Level Of Histone H3K9 Acetylation Research Articles

SIRT1 mediates nutritional regulation of SREBP-1c-driven hepatic PNPLA3 transcription via modulation of H3k9 acetylation

BackgroundPatatin-like phospholipase domain containing 3 (PNPLA3) is the main nonalcoholic fatty liver disease (NAFLD) susceptibility. Its expression is regulated tightly by nutritional and energy status, but the mechanism of epigenetic regulation of PNPLA3 gene by nutritional dietary factors has not been reported. Here, we investigated the effect and mechanism of Sirtuin 1 (SIRT1) regulated H3K9 deacetylation on PNPLA3 transcriptional expression in vivo and in vitro.MethodsMouse models of fasting/re-feeding transition and nonalcoholic fatty liver induced by high Sucrose diet were constructed; and HepG2 cells were treated with serum- and glucose-free medium or exposed to high glucose and high insulin, to generate fasting and high-glucose-induced lipid deposition cell states. Enrichment levels of histone H3K9 acetylation and sterol responsive element binding protein-1c (SREBP-1c) at the PNPLA3 promoter were observed by ChIP-qPCR. PNPLA3 gene expression was detected by real-time PCR; SIRT1 protein expression was detected by western blot. And lipid deposition was detected by Oil Red O.ResultsH3K9ac levels at SRE regions of PNPLA3 promoter were found to be decreased in mice during fasting and increase during refeeding, and increased in mice with NAFLD induced by high-sucrose diet. The change pattern of PNPLA3 promoter H3K9Ac physiologically (fasting/refeeding) and pathologically was consistent with that of PNPLA3 gene expression, but opposite to that of SIRT1 protein expression. In HepG2 cells, overexpression of SIRT1 inhibited high-glucose induced hyper-acetylation of H3K9 at PNPLA3 promoter, and silent expression of SIRT1 suppressed fasting-induced hypo-acetylation of H3K9. Overexpression of SIRT1 prevented basal and SREBP-1c-driven PNPLA3 gene expression and also prevented the endogenous binding of SREBP-1c to PNPLA3.ConclusionsWe first preliminarily revealed SIRT1 may regulate PNPLA3 gene expression by affecting SREBP-1-driven transcription via acetylation modification of H3K9.

FK228 sensitizes radioresistant small cell lung cancer cells to radiation

BackgroundConcurrent thoracic radiation plus chemotherapy is the mainstay of first-line treatment for limited-stage small cell lung cancer (LS-SCLC). Despite initial high responsiveness to combined chemo- and radiotherapy, SCLC almost invariably relapses and develops resistance within one year, leading to poor prognosis in patients with LS-SCLC. Developing new chemical agents that increase ionizing radiation’s cytotoxicity against SCLC is urgently needed.ResultsDual histone deacetylase (HDAC) and PI3K inhibitor FK228 not only displayed potent anticancer activity, but also enhanced the therapeutic effects of radiotherapy in SCLC cells. Mechanistically, radioresistant SCLC cells exhibit a lower level of histone H3K9 acetylation and a higher expression level of the MRE11-RAD50-NBS1 (MRN) complex and show more efficient and redundant DNA damage repair capacities than radiosensitive SCLC cells. FK228 pretreatment resulted in marked induction of H3k9 acetylation, attenuated homologous recombination (HR) repair competency and impaired non-homologous end joining (NHEJ) repair efficacy, leading to the accumulation of radiation-induced DNA damage and radiosensitization.ConclusionThe study uncovered that FK228 sensitized human radioresistant SCLC cells to radiation mainly through induction of chromatin decondensation and suppression of DNA damage signaling and repair. Our study provides a rational basis for a further clinical study to test the potential of FK228 as a radiosensitizing agent to increase the radiation-induced tumor cell kill in LS-SCLC patients.

SIRT1 alleviates isoniazid-induced hepatocyte injury by reducing histone acetylation in the IL-6 promoter region

Silent information regulator 1 (SIRT1) is a type III histone deacetylase that is related to the inhibition of the inflammatory response. The aim of this study was to investigate the regulation of SIRT1 on isoniazid-induced hepatocyte injury and the possible mechanism of histone modification. We found that compared with the blank control group, expression of SIRT1 was decreased in the isoniazid group and that expression of NF-κB p65 was increased, leading to an increase of the expression of inflammatory cytokines Interleukin-6 (IL-6) and Tumour necrosis factor alpha (TNF-α). The level of histone H3K9 acetylation in the promoter region of IL-6 was increased as well. Addition of a SIRT1 agonist (SRT1720) alleviated the inflammatory reaction caused by isoniazid, while the use of a SIRT1 inhibitor (EX527) aggravated the inflammatory damage to cells. In conclusion, these findings indicated that during the period of isoniazid-induced hepatocyte injury, SIRT1 levels were decreased and inflammatory factor levels were increased. Activation of SIRT1 may reduce hepatocyte injury by reducing the level of histone H3K9 acetylation in the promoter region of the IL-6 gene.

Inhibition of histone acetylation by curcumin reduces alcohol-induced fetal cardiac apoptosis

BackgroundPrenatal alcohol exposure may cause cardiac development defects, however, the underlying mechanisms are not yet clear. In the present study we have investigated the roles of histone modification by curcumin on alcohol induced fetal cardiac abnormalities during the development.Methods and resultsQ-PCR and Western blot results showed that alcohol exposure increased gene and active forms of caspase-3 and caspase-8, while decreased gene and protein of bcl-2. ChIP assay results showed that, alcohol exposure increased the acetylation of histone H3K9 near the promoter region of caspase-3 and caspase-8, and decreased the acetylation of histone H3K9 near the promoter region of bcl-2. TUNEL assay data revealed that alcohol exposure increased the apoptosis levels in the embryonic hearts. In vitro experiments demonstrated that curcumin treatment could reverse the up-regulation of active forms of caspase-3 and caspase-8, and down-regulation of bcl-2 induced by alcohol treatment. In addition, curcumin also corrected the high level of histone H3K9 acetylation induced by alcohol. Moreover, the high apoptosis level induced by alcohol was reversed after curcumin treatment in cardiac cells.ConclusionsThese findings indicate that histone modification may play an important role in mediating alcohol induced fetal cardiac apoptosis, possibly through the up-regulation of H3K9 acetylation near the promoter regions of apoptotic genes. Curcumin treatment may correct alcohol-mediated fetal cardiac apoptosis, suggesting that curcumin may play a protective role against alcohol abuse caused cardiac damage during pregnancy.

Chronic stress leads to epigenetic dysregulation in the neuropeptide-Y and cannabinoid CB1 receptor genes in the mouse cingulate cortex

Persistent stress triggers a variety of mechanisms, which may ultimately lead to the occurrence of anxiety- and depression-related disorders. Epigenetic modifications represent a mechanism by which chronic stress mediates long-term effects. Here, we analyzed brain tissue from mice exposed to chronic unpredictable stress (CUS), which induced impaired emotional and nociceptive behaviors. As endocannabinoid (eCB) and neuropeptide-Y (Npy) systems modulate emotional processes, we hypothesized that CUS may affect these systems through epigenetic mechanisms. We found reduced Npy expression and Npy type 1 receptor (Npy1r) signaling, and decreased expression of the cannabinoid type 1 receptor (CB1) in the cingulate cortex of CUS mice specifically in low CB1-expressing neurons. Epigenetic investigations revealed reduced levels of histone H3K9 acetylation (H3K9ac) associated to Npy and CB1 genes, which may represent a factor determining the dysregulation occurring at expression and signaling level. CUS mice also showed increased nuclear protein levels and activity of the histone deacetylase type 2 (HDAC2) in the cingulate cortex as compared to controls. Chronic administration of URB597, an inhibitor of anandamide degradation, which is known to induce anxiolysis in CUS mice, reversed the epigenetic changes found in the Npy gene, but was ineffective in alleviating the dysregulation of Npy at transcriptional and signaling level. Our findings suggest that epigenetic alterations in the Npy and CB1 genes represent one of the potential mechanisms contributing to the emotional imbalance induced by CUS in mice, and that the Npy and eCB systems may represent therapeutic targets for the treatment of psychopathologies associated with or triggered by chronic stress states.

Histone deacetylase HDA9 negatively regulates salt and drought stress responsiveness in Arabidopsis.

Histone modification is an important epigenetic regulation in higher plants adapting to environment changes including salt and drought stresses. In this report, we show that the Arabidopsis RPD3-type histone deacetylase HDA9 is involved in modulating plant responses to salt and drought stresses in Arabidopsis. Loss-of-function mutants of the gene displayed phenotypes (such as seedling root growth and seed germination) insensitive to NaCl and polyethylene glycol (PEG) treatments. HDA9 mutation led to up-regulation of many genes, among which those involved in response to water deprivation stress (GO: 0009414) were enriched. These genes were much more induced in the mutants than wild-type plants when treated with PEG and NaCl. In addition, we found that in the mutants, salt and drought stresses led to much higher levels of histone H3K9 acetylation at promoters of 14 genes randomly selected from those that respond to water-deprivation stress than in wild-type plants. Our study suggested that HDA9 might be a novel chromatin protein that negatively regulates plant sensitivity to salt and drought stresses by regulating histone acetylation levels of a large number of stress-responsive genes in Arabidopsis.

MYBD employed by HY5 increases anthocyanin accumulation via repression of MYBL2 in Arabidopsis.

Photomorphogenesis is an essential program in plant development. This process is effected by the balanced cooperation of many factors under light and dark conditions. In a previous study, we showed that MYB hypocotyl elongation-related (MYBH) is involved in cell elongation. To expand our understanding of MYBH function, we performed a yeast two-hybrid assay and identified an MYB-like Domain transcription factor (MYBD). In this study, we investigated the function of MYBD, which is an MYBH homolog involved in anthocyanin accumulation. MYBD expression increased in response to light or cytokinin, and MYBD enhanced anthocyanin biosynthesis via repression of MYBL2, which encodes a transcription factor that has a negative effect on this process. In addition, MYBD binding in vivo to the MYBL2 promoter and the lower level of histone H3K9 acetylation at the upstream region of MYBL2 in MYBD over-expressing plants in comparison with wild-type plants imply that MYBD represses MYBL2 expression via an epigenetic mechanism. HY5 directly binds to the MYBD promoter, which indicates that MYBD acts on HY5-downstream in light- or cytokinin-triggered signaling pathways, leading to anthocyanin accumulation. Our results suggest that, although MYBD and MYBH are homologs, they act in opposite ways during plant photomorphogenesis, and these functions should be examined in further studies.

Knockdown of Rad9A enhanced DNA damage induced by trichostatin A in esophageal cancer cells.

Histone deacetylase (HDAC) inhibitors have recently emerged as a new class of anticancer agents. As a classical HDAC inhibitor, trichostatin A (TSA) has been shown to possess many anticancer activities such as induction of cell cycle arrest, promotion of cell death, and enhancement of radiosensitity. In our previous work, we found that TSA treatment induced Rad9 gene expression, which suggested that Rad9 might play a role in TSA-induced biological effects. As Rad9 is involved in maintaining genomic integrity, we further analyzed the DNA damage induced by TSA and combined with Rad9 knockdown in esophageal cancer cells (ESCCs). Our results showed that TSA treatment alone induced significantly DNA damage in ESCC cells. Simultaneously, TSA also induced Rad9 gene expression both at transcriptional and translational levels in EC109 cells, but not in KYSE150 cells. Further, the induction of Rad9 by TSA was accompanied with increased level of histone H3K9 acetylation in Rad9 promoter region. To understand the role of Rad9 in TSA-induced DNA damage, Rad9 gene expression was efficiently knocked down by small interfering RNA (siRNA), which led to enhanced DNA damage and cell death induced by TSA. Our data suggested that Rad9 plays an important role in DNA damage, which is related to the biological effects of TSA.

The chemopreventive activity of the histone deacetylase inhibitor tributyrin in colon carcinogenesis involves the induction of apoptosis and reduction of DNA damage

The chemopreventive activity of the histone deacetylase inhibitor (HDACi) tributyrin (TB), a prodrug of butyric acid (BA), was evaluated in a rat model of colon carcinogenesis. The animals were treated with TB (TB group: 200mg/100g of body weight, b.w.) or maltodextrin (MD isocaloric control group: 300mg/100g b.w.) daily for 9 consecutive weeks. In the 3rd and 4th weeks of treatment, the rats in the TB and MD groups were given DMH (40mg/kg b.w.) twice a week. After 9weeks, the animals were euthanized, and the distal colon was examined. Compared with the control group (MD group), TB treatment reduced the total number of aberrant crypt foci (ACF; p<0.05) as well as the ACF with ≥4 crypts (p<0.05), which are considered more aggressive, but not inhibited the formation of DMH-induced O6-methyldeoxyguanosine DNA adducts. The TB group also showed a higher apoptotic index (p<0.05) and reduced DNA damage (p<0.05) compared with MD group. TB acted as a HDACi, as rats treated with the prodrug of BA had higher levels of histone H3K9 acetylation compared with the MD group (p<0.05). TB administration resulted in increased colonic tissue concentrations of BA (p<0.05) compared with the control animals. These results suggest that TB can be considered a promising chemopreventive agent for colon carcinogenesis because it reduced the number of ACF, including those that were more aggressive. Induction of apoptosis and reduction of DNA damage are cellular mechanisms that appear to be involved in the chemopreventive activity of TB.

Efficacy of the dietary histone deacetylase inhibitor butyrate alone or in combination with vitamin A against proliferation of MCF-7 human breast cancer cells

The combined treatment with histone deacetylase inhibitors (HDACi) and retinoids has been suggested as a potential epigenetic strategy for the control of cancer. In the present study, we investigated the effects of treatment with butyrate, a dietary HDACi, combined with vitamin A on MCF-7 human breast cancer cells. Cell proliferation was evaluated by the crystal violet staining method. MCF-7 cells were plated at 5 x 104 cells/mL and treated with butyrate (1 mM) alone or combined with vitamin A (10 µM) for 24 to 120 h. Cell proliferation inhibition was 34, 10 and 46% following treatment with butyrate, vitamin A and their combination, respectively, suggesting that vitamin A potentiated the inhibitory activities of butyrate. Furthermore, exposure to this short-chain fatty acid increased the level of histone H3K9 acetylation by 9.5-fold (Western blot), but not of H4K16, and increased the expression levels of p21WAF1 by 2.7-fold (Western blot) and of RARβ by 2.0-fold (quantitative real-time PCR). Our data show that RARβ may represent a molecular target for butyrate in breast cancer cells. Due to its effectiveness as a dietary HDACi, butyrate should be considered for use in combinatorial strategies with more active retinoids, especially in breast cancers in which RARβ is epigenetically altered.

Comparative evaluation of the effects of 5-Aza-2’-deoxycytidine and Trichostatin A on reactivation of hMLH1 in COC1/DDP ovarian cancer cell line

ObjectivehMLH1 protein serves to detect the DNA damage caused by cisplatin (DDP) and destroys the cell. The absence of hMLH1 expression has been correlated with acquired resistance of ovarian cancer cells to platinum. The aim of this study was to determine the possible role of DNA methylation and histone H3 lysine 9 (H3-K9) acetylation on the loss of hMLH1 expression, and to evaluate the reversal effects of 5-Aza-2′-deoxycytidine (5-Aza-dC) and Trichostatin A (TSA) on DDP-resistance in ovarian cancer cell lines.MethodsTwo human ovarian cancer cell lines, COC1 and its DDP-resistant subline, COC1/DDP were cultured. The two cancer cells were treated with 5-Aza-dC or TSA. Using COC1 cells as a control, we used methylation-specific PCR (MSP) to analyze DNA methylation at hMLH1 gene promoter. hMLH1 mRNA and protein expressions were analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot. Chromatin immunoprecipitation assay (ChIP) was used to test the levels of histone H3-K9 acetylation at hMLH1 gene promoter.ResultsIn COC1 cells, there was no DNA methylation at hMLH1 gene promoter, while there were hMLH1 mRNA and protein expression. In COC1/DDP cells, there was DNA hypermethylation at hMLH1 gene promoter, while there was no hMLH1 mRNA or protein expression. The treatment with 5-Aza-dC resulted in DNA demethylation at the promoter region, as well as restoration of hMLH1 expression in COC1/DDP cells. The treatment with TSA had no effects on DNA demethylation or restoration of hMLH1 expression in COC1/DDP cells.ConclusionHypermethylation of DNA at the promoter is related to the silencing of hMLH1 in COC1/DDP ovarian cancer cells. DNA methylation at hMLH1 promoter could play a significant role in determining the sensitivity of ovarian cancer to DDP. The drug resistance mediated by methylation of hMLH1 could be overcome by 5-Aza-dC.

The KAP1 Corepressor Functions To Coordinate the Assembly of De Novo HP1-Demarcated Microenvironments of Heterochromatin Required for KRAB Zinc Finger Protein-Mediated Transcriptional Repression

KAP1/TIF1beta is proposed to be a universal corepressor protein for the KRAB zinc finger protein (KRAB-zfp) superfamily of transcriptional repressors. To characterize the role of KAP1 and KAP1-interacting proteins in transcriptional repression, we investigated the regulation of stably integrated reporter transgenes by hormone-responsive KRAB and KAP1 repressor proteins. Here, we demonstrate that depletion of endogenous KAP1 levels by small interfering RNA (siRNA) significantly inhibited KRAB-mediated transcriptional repression of a chromatin template. Similarly, reduction in cellular levels of HP1alpha/beta/gamma and SETDB1 by siRNA attenuated KRAB-KAP1 repression. We also found that direct tethering of KAP1 to DNA was sufficient to repress transcription of an integrated transgene. This activity is absolutely dependent upon the interaction of KAP1 with HP1 and on an intact PHD finger and bromodomain of KAP1, suggesting that these domains function cooperatively in transcriptional corepression. The achievement of the repressed state by wild-type KAP1 involves decreased recruitment of RNA polymerase II, reduced levels of histone H3 K9 acetylation and H3K4 methylation, an increase in histone occupancy, enrichment of trimethyl histone H3K9, H3K36, and histone H4K20, and HP1 deposition at proximal regulatory sequences of the transgene. A KAP1 protein containing a mutation of the HP1 binding domain failed to induce any change in the histone modifications associated with DNA sequences of the transgene, implying that HP1-directed nuclear compartmentalization is required for transcriptional repression by the KRAB/KAP1 repression complex. The combination of these data suggests that KAP1 functions to coordinate activities that dynamically regulate changes in histone modifications and deposition of HP1 to establish a de novo microenvironment of heterochromatin, which is required for repression of gene transcription by KRAB-zfps.

Neural induction promotes large-scale chromatin reorganisation of theMash1locus

Determining how genes are epigenetically regulated to ensure their correct spatial and temporal expression during development is key to our understanding of cell lineage commitment. Here we examined epigenetic changes at an important proneural regulator gene Mash1 (Ascl1), as embryonic stem (ES) cells commit to the neural lineage. In ES cells where the Mash1 gene is transcriptionally repressed, the locus replicated late in S phase and was preferentially positioned at the nuclear periphery with other late-replicating genes (Neurod, Sprr2a). This peripheral location was coupled with low levels of histone H3K9 acetylation at the Mash1 promoter and enhanced H3K27 methylation but surprisingly location was not affected by removal of the Ezh2/Eed HMTase complex or several other chromatin-silencing candidates (G9a, SuV39h-1, Dnmt-1, Dnmt-3a and Dnmt-3b). Upon neural induction however, Mash1 transcription was upregulated (>100-fold), switched its time of replication from late to early in S phase and relocated towards the interior of the nucleus. This spatial repositioning was selective for neural commitment because Mash1 was peripheral in ES-derived mesoderm and other non-neural cell types. A bidirectional analysis of replication timing across a 2 Mb region flanking the Mash1 locus showed that chromatin changes were focused at Mash1. These results suggest that Mash1 is regulated by changes in chromatin structure and location and implicate the nuclear periphery as an important environment for maintaining the undifferentiated state of ES cells.

102 RABBIT CLONING: HISTONE ACETYLATION STATUS OF DONOR CELLS AND CLONED EMBRYOS

Epigenetic status of the genome of a donor nucleus is likely to be associated with the developmental potential of cloned embryos produced by somatic cell nuclear transfer (SCNT). Prevention of epigenetic errors by manipulation of the epigenetic status of donor cells is expected to result in improvement of cloning efficiency. In this study, we transferred cultured rabbit cumulus cells (RCC) and fetal fibroblasts (RFF) from genetically marked rabbits (Ali/Bas) into metaphase II (MII) oocytes and analyzed the levels of histone H3K9 acetylation in donor cells and cloned embryos. We also assessed the correlation between the histone acetylation status of donor cells and cloned embryos and their developmental potential. To test whether alteration of the histone acetylation status affects development of cloned embryos, we treated donor cells with sodium butyrate (NaBu), a histone deacetylase inhibitor. Further, we tried to improve cloning efficiency by chimeric complementation of cloned embryos with one or two blastomeres from in vitro-fertilized or parthenogenetic embryos. Histone acetylation in donor cells and cloned embryos was detected by anti-acH3K9 antibody using Western immunoblot analysis or immunochemistry, respectively. Data were analyzed by chi-square (developmental rates) or Student-Newman-Keuls (histone acetylation) test. The levels of acetylated histone H3K9 were higher in RCCs than in RFFs (P &amp;lt; 0.05). Although the type of donor cells did not affect development to blastocyst, after transfer into recipients, RCC-cloned embryos induced a higher initial pregnancy rate as compared to RFF-cloned embryos (40% vs. 20%; P &amp;lt; 0.05). However, almost all pregnancies with either type of cloned embryos were lost by the middle of gestation and only one fully developed; a live RCC-derived rabbit was obtained. Treatment of RFFs with NaBu significantly (P &amp;lt; 0.05) increased the level of histone H3K9/14 acetylation and the proportion of nuclear transfer embryos developing to blastocyst (49% vs. 33% with non-treated RFF; P &amp;lt; 0.05). The distribution of signals for acH3K9 in either group of cloned embryos did not resemble that in in vivo-fertilized embryos, suggesting that reprogramming of this epigenetic mark is aberrant in cloned rabbit embryos and cannot be corrected by treatment of donor cells with NaBu. Aggregation of embryos cloned from NaBu-treated RFFs with blastomeres from in vivo-derived embryos improved development to blastocyst, but no cloned offspring were obtained. Two live cloned rabbits were produced from this donor cell type only after aggregation of cloned embryos with a parthenogenetic blastomere. Our study demonstrates that the levels of histone acetylation in donor cells and cloned embryos correlate with their developmental potential and can be a useful epigenetic mark to predict efficiency of SCNT rabbits. This work was supported by the Bayerische Forschungsstiftung and by Therapeutic Human Polyclonals, Inc.