We studied the effects of suberoylanilide hydroxamic acid (SAHA), a histone deacetylase (HDAC) inhibitor, on colon cancer. The expression of HDACs in colorectal cancer specimens and the effects of SAHA on colon cancer cells and tumors of nude mice were assessed. Treatment with SAHA (3 μm) for 72 h induced downregulation of different subtypes of HDAC proteins and also induced acetylation of histone 3 and histone 4. SAHA significantly inhibited the expression of the oncogenic protein c-myc and also increased the expression of the p53 and Rb proteins. The immunohistochemical staining of HDACs, including HDAC1, HDAC2, HDAC3, and HDAC4, was significantly increased in colorectal adenocarcinoma specimens compared to healthy control tissues. In addition, murine studies showed that 100 mg/kg SAHA administered by intraperitoneal injection significantly induced tumor necrosis and inhibited the growth of colon tumors. Immunohistochemistry of the tumor tissues from nude mice revealed that SAHA inhibited the expression of different subtypes of histone deacetylase, the anti-apoptotic proteins cyclin D1, survivin, and also inhibited cell proliferative as determined by Ki67 expression. SAHA inhibited the growth of colon tumors by decreasing histone deacetylases and the expression of cyclin D1 and survivin in nude mice.

Dysregulation of DNA methyltransferase (DNMT)1 expression is associated with cellular transformation, and inhibition of DNMT1 exerts antitumorigenic effects. Here, we report that DNMT1 abnormally expressed in HeLa cells is downregulated by a histone deacetylase (HDAC) inhibitor apicidin, which is correlated with induction of repressive histone modifications on the promoter site. Apicidin selectively represses the expression of DNMT1 among DNMTs in HeLa cells, independent of cell cycle arrest at G0/G1. Furthermore, apicidin causes a significant reduction in the recruitment of RNA polymerase II into the promoter. Chromatin immunoprecipitation analysis shows that even though apicidin causes global hyperacetylation of histone H3 and H4, localized deacetylation of histone H3 and H4 occurs at the E2F binding site, which is accompanied by the recruitment of pRB and the replacement of P/CAF with HDAC1 into the sites. In addition, K4-trimethylated H3 on nucleosomes associated with the transcriptional start site is depleted following apicidin treatment, whereas repressive markers, K9- and K27-trimethylation of H3 are enriched on the site. The downregulation of DNMT1 expression seems to require de novo protein synthesis, because the apicidin effect is antagonized by cycloheximide treatment. Moreover, knock down of DNMT1 with siRNA induces the apoptosis of HeLa cells, indicating that downregulation of DNMT1 might be a good strategy for therapeutics of human cervix cancer. Collectively, our findings will provide a mechanistic rationale for the use of HDAC inhibitors in cancer therapeutics.

The sensory hair cells of the cochlea and vestibular organs are essential for normal hearing and balance function. The mammalian ear possesses a very limited ability to regenerate hair cells and their loss can lead to permanent sensory impairment. In contrast, hair cells in the avian ear are quickly regenerated after acoustic trauma or ototoxic injury. The very different regenerative abilities of the avian vs. mammalian ear can be attributed to differences in injury-evoked expression of genes that either promote or inhibit the production of new hair cells. Gene expression is regulated both by the binding of cis-regulatory molecules to promoter regions as well as through structural modifications of chromatin (e.g., methylation and acetylation). This study examined effects of histone deacetylases (HDACs), whose main function is to modify histone acetylation, on the regulation of regenerative proliferation in the chick utricle. Cultures of regenerating utricles and dissociated cells from the utricular sensory epithelia were treated with the HDAC inhibitors valproic acid, trichostatin A, sodium butyrate, and MS-275. All of these molecules prevent the enzymatic removal of acetyl groups from histones, thus maintaining nuclear chromatin in a "relaxed" (open) configuration. Treatment with all inhibitors resulted in comparable decreases in supporting cell proliferation. We also observed that treatment with the HDAC1-, 2-, and 3-specific inhibitor MS-275 was sufficient to reduce proliferation and that two class I HDACs--HDAC1 and HDAC2--were expressed in the sensory epithelium of the utricle. These results suggest that inhibition of specific type I HDACs is sufficient to prevent cell cycle entry in supporting cells. Notably, treatment with HDAC inhibitors did not affect the differentiation of replacement hair cells. We conclude that histone deacetylation is a positive regulator of regenerative proliferation but is not critical for avian hair cell differentiation.

In eukaryotic cells, covalent modifications to core histones contribute to the establishment and maintenance of cellular phenotype via regulation of gene expression. Histone acetyltransferases (HATs) cooperate with histone deacetylases (HDACs) to establish and maintain specific patterns of histone acetylation. HDAC inhibitors can cause pluripotent stem cells to cease proliferating and enter terminal differentiation pathways in culture. To better define the roles of individual HDACs in stem cell differentiation, we have constructed "dominant-negative" stem cell lines expressing mutant, Flag-tagged HDACs with reduced enzymatic activity. Replacement of a single residue (His->>Ala) in the catalytic center reduced the activity of HDACs 1 and 2 by 80%, and abolished HDAC3 activity; the mutant HDACs were expressed at similar levels and in the same multiprotein complexes as wild-type HDACs. Hexamethylene bisacetamide-induced MEL cell differentiation was potentiated by the individual mutant HDACs, but only to 2%, versus 60% for an HDAC inhibitor, sodium butyrate, suggesting that inhibition of multiple HDACs is required for full potentiation. Cultured E14.5 cortical stem cells differentiate to neurons, astrocytes, and oligodendrocytes upon withdrawal of basic fibroblast growth factor. Transduction of stem cells with mutant HDACs 1, 2, or 3 shifted cell fate choice toward oligodendrocytes. Mutant HDAC2 also increased differentiation to astrocytes, while mutant HDAC1 reduced differentiation to neurons by 50%. These results indicate that HDAC activity inhibits differentiation to oligodendrocytes, and that HDAC2 activity specifically inhibits differentiation to astrocytes, while HDAC1 activity is required for differentiation to neurons.

In utero exposure of rodents to valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, has been proposed to induce an adult phenotype with behavioural characteristics reminiscent of those observed in autism spectrum disorder (ASD). We have evaluated the face validity of this model in terms of social cognition deficits which are a major core symptom of ASD. We employed the social approach avoidance paradigm as a measure of social reciprocity, detection of biological motion that is crucial to social interactions, and spatial learning as an indicator of dorsal stream processing of social cognition and found each parameter to be significantly impaired in Wistar rats with prior in utero exposure to VPA. We found no significant change in the expression of neural cell adhesion molecule polysialylation state (NCAM PSA), a measure of construct validity, but a complete inability to increase its glycosylation state which is necessary to mount the neuroplastic response associated with effective spatial learning. Finally, in all cases, we found chronic HDAC inhibition, with either pan-specific or HDAC1-3 isoform-specific inhibitors, to significantly ameliorate deficits in both social cognition and its associated neuroplastic response. We conclude that in utero exposure to VPA provides a robust animal model for the social cognitive deficits of ASD and a potential screen for the development of novel therapeutics for this condition.

The ability to fully restore damaged or lost organs is present in only a subset of animals. The Xenopus tadpole tail is a complex appendage, containing epidermis, muscle, nerves, spinal cord, and vasculature, which regenerates after amputation. Understanding the mechanisms of tail regeneration may lead to new insights to promote biomedical regeneration in non-regenerative tissues. Although chromatin remodeling is known to be critical for stem cell pluripotency, its role in complex organ regeneration in vivo remains largely uncharacterized. Here we show that histone deacetylase (HDAC) activity is required for the early stages of tail regeneration. HDAC1 is expressed during the 1(st) two days of regeneration. Pharmacological blockade of HDACs using Trichostatin A (TSA) increased histone acetylation levels in the amputated tail. Furthermore, treatment with TSA or another HDAC inhibitor, valproic acid, specifically inhibited regeneration. Over-expression of wild-type Mad3, a transcriptional repressor known to associate in a complex with HDACs via Sin3, inhibited regeneration. Similarly, expression of a Mad3 mutant lacking the Sin3-interacting domain that is required for HDAC binding also blocks regeneration, suggesting that HDAC and Mad3 may act together to regulate regeneration. Inhibition of HDAC function resulted in aberrant expression of Notch1 and BMP2, two genes known to be required for tail regeneration. Our results identify a novel early role for HDAC in appendage regeneration and suggest that modulation of histone acetylation is important in regenerative repair of complex appendages.

Dietary supplementation of butyrate can prevent diet-induced obesity through increasing mitochondrial function in mice, yet the up-stream signaling pathway remains elusive. In this study, weaned mice were divided into two groups, fed control (CON) and high-fat diet (HF, 45% energy from fat), respectively, for 8 weeks. HF-induced obese mice, maintained on HF diet, were then divided into two groups; HFB group was gavaged with 80 mg sodium butyrate (SB) per mice every other day for 10 days, while the HF group received vehicle. It was shown that five gavage doses of SB significantly alleviated HF diet-induced obesity and restored plasma glucose, insulin and leptin to control levels. Muscle contents of ADP and AMP were significantly increased, which was associated with enhanced mitochondrial oxidative phosphorylation and up-regulated expression of fatty acid oxidation enzymes and uncoupling proteins, UCP2 and UCP3 in the skeletal muscle. SB significantly enhanced the expression of adiponectin receptors (adipoR1/2) and AMP kinase (AMPK), while diminished the expression of histone deacetylase 1 (HDAC1). Higher H3K9Ac, a gene activation histone mark, was detected on the promoter of Adipor1/2, Ucp2 and Ucp3 genes that were activated in the muscle of SB-treated obese mice. Our results indicate that short-term oral administration of SB can alleviate diet-induced obesity and insulin resistance in mice through activation of adiponectin-mediated pathway and stimulation of mitochondrial function in the skeletal muscle.

Histone deacetylase (HDAC) inhibitors induce cell cycle arrest, differentiation or apoptosis in tumour cells and are, therefore, promising anti-cancer reagents. However, the specific HDAC isoforms that mediate these effects are not yet identified. To explore the role of HDAC1 in tumourigenesis and tumour proliferation, we established an experimental teratoma model using wild-type and HDAC1-deficient embryonic stem cells. HDAC1-deficient teratomas showed no significant difference in size compared with wild-type teratomas. Surprisingly, loss of HDAC1 was not only linked to increased apoptosis, but also to significantly enhanced proliferation. Epithelial structures showed reduced differentiation as monitored by Oct3/4 expression and changed E-cadherin localization and displayed up-regulated expression of SNAIL1, a regulator of epithelial cell plasticity. Increased levels of the transcriptional regulator SNAIL1 are crucial for enhanced proliferation and reduced differentiation of HDAC1-deficient teratoma. Importantly, the analysis of human teratomas revealed a similar link between loss of HDAC1 and enhanced tumour malignancy. These findings reveal a novel role for HDAC1 in the control of tumour proliferation and identify HDAC1 as potential marker for benign teratomas.

OBJECTIVE

Overexpression of histone deacetylase (HDAC)1, which controls the expression of genes related to cell cycle and apoptosis, has recently been reported in gastric cancer (GC) tissues. In the present study, the pattern of gelsolin and retinoic acid receptor (RAR)beta expression in GC tissues showing HDAC1 overexpression was investigated.

METHODS

Expression profiles of HDAC1, gelsolin, and RARbeta were evaluated and compared using reverse transcription-polymerase chain reaction, immunoblotting, and immunohistochemical analyses with 22 paired primary human GC tissues and corresponding normal tissues.

RESULTS

Compared with normal gastric tissue, increased expression of HDAC1 mRNA and protein was detected in 17 (77.3%) of 22 GC tissues, while decreased expressions of gelsolin mRNA and protein were shown in 15 (68.1%) samples. Concomitantly, expressions of RARbeta mRNA and protein were decreased in 16 (72.7%) and 17 (77.3%), respectively. Among 17 GC tissues with increased HDAC1 expression, the expressions of gelsolin and RARbeta were simultaneously decreased in 14 (82.4%) and 15 (88.2%) GC tissues, which indicates a strong inverse correlation between HDAC1 and gelsolin/RARbeta expressions. Correlation between HDAC1 and gelsolin/RARbeta was also confirmed by immunohistochemistry.

CONCLUSIONS

Taken together, the results of the present study reveal that silencing of gelsolin and RARbeta occurs in GC tissues probably through HDAC1 overexpression and might play some role in gastric carcinogenesis.

BACKGROUND

Upregulation of glucocorticoid receptor β (GRβ) has been implicated in steroid resistance in severe asthma, although previous studies are conflicting. GRβ has been proposed as a dominant negative isoform of glucocorticoid receptor α (GRα) but it has also been suggested that GRβ can cause steroid resistance via reduced expression of histone deacetylase 2 (HDAC2), a key regulator of steroid responsiveness in the airway.

OBJECTIVE

To examine GRβ, GRα, HDAC1 and HDAC2 expression at transcript and protein levels in bronchial biopsies from a large series of patients with severe asthma, and to compare the findings with those of patients with mild to moderate asthma and healthy volunteers.

METHODS

Bronchoscopic study in two UK centres with real-time PCR and immunohistochemistry performed on biopsies, western blotting of bronchial epithelial cells and immunoprecipitation with anti-GRβ antibody.

RESULTS

Protein and mRNA expression for GRα and HDAC2 did not differ between groups. GRβ mRNA was detected in only 13 of 73 samples (seven patients with severe asthma), however immunohistochemistry showed widespread epithelial staining in all groups. Western blotting of bronchial epithelial cells with GRβ antibody detected an additional 'cross-reacting' protein, identified as clathrin. HDAC1 expression was increased in patients with severe asthma compared with healthy volunteers.

CONCLUSIONS

GRβ mRNA is expressed at low levels in a minority of patients with severe asthma. HDAC1 and HDAC2 expression was not downregulated in severe asthma. These data do not support upregulated GRβ and resultant reduced HDAC expression as the principal mechanism of steroid resistance in severe asthma. Conflicting GRβ literature may be explained in part by clathrin cross-reactivity with commercial antibodies.

OBJECTIVE

The retinoblastoma-interacting zinc finger gene RIZ1 is inactivated in many cancers, but the underlying mechanisms remain unknown. This study aimed to investigate the epigenetic mechanisms of RIZ1 inactivation by analyzing the relationship between DNA methylation and histone modifications during regulation of RIZ1 expression.

METHODS

Methylation-specific PCR, RT-PCR, and immunohistochemistry were performed to examine RIZ1 methylation and expression. Dynamic changes in histone H3 lysine 9 (H3K9) modifications and histone deacetylases (HDACs) associated with the promoter were analyzed by chromatin immunoprecipitation (ChIP).

RESULTS

RIZ1 methylation was detected in 66.7% (32/48) HCC tissues, 6.3% (3/48) corresponding non-cancerous tissues, and 66.7% (4/6) HCC cell lines. All 32 HCC tissues with promoter methylation showed complete loss of RIZ1 protein, whereas RIZ1 protein was present in all the corresponding non-cancerous tissues. Neither 5-aza-2-deoxycitidine (5-Aza-dC) nor Trichostatin A (TSA) reversed promoter methylation, but did restore RIZ1 mRNA and resulted in the downregulation of HDAC1 but not HDAC3. However, 5-Aza-dC+TSA induced a partial reversal of promoter methylation and a markedly synergistic reactivation of RIZ1. Moreover, both HDAC1 and HDAC3 were downregulated. The ChIP assays showed 5-Aza-dC and/or TSA also contributed to the dynamic conversion of trimethylated to acetylated H3K9 at the promoter. Furthermore, a decrease in H3K9 trimethylation preceded an increase in H3K9 acetylation.

CONCLUSIONS

Our results suggest that promoter methylation and H3K9 modifications work together to silence the RIZ1 gene in HCC. 5-Aza-dC can restore the expression of RIZ1, as reflected by its effects on histone modification levels. This finding indicates that cooperative effects between these epigenetic modifications exist.

Psammaplin A (11c) is a marine metabolite previously reported to be a potent inhibitor of two classes of epigenetic enzymes: histone deacetylases and DNA methyltransferases. The design and synthesis of a focused library based on the psammaplin A core has been carried out to probe the molecular features of this molecule responsible for its activity. By direct in vitro assay of the free thiol generated upon reduction of the dimeric psammaplin scaffold, we have unambiguously demonstrated that 11c functions as a natural prodrug, with the reduced form being highly potent against HDAC1 in vitro (IC(50) 0.9 nM). Furthermore, we have shown it to have high isoform selectivity, being 360-fold selective for HDAC1 over HDAC6 and more than 1000-fold less potent against HDAC7 and HDAC8. SAR around our focused library revealed a number of features, most notably the oxime functionality to be important to this selectivity. Many of the compounds show significant cytotoxicity in A549, MCF7, and W138 cells, with the SAR of cytotoxicity correlating to HDAC inhibition. Furthermore, compound treatment causes upregulation of histone acetylation but little effect on tubulin acetylation. Finally, we have found no evidence for 11c functioning as a DNMT inhibitor.

To better understand the link between chromatin modification and manganese superoxide dismutase (Mn-SOD) gene expression, we have investigated the level of histone acetylation at Mn-SOD proximal promoter. TSA induced the expression of Mn-SOD mRNA and its transcriptional activity in C2C12 cells. Sp1 binding sites in the proximal promoter region of Mn-SOD were transcriptionally responsive to TSA by transfection studies. We have detected a localized acetylation of histones H3 and H4, in vivo occupation by Sp1, early growth responsive-1 (Egr-1), and histone deacetylase-1 (HDAC1) in the proximal promoter region of Mn-SOD gene using chromatin immunoprecipitation assays. Our findings indicate that Mn-SOD gene expression is repressed by Sp1-HDAC1 complex. This repression is released by a localized histone acetylation and at least in parts a displacement by Egr-1 in response to TSA.

Histidine triad nucleotide-binding protein 1 (HINT1) is a haploinsufficient tumor suppressor gene that inhibits the Wnt/β-catenin pathway in colon cancer cells and Microphthalmia-associated transcription factor (MITF) activity in human mast cells. MITF and β-catenin play a central role in melanocyte and melanoma cell survival, and this study aimed to investigate the effects of HINT1 on the MITF and β-catenin pathways in malignant melanoma cells. We found that HINT1 inhibits MITF and β-catenin transcriptional activity, and both proteins can be co-immunoprecipitated with an anti-HINT1-specific antibody in melanoma cell lines. Stable, constitutive overexpression of the HINT1 protein in human melanoma cells significantly impaired cell proliferation in vitro and tumorigenesis in vivo. These effects were associated with a decreased expression of cyclin D1 and BCL2, well known MITF and β-catenin transcription targets, respectively. We also demonstrated that BCL2 and cyclin D1 can partially rescue the HINT1-driven phenotype. Moreover, we found in ChIP assays that HINT1 binds the chromatin at MITF and β-catenin sites in BCL2 and cyclin D1 promoters, respectively, and that mSIN3a and HDAC1, well known transcriptional repressors, can be co-immunoprecipitated with an anti-HINT1-specific antibody. These findings support the tumor suppressor activity of HINT1 gene in melanoma cells by promoting the formation of non-functional complexes with oncogenic transcription factors like MITF and β-catenin.

Histone deacetylases (HDACs) are enzymes that are able to deacetylate lysine side chains in histones and certain non-histone proteins which leads to altered states of conformation and activity for the proteins in question. Three classes of histone deacetylases have been recognized in humans. Class I and II are zinc-dependent amidohydrolases and eleven subtypes have been discovered (HDAC1-11). Class III enzymes depend in their catalysis on NAD(+) and subsequently, O-acetyl ADP ribose and nicotinamide are formed as a consequence of the acetyl transfer. Due to the homology to the yeast histone deacetylase Sir2p the NAD(+)-dependent deacetylases are also termed sirtuins and seven members (Sirt1-7) are known in humans. Sirtuins are found from bacteria to eukaryotes and altogether about 60 isoforms have been characterized in different organisms. Sirtuins have been implicated in the regulation of molecular mechanisms of aging. The overexpression of sirtuin enzymatic activity leads to an increase of lifespan in Saccharomyces cerevisiae and Caenorhabditis elegans that can also be reached by calorie restriction. Sirtuins have been proposed to act as sensors for glucose uptake that respond to the levels of NAD(+) but more complex ways of action have been suggested as well. This article will present the members of the human sirtuin family with their respective functions and review the existing druglike inhibitors and activators of sirtuin activity.

Histone deacetylases are important targets for cancer therapeutics, but their regulation is poorly understood. Our data show coordinated transcription of HDAC1 and HDAC2 in lung cancer cell lines, but suggest HDAC2 protein expression is cell-context specific. Through an unbiased siRNA screen we found that BRCA1-associated protein 1 (BAP1) regulates their expression, with HDAC2 reduced and HDAC1 increased in BAP1 depleted cells. BAP1 loss-of-function is increasingly reported in cancers including thoracic malignancies, with frequent mutation in malignant pleural mesothelioma. Endogenous HDAC2 directly correlates with BAP1 across a panel of lung cancer cell lines, and is downregulated in mesothelioma cell lines with genetic BAP1 inactivation. We find that BAP1 regulates HDAC2 by increasing transcript abundance, rather than opposing its ubiquitylation. Importantly, although total cellular HDAC activity is unaffected by transient depletion of HDAC2 or of BAP1 due to HDAC1 compensation, this isoenzyme imbalance sensitizes MSTO-211H cells to HDAC inhibitors. However, other established mesothelioma cell lines with low endogenous HDAC2 have adapted to become more resistant to HDAC inhibition. Our work establishes a mechanism by which BAP1 loss alters sensitivity of cancer cells to HDAC inhibitors. Assessment of BAP1 and HDAC expression may ultimately help identify patients likely to respond to HDAC inhibitors.

miR-34a is an important molecule that can inhibit the tumor growth. This study aimed to investigate the functional role of miR-34a in hepatocellular carcinoma (HCC) and explore the interaction between miR-34a and histone deacetylase 1 (HDAC1). RT-qPCR was employed to detect the mRNA expression of miR-34a and HDAC1 in 60 HCC tissues. Results showed miR-34a expression in HCC tissues was significantly lower than in normal tissues (P<0.05), but HDAC1 expression in HCC tissues was markedly higher than in normal tissues (P<0.05). In addition, miR-34a expression was negatively related to HDAC1 expression. miR-34a mimic was transfected into HCC cell lines (HepB3 and HepG2). CCK8 assay, colony formation assay and flow cytometry showed miR-34a over-expression could inhibit the proliferation of HCC cells and induce their apoptosis. Western blotting indicated miR-34a over-expression down-regulated the expression of Bcl-2, procaspase-3, procaspase-9 and c-Myc, but up-regulate p21 expression. Bioinformatics analysis indicated HDAC1 was a target gene of miR-34a. Dual Luciferase Reporter Gene Assay and retrieval assay showed miR-34a could act at the 3'UTR of HDAC1 gene to regulate its expression. Thus, miR-34a may inhibit the proliferation of HCC cells and induce their apoptosis via regulating HDAC1 expression. Our findings provide evidence for the diagnosis and therapeutic target of HCC.

Real space flight and modeled microgravity conditions result in changes in the expression of genes that control important cellular functions. However, the mechanisms for microgravity-induced gene expression changes are not clear. The epigenetic changes of DNA methylation and chromatin histones modifications are known to regulate gene expression. The objectives of this study were to investigate whether simulated microgravity alters (a) the DNA methylation and histone acetylation, and (b) the expression of DNMT1, DNMT3a, DNMT3b, and HDAC1 genes that regulate epigenetic events. To achieve these objectives, human T-lymphocyte cells were grown in a rotary cell culture system (RCCS) that simulates microgravity, and in parallel under normal gravitational conditions as control. The microgravity-induced DNA methylation changes were detected by methylation sensitive-random amplified polymorphic DNA (MS-RAPD) analysis of genomic DNA. The gene expression was measured by Quantitative Real-time PCR. The expression of DNMT1, DNMT3a, and DNMT3b was found to be increased at 72 h, and decreased at 7 days in microgravity exposed cells. The MS-RAPD analysis revealed that simulated microgravity exposure results in DNA hypomethylation and mutational changes. Gene expression analysis revealed microgravity exposure time-dependent decreased expression of HDAC1. Decreased expression of HDAC1 should result in increased level of acetylated histone H3, however a decreased level of acetylated H3 was observed in microgravity condition, indicating thereby that other HDACs may be involved in regulation of H3 deacetylation. The findings of this study suggest that epigenetic events could be one of the mechanistic bases for microgravity-induced gene expression changes and associated adverse health effects.

Small Heterodimer Partner (SHP) inhibits numerous transcription factors that are involved in diverse biological processes, including lipid and glucose metabolism. In response to increased hepatic bile acids, SHP gene expression is induced and the SHP protein is stabilized. We now show that the activity of SHP is also increased by posttranslational methylation at Arg-57 by protein arginine methyltransferase 5 (PRMT5). Adenovirus-mediated hepatic depletion of PRMT5 decreased SHP methylation and reversed the suppression of metabolic genes by SHP. Mutation of Arg-57 decreased SHP interaction with its known cofactors, Brm, mSin3A, and histone deacetylase 1 (HDAC1), but not with G9a, and decreased their recruitment to SHP target genes in mice. Hepatic overexpression of SHP inhibited metabolic target genes, decreased bile acid and hepatic triglyceride levels, and increased glucose tolerance. In contrast, mutation of Arg-57 selectively reversed the inhibition of SHP target genes and metabolic outcomes. The importance of Arg-57 methylation for the repression activity of SHP provides a molecular basis for the observation that a natural mutation of Arg-57 in humans is associated with the metabolic syndrome. Targeting posttranslational modifications of SHP may be an effective therapeutic strategy by controlling selected groups of genes to treat SHP-related human diseases, such as metabolic syndrome, cancer, and infertility.

Mutation of p53 is a frequent genetic lesion in pancreatic cancer being an unmet clinical challenge. Mutants of p53 have lost the tumour-suppressive functions of wild type p53. In addition, p53 mutants exert tumour-promoting functions, qualifying them as important therapeutic targets. Here, we show that the class I histone deacetylases HDAC1 and HDAC2 contribute to maintain the expression of p53 mutants in human and genetically defined murine pancreatic cancer cells. Our data reveal that the inhibition of these HDACs with small molecule HDAC inhibitors (HDACi), as well as the specific genetic elimination of HDAC1 and HDAC2, reduce the expression of mutant p53 mRNA and protein levels. We further show that HDAC1, HDAC2 and MYC directly bind to the TP53 gene and that MYC recruitment drops upon HDAC inhibitor treatment. Therefore, our results illustrate a previously unrecognized class I HDAC-dependent control of the TP53 gene and provide evidence for a contribution of MYC. A combined approach targeting HDAC1/HDAC2 and MYC may present a novel and molecularly defined strategy to target mutant p53 in pancreatic cancer.

Atrial natriuretic peptide (ANP) binds guanylyl cyclase-A/natriuretic peptide receptor-A (GC-A/NPRA) and produces the intracellular second messenger, cGMP, which regulates cardiovascular homeostasis. We sought to determine the function of histone deacetylases (HDACs) in regulating Npr1 (coding for GC-A/NPRA) gene transcription, using primary mouse mesangial cells treated with class-specific HDAC inhibitors (HDACi). Trichostatin A, a pan inhibitor, and mocetinostat (MGCD0103), a class I HDAC inhibitor, significantly enhanced Npr1 promoter activity (by 8- and 10-fold, respectively), mRNA levels (4- and 5.3-fold, respectively), and NPRA protein (2.7- and 3.5-fold, respectively). However, MC1568 (class II HDAC inhibitor) had no discernible effect. Overexpression of HDAC1 and HDAC2 significantly attenuated Npr1 promoter activity, whereas HDAC3 and HDAC8 had no effect. HDACi-treated cultured cells in vitro and intact animals in vivo showed significantly reduced binding of HDAC1 and -2 and increased accumulation of acetylated H3-K9/14 and H4-K12 at the Npr1 promoter. Deletional analyses of the Npr1 promoter along with ectopic overexpression and inhibition of Sp1 confirmed that HDACi-induced Npr1 gene transcription is accomplished by Sp1 activation. Furthermore, HDACi attenuated the interaction of Sp1 with HDAC1/2 and promoted Sp1 association with p300 and p300/cAMP-binding protein-associated factor; it also promoted the recruitment of p300 and p300/cAMP-binding protein-associated factor to the Npr1 promoter. Our results demonstrate that trichostatin A and MGCD0103 enhanced Npr1 gene expression through inhibition of HDAC1/2 and increased both acetylation of histones (H3-K9/14, H4-K12) and Sp1 by p300, and their recruitment to Npr1 promoter. Our findings define a novel epigenetic regulatory mechanism that governs Npr1 gene transcription.

The clinical promise of cancer immunotherapy relies on the premise that the immune system can recognize and eliminate tumor cells identified as non-self. However, tumors can evade host immune surveillance through multiple mechanisms, including epigenetic silencing of genes involved in antigen processing and immune recognition. Hence, there is an unmet clinical need to develop effective therapeutic strategies that can restore tumor immune recognition when combined with immunotherapy, such as immune checkpoint blockade and therapeutic cancer vaccines. We sought to examine the potential of clinically relevant exposure of prostate and breast human carcinoma cells to histone deacetylase (HDAC) inhibitors to reverse tumor immune escape to T-cell mediated lysis. Here we demonstrate that prostate (LNCAP) and breast (MDA-MB-231) carcinoma cells are more sensitive to T-cell mediated lysis in vitro after clinically relevant exposure to epigenetic therapy with either the pan-HDAC inhibitor vorinostat or the class I HDAC inhibitor entinostat. This pattern of immunogenic modulation was observed against a broad range of tumor-associated antigens, such as CEA, MUC1, PSA, and brachyury, and associated with augmented expression of multiple proteins involved in antigen processing and tumor immune recognition. Genetic and pharmacological inhibition studies identified HDAC1 as a key determinant in the reversal of carcinoma immune escape. Further, our findings suggest that the observed reversal of tumor immune evasion is driven by a response to cellular stress through activation of the unfolded protein response. This offers the rationale for combining HDAC inhibitors with immunotherapy, including therapeutic cancer vaccines.

Control of host epigenetics is becoming evident as a mechanism by which symbionts and pathogens survive. Anaplasma phagocytophilum, an obligate intracellular bacterium, down-regulates multiple host defence genes where histone deacetylase 1 (HDAC1) binds and histone 3 is deacetylated at their promoters, including the NADPH oxidase component, CYBB. How HDAC1 is targeted to defence gene promoters is unknown. Ankyrin A (AnkA), an A. phagocytophilum type IV secretion system effector, enters the granulocyte nucleus, binds stretches of AT-rich DNA and alters transcription of antimicrobial defence genes, including down-regulation of CYBB. Here we found AnkA binds to a predicted matrix attachment region in the proximal CYBB promoter. Using the CYBB promoter as a model of cis-gene silencing, we interrogated the mechanism of AnkA-mediated CYBB repression. The N-terminus of AnkA was critical for nuclear localization, the central ANK repeats and C-terminus were important for DNA binding, and most promoter activity localized to the central ANK repeats. Furthermore, a direct interaction between AnkA and HDAC1 was detected at the CYBB promoter, and was critical for AnkA-mediated CYBB repression. This novel microbial manipulation of host chromatin and gene expression provides important evidence of the direct effects that prokaryotic nuclear effectors can exert over host transcription and function.

Atrichia with papular lesions (APL) and hereditary vitamin D-resistant rickets have a similar congenital hair loss disorder caused by mutations in hairless (HR) and vitamin D receptor (VDR) genes, respectively. HR is a VDR corepressor, and it has been hypothesized that VDR.HR suppress gene expression during specific phases of the hair cycle. In this study, we examined the corepressor activity of HR mutants (E583V, C622G, N970S, V1056M, D1012N, V1136D, and Q1176X) previously described as the molecular cause of APL as well as HR variants (P69S, C397Y, A576V, E591G, R620Q, T1022A) due to non-synonymous polymorphisms in the HR gene. We found that the corepressor activities of all but one of the pathogenic HR mutants were completely abolished. HR mutant E583V exhibited normal corepressor activity, suggesting that it may not be pathogenic. In co-immunoprecipitation assays, all of the pathogenic HR mutants bound VDR but exhibited reduced binding to histone deacetylase 1 (HDAC1), suggesting that the impaired corepressor activity is due in part to defective interactions with HDACs. The HR variants exhibited two classes of corepressor activity, those with normal activity (C397Y, E591G, R620Q) and those with partially reduced activity (P69S, A576V, T1022A). All of the variants interacted with VDR and HDAC1 with the exception of P69S, which was degraded. When coexpressed with VDR, all of the HR pathogenic mutants and variants increased the level of VDR protein, demonstrating that this function of HR was not impaired by these mutations. This study of HR mutations provides evidence for the molecular basis of APL.