC) inhibitors (HDACi) cause cancer cell growth arrest and/or apoptosis in vivo and in vitro. The HDACi suberoylanilide hydroxamic acid (SAHA) is in phase I/II clinical trials showing significant anticancer activity. Despite wide distribution of HDACs in chromatin, SAHA alters the expression of few genes in transformed cells. p21(WAF1) is one of the most commonly induced. SAHA does not alter the expression of p27(KIPI), an actively transcribed gene, or globin, a silent gene, in ARP-1 cells. Here we studied SAHA-induced changes in the p21(WAF1) promoter of ARP-1 cells to better understand the mechanism of HDACi gene activation. Within 1 h, SAHA caused modifications in acetylation and methylation of core histones and increased DNase I sensitivity and restriction enzyme accessibility in the p21(WAF1) promoter. These changes did not occur in the p27(KIPI) or epsilon-globin gene-related histones. The HDACi caused a marked decrease in HDAC1 and Myc and an increase in RNA polymerase II in proteins bound to the p21(WAF1) promoter. Thus, this study identifies effects of SAHA on p21(WAF1)-associated proteins that explain, at least in part, the selective effect of HDACi in altering gene expression.

GABAergic dysfunction is present in the hippocampus in schizophrenia (SZ) and bipolar disorder (BD). The trisynaptic pathway was "deconstructed" into various layers of sectors CA3/2 and CA1 and gene expression profiling performed. Network association analysis was used to uncover genes that may be related to regulation of glutamate decarboxylase 67 (GAD(67)), a marker for this system that has been found by many studies to show decreased expression in SZs and BDs. The most striking change was a down-regulation of GAD(67) in the stratum oriens (SO) of CA2/3 in both groups; CA1 only showed changes in the SO of schizophrenics. The network generated for GAD(67) contained 25 genes involved in the regulation of kainate receptors, TGF-beta and Wnt signaling, as well as transcription factors involved in cell growth and differentiation. In SZs, IL-1beta, (GRIK2/3), TGF-beta2, TGF-betaR1, histone deacetylase 1 (HDAC1), death associated protein (DAXX), and cyclin D2 (CCND2) were all significantly up-regulated, whereas in BDs, PAX5, Runx2, LEF1, TLE1, and CCND2 were significantly down-regulated. In the SO of CA1 of BDs, where GAD67 showed no expression change, TGF-beta and Wnt signaling genes were all up-regulated, but other transcription factors showed no change in expression. In other layers/sectors, BDs showed no expression changes in these GAD(67) network genes. Overall, these results are consistent with the hypothesis that decreased expression of GAD(67) may be associated with an epigenetic mechanism in SZ. In BD, however, a suppression of transcription factors involved in cell differentiation may contribute to GABA dysfunction.

Low oxygen tension influences tumor progression by enhancing angiogenesis; and histone deacetylases (HDAC) are implicated in alteration of chromatin assembly and tumorigenesis. Here we show induction of HDAC under hypoxia and elucidate a role for HDAC in the regulation of hypoxia-induced angiogenesis. Overexpressed wild-type HDAC1 downregulated expression of p53 and von Hippel-Lindau tumor suppressor genes and stimulated angiogenesis of human endothelial cells. A specific HDAC inhibitor, trichostatin A (TSA), upregulated p53 and von Hippel-Lindau expression and downregulated hypoxia-inducible factor-1alpha and vascular endothelial growth factor. TSA also blocked angiogenesis in vitro and in vivo. TSA specifically inhibited hypoxia-induced angiogenesis in the Lewis lung carcinoma model. These results indicate that hypoxia enhances HDAC function and that HDAC is closely involved in angiogenesis through suppression of hypoxia-responsive tumor suppressor genes.

Here we describe the components of a histone deacetylase (HDAC) complex that we term the CoREST-HDAC complex. CoREST-HDAC is composed of polypeptides distinct from previously characterized HDAC1/2-containing complexes such as the mSin3 and nucleosome remodeling and deacetylating (NRD, also named NURD, NuRD) complex. Interestingly, we do not observe RbAp46 and RbAp48 in this complex, although these proteins have been observed in all previously identified complexes and are thought to be part of an HDAC1/2 core. We identify the transcriptional corepressor CoREST and a protein with homology to polyamine oxidases as components of CoREST-HDAC. The HDAC1/2-interacting region of CoREST is mapped to a 179-aa region containing a SANT domain, a domain found in other HDAC1/2-interacting proteins such as NCoR, MTA1, and MTA2. Furthermore, we demonstrate that the corepressor function of CoREST depends on this region. Although CoREST initially was cloned as a corepressor to REST (RE1 silencing transcription factor/neural restrictive silencing factor), we find no evidence for the existence of the eight-zinc finger REST transcription factor as an interacting partner in this complex; however, we do find evidence for association of the putative oncogene ZNF 217 that contains eight zinc fingers.