Trimethylation profile of histones H3 lysine 4 and 9 in late preantral and early antral caprine follicles grown in vivo versus in vitro in the presence of anethole.

This study assessed the histones methylation profile (H3K4me3 and H3K9me3) in late preantral (PA) and early antral (EA) caprine follicles grown in vivo and in vitro, and the anethole effect during in vitro culture of PA follicles. Uncultured in vivo-grown follicles (PA, n = 64; EA, n = 73) were used as controls to assess the methylation profile and genes' expression related to apoptosis cascade (BAX, proapoptotic; BCL2, antiapoptotic), steroidogenesis (CYP17, CYP19A1), and demethylation (KDM1AX1, KDM1AX2, KDM3A). The isolated PA follicles (n = 174) were cultured in vitro for 6 days in α-MEM+ in either absence (control) or presence of anethole. After culture, EA follicles were evaluated for methylation, mRNA abundance, and morphometry. Follicle diameter increased after culture, regardless of treatment. The methylation profile and the mRNA abundance were similar between in vivo-grown PA and EA follicles. Anethole treatment led to higher H3K4me3 fluorescence intensity in EA follicles. The mRNA abundances of BAX, CYP17, and CYP19A1 were higher, and BCL2 and KDM3A were lower in in vitro-grown EA follicles than in vivo-grown follicles. In conclusion, in vitro follicle culture affected H3K4me3 fluorescence intensity, mRNA abundance of apoptotic genes, and steroidogenic and demethylase enzymes compared with in vivo-grown follicles.

Epigenetic inhibition of lncRNA GMDS-AS1 by methyltransferase ESET promoted cell viability and metastasis of hepatocellular carcinoma.

BACKGROUND: Long noncoding RNA (lncRNAs) GMDS-AS1 has been reported as a tumor regulator in tumor growth and metastasis, but its effect in hepatocellular carcinoma (HCC) remains unclear. ESET, a histone H3K9 methyl-transferase, is involved in epigenomic regulation of tumor progression in multiple cancers. However, the correlation between ESET and lncRNA in HCC is less reported. METHODS: Quantitative real-time PCR (qRT-PCR) was taken to determine the expression of ESET and GMDS-AS1. Western blot was taken to determine the target protein levels of ESET and GMDS-AS1. Online database and bioinformatics analysis were used to screen abnormally expressed genes. Luciferase assay was performed to confirm the binding of GMDS-AS1 and PSMB1. Ki67 and Edu were used for evaluated the proliferation of tumor cells. ChIP assay was performed to verify the relationship between H3K9me1 and lncRNA GMDS-AS1 promoter. Transwell was taken to determine the migration and invasion ability of tumor cells. CCK-8 was used for determining the viability of tumor cells. Flow cytometry was performed to detect the cell cycle of tumor cells. RESULTS: The expression of GMDS-AS1 was decreased and the expression of ESET was increased in HCC. GMDS-AS1 inhibition contributed to tumor development, and this effect was closely related to epigenetic inhibition of GMDS-AS1 by ESET. PSMB1, a downstream target of GMDS-AS1, promoted the tumor proliferation and was negatively regulated by GMDS-AS1. CONCLUSION: Our result demonstrates anti-tumorigenic traits of lncRNA GMDS-AS1 in HCC and explains its pattern of regulation mediated by ESET. Our work unmasked an essential role of GMDS-AS1 in HCC progression and detected a novel pathway for ESET to promote HCC.

Convergent Epigenetic Mechanisms Avoid Constitutive Expression of Immune Receptor Gene Subsets.

The gene pool encoding PRR and NLR immune receptors determines the ability of a plant to resist microbial infections. Basal expression of these genes is prevented by diverse mechanisms since their hyperactivity can be harmful. To approach the study of epigenetic control of PRR/NLR genes we here analyzed their expression in mutants carrying abnormal repressive 5-methyl cytosine (5-mC) and histone 3 lysine 9 dimethylation (H3K9me2) marks, due to lack of MET1, CMT3, MOM1, SUVH4/5/6, or DDM1. At optimal growth conditions, none of the mutants showed basal expression of the defense gene marker PR1, but all of them had greater resistance to Pseudomonas syringae pv. tomato than wild type plants, suggesting they are primed to stimulate immune cascades. Consistently, analysis of available transcriptomes indicated that all mutants showed activation of particular PRR/NLR genes under some growth conditions. Under low defense activation, 37 PRR/NLR genes were expressed in these plants, but 29 of them were exclusively activated in specific mutants, indicating that MET1, CMT3, MOM1, SUVH4/5/6, and DDM1 mediate basal repression of different subsets of genes. Some epigenetic marks present at promoters, but not gene bodies, could explain the activation of these genes in the mutants. As expected, suvh4/5/6 and ddm1 activated genes carrying 5-mC and H3K9me2 marks in wild type plants. Surprisingly, all mutants expressed genes harboring promoter H2A.Z/H3K27me3 marks likely affected by the chromatin remodeler PIE1 and the histone demethylase REF6, respectively. Therefore, MET1, CMT3, MOM1, SUVH4/5/6, and DDM1, together with REF6, seemingly contribute to the establishment of chromatin states that prevent constitutive PRR/NLR gene activation, but facilitate their priming by modulating epigenetic marks at their promoters.

Pilocarpine-induced seizures associate with modifications of LSD1/CoREST/HDAC1/2 epigenetic complex and repressive chromatin in mice hippocampus.

Epilepsy is a neurological disorder of genetic or environmental origin characterized by recurrent spontaneous seizures. A rodent model of temporal lobe epilepsy is induced by a single administration of pilocarpine, a non-selective cholinergic muscarinic receptor agonist. The molecular changes associated with pilocarpine-induced seizures are still poorly described. Epigenetic multiprotein complexes that regulate gene expression by changing the structure of chromatin impose transcriptional memories. Among the epigenetic enzymes relevant to the epileptogenic process is lysine-specific demethylase 1 (LSD1, KDM1A), which regulates the expression of genes that control neuronal excitability. LSD1 forms complexes with the CoREST family of transcriptional corepressors, which are molecular bridges that bring HDAC1/2 and LSD1 enzymes to deacetylate and demethylate the tail of nucleosomal histone H3. To test the hypothesis that LSD1-complexes are involved in initial modifications associated with pilocarpine-induced epilepsy, we studied the expression of main components of LSD1-complexes and the associated epigenetic marks on isolated neurons and the hippocampus of pilocarpine-treated mice. Using a single injection of 300 mg/kg of pilocarpine and after 24 h, we found that protein levels of LSD1, CoREST2, and HDAC1/2 increased, while CoREST1 decreased in the hippocampus. In addition, we observed increased histone H3 lysine 9 di- and trimethylation (H3K9me2/3) and decreased histone H3 lysine 4 di and trimethylation (H3K4me2/3). Similar findings were observed in cultured hippocampal neurons and HT-22 hippocampal cell line treated with pilocarpine. In conclusion, our data show that muscarinic receptor activation by pilocarpine induces a global repressive state of chromatin and prevalence of LSD1-CoREST2 epigenetic complexes, modifications that could underlie the pathophysiological processes leading to epilepsy.

H3K9 Methyltransferases Suv39h1 and Suv39h2 Control the Differentiation of Neural Progenitor Cells in the Adult Hippocampus.

In the dentate gyrus of the adult hippocampus new neurons are generated from neural precursor cells through different stages including proliferation and differentiation of neural progenitor cells and maturation of newborn neurons. These stages are controlled by the expression of specific transcription factors and epigenetic mechanisms, which together orchestrate the progression of the neurogenic process. However, little is known about the involvement of histone posttranslational modifications, a crucial epigenetic mechanism in embryonic neurogenesis that regulates fate commitment and neuronal differentiation. During embryonic development, the repressive modification trimethylation of histone H3 on lysine 9 (H3K9me3) contributes to the cellular identity of different cell-types. However, the role of this modification and its H3K9 methyltransferases has not been elucidated in adult hippocampal neurogenesis. We determined that during the stages of neurogenesis in the adult mouse dentate gyrus and in cultured adult hippocampal progenitors (AHPs), there was a dynamic change in the expression and distribution of H3K9me3, being enriched at early stages of the neurogenic process. A similar pattern was observed in the hippocampus for the dimethylation of histone H3 on lysine 9 (H3K9me2), another repressive modification. Among H3K9 methyltransferases, the enzymes Suv39h1 and Suv39h2 exhibited high levels of expression at early stages of neurogenesis and their expression decreased upon differentiation. Pharmacological inhibition of these enzymes by chaetocin in AHPs reduced H3K9me3 and concomitantly decreased neuronal differentiation while increasing proliferation. Moreover, Suv39h1 and Suv39h2 knockdown in newborn cells of the adult mouse dentate gyrus by retrovirus-mediated RNA interference impaired neuronal differentiation of progenitor cells. Our results indicate that H3K9me3 and H3K9 methyltransferases Suv39h1 and Suv39h2 are critically involved in the regulation of adult hippocampal neurogenesis by controlling the differentiation of neural progenitor cells.

LINE-1 specific nuclear organization in mice olfactory sensory neurons.

Long interspersed nuclear elements-1 (LINE-1) are mobile DNA elements that comprise the majority of interspersed repeats in the mammalian genome. During the last decade, these transposable sequences have been described as controlling elements involved in transcriptional regulation and genome plasticity. Recently, LINE-1 have been implicated in neurogenesis, but to date little is known about their nuclear organization in neurons. The olfactory epithelium is a site of continuous neurogenesis, and loci of olfactory receptor genes are enriched in LINE-1 copies. Olfactory neurons have a unique inverted nuclear architecture and constitutive heterochromatin forms a block in the center of the nuclei. Our DNA FISH images show that, even though LINE-1 copies are dispersed throughout the mice genome, they are clustered forming a cap around the central heterochromatin block and frequently occupy the same position as facultative heterochromatin in olfactory neurons nuclei. This specific LINE-1 organization could not be observed in other olfactory epithelium cell types. Analyses of H3K27me3 and H3K9me3 ChIP-seq data from olfactory epithelium revealed that LINE-1 copies located at OR gene loci show different enrichment for these heterochromatin marks. We also found that LINE-1 are transcribed in mouse olfactory epithelium. These results suggest that LINE-1 play a role in the olfactory neurons' nuclear architecture. SIGNIFICANCE STATEMENT: LINE-1 are mobile DNA elements and comprise almost 20% of mice and human genomes. These retrotransposons have been implicated in neurogenesis. We show for the first time that LINE-1 retrotransposons have a specific nuclear organization in olfactory neurons, forming aggregates concentric to the heterochromatin block and frequently occupying the same region as facultative heterochromatin. We found that LINE-1 at olfactory receptor gene loci are differently enriched for H3K9me3 and H3K27me3, but LINE-1 transcripts could be detected in the olfactory epithelium. We speculate that these retrotransposons play an active role in olfactory neurons' nuclear architecture.

Ski Is Required for Tri-Methylation of H3K9 in Major Satellite and for Repression of Pericentromeric Genes: Mmp3, Mmp10 and Mmp13, in Mouse Fibroblasts.

Several mechanisms directing a rapid transcriptional reactivation of genes immediately after mitosis have been described. However, little is known about the maintenance of repressive signals during mitosis. In this work, we address the role of Ski in the repression of gene expression during M/G1 transition in mouse embryonic fibroblasts (MEFs). We found that Ski localises as a distinct pair of dots at the pericentromeric region of mitotic chromosomes, and the absence of the protein is related to high acetylation and low tri-methylation of H3K9 in pericentromeric major satellite. Moreover, differential expression assays in early G1 cells showed that the presence of Ski is significantly associated with repression of genes localised nearby to pericentromeric DNA. In mitotic cells, chromatin immunoprecipitation assays confirmed the association of Ski to major satellite and the promoters of the most repressed genes: Mmp3, Mmp10 and Mmp13. These genes are at pericentromeric region of chromosome 9. In these promoters, the presence of Ski resulted in increased H3K9 tri-methylation levels. This Ski-dependent regulation is also observed during interphase. Consequently, Mmp activity is augmented in Ski-/- MEFs. Altogether, these data indicate that association of Ski with the pericentromeric region of chromosomes during mitosis is required to maintain the silencing bookmarks of underlying chromatin.

Widespread loss of the silencing epigenetic mark H3K9me3 in astrocytes and neurons along with hippocampal-dependent cognitive impairment in C9orf72 BAC transgenic mice.

BACKGROUND: Hexanucleotide repeat expansions of the G4C2 motif in a non-coding region of the C9ORF72 gene are the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Tissues from C9ALS/FTD patients and from mouse models of ALS show RNA foci, dipeptide-repeat proteins, and notably, widespread alterations in the transcriptome. Epigenetic processes regulate gene expression without changing DNA sequences and therefore could account for the altered transcriptome profiles in C9ALS/FTD; here, we explore whether the critical repressive marks H3K9me2 and H3K9me3 are altered in a recently developed C9ALS/FTD BAC mouse model (C9BAC). RESULTS: Chromocenters that constitute pericentric constitutive heterochromatin were visualized as DAPI- or Nucblue-dense foci in nuclei. Cultured C9BAC astrocytes exhibited a reduced staining signal for H3K9me3 (but not for H3K9me2) at chromocenters that was accompanied by a marked decline in the global nuclear level of this mark. Similar depletion of H3K9me3 at chromocenters was detected in astrocytes and neurons of the spinal cord, motor cortex, and hippocampus of C9BAC mice. The alterations of H3K9me3 in the hippocampus of C9BAC mice led us to identify previously undetected neuronal loss in CA1, CA3, and dentate gyrus, as well as hippocampal-dependent cognitive deficits. CONCLUSIONS: Our data indicate that a loss of the repressive mark H3K9me3 in astrocytes and neurons in the central nervous system of C9BAC mice represents a signature during neurodegeneration and memory deficit of C9ALS/FTD.

Expression of tissue-specific imprinted gene tumor Suppressing Subtransferable Candidate 4 (TSSC4) is altered in placentae produced by nuclear transfer in cattle.

Embryonic and placental development is highly orchestrated by epigenetic processes. Disruptions in normal placental development, commonly observed in pregnancies produced by nuclear transfer, are associated with abnormal gene expression and altered epigenetic regulation of imprinted and vital placental genes. The objective of this study was to evaluate expression and epigenetic regulation of the imprinted gene TSSC4 in cotyledonary and intercotyledonary tissues from day 60 pregnancies produced by embryo transfer (ET), in vitro fertilization (IVF) and nuclear transfer (NT) in cattle. TSSC4 expression was reduced by 30% in cotyledons at 60days of gestation in the NT group. The proximal promoter region of TSSC4 showed an increase in the permissive histone mark (H3K4me2) and a reduction in the inhibitory histone mark (H3K9me2) in the cotyledons produced by NT, in relation to cotyledons produced by embryo transfer. Interestingly, H3K9me2 was also significantly reduced in cotyledons produced by IVF, compared to the ET controls. DNA methylation, in CpG-rich regions located at the proximal promoter region and the coding region of TSSC4 did not differ. These results suggest that the reduction in TSSC4 expression, observed following NT, can not be explained by the histone changes investigated in the proximal promoter region of the gene, or by changes in methylation in three regions evaluated. Also, a decrease in the levels of H3K9 dimethylation in IVF samples, indicate that in vitro culturing could corroborate with the alterations seen in the NT group.

The tumor suppressor phosphatase and tensin homolog protein (PTEN) is negatively regulated by NF-κb p50 homodimers and involves histone 3 methylation/deacetylation in UROtsa cells chronically exposed to monomethylarsonous acid.

UROtsa cells have been accepted as a model to study carcinogenicity mechanisms of arsenic-associated human bladder cancer. In vitro continuous exposure to monomethylarsonous acid (MMAIII), leads UROtsa cells to commit to malignant transformation. In this process, NF-κß-associated inflammatory response seems to play an important role since this transcription factor activates some minutes after cells are exposed in vitro to MMAIII and keeps activated during the cellular malignant transformation. It is known that a slight decrease in the protein phosphatase and tensin homologue (PTEN) gene expression is enough for some cells to become malignantly transformed. Interestingly, this tumor suppressor has been proven to be negatively regulated by NF-κß through binding to its gene promoter. Based on these observations we propose that NF-κß may be involved in arsenic associated carcinogenesis through the negative regulation of PTEN gene expression. Changes in PTEN expression and the binding of p50 NF-κß subunit to PTEN promoter were evaluated in UROtsa cells exposed for 4, 12, 20, or 24 wk to 50nM MMAIII. Results showed that MMAIII induced a significant decrease in PTEN expression around 20 wk exposure to MMAIII,which correlated with increased binding of p50 subunit to the PTEN promoter. Consistent with these results, ChIP assays also showed a significant decrease in H3 acetylation (H3ac) but an increase in the repression marks H3k9me3 and H327me3 in PTEN promoter when compared with not treated cells. These results suggest that the activation of NF-κß by MMAIII may participate in UROtsa cells malignant transformation through the negative regulation of PTEN expression involving p50 homodimers-mediated chromatin remodeling around the PTEN promoter.

Histone methylation, alternative splicing and neuronal differentiation.

Alternative splicing, as well as chromatin structure, greatly contributes to specific transcriptional programs that promote neuronal differentiation. The activity of G9a, the enzyme responsible for mono- and di-methylation of lysine 9 on histone H3 (H3K9me1 and H3K9me2) in mammalian euchromatin, has been widely implicated in the differentiation of a variety of cell types and tissues. In a recent work from our group (Fiszbein et al., 2016) we have shown that alternative splicing of G9a regulates its nuclear localization and, therefore, the efficiency of H3K9 methylation, which promotes neuronal differentiation. We discuss here our results in the light of a report from other group (Laurent et al. 2015) demonstrating a key role for the alternative splicing of the histone demethylase LSD1 in controlling specific gene expression in neurons. All together, these results illustrate the importance of alternative splicing in the generation of a proper equilibrium between methylation and demethylation of histones for the regulation of neuron-specific transcriptional programs.

Oxygen tension affects histone remodeling of in vitro-produced embryos in a bovine model.

In vitro production of bovine embryos is a biotechnology of great economic impact. Epigenetic processes, such as histone remodeling, control gene expression and are essential for proper embryo development. Given the importance of IVP as a reproductive biotechnology, the role of epigenetic processes during embryo development, and the important correlation between culture conditions and epigenetic patterns, the present study was designed as a 2 × 2 factorial to investigate the influence of varying oxygen tensions (O2; 5% and 20%) and concentrations of fetal bovine serum (0% and 2.5%), during IVC, in the epigenetic remodeling of H3K9me2 (repressive) and H3K4me2 (permissive) in bovine embryos. Bovine oocytes were used for IVP of embryos, cleavage and blastocyst rates were evaluated, and expanded blastocysts were used for evaluation of the histone marks H3K9me2 and H3K4me2. Morulae and expanded blastocysts were also used to evaluate the expression of remodeling enzymes, specific to the aforementioned marks, by real-time polymerase chain reaction. Embryos produced in the presence of fetal bovine serum (2.5%) had a 10% higher rate of blastocyst formation. Global staining for the residues H3K9me2 and H3K4me2 was not affected significantly by the presence of serum. Notwithstanding, the main effect of oxygen tension was significant for both histone marks, with both repressive and permissive marks being higher in embryos cultured at the higher oxygen tension; however, expression of the remodeling enzymes did not differ in morulae or blastocysts in response to the varying oxygen tension. These results suggest that the use of serum during IVC of embryos increases blastocyst rate without affecting the evaluated histone marks and that oxygen tension has an important effect on the histone marks H3K9me2 and H3K4me2 in bovine blastocysts.

ChIP-seq analysis of histone H3K9 trimethylation in peripheral blood mononuclear cells of membranous nephropathy patients

Membranous nephropathy (MN), characterized by the presence of diffuse thickening of the glomerular basement membrane and subepithelial in situ immune complex disposition, is the most common cause of idiopathic nephrotic syndrome in adults, with an incidence of 5-10 per million per year. A number of studies have confirmed the relevance of several experimental insights to the pathogenesis of human MN, but the specific biomarkers of MN have not been fully elucidated. As a result, our knowledge of the alterations in histone methylation in MN is unclear. We used chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) to analyze the variations in a methylated histone (H3K9me3) in peripheral blood mononuclear cells from 10 MN patients and 10 healthy subjects. There were 108 genes with significantly different expression in the MN patients compared with the normal controls. In MN patients, significantly increased activity was seen in 75 H3K9me3 genes, and decreased activity was seen in 33, compared with healthy subjects. Five positive genes, DiGeorge syndrome critical region gene 6 (DGCR6), sorting nexin 16 (SNX16), contactin 4 (CNTN4), baculoviral IAP repeat containing 3 (BIRC3), and baculoviral IAP repeat containing 2 (BIRC2), were selected and quantified. There were alterations of H3K9me3 in MN patients. These may be candidates to help explain pathogenesis in MN patients. Such novel findings show that H3K9me3 may be a potential biomarker or promising target for epigenetic-based MN therapies.