Histone Acetylation Levels Research Articles

Disruption of Broad Epigenetic Domains in PDAC Cells by HAT Inhibitors.

The spreading of epigenetic domains has emerged as a distinguishing epigenomic phenotype for diverse cell types. In particular, clusters of H3K27ac- and H3K4me3-marked elements, referred to as super-enhancers, and broad H3K4me3 domains, respectively, have been linked to cell identity and disease states. Here, we characterized the broad domains from different pancreatic ductal adenocarcinoma (PDAC) cell lines that represent distinct histological grades. Our integrative genomic analysis found that human derived cell line models for distinct PDAC grades exhibit characteristic broad epigenetic features associated with gene expression patterns that are predictive of patient prognosis and provide insight into pancreatic cancer cell identity. In particular, we find that genes marked by overlapping Low-Grade broad domains correspond to an epithelial phenotype and hold potential as markers for patient stratification. We further utilize ChIP-seq to compare the effects of histone acetyltransferase (HAT) inhibitors to detect global changes in histone acetylation and methylation levels. We found that HAT inhibitors impact certain broad domains of pancreatic cancer cells. Overall, our results reveal potential roles for broad domains in cells from distinct PDAC grades and demonstrate the plasticity of particular broad epigenomic domains to epigenetic inhibitors.

1973-P: Deficiency of Protein Deacetylase Sirt3 Enhances Acetylation of Histone H3 and H4 as to Affect Glucose Metabolism through Regulation of Dipeptidyl Peptidase-4 in Mice

Background: Sirt3 is a NAD-dependent deacetylase that regulates lipid metabolism in mice and is related to longevity through modification of epigenome. Sirt3-deficient knockout (KO) mice fed a high-fat diet have been shown to develop obesity and metabolic syndrome-like phenotype. However, the precise mechanism by which they cause glucose intolerance has not been known. To reveal the regulatory mechanism of glucose metabolism through Sirt3, the metabolic parameters in the Sirt3-KO mice were examined. Objective and Methods: The histone acetylation levels in various organs of the Sirt3-KO mice were examined by immunostaining of acetylated histone H3 (H3Ac) and H4 (H4Ac). Blood glucose levels in the Sirt3-KO mice after glucose loading were examined, associated with blood glucagon-like peptide-1 (GLP-1) levels and dipeptidyl peptidase-4 (DPP-4) activity. Gene expressions which were associated with the expressions and functions of GLP-1 and DPP-4 were quantified by qPCR. Results: The Sirt3-KO mice were subjected to an oral glucose tolerance test (OGTT), together with an intraperitoneal glucose tolerance test (ipGTT). OGTT revealed an increase in the blood glucose levels in the KO mice, although ipGTT did not show the increase after the glucose loading. The blood GLP-1 and insulin levels decreased, and the DPP-4 activity increased significantly in the KO mice. qPCR analysis revealed a predominance of Sirt3 expression in the intestinal tract in the wild type mice. The immunostaining of H3Ac and H4Ac showed enhanced intensity in the small intestine and colon, and in the vascular endothelial cells in the KO mice. Conclusion: These results suggest that Sirt3 deficiency impairs the incretin effect by a suppression of the blood GLP-1 levels and causes glucose intolerance. Enhanced acetylation of histone H3 and H4 in the KO mice might relate to the increase in the DPP-4 activity, which lead to the decrease in the GLP-1 levels. Disclosure A. Uto: None. K. Miyashita: Research Support; Self; Johnson & Johnson, Merck & Co., Inc., Novo Nordisk Inc.

Redox-Dependent Chromatin Remodeling: A New Function of Nitric Oxide as Architect of Chromatin Structure in Plants

Nitric oxide (NO) is a key signaling molecule in all kingdoms. In plants, NO is involved in the regulation of various processes of growth and development as well as biotic and abiotic stress response. It mainly acts by modifying protein cysteine or tyrosine residues or by interacting with protein bound transition metals. Thereby, the modification of cysteine residues known as protein S-nitrosation is the predominant mechanism for transduction of NO bioactivity. Histone acetylation on N-terminal lysine residues is a very important epigenetic regulatory mechanism. The transfer of acetyl groups from acetyl-coenzyme A on histone lysine residues is catalyzed by histone acetyltransferases. This modification neutralizes the positive charge of the lysine residue and results in a loose structure of the chromatin accessible for the transcriptional machinery. Histone deacetylases, in contrast, remove the acetyl group of histone tails resulting in condensed chromatin with reduced gene expression activity. In plants, the histone acetylation level is regulated by S-nitrosation. NO inhibits HDA complexes resulting in enhanced histone acetylation and promoting a supportive chromatin state for expression of genes. Moreover, methylation of histone tails and DNA are important epigenetic modifications, too. Interestingly, methyltransferases and demethylases are described as targets for redox molecules in several biological systems suggesting that these types of chromatin modifications are also regulated by NO. In this review article, we will focus on redox-regulation of histone acetylation/methylation and DNA methylation in plants, discuss the consequences on the structural level and give an overview where NO can act to modulate chromatin structure.

As a Histone Deacetylase Inhibitor, γ-Aminobutyric Acid Upregulates GluR2 Expression: An In Vitro and In Vivo Study.

γ-Aminobutyric acid (GABA) possesses extensive physiological functions and can be directly obtained from foods. GABA-enriched functional foods have been developed and the commercial demands for GABA are increasing. GABA is widely recognized as a central nervous system inhibitory neurotransmitter and plays an important role in some diseases by binding to its receptors. However, some of the functions of GABA are not explained by neurotransmission or GABA receptor pathways. Therefore, this study investigates whether GABA has the potential to inhibit histone deacetylase (HDAC). It is found that GABA inhibits HDAC1/2/3 expression and upregulates histone acetylation levels (Ace-H3K9/Ace-H4K12) in SH-SY5Y cells (which express GABA receptors), 3T3-L1 cells (which do not express GABA receptors), and the cerebral cortex in mice. Glutamate receptor 2 (GluR2) is a subunit of the α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor and is implicated in the pathogenesis of some neurological diseases. It is also found that GABA increases GluR2 expression by inhibiting HDAC1/2 but not HDAC3. A novel role for GABA is demonstrated in which it acts as an HDAC inhibitor. The present study expands the horizons for exploring the non-neurotransmitter functions of GABA.

Exceptional responders to abexinostat (ABX) plus pazopanib (PAZ) in pretreated renal cell carcinoma (RCC) and other solid tumors: Long-term follow-up of a phase 1b study.

3022 Background: We previously reported the initial phase 1b study results of PAZ + ABX, a potent pan-HDAC inhibitor, demonstrating acceptable toxicity profile and encouraging anti-tumor activity (Aggarwal et al. JCO 2017). We report the long-term follow up of exceptional responders and additional correlative analyses associated with clinical outcomes. Methods: Key efficacy endpoints included objective response rate and duration of response. Peripheral blood histone acetylation, HDAC expression, and plasma VEGF levels were analyzed and associated with clinical outcomes. Results: 51 pts (RCC subset; N = 22) were enrolled between June 2012 and October 2015. 10 pts (20%) had experienced disease progression on prior PAZ; 59% had received any prior VEGF-targeting therapy. 9 evaluable pts (18%) (N = 6 RCC; 2 thyroid; 1 mesothelioma) achieved partial tumor response (PR), of which 6 had prior progression on VEGF-targeting therapy. 7/10 (70%) of pts with prior disease progression on PAZ monotherapy had reduction in tumor burden on study. The median duration of response was 9.1 months (range 1.2 to 70+), and clinical benefit rate (PR or stable disease > 6 months) was 33%. Five treatment-refractory pts achieved durable PRs lasting for > 2 years duration, and one previously PAZ-refractory patient with RCC remains on treatment with ongoing PR for > 6 years. Higher HDAC2 expression was associated with prolonged progression-free survival (median PFS 5.9 vs. 3.5 months, log-rank p = 0.02). Induction of histone acetylation on ABX lead-in treatment was associated with subsequent time to progression (p = 0.002). On-treatment plasma VEGF levels were inversely correlated with PBMC histone acetylation (p = 0.02). Conclusions: Markedly durable responses with PAZ + ABX are achievable, including in pts with PAZ- and VEGF-refractory RCC and other solid tumor malignancies. Host factors including HDAC expression and acetylation status may identify those most likely to benefit. A randomized phase 3 study is underway of PAZ + ABX as a first- or second-line therapy in pts with locally advanced or metastatic RCC (RENAVIV; NCT03592472). Clinical trial information: NCT01543763.

Phase Ib study: Selective histone deacetylase (HDAC) inhibitor ACY-241 + nivolumab (Nivo) in advanced non-small cell lung cancer (NSCLC).

9029 Background: Identification of new regimens for patients (pts) with relapse/refractory NSCLC is an ongoing need. HDAC inhibitor monotherapy has cytostatic activity in pts with NSCLC. The HDAC6 inhibitor ACY-241 has immunomodulatory activity and may synergize with immune checkpoint inhibitors (ICI). This phase Ib study investigated ACY-241 + nivo in metastatic NSCLC. Methods: Previously platinum-treated, ICI-naïve pts with advanced NSCLC and ECOG PS ≤ 1 were eligible. In 3 + 3 dose-escalation, pts received ACY-241 (180, 360, or 480 mg PO; d 1-28) + nivo 240 mg on d 15 of cycle 1 and d 1,15 subsequently in 28-d cycles. Primary outcomes: safety and RP2D. Secondary outcome: antitumor activity. Exploratory outcome: biomarker analyses; peripheral blood samples and tumor biopsies were collected pre- and on-treatment. Results: As of Jan 17, 2019, 18 pts were treated. Median age 66 y; 56% male, 78% PS 1, 93% stage IV, 28% PD-L1–negative, and 89% adenocarcinoma (11% squamous). No DLTs were observed at ACY-241 180 or 360 mg, and 2 DLTs were observed at 480 mg (gr 3 nausea lasting > 72 h despite medication; gr 5 cardiac arrest); 360 mg was declared RP2D. Common all-grade AEs: fatigue (22%), arthralgia (17%), and cough (17%); 1 gr 3 cerebrovascular accident (related to brain metastasis unrelated to study drug) and 1 additional gr 5 AE (myasthenia gravis, possibly related to ACY-241 and nivo) occurred. Of 13 response-evaluable pts, 8 had clinical benefit (1 CR, 3 PR [1 PD-L1–negative], 1 unconfirmed PR [tumor shrinkage followed by new lesion], 3 SD); 5 had PD. At data cutoff, treatment was ongoing in 5 pts (12 – 30 cycles). Histone and tubulin acetylation levels were transiently increased after treatment. Circulating IL-2, IL-1β, MMP-9, and IL-10 levels increased, with undetectable TNF-α and IFN-γ modulation. Treatment-related changes in tumor-infiltrating Treg, MDSC, TCM, and macrophages varied between pts but revealed a trend of increased infiltrating cytotoxic T and NK cells. Conclusions: The ACY-241 + nivo combination is feasible and can be considered for further research as a potential NSCLC treatment option. Biomarker analyses may help identify an optimal population for the combination. Clinical trial information: NCT02635061.

Epigenetic and autophagic changes after nerve agent exposure in the rat piriform cortex and hippocampus

The detrimental effects of organophosphate (OP) nerve agents have been reported but the mechanisms mediating these multiple effects are not well understood. Recent use of nerve agents in Syria and the UK illustrate their continuous threat to the modern world. Epigenetic and autophagy studies are useful to address the issues related to regulation of gene and protein expression by which nerve agents could impact on human health. These studies help to understand molecular mechanisms underlying the multiple neurotoxic effects of nerve agents. In the present study, changes in epigenetic (global DNA methylation and histone acetylation) and autophagic marker proteins were studied in the nerve agent sensitive rat brain areas (piriform cortex and hippocampus) after soman (1xLD50) exposure. Global DNA methylation analysis revealed that nerve agent induced hypomethylation in the brain regions at 1 and 7 days post exposure. In contrast, DNA hypermethylation was observed at 30 days post soman exposure, demonstrating a possible compensatory mechanism. Western blot analysis showed significant increase in the histone acetylation levels after soman exposure in the piriform cortex and hippocampus. The present study observed the changes in autophagic proteins of nerve agent poisoning for the first time to the best of our knowledge. Immunoreactivity levels of autophagic proteins (LC3-II, ATG-5 and p62) were transiently increased in the rat piriform cortex and hippocampus after soman exposure. In conclusion, this study provides insight into the epigenetic and autophagic changes in the brain following soman exposure and their possible role in the neuronal damage and development of multiple neurological effects.

Retraction: Antitumor Effects of a Novel Phenylbutyrate-based Histone Deacetylase Inhibitor, (S)-HDAC-42, in Prostate Cancer.

Purpose: To assess the antitumor effects of a novel phenylbutyrate-derived histone deacetylase (HDAC) inhibitor, ( S )-HDAC-42, vis-a-vis suberoylanilide hydroxamic acid (SAHA) in in vitro and in vivo models of human prostate cancer. Experimental Design: The in vitro effects of ( S )-HDAC-42 and SAHA were evaluated in PC-3, DU-145, or LNCaP human prostate cancer cell lines. Cell viability, apoptosis, and indicators of HDAC inhibition were assessed. Effects on Akt and members of the Bcl-2 and inhibitor of apoptosis protein families were determined by immunoblotting. Immunocompromised mice bearing established s.c. PC-3 xenograft tumors were treated orally with ( S )-HDAC-42 (50 mg/kg q.o.d. or 25 mg/kg q.d.) or SAHA (50 mg/kg q.d.) for 28 days. In vivo end points included tumor volumes and intratumoral changes in histone acetylation, phospho-Akt status, and protein levels of Bcl-xL and survivin. Results: ( S )-HDAC-42 was more potent than SAHA in suppressing the viability of all cell lines evaluated with submicromolar IC 50 values. Relative to SAHA, ( S )-HDAC-42 exhibited distinctly superior apoptogenic potency, and caused markedly greater decreases in phospho-Akt, Bcl-xL, and survivin in PC-3 cells. The growth of PC-3 tumor xenografts was suppressed by 52% and 67% after treatment with ( S )-HDAC-42 at 25 and 50 mg/kg, respectively, whereas SAHA at 50 mg/kg suppressed growth by 31%. Intratumoral levels of phospho-Akt and Bcl-xL were markedly reduced in ( S )-HDAC-42-treated mice, in contrast to mice treated with SAHA. Conclusions: ( S )-HDAC-42 is a potent orally bioavailable inhibitor of HDAC, as well as targets regulating multiple aspects of cancer cell survival, which might have clinical value in prostate cancer chemotherapy and warrants further investigation in this regard.

Reduced acetylation of histones H3 in peripheral blood mononuclear cells of patients with Primary Sjögren’s syndrome

Abstract Sjögren’s syndrome (SS) is an autoimmune disease characterized by an exocrine glands epithelium injury with dense lymphocytic infiltrates, mainly composed of activated T and B cells, involvement of the peripheral nervous, pulmonary, blood, kidney, skin, and joint. Present at the interface of genetic and environmental risk factors, histone acetylation is suspected to play a key role in SS. To clarify this point, we investigated the expression of histone deacetylases (HDACs1–10) and histone acetyltransferase (HAT-p300 CREEBP PCAF) by real time PCR in peripheral blood mononuclear cells (PBMCs) from SS patients. Elisa measured HDACs\HATs and histone H3/ H4 acetylation activity. Flow Cytometry and western blot verified histone acetylation level. The expression of HDACs1-10, p300, CREEBP and PCAF in PBMCs from SS patients was decreased in mRNA comparison with healthy controls (HCs). Total histone acetylation was reduced in PBMCs from SS patients, while no difference was observed in histone deacetylation. The extent of total histone H3, but not H4, acetylation in PBMCs from SS patients was decreased compared to that in HCs. An altered balance between HDAC and HAT activity was found in SS PBMCs. As a consequence, part of the pathogenic mechanisms in SS may be linked to epigenetic modifications, thus opening new therapeutic perspectives in SS.

Histone acetylation promotes long-lasting defense responses and longevity following early life heat stress.

Early exposure to some mild stresses can slow down the aging process and extend lifespan, raising the question of how early life stress might impact the somatic health of aged animals. Here, we reveal that early life heat experience triggers the establishment of epigenetic memory in soma, which promotes long-lasting stress responses and longevity in C. elegans. Unlike lethal heat shock, mild heat activates a unique transcriptional program mimicking pathogen defense responses, characterized by the enhanced expression of innate immune and detoxification genes. Surprisingly, the expression of defense response genes persists long after heat exposure, conferring enhanced stress resistance even in aged animals. Further studies identify the histone acetyltransferase CBP-1 and the chromatin remodeling SWI/SNF complex as epigenetic modulators of the long-lasting defense responses. Histone acetylation is elevated by heat stress and maintained into agedness thereafter. Accordingly, histone acetylation levels were increased on the promoters of defense genes. Moreover, disruption of epigenetic memory abrogates the longevity response to early hormetic heat stress, indicating that long-lasting defense responses are crucial for the survival of aged animals. Together, our findings provide mechanistic insights into how temperature stress experienced in early life provides animals with lifetime health benefits.

Epigenetic Modifications in Placenta are Associated with the Child's Sensitization to Allergens.

Prenatal environmental exposures are considered to contribute to the development of allergic sensitization by epigenetic mechanisms. The role of histone acetylation in the placenta has not been examined yet. We hypothesized that placental histone acetylation at the promoter regions of allergy-related immune regulatory genes is associated with the development of sensitization to allergens in the child. Histones H3 and H4 acetylation at the promoter regions of 6 selected allergy-related immune regulatory genes was assessed by a chromatin immunoprecipitation assay in 173 term placentas collected in the prospective birth-cohort ALADDIN. The development of IgE sensitization to allergens in the children was followed from 6 months up to 5 years of age. We discovered significant associations of histone acetylation levels with decreased risk of allergic sensitization in 3 genes. Decreased risk of sensitization to food allergens was associated with higher H3 acetylation levels in placentas at the IFNG and SH2B3 genes, and for H4 acetylation in HDAC4. Higher HDAC4 H4 acetylation levels were also associated with a decreased risk of sensitization to aeroallergens. In conclusion, our results suggest that acetylation of histones in placenta has a potential to predict the development of sensitization to allergens in children.

Copy number variation (CNV) in the <i>IGF1R</i> gene across four cattle breeds and its association with economic traits

The insulin-like growth factor 1 receptor (IGF1R) plays a vital role in immunomodulation and muscle and bone growth. The copy number variation (CNV) is believed to the reason for many complex phenotypic variations. In this paper, we statistically analyzed the copy number and the expression profiling in different tissue types of the IGF1R gene using the 422 samples from four Chinese beef cattle breeds, and the mRNA of IGF1R was widely expressed in nine tissue types of adult cattle (heart, liver, kidney, muscle, fat, stomach, spleen, lung and testis). Results of CNV and growth traits indicated that the IGF1R CNV was significantly associated with body weight and body height of Jinnan (JN) cattle and was significantly associated with body height and hucklebone width of Qinchuan (QC) cattle, making IGF1R CNV a promising molecular marker to improve meat production in beef cattle breeding. Bioinformatics predictions show that the CNV region is highly similar to the human genome, and there are a large number of transcription factors, DNase I hypersensitive sites, and high levels of histone acetylation, suggesting that this region may play a role in transcriptional regulation, providing directions for further study of the role of bovine CNV and economic traits.

Molecular cloning and subcellular localization of six HDACs and their roles in response to salt and drought stress in kenaf (Hibiscus cannabinus L.).

BackgroundHistone acetylation is an important epigenetic modification that regulates gene activity in response to stress. Histone acetylation levels are reversibly regulated by histone acetyltransferases (HATs) and histone deacetylases (HDACs). The imperative roles of HDACs in gene transcription, transcriptional regulation, growth and responses to stressful environment have been widely investigated in Arabidopsis. However, data regarding HDACs in kenaf crop has not been disclosed yet.ResultsIn this study, six HDACs genes (HcHDA2, HcHDA6, HcHDA8, HcHDA9, HcHDA19, and HcSRT2) were isolated and characterized. Phylogenetic tree revealed that these HcHDACs shared high degree of sequence homology with those of Gossypium arboreum. Subcellular localization analysis showed that GFP-tagged HcHDA2 and HcHDA8 were predominantly localized in the nucleus, HcHDA6 and HcHDA19 in nucleus and cytosol. The HcHDA9 was found in both nucleus and plasma membranes. Real-time quantitative PCR showed that the six HcHDACs genes were expressed with distinct expression patterns across plant tissues. Furthermore, we determined differential accumulation of HcHDACs transcripts under salt and drought treatments, indicating that these enzymes may participate in the biological process under stress in kenaf. Finally, we showed that the levels of histone H3 and H4 acetylation were modulated by salt and drought stress in kenaf.ConclusionsWe have isolated and characterized six HDACs genes from kenaf. These data showed that HDACs are imperative players for growth and development as well abiotic stress responses in kenaf.

Spatial intratumor heterogeneity in uveal melanoma: Tumor cell subtypes with a presumed invasive potential exhibit a particular epigenetic staining reaction

Cancer evolves from a combination of genetic and epigenetic abnormalities resulting in aberrant gene expression profiles as well as altered epigenomic patterns. Epigenetic alterations such as DNA methylation and histone modification play an important role in tumorigenesis. While in the pathobiology of uveal melanoma (UM) genetic changes have been well characterized, there is growing evidence suggesting that epigenetic changes are also involved. We investigated whether epigenetic modifications (global levels of histone acetylation, DNA methylation, ubiquitination) are detectable in UM tissues compared to healthy controls with respect to inter- and intratumoral heterogeneity. Formalin-fixed paraffin-embedded tissues of primary UM (n = 15), UM metastasis (n = 13), and control choroid (n = 12) were immunohistochemically investigated by two ophthalmic pathologists for global levels of histone acetylation (Histone 3 acetylation, H3Ac; Histone 4 acetylation, H4Ac), DNA methylation (5-methylcytosine, 5-MeC; 5′-hydroxymethylcytosine, 5-hMeC), global ubiquitination (UBC) as well as Ubiquityl-Histone H2A (H2Aub). The nuclear staining intensity of primary tumors, metastases and control choroids was evaluated using a score from 0 to 3, which was multiplied with the percentage of stained cells (score from 0 to 4). The control choroid and the choroid next to the tumor showed a more intense nuclear staining than the primary tumor tissue. The choroid next to the tumor was stained less than the control choroid. The nuclear staining intensity in the tumor was comparable to that in the metastases. The tumor tissue itself often exhibited a heterogeneous staining pattern, as nuclei in the tumor center were less intensely stained than in the periphery. Cells with a presumed invasive potential (extraocular extension, growth along emissary canals) showed also an intense staining reaction. Although no prognostically relevant pattern of global epigentic markers could be identified, our results suggest that epigenetic changes play a role in UM pathogenesis and metastasis. In particular the staining reaction of tumor cell subtypes with a presumed invasive potential warrants further attention. The role of epigenetically relevant interactions with the tumor micromilieu should be further investigated as immune cells are predominantly located in the tumor periphery which showed a different staining intensity than the tumor center. However, as considerable epigenetic diversity exists in primary tumors, studies on biopsy tissue are not recommended for the immunohistochemical investigation of epigenetic markers.

Depletion of ATP-Citrate Lyase (ATPCL) Affects Chromosome Integrity Without Altering Histone Acetylation in Drosophila Mitotic Cells.

The Citrate Lyase (ACL) is the main cytosolic enzyme that converts the citrate exported from mitochondria by the SLC25A1 carrier in Acetyl Coenzyme A (acetyl-CoA) and oxaloacetate. Acetyl-CoA is a high-energy intermediate common to a large number of metabolic processes including protein acetylation reactions. This renders ACL a key regulator of histone acetylation levels and gene expression in diverse organisms including humans. We have found that depletion of ATPCL, the Drosophila ortholog of human ACL, reduced levels of Acetyl CoA but, unlike its human counterpart, does not affect global histone acetylation and gene expression. Nevertheless, reduced ATPCL levels caused evident, although moderate, mitotic chromosome breakage suggesting that this enzyme plays a partial role in chromosome stability. These defects did not increase upon X-ray irradiation, indicating that they are not dependent on an impairment of DNA repair. Interestingly, depletion of ATPCL drastically increased the frequency of chromosome breaks (CBs) associated to mutations in scheggia, which encodes the ortholog of the mitochondrial citrate carrier SLC25A1 that is also required for chromosome integrity and histone acetylation. Our results indicate that ATPCL has a dispensable role in histone acetylation and prevents massive chromosome fragmentation when citrate efflux is altered.

Epigenetic Changes in Alveolar Type II Lung Cells of A/J Mice Following Intranasal Treatment with Lipopolysaccharide.

Lipopolysaccharide (LPS) is a bacterial endotoxin present in cigarette smoke. LPS is known to induce inflammation and to increase the size and the multiplicity of lung tumors induced by tobacco-specific nitrosamines. However, the means by which LPS contributes to pulmonary carcinogenesis are not known. One possible mechanism includes LPS-mediated epigenetic deregulation, which leads to aberrant expression of genes involved in DNA repair, tumor suppression, cell cycle progression, and cell growth. In the present work, epigenetic effects of LPS were examined in alveolar type II lung cells of A/J mice. Type II cells were selected because they serve as progenitors of lung adenocarcinomas in smoking induced lung cancer. A/J mice were intranasally treated with LPS, followed by isolation of alveolar type II cells from the lung using cell panning. Global levels of DNA methylation and histone acetylation were quantified by mass spectrometry, while genome-wide transcriptomic changes were characterized by RNA-Seq. LPS treatment was associated with epigenetic changes including decreased cytosine formylation and reduced histone H3K14 and H3K23 acetylation, as well as altered expression levels of genes involved in cell adhesion, inflammation, immune response, and epigenetic regulation. These results suggest that exposure to inflammatory agents in cigarette smoke leads to early epigenetic changes in the lung, which may collaborate with genetic changes to drive the development of lung cancer.

Identification of isoform-selective hydroxamic acid derivatives that potently reactivate HIV from latency

ObjectivesCurrent antiretroviral therapy can suppress HIV replication, increase CD4 count and result in increased lifespan. However, it cannot eradicate the virus due to the presence of latent provirus in cellular reservoirs, such as resting CD4+ T cells. Using combination latency-reversing agents to shock the virus out of latency for elimination through immune clearance or viral cytopathic effect is one of the most promising strategies for HIV eradication. Specifically, recent evidence shows that isoform-selective histone deacetylase inhibitors may be more effective than their non-selective counterparts. Therefore, identification and characterisation of new isoform-selective compounds are of prime importance. Here, we sought to determine the ability of two new isoform-targeted hydroxamic acid derivatives to reactivate HIV from latency. MethodsWe used cell lines and infected primary resting CD4+ T cells. These were treated with these compounds with HIV reactivation measured using fluorescence-activated cell sorting, Western blots and luciferase luminescence. Isoform selectivity and acetylation of the HIV promoter were measured by Western blotting and chromatic immunoprecipitation. Results and conclusionsThe two new hydroxamic acid derivatives, MC2625 and MC1742, potently reactivate HIV from latency. These compounds are isoform-selective histone deacetylate inhibitors that increase the levels of histone acetylation at the HIV promoter. In addition, they synergise effectively with the protein kinase C modulators bryostatin-1 and INDY, an inhibitor of the dual-specificity tyrosine phosphorylation regulated kinase 1A. We conclude that the combinations of new hydroxamic acid derivatives and bryostatin-1 or INDY could be a new tool for HIV reactivation in the cure efforts.

Multiple superovulations alter histone modifications in mouse early embryos.

It is demonstrated that repeated superovulation has deleterious effects on mouse ovaries and cumulus cells. However, little is known about the effects of repeated superovulation on early embryos. Epigenetic reprogramming is an important event in early embryonic development and could be easily disrupted by the environment. Thus, we speculated that multiple superovulations may have adverse effects on histone modifications in the early embryos. Female CD1 mice were randomly divided into four groups: (a) spontaneous estrus cycle (R0); (b) with once superovulation (R1); (c) with three times superovulation at a 7-day interval (R3) and (d) with five times superovulation at a 7-day interval (R5). We found that repeated superovulation remarkably decreased the fertilization rate. With the increase of superovulation times, the rate of early embryo development was decreased. The expression of Oct4, Sox2 and Nanog was also affected by superovulation in blastocysts. The immunofluorescence results showed that the acetylation level of histone 4 at lysine 12 (H4K12ac) was significantly reduced by repeated superovulation in mouse early embryos (P < 0.01). Acetylation level of histone 4 at lysine 16 (H4K16ac) was also significantly reduced in pronuclei and blastocyst along with the increase of superovulation times (P < 0.01). H3K9me2 and H3K27me3 were significantly increased in four-cell embryos and blastocysts. We further found that repeated superovulation treatment increased the mRNA level of histone deacetylases Hdac1, Hdac2 and histone methyltransferase G9a, but decreased the expression level of histone demethylase-encoding genes Kdm6a and Kdm6b in early embryos. In a word, multiple superovulations alter histone modifications in early embryos.

Loss of genomic integrity induced by lysosphingolipid imbalance drives ageing in the heart.

Cardiac dysfunctions dramatically increase with age. Revealing a currently unknown contributor to cardiac ageing, we report the age-dependent, cardiac-specific accumulation of the lysosphingolipid sphinganine (dihydrosphingosine, DHS) as an evolutionarily conserved hallmark of the aged vertebrate heart. Mechanistically, the DHS-derivative sphinganine-1-phosphate (DHS1P) directly inhibits HDAC1, causing an aberrant elevation in histone acetylation and transcription levels, leading to DNA damage. Accordingly, the pharmacological interventions, preventing (i) the accumulation of DHS1P using SPHK2 inhibitors, (ii) the aberrant increase in histone acetylation using histone acetyltransferase (HAT) inhibitors, (iii) the DHS1P-dependent increase in transcription using an RNA polymerase II inhibitor, block DHS-induced DNA damage in human cardiomyocytes. Importantly, an increase in DHS levels in the hearts of healthy young adult mice leads to an impairment in cardiac functionality indicated by a significant reduction in left ventricular fractional shortening and ejection fraction, mimicking the functional deterioration of aged hearts. These molecular and functional defects can be partially prevented invivo using HAT inhibitors. Together, we report an evolutionarily conserved mechanism by which increased DHS levels drive the decline in cardiac health.

DNA replication dynamics of vole genome and its epigenetic regulation

BackgroundThe genome of some vole rodents exhibit large blocks of heterochromatin coupled to their sex chromosomes. The DNA composition and transcriptional activity of these heterochromatin blocks have been studied, but little is known about their DNA replication dynamics and epigenetic composition.ResultsHere, we show prominent epigenetic marks of the heterochromatic blocks in the giant sex chromosomes of female Microtus cabrerae cells. While the X chromosomes are hypoacetylated and cytosine hypomethylated, they are either enriched for macroH2A and H3K27me3 typical for facultative heterochromatin or for H3K9me3 and HP1 beta typical for constitutive heterochromatin. Using pulse-chase replication labeling and time-lapse microscopy, we found that the heterochromatic block enriched for macroH2A/H3K27me3 of the X chromosome is replicated during mid-S-phase, prior to the heterochromatic block enriched for H3K9me3/HP1 beta, which is replicated during late S-phase. To test whether histone acetylation level regulates its replication dynamics, we induced either global hyperacetylation by pharmacological inhibition or by targeting a histone acetyltransferase to the heterochromatic region of the X chromosomes. Our data reveal that histone acetylation level affects DNA replication dynamics of the sex chromosomes’ heterochromatin and leads to a global reduction in replication fork rate genome wide.ConclusionsIn conclusion, we mapped major epigenetic modifications controlling the structure of the sex chromosome-associated heterochromatin and demonstrated the occurrence of differences in the molecular mechanisms controlling the replication timing of the heterochromatic blocks at the sex chromosomes in female Microtus cabrerae cells. Furthermore, we highlighted a conserved role of histone acetylation level on replication dynamics across mammalian species.