HDAC1 is a member of the histone deacetylase family, which plays an important role in modulating the eukaryotic chromatin structure. Numerous studies have demonstrated its involvement in transcription and in tumorigenesis. To better understand the functions and regulation of HDAC1, a yeast two-hybrid screening approach was chosen to identify novel interactions involving HDAC1. Human HDAC1 was found to interact specifically in yeast, mammalian cells, and in vitro with the human Hus1 gene product, whose Schizosaccharomyces pombe homolog has been implicated in G(2)/M checkpoint control. Both HDAC1 and Hus1 proteins localize to the nuclei. Furthermore, HDAC1 and Hus1 were found to exist in a complex with Rad9, a known Hus1-interacting factor. In addition, bioinformatics analysis of the protein sequences of Hus1, Rad1, and Rad9, three checkpoint Rad proteins that form a complex, revealed that they all contain a putative proliferating cell nuclear antigen (PCNA) fold, raising the possibility that these factors may bind to DNA in a PCNA-like ring structure. The results reported in this study strongly suggest a novel pathway involving HDAC1 in G(2)/M checkpoint control through the interaction with a functional Rad complex that may utilize a PCNA-like structure. Therefore, physically and functionally similar apparatus may function during G(2)/M checkpoint and DNA replication.

HIV-1 integrase (IN) is a virally encoded protein required for integration of viral cDNA into host chromosomes. INI1/hSNF5 is a component of the SWI/SNF complex that interacts with HIV-1 IN, is selectively incorporated into HIV-1 (but not other retroviral) virions, and modulates multiple steps, including particle production and infectivity. To gain further insight into the role of INI1 in HIV-1 replication, we screened for INI1-interacting proteins using the yeast two-hybrid system. We found that SAP18 (Sin3a associated protein 18 kD), a component of the Sin3a-HDAC1 complex, directly binds to INI1 in yeast, in vitro and in vivo. Interestingly, we found that IN also binds to SAP18 in vitro and in vivo. SAP18 and components of a Sin3A-HDAC1 complex were specifically incorporated into HIV-1 (but not SIV and HTLV-1) virions in an HIV-1 IN-dependent manner. Using a fluorescence-based assay, we found that HIV-1 (but not SIV) virion preparations harbour significant deacetylase activity, indicating the specific recruitment of catalytically active HDAC into the virions. To determine the requirement of virion-associated HDAC1 to HIV-1 replication, an inactive, transdominant negative mutant of HDAC1 (HDAC1(H141A)) was utilized. Incorporation of HDAC1(H141A) decreased the virion-associated histone deacetylase activity. Furthermore, incorporation of HDAC1(H141A) decreased the infectivity of HIV-1 (but not SIV) virions. The block in infectivity due to virion-associated HDAC1(H141A) occurred specifically at the early reverse transcription stage, while entry of the virions was unaffected. RNA-interference mediated knock-down of HDAC1 in producer cells resulted in decreased virion-associated HDAC1 activity and a reduction in infectivity of these virions. These studies indicate that HIV-1 IN and INI1/hSNF5 bind SAP18 and selectively recruit components of Sin3a-HDAC1 complex into HIV-1 virions. Furthermore, HIV-1 virion-associated HDAC1 is required for efficient early post-entry events, indicating a novel role for HDAC1 during HIV-1 replication.

Gene fusions between prostate-specific, androgen responsive TMPRSS2 gene and oncogenic ETS factors, such as ERG, occur in up to 50% of all prostate cancers. We recently defined a gene signature that was characteristic to prostate cancers with ERG activation. This suggested epigenetic reprogramming, such as upregulation of histone deactylase 1 (HDAC1) gene and downregulation of its target genes. We then hypothesized that patients with ERG-positive prostate cancers may benefit from epigenetic therapy such as HDAC inhibition (HDACi), especially in combination with antiandrogens. Here, we exposed ERG-positive prostate cancer cell lines to HDAC inhibitors Trichostatin A (TSA), MS-275 and suberoylanilide hydroxamic acid (SAHA) with or without androgen deprivation. We explored the effects on cell phenotype, gene expression as well as ERG and androgen receptor (AR) signaling. When compared with 5 other prostate cell lines, ERG-positive VCaP and DuCap cells were extremely sensitive to HDACi, in particular TSA, showing synergy with concomitant androgen deprivation increasing apoptosis. Both of the HDAC inhibitors studied caused repression of the ERG-fusion gene, whereas the pan-HDAC inhibitor TSA prominently repressed the ERG-associated gene signature. Additionally, HDACi and flutamide caused retention of AR in the cytoplasm, indicating blockage of androgen signaling. Our results support the hypothesis that HDACi, especially in combination with androgen deprivation, is effective against TMPRSS2-ERG-fusion positive prostate cancer in vitro. Together with our previous in vivo observations of an "epigenetic reprogramming gene signature" in clinical ERG-positive prostate cancers, these studies provide mechanistic insights to ERG-associated tumorigenesis and suggest therapeutic paradigms to be tested in vivo.

Human GCMa transcription factor regulates expression of syncytin, a placental fusogenic protein mediating trophoblastic fusion. Recently, we have demonstrated that CBP-mediated GCMa acetylation underlies the activated cAMP/PKA signaling pathway that stimulates trophoblastic fusion. Because protein acetylation is a reversible modification governed by histone acetyltransferases (HATs) and histone deacetylase (HDACs), in this study we investigated the key HDACs responsible for deacetylation of GCMa and thus the reduction in GCMa activity to avoid unwanted fusion events that may have adverse effects on placental morphogenesis. We herein demonstrate that the HDAC inhibitor, trichostatin A (TSA), increases the level of acetylated GCMa and that HDAC1, 3, 4 and 5 interact with and deacetylate GCMa. Glutathione S-transferase (GST) pull-down assays further verified direct interaction between GCMa and HDAC3 or CBP and HDAC3. HDAC3 counteracts the transcriptional coactivator activity of CBP and the enhancement effect of CBP on GCMa-mediated transcriptional activation. Correlatively, we found in placental cells that HDAC3 associates with the proximal GCMa-binding site (pGBS) in the syncytin promoter and dissociates from pGBS in the presence of forskolin, which stimulates the association of CBP and GCMa with pGBS. Our studies support that trophoblastic fusion in placental morphogenesis depends on the regulation of GCMa activity by HAT and HDAC.

BACKGROUND

HIV-1 is a retrovirus that has infected millions in recent decades. The level of life cycle complexity and host control exerted by this small virus with only nine proteins is astonishing. An interesting direction that has emerged in recent years is the role of small non-coding RNAs in viral gene expression.

METHODS

We focus on HIV-1 produced microRNAs (miRNAs), namely, TAR, Nef and miR-H1, and their roles in HIV-1 biogenesis. The article provides insights into TAR miRNA-mediated downregulation of viral and host gene expression by recruitment of chromatin remodeling components (HDAC1).

RESULTS

We address the influence of TAR miRNA on host cell cycle progression and apoptosis, and the role of Nef miRNA in the regulation of viral and host gene expression. The review also highlights an intriguing connection between miR-H1 and HIV-1-associated neurological pathogenesis, and the influence of the miRNA machinery in the establishment of latency. In the Expert Opinion section, we analyze the issue of host-based therapeutics against HIV-1 and how transcription inhibitors are influenced by viral miRNA production.

CONCLUSIONS

HIV-derived miRNAs are of significance not only to understand host-virus interactions, but also for the design of effective therapeutics.

Activating transcription factor (ATF) 3 plays a role in determining cell fate and generates a variety of alternatively spliced isoforms in stress response. We have reported previously that splice variant ATF3deltaZip2, which lacks the leucine zipper region, is induced in response to various stress stimuli. However, its biological function has not been elucidated. By using cells treated with tumor necrosis factor-alpha and actinomycin D or cells overexpressing ATF3deltaZip2, we showed that ATF3deltaZip2 sensitizes cells to apoptotic cell death in response to tumor necrosis factor-alpha, at least in part through suppressing nuclear factor (NF)-kappaB-dependent transcription of anti-apoptotic genes such as cIAP2 and XIAP. ATF3deltaZip2 interacts with a p65 (RelA)-cofactor complex containing CBP/p300 and HDAC1 at NF-kappaB sites of the proximal promoter region of the cIAP2 gene in vivo and down-regulates the recruitment of CBP/p300. Our study revealed that ATF3deltaZip2 counteracts anti-apoptotic activity of NF-kappaB, at least in part, by displacing positive cofactor CBP/p300 and provides insight into the mechanism by which ATF3 regulates cell fate through alternative splicing in stress response.

The temporal regulation of DNA replication is thought to be important for chromosome organization and genome stability. We show here that Epstein-Barr virus (EBV) genomes replicate in mid- to late S phase and that agents that accelerate replication timing of EBV reduce viral genome stability. Hydroxyurea (HU) treatment, which is known to eliminate EBV episomes, shifted EBV replication to earlier times in the cell cycle. HU treatment correlated with hyperacetylation of histone H3 and loss of telomere repeat factor 2 (TRF2) binding at the EBV origin of plasmid replication (OriP). Deletion of TRF2 binding sites within OriP or short hairpin RNA depletion of TRF2 advanced the replication timing of OriP-containing plasmids. Inhibitors of class I histone deacetylases (HDACs) increased histone acetylation at OriP, advanced the replication timing of EBV, and reduced EBV genome copy number. We also show that HDAC1 and -2 form a stable complex with TRF2 at OriP and that HU treatment inhibits HDAC activity. We propose that the TRF2-HDAC complex enhances EBV episome stability by providing a checkpoint that delays replication initiation at OriP.

Vimentin, a member of the intermediate filament (IF) protein family, exhibits a complex pattern of tissue- and developmental-specific expression. Although vimentin is widely expressed in the embryo, its expression becomes restricted during terminal differentiation. Moreover, it is often expressed in tissue culture cells despite their embryological origin and is a marker for the metastatic tumor cell. Previously, the vimentin promoter has been shown to contain several positive- and negative-acting cis-elements. The negative elements bind the transcription factor ZBP-89. Interestingly, ZBP-89 can be either an activator or a repressor of gene expression. For instance, ZBP-89 has been shown to activate p21(waf1/cip1) expression by recruiting p300 to the p21 promoter. Here, we have investigated the mechanism of ZBP-89 repression. The histone deacetylase (HDAC) inhibitor TSA enhances vimentin gene expression requiring the proximal promoter region including GC-box 1, a known Sp1/Sp3 binding site. Chromatin immunoprecipitation (ChIP) assays document an increase in the acetylation status of histone H3 on the endogenous vimentin gene concomitant with TSA treatment. However, EMSAs, DNA precipitation, co-immunoprecipitation and ChIP data show that it is not Sp1, but rather ZBP-89, which recruits HDAC1. From these studies we conclude that ZBP-89 functions as a repressor by recruiting HDAC1 to the vimentin promoter.

OBJECTIVE

Cytokine-induced beta cell toxicity is abrogated by non-selective inhibitors of lysine deacetylases (KDACs). The KDAC family consists of 11 members, namely histone deacetylases <em>HDAC1</em> to <em>HDAC1</em>1, but it is not known which KDAC members play a role in cytokine-mediated beta cell death. The aim of the present study was to examine the KDAC gene expression profile of the beta cell and to investigate whether KDAC expression is regulated by cytokines. In addition, the protective effect of the non-selective KDAC inhibitor ITF2357 and interdependent regulation of four selected KDACs were investigated.

METHODS

The beta cell line INS-1 and intact rat and human islets were exposed to cytokines with or without ITF2357. Expression of mRNA was assessed by real-time PCR and selected targets validated at the protein level by immunoblotting. Effects on cytokine-induced toxicity were investigated by in vitro assays.

RESULTS

Hdac1 to Hdac11 were expressed and differentially regulated by cytokines in INS-1 cells and rat islets. HDAC1, -2, -6 and -11 were found to be expressed and regulated by cytokines in human islets. ITF2357 protected against cytokine-induced beta cell apoptosis and counteracted cytokine-induced attenuation of basal insulin secretion. In addition, cytokine-induced regulation of Hdac2 and Hdac6, but not Hdac1 and Hdac11, was reduced by KDAC inhibition.

CONCLUSIONS

All classical KDAC genes are expressed by beta cells and differentially regulated by cytokines. Based on the relative expression levels and degree of regulation by cytokines, we propose that HDAC1, -2, -6 and -11 are of particular importance for beta cell function. These observations may help in the design of specific KDAC inhibitors to prevent beta cell destruction in situ and in islet grafts.

Histone deacetylases (HDACs) are enzymes that deacetylate acetyl lysines in histones and various non-histone proteins. Three classes of histone deacetylases have been described in humans: class I, II and IV were shown to be zinc dependent amidohydrolases and eleven subtypes are known today (HDAC1-11). Class III enzymes depend in their catalysis on NAD+ with the subsequent formation of nicotinamide and O acetyl-ADP ribose. Based on the homology to the yeast histone deacetylase Sir2p the NAD+-dependent deacetylases have been termed sirtuins and seven members (SIRT1-7) have been described in humans. Whereas class I and II HDACs have been identified as valid anticancer targets and clinical studies of their inhibitors as new anticancer agents are under way much less is known about the consequences of class III histone deacetylase inhibition. Sirtuins have been linked to ageing and overexpression of sirtuins leads to a prolonged lifespan in yeast. Lately, sirtuin activity has been tied to the pathogenesis of HIV, cancer and neurodegenerative disease. In the last two years several reports of new sirtuin inhibitors have emerged. Additionally, sirtuin activators have been identified and have been implicated as potential drugs for the amelioration of metabolic diseases. Thus, the field of sirtuin biology can be investigated with these new tools which will allow in turn to assess the therapeutic potential of those compounds. We will present an overview over sirtuins and their available inhibitors and activators.

Metastasis-associated antigens 1/2/3 (Mta1/2/3) are components of nucleosome remodeling and deacetylase (NuRD) complexes and have been found to play roles in embryonic development and homeostasis. However, their functions in primitive hematopoiesis are unknown. In this study, we demonstrate that knockdown of mta3 by antisense morpholinos abolishes primitive hematopoietic lineages and causes abnormal angiogenesis in zebrafish embryos. However, the expression of the pronephric duct and paraxial mesoderm markers is unaltered and the specification of angioblasts is unaffected in mta3 morphants. The results suggest that mta3 is specifically required for primitive hematopoiesis. Furthermore, inhibition of deacetylase activity with the inhibitors valproic acid (VPA) or trichostatin A (TSA) in zebrafish embryos completely blocks primitive hematopoiesis, resulting in hematopoietic defects almost identical to those seen in mta3 morphants. Importantly, overexpression of scl or scl and lmo2, 2 master genes for primitive hematopoiesis, is able to overturn effects of mta3 knockdown or VPA/TSA treatment; and overexpression of mta3, and human MBD3 or HDAC1, 2 other components of NuRD complex, enhances the expression of scl and lmo2 in the posterior lateral plate mesoderm during early primitive hematopoiesis. We conclude that Mta3-NuRD complex is essential for the initiation of primitive hematopoiesis. Thus, our findings provide new insight into the regulatory hierarchy of primitive hematopoiesis in vertebrates.

Histone deacetylases (HDACs) represent a large family of enzymes identified as key regulators of nucleosomal histone acetylation, a major epigenetic event that controls eukaryotic gene transcription. Inappropriate deacetylation mediated by HDACs has been associated with profound alterations in cellular biology. We have thus hypothesized that an altered HDAC expression may favor cancer development/progression. To test this possibility, we have sought to screen the expression profiles of several class I and class II HDACs (HDAC1-8) in DU-145, PC-3 and LNCaP human prostate cancer cell lines as well as in matched malignant and non-malignant prostate tissues by use of real time RT-PCR, immunoblot and immunohistochemistry. All HDAC transcripts tested were detected at various levels in all prostate cancer cell lines and tissue samples analyzed. In prostate tissues, the abundance of HDAC1 protein, which was exclusively expressed in the cell nucleus, was similar in normal and malignant epithelial cells, but was usually lower in stromal cells. Unexpectedly, HDAC8, another class I HDAC, was not detected in epithelial cells but was uniquely expressed in the cytoplasm of stromal cells. HDAC5, a class II HDAC involved in myogenesis, was not detected in the tissues. Altogether, our findings indicate that epithelial and stromal cells exhibit distinct class I HDAC expression profiles, and the abundance of HDAC1 is not altered in human prostate cancer. In addition, our observations are the first to demonstrate the prominently cytosolic distribution of a class I HDAC, HDAC8.

BACKGROUND

Activation and differentiation of fibroblasts into contractile protein-expressing myofibroblasts and their acquired apoptosis-resistant phenotype are critical factors towards the development of idiopathic pulmonary fibrosis (IPF), a fatal disease characterised by distorted pulmonary structure and excessive extracellular matrix (ECM) deposition. The molecular mechanisms underlying these processes in IPF remain incompletely understood. We investigated the possible implication of aberrant overexpression and activity of histone deacetylases (HDACs) in IPF.

METHODS

We analysed lung tissues from patients with sporadic IPF (n=26) and non-diseased control lungs (n=16) for expression of class I and II HDACs. Primary IPF fibroblasts were treated with HDAC inhibitors (HDACi) LBH589 or valproic acid (VPA).

RESULTS

Compared to control lungs, protein levels of class I (HDAC1, HDAC2, HDAC3, HDAC8) and class II HDACs (HDAC4, HDAC 5, HDAC 7, HDAC 9) were significantly elevated in IPF lungs. Using immunohistochemistry, strong induction of nearly all HDAC enzymes was observed in myofibroblasts of fibroblast foci and in abnormal bronchiolar basal cells at sites of aberrant re-epithelialisation in IPF lungs, but not in controls. Treatment of primary IPF fibroblasts with the pan-HDACi LBH589 resulted in significantly reduced expression of genes associated with ECM synthesis, proliferation and cell survival, as well as in suppression of HDAC7, and was paralleled by induction of endoplasmic reticulum stress and apoptosis. The profibrotic and apoptosis-resistant phenotype of IPF fibroblasts was also partly attenuated by the class I HDACi VPA.

CONCLUSIONS

Aberrant overexpression of HDACs in basal cells of IPF lungs may contribute to the bronchiolisation process in this disease. Similarly, generation and apoptosis resistance of IPF fibroblasts are mediated by enhanced activity of HDAC enzymes. Therefore, pan-HDAC inhibition by LBH589 may present a novel therapeutic option for patients with IPF.

We previously identified a novel rat candidate metastasis-associated gene, mta1, based on its differential expression in highly metastatic cells compared to nonmetastatic cells. Furthermore, we showed that overexpression of its human counterpart, MTA1, correlated with the invasiveness or lymph node metastasis of gastric, colorectal and esophageal carcinomas. The aim of this study was to analyze the domains of the MTA1 and investigate the function(s) of this protein. Structural analysis revealed that the MTA1 protein contained a GATA-like zinc-finger domain, a leucine zipper domain, a SANT domain similar to the DNA binding domain of myb-related proteins, a src homology 3-binding domain important in protein-protein interactions, two highly acidic regions characteristic of the acidic activation domains of many transcription factors, and nuclear localization signals. Immunofluorescence staining of COS-7 cells transfected with a myc-epitope-tagged MTA1 expression vector clearly showed nuclear localization of MTA1. Coimmunoprecipitation of myc-tagged MTA1 and FLAG-tagged histone deacetylase 1 (HDAC1), followed by western blot analysis using anti-myc and anti-FLAG monoclonal antibodies showed that MTA1 physically bound with HDAC1 in COS-7 cells. Together with the recent finding that the NURD (nucleosome remodeling and histone deacetylase activities) complex contains an MTA1-related gene product, named MTA2, MTA1 may be another component of this complex and be involved in the alteration of chromatin structure and transcription repression.

Tumours are comprised of a highly heterogeneous population of cells, of which only a small subset of stem-like cells possess the ability to regenerate tumours in vivo. These cancer stem cells (CSCs) represent a significant clinical challenge as they are resistant to conventional cancer therapies and play essential roles in metastasis and tumour relapse. Despite this realization and great interest in CSCs, it has been difficult to develop CSC-targeted treatments due to our limited understanding of CSC biology. Here, we present evidence that specific histone deacetylases (HDACs) play essential roles in the CSC phenotype. Utilizing a novel CSC model, we discovered that the HDACs, HDAC1 and HDAC7, are specifically over-expressed in CSCs when compared to non-stem-tumour-cells (nsTCs). Furthermore, we determine that HDAC1 and HDAC7 are necessary to maintain CSCs, and that over-expression of HDAC7 is sufficient to augment the CSC phenotype. We also demonstrate that clinically available HDAC inhibitors (HDACi) targeting HDAC1 and HDAC7 can be used to preferentially target CSCs. These results provide actionable insights that can be rapidly translated into CSC-specific therapies.

The ING family of tumor suppressor proteins affects cell growth, apoptosis and response to DNA damage by modulating chromatin structure through association with different HAT and HDAC complexes. The major splicing isoforms of the ING1 locus are ING1a and INGlb. While INGlb plays a role in inducing apoptosis, the function of ING1a is currently unknown. Here we show that alternative splicing of the ING1 message alters the INGla:INGlb ratio by approximately 30-fold in senescent compared to low passage primary fibroblasts. INGla antagonizes INGlb function in apoptosis, induces the formation of structures resembling senescence-associated heterochromatic foci containing heterochromatin protein 1 gamma, the accumulation of senescence-associated beta-galactosidase activity and promotes senescent cell morphology and cell cycle arrest. Phenotypic effects may result from differential effects on gene expression since ING1a increases levels of both retinoblastoma and the p16 cyclin-dependent kinase inhibitor and ING1a and ING1b have opposite effects on the expression of proliferating nuclear cell antigen (PCNA), which is required for cell growth. Gene expression appears to be altered by targeting of HDAC complexes to gene promoters since INGla associates with several-fold higher levels of HDAC1 in senescent, compared to replication-competent cells and ING1 is found on the PCNA promoter by chromatin immunoprecipitation analysis. These data demonstrate a novel role for the ING1 proteins in differentially regulating senescence-associated chromatin remodeling vs. apoptosis and support the idea that altered ratios of the ING1 splicing isoforms may contribute to establishing the senescent phenotype through HDAC and HAT complex-mediated effects on chromatin structure.

Early neurodevelopment requires cell fate commitment from pluripotent stem cells to restricted neural lineages, which involves the epigenetic regulation of chromatin structure and lineage-specific gene transcription. However, it remains unclear how histone H3 lysine 9 acetylation (H3K9Ac), an epigenetic mark representing transcriptionally active chromatin, is involved in the neural commitment from pluripotent embryonic stem cells (ESCs). In this study, we demonstrate that H3K9Ac gradually declines during the first 4 days of in vitro neural differentiation of human ESCs (hESCs) and then increases during days 4-8. Consistent with this finding, the H3K9Ac enrichment at several pluripotency genes was decreased, and H3K9Ac occupancies at the loci of neurodevelopmental genes increased during hESC neural commitment. Inhibiting H3K9 deacetylation on days 0-4 by histone deacetylase inhibitors (HDACis) promoted hESC pluripotency and suppressed its neural differentiation. Conversely, HDACi-elicited up-regulation of H3K9 acetylation on days 4-8 enhanced neural differentiation and activated multiple neurodevelopmental genes. Mechanistically, HDACis promote pluripotency gene transcription to support hESC self-renewal through suppressing HDAC3 activity. During hESC neural commitment, HDACis relieve the inhibitory activities of HDAC1/5/8 and thereby promote early neurodevelopmental gene expression by interfering with gene-specific histone acetylation patterns. Furthermore, p300 is primarily identified as the major histone acetyltransferase involved in both hESC pluripotency and neural differentiation. Our results indicate that epigenetic modification plays pivotal roles during the early neural specification of hESCs. The histone acetylation, which is regulated by distinct HDAC members at different neurodevelopmental stages, plays dual roles in hESC pluripotency maintenance and neural differentiation.

BACKGROUND

Genes that are overexpressed in multidrug-resistant neuroblastomas relative to drug-sensitive neuroblastomas may provide targets for modulating drug resistance.

METHODS

We used microarrays to compare the gene expression profile of two drug-sensitive neuroblastoma cell lines with that of three multidrug-resistant neuroblastoma cell lines. RNA expression of selected overexpressed genes was quantified in 17 neuroblastoma cell lines by reverse transcription-polymerase chain reaction (RT-PCR). Small-interfering RNAs (siRNAs) were used for silencing gene expression. Cytotoxicity of melphalan, carboplatin, etoposide, and vincristine and cytotoxic synergy (expressed as combination index calculated by CalcuSyn software, where combination index < 1 indicates synergy and>> 1 indicates antagonism) were measured in cell lines with a fluorescence-based assay of cell viability. All statistical tests were two-sided.

RESULTS

A total of 94 genes were overexpressed in the multidrug-resistant cell lines relative to the drug-sensitive cell lines. Nine genes were selected for RT-PCR analysis, of which four displayed higher mRNA expression in the multidrug-resistant lines than in the drug-sensitive lines: histone deacetylase 1 (HDAC1; 2.3-fold difference, 95% confidence interval [CI] = 1.0-fold to 3.5-fold, P = .025), nuclear transport factor 2-like export factor (4.2-fold difference, 95% CI = 1.7-fold to 7.6-fold, P = .0018), heat shock 27-kDa protein 1 (2.5-fold difference, 95% CI = 1.0-fold to 87.7-fold, P = .028), and TAF12 RNA polymerase II, TATA box-binding protein-associated factor, 20 kDa (2.2-fold, 95% CI = 0.9-fold to 6.0-fold, P = .051). siRNA knockdown of HDAC1 gene expression sensitized CHLA-136 neuroblastoma cells to etoposide up to fivefold relative to the parental cell line or scrambled siRNA-transfected cells (P<.001). Cytotoxicity of the histone deacetylase inhibitor depsipeptide was tested in combination with melphalan, carboplatin, etoposide, or vincristine in five multidrug-resistant neuroblastoma cell lines, and synergistic cytotoxicity was demonstrated at a 90% cell kill of treated cells (combination index < 0.8) in all cell lines.

CONCLUSIONS

High HDAC1 mRNA expression was associated with multidrug resistance in neuroblastoma cell lines, and inhibition of HDAC1 expression or activity enhanced the cytotoxicity of chemotherapeutic drugs in multidrug-resistant neuroblastoma cell lines. Thus, HDAC1 is a potential therapeutic target in multidrug-resistant neuroblastoma.

The fundamental question posed here is why in dorsal root ganglia herpes simplex viruses (HSV) can establish a silent infection in which only latency associate transcripts (LAT) and miRNAs are expressed and the neuronal cell survives whereas in non-neuronal cells HSV replicates and destroys the infected cells. Current evidence indicates that in productive infection there are two checkpoints. The first is at activation of α genes and requires a viral protein (VP16) that recruits HCF-1, Oct1, LSD1, and the CLOCK histone acetyl transferase to demethylate histones and initiate transcription. The second checkpoint involves activation of β and γ genes. An α protein, ICP0, activates transcription by displacing HDAC1 or 2 from the HDAC/CoREST/LSD1/REST repressor complex at its DNA binding sites. Current data suggest that in dorsal root ganglia VP16 and HCF-1 are not translocated to neuronal nucleus and that the HDAC/CoREST/LSD1/REST complex is not suppressed-a first step in silencing of the viral genome and establishment of heterochromatin. The viral genome remains in a state of equilibrium with respect to viral gene expression. The function of both LAT and the micro RNAs is to silence low level expression of viral genes that could reactivate the latent genomes.

In the intestinal mucosa, retinoic acid (RA) is a critical signaling molecule. RA is derived from dietary vitamin A (retinol) through conversion by aldehyde dehydrogenases (aldh). Reduced levels of short-chain fatty acids (SCFAs) are associated with pathological microbial dysbiosis, inflammatory disease, and allergy. We hypothesized that SCFAs contribute to mucosal homeostasis by enhancing RA production in intestinal epithelia. With the use of human and mouse epithelial cell lines and primary enteroids, we studied the effect of SCFAs on the production of RA. Functional RA conversion was analyzed by Adlefluor activity assays. Butyrate (0-20 mM), in contrast to other SCFAs, dose dependently induced aldh1a1 or aldh1a3 transcript expression and increased RA conversion in human and mouse epithelial cells. Epithelial cell line data were replicated in intestinal organoids. In these organoids, butyrate (2-5 mM) upregulated aldh1a3 expression (36-fold over control), whereas aldh1a1 was not significantly affected. Butyrate enhanced maturation markers (Mucin-2 and villin) but did not consistently affect stemness markers or other Wnt target genes (lgr5, olfm4, ascl2, cdkn1). In enteroids, the stimulation of RA production by SCFA was mimicked by inhibitors of histone deacetylase 3 (HDAC3) but not by HDAC1/2 inhibitors nor by agonists of butyrate receptors G-protein-coupled receptor (GPR)43 or GPR109A, indicating that butyrate stimulates RA production via HDAC3 inhibition. We conclude that the SCFA butyrate inhibits HDAC3 and thereby supports epithelial RA production.

The regulatory protein HBx is essential for hepatitis B virus (HBV) replication in vivo and for transcription of the episomal HBV genome. We previously reported that in infected cells HBx activates genes targeted by the transcription factor CREB [cyclic adenosine monophosphate (cAMP) response element-binding protein]. cAMP induces phosphorylation and activation of CREB, and CREB inactivation is promoted by protein phosphatase 1 (PP1), which binds to CREB through histone deacetylase 1 (HDAC1). We showed that CREB was recruited to HBV DNA. Phosphorylation induced by cAMP had a longer half-life when CREB was bound to the episomal HBV genome compared to when it was bound to the promoter of a host target gene not regulated by HBx, suggesting that the virus has developed a mechanism to favor its own transcription. This mechanism required HBx, which interacted with and inhibited PP1 to extend the half-life of CREB phosphorylation. Silencing of PP1 rescued replication of an HBx-deficient HBV genome, suggesting that HBx enhances viral transcription in part by neutralizing PP1 activity. Our results illustrate a previously unknown mechanism of HBV transcriptional activation by HBx in which HBx interferes with the inactivation of CREB by the PP1 and HDAC1 complex.

We focused on the ability of the pan-histone deacetylase (HDAC) inhibitors AR42 and sodium valproate to alter the immunogenicity of melanoma cells. Treatment of melanoma cells with HDAC inhibitors rapidly reduced the expression of multiple HDAC proteins as well as the levels of PD-L1, PD-L2 and ODC, and increased expression of MHCA. In a cell-specific fashion, melanoma isolates released the immunogenic protein HMGB1 into the extracellular environment. Very similar data were obtained in ovarian and H&NSCC PDX isolates, and in established tumor cell lines from the lung and kidney. Knock down of <em>HDAC1</em>, HDAC3, HDAC8 and <em>HDAC1</em>0, but not HDAC6, recapitulated the effects of the HDAC inhibitors on the immunotherapy biomarkers. Using B16 mouse melanoma cells we discovered that pre-treatment with AR42 or sodium valproate enhanced the anti-tumor efficacy of an anti-PD-1 antibody and of an anti-CTLA4 antibody. In the B16 model, both AR42 and sodium valproate enhanced the anti-tumor efficacy of the multi-kinase inhibitor pazopanib. In plasma from animals exposed to [HDAC inhibitor + anti-PD-1], but not [HDAC inhibitor + anti-CTLA4], the levels of CCL2, CCL5, CXCL9 and CXCL2 were increased. The cytokine data from HDAC inhibitor plus anti-PD-1 exposed tumors correlated with increased activated T cell, M1 macrophage, neutrophil and NK cell infiltration. Collectively, our data support the use of pan-HDAC inhibitors in combination with kinase inhibitors or with checkpoint inhibitor antibodies as novel melanoma therapeutic strategies.

BACKGROUND

Histone deacetylases (HDACs) modulate chromatin and may influence the effect of DNA-damaging drugs. We investigated HDAC1 and -2 expression in gastric carcinomas (GCs) for an association of patient outcome with conventional neoadjuvant chemotherapy. In vitro, HDAC inhibitors were evaluated as alternative treatment options.

METHODS

HDAC1/2 expression was analyzed immunohistochemically in 127 pretherapeutic biopsy samples of neoadjuvant (platinum/5-fluorouracil) chemotherapy-treated GC patients and correlated with response and overall survival (OS). Chemosensitivity of four GC cell lines to cisplatin and the HDAC inhibitors suberoylanilide hydroxamic acid (SAHA) and valproic acid was determined by XTT assays. Efficiencies of combined drug schedules were analyzed.

RESULTS

High expression of HDAC1/2 was found in 69 (54%) of 127 and 108 (85%) of 127 carcinomas, respectively, and was not associated with response or OS. In patients whose disease responded to therapy, high HDAC1 expression was associated with worse OS (P = 0.005). In cell lines, sequential treatment with SAHA and cisplatin showed synergistic effects irrespective of the initial cisplatin sensitivity.

CONCLUSIONS

HDAC1 and -2 expression is not suitable to predict response or survival for neoadjuvant-treated GC patients, but HDAC1 expression may be used for risk stratification in patients whose disease responds to therapy. Sequential treatment with SAHA and cisplatin may represent an alternative treatment option for GC patients.

Different mechanisms for CBFβ-MYH11 function in acute myeloid leukemia with inv(16) have been proposed such as tethering of RUNX1 outside the nucleus, interference with transcription factor complex assembly and recruitment of histone deacetylases, all resulting in transcriptional repression of RUNX1 target genes. Here, through genome-wide CBFβ-MYH11-binding site analysis and quantitative interaction proteomics, we found that CBFβ-MYH11 localizes to RUNX1 occupied promoters, where it interacts with TAL1, FLI1 and TBP-associated factors (TAFs) in the context of the hematopoietic transcription factors ERG, GATA2 and PU.1/SPI1 and the coregulators EP300 and HDAC1. Transcriptional analysis revealed that upon fusion protein knockdown, a small subset of the CBFβ-MYH11 target genes show increased expression, confirming a role in transcriptional repression. However, the majority of CBFβ-MYH11 target genes, including genes implicated in hematopoietic stem cell self-renewal such as ID1, LMO1 and JAG1, are actively transcribed and repressed upon fusion protein knockdown. Together these results suggest an essential role for CBFβ-MYH11 in regulating the expression of genes involved in maintaining a stem cell phenotype.