Acetylation Profiles Research Articles

Mitochondrial Hyperacetylation in the Failing Hearts of Obese Patients Mediated Partly by a Reduction in SIRT3: The Involvement of the Mitochondrial Permeability Transition Pore.

Cyclophilin D (CypD) mediates the mitochondrial permeability transition pore (mPTP) opening that contributes to mitochondrial dysfunction. CypD is regulated by its acetylation/deacetylation state that depends on Sirtuin-3 (SIRT3) mitochondrial deacetylase. Since obesity and metabolic syndrome decrease SIRT3 activity and expression, we tested the hypothesis that CypD hyperacetylation promotes mitochondrial dysfunction under this pathophysiological state, which is associated with ventricular dysfunction and heart failure. Myocardial tissue samples from patients with left ventricular heart failure, with either obesity or normal weight, were processed for the expression of SIRT3 and acetylation profile by Western Blot (WB). In addition, a rat model of obesity and metabolic syndrome induced by 30% (w/v) of sucrose was conducted. The WB analysis was used to determine the levels of mitochondrial expression of SIRT3, Adenine Nucleotide Translocator (ANT), CypD and the acetylation profile, as well as immunoprecipitation to establish the acetylation levels of CypD. Mitochondrial function was assessed by oxygen consumption analysis and maximum Ca2+ retention capacity. Oxidative stress was assessed by aconitase activity, protein carbonyl and thiol groups content. SIRT3 expression in the biopsies of the failing human hearts showed a 46% decrease in the expression levels of obese patients in comparison to the non-obese patients (p=0.0219). Remarkably, body mass index was associated with protein acetylation (0.627; p = 0.035), suggesting that the acetylation profiles of the failing hearts of obese patients are partly mediated by a reduction in SIRT3, which is also associated with higher BNP levels, indicating a more severe ventricular dysfunction (-0.636; p = 0.043). Accordingly, obese rats demonstrated a SIRT3 mitochondrial expression decrease of 22% concomitantly with a hyperacetylated mitochondrial profile, including CypD. Cardiac mitochondria from obese animals were 2.5-fold more prone to mPTP opening than the controls. Our results indicate that obesity reduces SIRT3 expression and that CypD hyperacetylation increases mPTP opening, suggesting that the activation of SIRT3 might be a potential target to decrease ventricular dysfunction and slow the progression of heart failure.

N-acetyltransferase 2 polymorphism and acetylation profiles in Buginese ethnics of Indonesia.

N-acetyltransferase 2 (NAT2) is a key enzyme involved in the phase II metabolism of aromatic amines and heterocyclic aromatic amines present in a wide range of xenobiotics. The aim of this study was to investigate the NAT2 polymorphism in the Buginese ethnic group of Indonesia to determine the frequency of NAT2 alleles in this population. We found six haplotypes consisting of six single-nucleotide polymorphisms and 12 NAT2 genotype variations. NAT2*6A haplotype (42%) showed the highest frequency, followed by NAT2*4 (33%), NAT2*7B (15%), NAT2*5B (5%), NAT2*12A (3%), and NAT2*13 (2%). In terms of phenotypes, the Buginese population comprised 18% rapid acetylators, 40% intermediate acetylators, and 42% slow acetylators. We confirmed the high-frequency slow acetylator phenotype in the Buginese population. The NAT2*6A/*6A genotype was the most frequent slow acetylator genotype, followed by NAT2*6A/*7B. The pattern of NAT2 alleles of Buginese is similar to Southeast Asian populations but not Northeast Asian populations. However, the slow acetylator frequencies in the Buginese population were higher than those in Northeast Asian populations and lower than those in Caucasians and some American populations.

Tu1765 – Histone 3 Lysine 27 Acetylation Profiling Reveals Its Role in the Chronic Dss-Induced Colitis Mouse Model

Tu1765 – Histone 3 Lysine 27 Acetylation Profiling Reveals Its Role in the Chronic Dss-Induced Colitis Mouse Model

Arabinosyl Deacetylase Modulates the Arabinoxylan Acetylation Profile and Secondary Wall Formation.

Acetylation, a prevalent modification of cell-wall polymers, is a tightly controlled regulatory process that orchestrates plant growth and environmental adaptation. However, due to limited characterization of the enzymes involved, it is unclear how plants establish and dynamically regulate the acetylation pattern in response to growth requirements. In this study, we identified a rice (Oryza sativa) GDSL esterase that deacetylates the side chain of the major rice hemicellulose, arabinoxylan. Acetyl esterases involved in arabinoxylan modification were screened using enzymatic assays combined with mass spectrometry analysis. One candidate, DEACETYLASE ON ARABINOSYL SIDECHAIN OF XYLAN1 (DARX1), is specific for arabinosyl residues. Disruption of DARX1 via Tos17 insertion and CRISPR/Cas9 approaches resulted in the accumulation of acetates on the xylan arabinosyl side chains. Recombinant DARX1 abolished the excess acetyl groups on arabinoxylan-derived oligosaccharides of the darx1 mutants in vitro. Moreover, DARX1 is localized to the Golgi apparatus. Two-dimensional 13C-13C correlation spectroscopy and atomic force microscopy further revealed that the abnormal acetylation pattern observed in darx1 interrupts arabinoxylan conformation and cellulose microfibril orientation, resulting in compromised secondary wall patterning and reduced mechanical strength. This study provides insight into the mechanism controlling the acetylation pattern on arabinoxylan side chains and suggests a strategy to breed robust elite crops.

1815P - Comparative proteomic analysis of acetylation profiles in esophageal squamous carcinoma cells

1815P - Comparative proteomic analysis of acetylation profiles in esophageal squamous carcinoma cells

Novel histone deacetylase inhibitors and embryo aggregation enhance cloned embryo development and ES cell derivation in pigs.

The histone deacetylase inhibitor (HDACi) has been investigated for treating cancers and many other diseases as well as enhancing the reprogramming efficiency in cloned embryos for decades. In the present study, we investigated the effects of two novel HDAC inhibitors, i.e., HDACi-14 and -79, at the concentrations of 0, 1, 2, or 4 μM on the development of embryos cloned by the oocyte bisection cloning technique (OBCT). Blastocyst rates for the reconstructed embryos reached 60% in the 2 μM HDACi-14-treated groups, which was higher (P < 0.05) compared to the untreated group (36.9%). Similarly, HDACi-79 treatment at 2 and 4 μM also conferred higher (P < 0.05) blastocyst rates than that of the untreated group (79.4, 74.2, and 50.0%, respectively). Both HDACi-14 and -79 treatments had no beneficial effect on total cell numbers and apoptotic indices of cloned embryos (P > 0.05). Histone acetylation profile by both HDACi-14 (2 μM) and -79 (2 μM) treatments demonstrated a drastic increase (P < 0.05) mainly in two-cell stage embryos when compared to the control group. After seeding on the feeder cells, the aggregated cloned blastocysts produced by the HDACi-79 treatment showed a significant increase of primary outgrowths compared to the control group (60.0% vs. 42.9%; P < 0.05). Finally, the cloned embryo-derived ES cell lines from aggregated cloned embryos produced from the HDACi-79-treated, HDACi-14-treated and control groups were established (5, 3, and 2 lines, respectively). In conclusion, the novel histone deacetylation inhibitors improve blastocyst formation and potentially increase the derivation efficiency of ES cell lines from the cloned porcine embryos produced in vitro. Depending on the purposes, some fine-tuning may be required to maximize its beneficial effects of these newly synthesized chemicals.

Global Profiling of Lysine Acetylation in Borrelia burgdorferi B31 Reveals Its Role in Central Metabolism.

The post-translational modification of proteins has been shown to be extremely important in prokaryotes. Using a highly sensitive mass spectrometry-based proteomics approach, we have characterized the acetylome of B. burgdorferi. As previously reported for other bacteria, a relatively low number (5%) of the potential genome-encoded proteins of B. burgdorferi were acetylated. Of these, the vast majority were involved in central metabolism and cellular information processing (transcription, translation, etc.). Interestingly, these critical cell functions were targeted during both ML (mid-log) and S (stationary) phases of growth. However, acetylation of target proteins in ML phase was limited to single lysine residues while these same proteins were acetylated at multiple sites during S phase. To determine the acetyl donor in B. burgdorferi, we used mutants that targeted the sole acetate metabolic/anabolic pathway in B. burgdorferi (lipid I synthesis). B. burgdorferi strains B31-A3, B31-A3 ΔackA (acetyl-P- and acetyl-CoA-) and B31-A3 Δpta (acetyl-P+ and acetyl-CoA-) were grown to S phase and the acetylation profiles were analyzed. While only two proteins were acetylated in the ΔackA mutant, 140 proteins were acetylated in the Δpta mutant suggesting that acetyl-P was the primary acetyl donor in B. burgdorferi. Using specific enzymatic assays, we were able to demonstrate that hyperacetylation of proteins in S phase appeared to play a role in decreasing the enzymatic activity of at least two glycolytic proteins. Currently, we hypothesize that acetylation is used to modulate enzyme activities during different stages of growth. This strategy would allow the bacteria to post-translationally stimulate the activity of key glycolytic enzymes by deacetylation rather than expending excessive energy synthesizing new proteins. This would be an appealing, low-energy strategy for a bacterium with limited metabolic capabilities. Future work focuses on identifying potential protein deacetylase(s) to complete our understanding of this important biological process.

CBP/p300 inhibitor C646 prevents high glucose exposure induced neuroepithelial cell proliferation.

Maternal diabetes related neural tube defects (NTDs) are a result of oxidative stress and apoptosis. However, the molecular mechanism behind the pathogenesis is not fully understood. Here, we report that high glucose exposure-induced epigenetic changes influence histone H4 acetylation and neuroepithelial cell proliferation. We also show that the acetyltransferase inhibitor C646 can prevent high glucose induced changes in histone H4 acetylation and neuroepithelial cell proliferation. By using LC-MS/MS as an unbiased approach, we screened the histone acetylation profile in an E9 neuroepithelial cell line (NE-4C) under high glucose exposure. We further explored the mechanism in cells in vitro and in maternal diabetes-induced mouse embryos in vivo. We identified 35 core histone acetylation marks in normal E9 neuroepithelial cells, whereas high glucose exposure resulted in novel acetylation sites on H4K31 and H4K44. Acetylation levels of embryonic development associated H4K5/K8/K12/K16 increased in neuroepithelial cells exposed to high glucose in vitro and in brain tissue from maternal diabetes induced exencephalic embryos in vivo. Further, mRNA level of histone acetyltransferase CBP encoded gene Crebbp was significantly increased both in vitro and in vivo. The addition of C646, a selective inhibitor for CBP/p300, significantly rescued increase of H4K5/K8/K12/K16 acetylation levels and H3S10pi-labeled neuroepithelial cell proliferation induced by high glucose exposure. Our data provide complementary insights for potential mechanisms of maternal diabetes induced NTDs.

Mass spectrometry and DigiWest technology emphasize protein acetylation profile from Quisinostat-treated HuT78 CTCL cell line

Histone deacetylases (HDACs) are key enzymes involved in epigenetic modulation and were targeted by HDAC inhibitors (HDACis) for cancer treatment. The action of HDACis is not restricted to histones and also prevents deacetylation of other proteins, supporting their wide biological actions. The HuT78 cell line is recognized as a key tool to support and understand cutaneous T-cell lymphoma (CTCL) biology and was used as a predictive model since HDACi such as Vorinostat and Panobinostat have both demonstrated apoptotic activities in HuT78 cells and in primary blood CTCL cells. In this study, Quisinostat (JNJ-26481585) a novel second-generation HDACi with highest potency for HDAC1, was tested on HuT78 cell line. Quantitative mass spectrometry (MS)-based proteomics after acetylated-lysine peptide enrichment and a targeted antibody-based immunoassay (DigiWest) were used as complementary technologies to assess the modifications of the acetylated proteome. As expected, several acetylated lysines of histones were increased by the HDACi. Additional acetylated non-histone proteins were modulated after treatment with Quisinostat including the nucleolin (a major nucleolar protein), the replication protein A 70 kDa DNA-binding subunit, the phosphoglycerate kinase 1, the stress-70 protein, the proto-oncogene Myc and the serine hydroxymethyltransferase. A better knowledge of histone and non-histone acetylated protein profile after Quisinostat treatment can strongly support the understanding of non-clinical and clinical results of this HDACi. These technological tools can also help in designing new HDACis in a pharmaceutical drug discovery program. SignificanceA better knowledge of histone and non-histone acetylated protein profile after HDAC inhibitors (HDACis) treatment can strongly support the understanding of non-clinical and clinical investigations in a pharmaceutical drug discovery program. Relative quantification using mass spectrometry -based proteomics after acetylated-lysine peptide enrichment and a targeted antibody-based immunoassay (DigiWest) are proposed as complementary technologies to assess the modifications of the acetylated proteome. Quisinostat (JNJ-26481585) a novel second-generation HDACi with highest potency for HDAC1 was better characterized in vitro in HuT78 cells to support and understand cutaneous T-cell lymphoma (CTCL) therapeutic research program.

Abstract 2352: Defining a pediatric cancer dependency map

Abstract Many children with metastatic or recurrent pediatric solid tumors continue to have poor survival, and there is an immense need to identify novel therapeutic approaches. Moreover, these cancers typically have simple genomes with limited known druggable molecular events. In order to discover new vulnerabilities in pediatric solid tumors, we have performed genome-scale CRISPR-Cas9 loss-of-function screening and deep “omic” characterization in over 60 pediatric cancer cell lines to date, including neuroblastoma, medulloblastoma, Ewing sarcoma, malignant rhabdoid tumor and rhabdomyosarcoma lines, to begin defining a pediatric cancer dependency map. Global analyses of the pediatric dependency landscape have identified emerging classes of pediatric cancers, including epigenetic-driven, aberrant transcription factor-driven and receptor tyrosine kinase-driven malignancies. For example, the preferential dependencies identified in a subset of neuroblastoma, which has aberrantly high expression of the transcription factor MYCN, are highly enriched for an interconnected network of genes annotated to have transcription factor activity. In addition to the global evaluation, we have developed methods and tools for prioritizing targets for further validation within a cancer type. These tools computationally integrate the pediatric dependency data across multiple datasets to identify categories of genetic dependencies that are especially strong hits or enriched hits in a specific pediatric malignancy. As an example, the intersection of MYCN-amplified neuroblastoma specific dependencies and H3-lysine 27 acetylation (H3K27ac) profiling across MYCN-amplified neuroblastoma allowed us to identify a transcriptional core regulatory circuit (CRC) that may drive the malignant state. Furthermore, targeting transcription with the BRD4 inhibitor JQ1 and CDK7 inhibitor THZ1 caused synergistic killing of neuroblastoma cells suggesting a novel therapeutic approach to treating this disease. Thus, defining a comprehensive pediatric cancer dependency map and developing the methods and tools to prioritize vulnerabilities in different cancer types will allow us to discover both novel biology and new therapeutic opportunities in childhood malignancies. Citation Format: Neekesh V. Dharia, Clare Malone, Amanda Balboni Iniguez, Lillian Guenther, Liying Chen, Gabriela Alexe, Adam D. Durbin, Mark W. Zimmerman, Andrew Hong, Pratiti Bandopadhayay, Mariella G. Filbin, Thomas Howard, Brenton Paolella, Iris Fung, Josephine Lee, Phil Montgomery, John M. Krill-Burger, Brian J. Abraham, Jennifer Roth, David E. Root, Richard A. Young, A. Thomas Look, Rameen Beroukhim, Jesse S. Boehm, William C. Hahn, Todd R. Golub, Aviad Tsherniak, Francisca Vazquez, Kimberly Stegmaier. Defining a pediatric cancer dependency map [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2352.

Histone Acetyltransferases (HATs) in Chinese Cabbage: Insights from Histone H3 Acetylation and Expression Profiling of HATs in Response to Abiotic Stresses

Histone acetyltransferase (HAT) is known as an epigenetic enzyme that acetylates specific lysine residues on the histone tail to promote chromatin dynamics and gene expression. In higher plants, HATs have been recognized as playing a fundamental role in plant development, growth, and the response to diverse environmental stresses. In this study, using comprehensive bioinformatic analyses, we identified 15 HATs in genome of chinese cabbage [Brassica rapa (BraHATs)], which are divided into four families. In addition, evolution analysis suggested that the BraHAT genes were duplicated mainly via a segmental duplication event originating 3.05–18.39 million years ago. To determine the effects of abiotic stresses, such as salt, wounding, and drought, on histone H3 acetylation in chinese cabbage, histone H3 acetylation was analyzed via immunoblot analysis, suggesting that the acetylation level of histone H3 increased in response to wounding and salt stresses. Furthermore, the analysis of BraHAT expression patterns using quantitative real-time polymerase chain reaction (qRT-PCR) suggested that the increased acetylation of histone H3 was related to BraHAT transcripts and/or the functional interplay between HAT and histone deacetylase (HDAC) activities. Taken together, our comparative genomic analysis of HAT genes in this important vegetable crop will provide a solid foundation to further our understanding of epigenetically regulated processes in response to environmental stresses.

Abstract 1798: A detailed comparison between second-generation AR antagonists reveals differences in the overall impact on gene regulation patterns in prostate cancer cells

Abstract Androgen receptor (AR) signaling is essential at all prostate cancer stages and is clinically addressed by surgical or chemical castration, AR antagonists and androgen synthesis inhibitors. Second-generation AR antagonists include the approved drug enzalutamide and the late-stage clinical investigational oral compounds apalutamide and darolutamide. Comparing the gene expression patterns and the overarching epigenetic modifications elicited by AR antagonists will be essential for a detailed understanding of the molecular mode of action of these compounds. Methods: The transcriptome and AR binding patterns were analyzed by RNA-seq and qPCR, respectively. Genome-wide changes of the active transcription histone modification H3K27 acetylation were determined by ChIP-seq. Results: RNA-seq was performed in the androgen-dependent VCaP and LNCaP cell lines for a detailed analysis of the genome-wide transcriptional effects of the AR antagonists darolutamide, enzalutamide and apalutamide. Gene set enrichment analysis indicated that many genes involved in androgen response were up- or down-regulated by androgen treatment with R1881. Principal component analysis showed that treatment with pharmacologically relevant doses of AR antagonists had similar effects at the 8-hour time point, however some differences were visible at the 22-hour time point. A strong expression down-regulation of a number of genes from the Wnt and epithelial-mesenchymal transition (EMT) pathways was observed for darolutamide as compared to enzalutamide and apalutamide treatment in the VCaP cell line. In LNCaP cells the major differences between the AR antagonists were observed in the androgen-regulated genes, which is consistent with varying degrees of inhibition by the drugs in this cell line. Next we determined the global H3K27 acetylation profiles which characterize open chromatin and active transcription. ChIP-seq analysis performed in LNCaP cells revealed a strong impact of androgen treatment on the H3K27 acetylation profiles of numerous genes involved in hormonal regulation. Darolutamide significantly reversed genome-wide H3K27 acetylation patterns, almost to non-androgen stimulated levels. More specifically, a reduction of H3K27 peaks was observed at KLK3 and FKBP5 regulatory elements. In addition, we determined the impact of AR antagonists on AR binding at the KLK2 and KLK3 regulatory elements. Darolutamide and enzalutamide strongly reduced androgen-stimulated AR binding whereas the impact of apalutamide was slightly lower. Conclusions: AR antagonists strongly reversed the effects of the androgen R1881, both at the level of gene expression, and of H3K27 acetylation and AR binding. A detailed comparison between second-generation AR antagonists showed interesting differences in the regulation of genes in the Wnt and EMT pathways. Citation Format: Simon J. Baumgart, Ekaterina Nevedomskaya, Ralf Lesche, Henrik Seidel, Bernard Haendler. A detailed comparison between second-generation AR antagonists reveals differences in the overall impact on gene regulation patterns in prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1798.

Elevated histone H3 acetylation is associated with genes involved in T lymphocyte activation and glutamate decarboxylase antibody production in patients with type 1 diabetes.

Aims/IntroductionGenetic and epigenetic mechanisms have been implicated in the pathogenesis of type 1 diabetes, and histone acetylation is an epigenetic modification pattern that activates gene transcription. However, the genome‐wide histone H3 acetylation in new‐onset type 1 diabetes patients has not been well described. Accordingly, we aimed to unveil the genome‐wide promoter acetylation profile in CD4+ T lymphocytes from type 1 diabetes patients, especially for those who are glutamate decarboxylase antibody‐positive.Materials and MethodsA total of 12 patients with new‐onset type 1 diabetes who were glutamate decarboxylase antibody‐positive were enrolled, and 12 healthy individuals were recruited as controls. The global histone H3 acetylation level of CD4+ T lymphocytes from peripheral blood was detected by western blot, with chromatin immunoprecipitation linked to microarrays to characterize the promoter acetylation profile. Furthermore, we validated the results of particular genes from chromatin immunoprecipitation linked to microarrays by using chromatin immunoprecipitation quantitative polymerase chain reaction, and analyzed the transcription level by real‐time quantitative polymerase chain reaction.ResultsElevated global histone H3 acetylation level was observed in type 1 diabetes patients, with 607 differentially acetylated genes identified between type 1 diabetes patients and controls by chromatin immunoprecipitation linked to microarrays. The hyperacetylated genes were enriched in biological processes involved in immune cell activation and inflammatory response. Gene‐specific assessments showed that increased transcription of inducible T‐cell costimulator was in concordance with the elevated acetylation in its gene promoter, along with positive correlation with glutamate decarboxylase antibody titer in type 1 diabetes patients.ConclusionsThe present study generates a genome‐wide histone acetylation profile specific to CD4+ T lymphocytes in type 1 diabetes patients who are glutamic acid decarboxylase antibody‐positive, which is instrumental in improving our understanding of the epigenetic involvement in autoimmune diabetes.

H3K27 acetylation and gene expression analysis reveals differences in placental chromatin activity in fetal growth restriction

BackgroundPosttranslational modification of histone tails such as histone 3 lysine 27 acetylation (H3K27ac) is tightly coupled to epigenetic regulation of gene expression. To explore whether this is involved in placenta pathology, we probed genome-wide H3K27ac occupancy by chromatin immunoprecipitation sequencing (ChIP-seq) in healthy placentas and placentas from pathological pregnancies with fetal growth restriction (FGR). Furthermore, we related specific acetylation profiles of FGR placentas to gene expression changes.ResultsAnalysis of H3K27ac occupancy in FGR compared to healthy placentas showed 970 differentially acetylated regions distributed throughout the genome. Principal component analysis and hierarchical clustering revealed complete segregation of the FGR and control group. Next, we identified 569 upregulated genes and 521 downregulated genes in FGR placentas by RNA sequencing. Differential gene transcription largely corresponded to expected direction based on H3K27ac status. Pathway analysis on upregulated transcripts originating from hyperacetylated sites revealed genes related to the HIF-1-alpha transcription factor network and several other genes with known involvement in placental pathology (LEP, FLT1, HK2, ENG, FOS). Downregulated transcripts in the vicinity of hypoacetylated sites were related to the immune system and growth hormone receptor signaling. Additionally, we found enrichment of 141 transcription factor binding motifs within differentially acetylated regions. Of the corresponding transcription factors, four were upregulated, SP1, ARNT2, HEY2, and VDR, and two downregulated, FOSL and NR4A1.ConclusionWe demonstrate a key role for genome-wide alterations in H3K27ac in FGR placentas corresponding with changes in transcription profiles of regions relevant to placental function. Future studies on the role of H3K27ac in FGR and placental-fetal development may help to identify novel targets for therapy of this currently incurable disease.

Loci-specific histone acetylation profiles associated with transcriptional coactivator p300 during early myoblast differentiation

ABSTRACTMolecular regulation of stem cell differentiation is exerted through both genetic and epigenetic determinants over distal regulatory or enhancer regions. Understanding the mechanistic action of active or poised enhancers is therefore imperative for control of stem cell differentiation. Based on the genome-wide co-occurrence of different epigenetic marks in committed proliferating myoblasts, we have previously generated a 14-state chromatin state model to profile rexinoid-responsive histone acetylation in early myoblast differentiation. Here, we delineate the functional mode of transcription regulators during early myogenic differentiation using genome-wide chromatin state association. We define a role of transcriptional coactivator p300, when recruited by muscle master regulator MyoD, in the establishment and regulation of myogenic loci at the onset of myoblast differentiation. In addition, we reveal an enrichment of loci-specific histone acetylation at p300 associated active or poised enhancers, particularly when enlisted by MyoD. We provide novel molecular insights into the regulation of myogenic enhancers by p300 in concert with MyoD. Our studies present a valuable aptitude for driving condition-specific chromatin state or enhancers pharmacologically to treat muscle-related diseases and for the identification of additional myogenic targets and molecular interactions for therapeutic development.Abbreviations: MRF: Muscle regulatory factor; HAT: Histone acetyltransferase; CBP: CREB-binding protein; ES: Embryonic stem; ATCC: American type culture collection; DM: Differentiation medium; DMEM: Dulbecco’s Modified Eagle Medium; GM: Growth medium; GO: Gene ontology; GREAT: Genomic regions enrichment of annotations tool; FPKM: Fragments per kilobase of transcript per million; GEO: Gene expression omnibus; MACS: Model-based analysis for ChIP-seq

HDAC1 inhibition by MS-275 in mesothelial cells limits cellular invasion and promotes MMT reversal

Peritoneal fibrosis is a pathological alteration of the peritoneal membrane occurring in a variety of conditions including peritoneal dialysis (PD), post-surgery adhesions and peritoneal metastases. The acquisition of invasive and pro-fibrotic abilities by mesothelial cells (MCs) through induction of MMT, a cell-specific form of EMT, plays a main role in this process. Aim of this study was to evaluate possible effects of histone deacetylase (HDAC) inhibitors, key components of the epigenetic machinery, in counteracting MMT observed in MCs isolated from effluent of PD patients. HDAC inhibitors with different class/isoform selectivity have been used for pharmacological inhibition. While the effect of other inhibitors was limited to a partial E-cadherin re-expression, MS-275, a HDAC1-3 inhibitor, promoted: (i) downregulation of mesenchymal markers (MMP2, Col1A1, PAI-1, TGFβ1, TGFβRI) (ii) upregulation of epithelial markers (E-cadherin, Occludin), (iii) reacquisition of an epithelial-like morphology and (iv) marked reduction of cellular invasiveness. Results were confirmed by HDAC1 genetic silencing. Mechanistically, MS-275 causes: (i) increase of nuclear histone H3 acetylation (ii) rescue of the acetylation profile on E-cadherin promoter, (iii) Snail functional impairment. Overall, our study, pinpointing a role for HDAC1, revealed a new player in the regulation of peritoneal fibrosis, providing the rationale for future therapeutic opportunities.

Altered mitochondrial acetylation profiles in a kainic acid model of temporal lobe epilepsy

Impaired bioenergetics and oxidative damage in the mitochondria are implicated in the etiology of temporal lobe epilepsy, and hyperacetylation of mitochondrial proteins has recently emerged as a critical negative regulator of mitochondrial functions. However, the roles of mitochondrial acetylation and activity of the primary mitochondrial deacetylase, SIRT3, have not been explored in acquired epilepsy. We investigated changes in mitochondrial acetylation and SIRT3 activity in the development of chronic epilepsy in the kainic acid rat model of TLE. Hippocampal measurements were made at 48 h, 1 week and 12 weeks corresponding to the acute, latent and chronic stages of epileptogenesis. Assessment of hippocampal bioenergetics demonstrated a ≥ 27% decrease in the ATP/ADP ratio at all phases of epileptogenesis (p < 0.05), whereas cellular NAD+ levels were decreased by ≥ 41% in the acute and latent time points (p < 0.05), but not in chronically epileptic rats. In spontaneously epileptic rats, we found decreased protein expression of SIRT3 and a 60% increase in global mitochondrial acetylation, as well as enhanced acetylation of the known SIRT3 substrates MnSOD, Ndufa9 of Complex I and IDH2 (all p < 0.05), suggesting SIRT3 dysfunction in chronic epilepsy. Mass spectrometry-based acetylomics investigation of hippocampal mitochondria demonstrated a 79% increase in unique acetylated proteins from rats in the chronic phase vs. controls. Pathway analysis identified numerous mitochondrial bioenergetic pathways affected by mitochondrial acetylation. These results suggest SIRT3 dysfunction and aberrant protein acetylation may contribute to mitochondrial dysfunction in chronic epilepsy.

The KAT5-Acetyl-Histone4-Brd4 axis silences HIV-1 transcription and promotes viral latency.

The bromodomain protein Brd4 promotes HIV-1 latency by competitively inhibiting P-TEFb-mediated transcription induced by the virus-encoded Tat protein. Brd4 is recruited to the HIV LTR by interactions with acetyl-histones3 (AcH3) and AcH4. However, the precise modification pattern that it reads and the writer for generating this pattern are unknown. By examining a pool of latently infected proviruses with diverse integration sites, we found that the LTR characteristically has low AcH3 but high AcH4 content. This unusual acetylation profile attracts Brd4 to suppress the interaction of Tat with the host super elongation complex (SEC) that is essential for productive HIV transcription and latency reversal. KAT5 (lysine acetyltransferase 5), but not its paralogs KAT7 and KAT8, is found to promote HIV latency through acetylating H4 on the provirus. Antagonizing KAT5 removes AcH4 and Brd4 from the LTR, enhances the SEC loading, and reverses as well as delays, the establishment of latency. The pro-latency effect of KAT5 is confirmed in a primary CD4+ T cell latency model as well as cells from ART-treated patients. Our data thus indicate the KAT5-AcH4-Brd4 axis as a key regulator of latency and a potential therapeutic target to reactivate latent HIV reservoirs for eradication.

Oroxylin A prevents cardiac hypertrophy and dysfunction as a modulator of mitochondrial protein acetylation

Cardiac hypertrophy and remodeling are both well‐known consequences mediated by increased levels of Angiotensin II (Ang II) as a compensatory mechanism in non‐ischemic HF. In this sense, mitochondrial protein hyper‐acetylation and ROS production that induce mitochondrial dysfunction have previously been associated with this condition. Oroxylin A (OA) is a methylated flavone found on the root of Scutellaria baicalensis known for its antioxidant properties, and as a modulator of mitochondrial deacetylase activity. Nevertheless, OA effects have not been explored in cardiac remodeling mediated by exacerbated ROS production and mitochondrial hyper‐acetylation. Therefore, we explore the potential cardioprotective effect of OA in an in vitro model of cardiac hypertrophy induced by Ang II treatment in H9c2 myocytes. We found that OA reverted completely the Ang II mediated hypertrophic and cell death effect. Mitochondrial acetylation profile and sirtuin3 expression analyzed by western blot showed that Ang II promotes ~2‐fold increase in protein acetylation; however, OA pre‐treatment abrogates Ang II‐induced hyper‐acetylation in a dose‐dependent fashion. Remarkably, we found that the OA pre‐treatment prevents mitochondrial dysfunction by decreasing significantly permeability transition susceptibility and membrane potential depolarization. These results were consistent with a decrease in apoptosis, determined by caspase 9 and 3,7 activity. Overall, these findings suggest that OA promotes sirtuin3 activation, targeting pivot mitochondrial proteins. Accordingly, we evaluated the cardioprotective effect of OA in an in vivo model of HF induced by AngII+L‐NAME. In this model, we found an increased mitochondrial acetylation in the myocardium and found that OA was capable of prevent cardiac hypertrophy and dysfunction. Taken together, our results suggest that OA is a novel strategy to treat cardiac dysfunction in HF.Support or Funding InformationThis work was partially supported by Xignus Research Foundation as well endowed Chair in Cardiology/Grupo de Enfoque Medicina Cardiovascular Tec de Monterrey, CONACYT Grant CB‐256577, Fronteras de la Ciencias 0682 (Gerardo García‐Rivas), and Red Temática Farmoquímicos del CONACYT.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Histone Deacetylase Inhibition Leads to Dose‐Dependent Suppression of Oncogene‐Associated Super‐Enhancers

In cancer cells, decreased histone acetylation leads to gene expression patterns that drive oncogenic processes. Histone deacetylase (HDAC) inhibitors are therefore used as therapeutics to selectively kill cancer cells. The complex processes by which these chromatin‐remodeling agents affect gene expression and ultimately cell fate are still not entirely understood. The Liu group seeks to characterize the mechanisms of the HDAC inhibitor largazole by using epigenomic and transcriptomic studies to compare its effects on transformed and non‐transformed cells at different doses. From broad chromatin immunoprecipitation sequencing and RNA sequencing, we observed that largazole has a striking effect on super‐enhancer regions and the genes associated with them. Largazole decreases the accumulation of RNA polymerase II at super‐enhancer elements and limits the transcription of genes under the regulation of those super‐enhancers. Largazole also alters the expression of many genes by decreasing the histone acetylation of poised distal enhancers in a dose‐dependent manner. Many of these genes are transcriptionally upregulated after treatment, as is canonically associated with an increased histone acetylation profile, but some transcripts are repressed. These insights into the epigenetic reprogramming that drives oncogenic processes provide fundamental information about tumorigenesis and could potentially serve as a foundation for the development of further oncology therapies.Support or Funding InformationNational Institutes of Health [T32GM008759 to G.W.]; National Cancer Institute and National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health [CA107098, AR068254 to X.L.]; National Cancer Institute [CA107098S1 to G.S.];This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.