H2A Variant Research Articles

Histone variant H2A.J accumulates in senescent cells and promotes inflammatory gene expression

The senescence of mammalian cells is characterized by a proliferative arrest in response to stress and the expression of an inflammatory phenotype. Here we show that histone H2A.J, a poorly studied H2A variant found only in mammals, accumulates in human fibroblasts in senescence with persistent DNA damage. H2A.J also accumulates in mice with aging in a tissue-specific manner and in human skin. Knock-down of H2A.J inhibits the expression of inflammatory genes that contribute to the senescent-associated secretory phenotype (SASP), and over expression of H2A.J increases the expression of some of these genes in proliferating cells. H2A.J accumulation may thus promote the signalling of senescent cells to the immune system, and it may contribute to chronic inflammation and the development of aging-associated diseases.

SLY regulates genes involved in chromatin remodeling and interacts with TBL1XR1 during sperm differentiation

Sperm differentiation requires unique transcriptional regulation and chromatin remodeling after meiosis to ensure proper compaction and protection of the paternal genome. Abnormal sperm chromatin remodeling can induce sperm DNA damage, embryo lethality and male infertility, yet, little is known about the factors which regulate this process. Deficiency in Sly, a mouse Y chromosome-encoded gene expressed only in postmeiotic male germ cells, has been shown to result in the deregulation of hundreds of sex chromosome-encoded genes associated with multiple sperm differentiation defects and subsequent male infertility. The underlying mechanism remained, to date, unknown. Here, we show that SLY binds to the promoter of sex chromosome-encoded and autosomal genes highly expressed postmeiotically and involved in chromatin regulation. Specifically, we demonstrate that Sly knockdown directly induces the deregulation of sex chromosome-encoded H2A variants and of the H3K79 methyltransferase DOT1L. The modifications prompted by loss of Sly alter the postmeiotic chromatin structure and ultimately result in abnormal sperm chromatin remodeling with negative consequences on the sperm genome integrity. Altogether our results show that SLY is a regulator of sperm chromatin remodeling. Finally we identified that SMRT/N-CoR repressor complex is involved in gene regulation during sperm differentiation since members of this complex, in particular TBL1XR1, interact with SLY in postmeiotic male germ cells.

Identification of a variant-specific phosphorylation of TH2A during spermiogenesis

Tissue-specific histone variant incorporation into chromatin plays dynamic and important roles in tissue development. Testis is one such tissue, and a number of testis-specific histone variants are expressed that have unique roles. While it is expected that such variants acquire post-transcriptional modifications to be functional, identification of variant-specific histone modifications is challenging because of the high similarity of amino acid sequences between canonical and variant versions. Here we identified a novel phosphorylation on TH2A, a germ cell-specific histone H2A variant. TH2A-Thr127 is unique to the variant and phosphorylated concomitant with chromatin condensation including spermiogenesis and early embryonic mitosis. In sperm chromatin, phosphorylated TH2A-Thr127 (=pTH2A) is co-localized with H3.3 at transcriptional starting sites of the genome, and subsequently becomes absent from the paternal genome upon fertilization. Notably, pTH2A is recurrent and accumulated in the pericentromeric heterochromatin of both paternal and maternal chromosomes in the first mitosis of embryos, suggesting its unique regulation during spermiogenesis and early embryogenesis.

The histone variant H2A.Z promotes efficient cotranscriptional splicing in S. cerevisiae.

In eukaryotes, a dynamic ribonucleic protein machine known as the spliceosome catalyzes the removal of introns from premessenger RNA (pre-mRNA). Recent studies show the processes of RNA synthesis and RNA processing to be spatio-temporally coordinated, indicating that RNA splicing takes place in the context of chromatin. H2A.Z is a highly conserved histone variant of the canonical histone H2A. In Saccharomyces cerevisiae, H2A.Z is deposited into chromatin by the SWR-C complex, is found near the 5' ends of protein-coding genes, and has been implicated in transcription regulation. Here we show that splicing of intron-containing genes in cells lacking H2A.Z is impaired, particularly under suboptimal splicing conditions. Cells lacking H2A.Z are especially dependent on a functional U2 snRNP (small nuclear RNA [snRNA] plus associated proteins), as H2A.Z shows extensive genetic interactions with U2 snRNP-associated proteins, and RNA sequencing (RNA-seq) reveals that introns with nonconsensus branch points are particularly sensitive to H2A.Z loss. Consistently, H2A.Z promotes efficient spliceosomal rearrangements involving the U2 snRNP, as H2A.Z loss results in persistent U2 snRNP association and decreased recruitment of downstream snRNPs to nascent RNA. H2A.Z impairs transcription elongation, suggesting that spliceosome rearrangements are tied to H2A.Z's role in elongation. Depletion of disassembly factor Prp43 suppresses H2A.Z-mediated splice defects, indicating that, in the absence of H2A.Z, stalled spliceosomes are disassembled, and unspliced RNAs are released. Together, these data demonstrate that H2A.Z is required for efficient pre-mRNA splicing and indicate a role for H2A.Z in coordinating the kinetics of transcription elongation and splicing.

A “Tail” of Unusual Histone H2A Variants in Bdelloid Rotifers

In eukaryotes, the nucleosome is comprised of an octamer of four canonical core histones (two copies each of H2A, H2B, H3, and H4) around which ~147bp of DNA is wrapped. Histone variants (non‐allelic versions of the core histones) are also present in chromatin, and these proteins have diverse functions in DNA repair, meiotic recombination, chromosome segregation and transcription regulation. Bdelloid rotifers are aquatic microinvertebrates that possess H2A variants with C‐terminal tails ~20–40 residues longer than in other eukaryotes. While the genome project for the bdelloid Adineta vaga annotated three distinct histone H2A variants (H2Abd, H2Abd1 and H2Abd2), our search of the genome revealed a gene encoding a novel fourth H2A variant (hereafter called “H2A‐HDX”). The predicted translation of H2A‐HDX is 256 amino acids; this makes it unusual in that its C‐terminal tail is estimated to be over 120 residues longer than canonical H2A. The objective of this project was to characterize the sequence and function of histone H2A variants, including the newly discovered H2A‐HDX variant, found in bdelloid rotifers using phylogenetic, gene expression and mass spectrometry analyses. First, a phylogenetic analysis including H2A variants was done. We searched databases of protein and expressed sequence tag sequences for H2A variants from diverse eukaryotes. The analysis revealed that bdelloid H2A variants are present in a clade of other eukaryotic H2A homologs. In addition, H2A‐HDX represents an independent lineage of H2A variants distinct from other animals. Interestingly, bdelloids have the ability to effectively repair DNA double‐strand breaks (DSBs) induced by high doses of ionizing radiation. Since certain H2A variants (such as H2AX) function in DNA repair in other eukaryotes, we wanted to determine if H2A variants in bdelloid rotifers performed a similar role. We exposed the rotifers to 280 Gray of ionizing radiation to induce DNA damage, and used real‐time PCR to quantify expression levels of H2A variants. We found the expression of H2A variants in irradiated samples was not elevated relative to non‐irradiated samples, suggesting that upregulation is not associated with DNA repair. Next, mass spectrometry was used to test whether the long C‐terminal tails of the H2A variants are the targets of post‐translational modifications (PTMs) during DNA repair. An acid extraction protocol was used to isolate histones from irradiated and non‐irradiated rotifers. High‐resolution mass spectrometric analysis done using a Thermo Velos Orbitrap mass spectrometer identified all four bdelloid‐specific H2A variants, and validated the long C‐terminal tails of H2Abd, H2Abd1 and H2Abd2. H2A‐HDX was present in a lower abundance relative to other H2A variants, and only the first 194 of the predicted 256 amino acids were confirmed. However, reverse transcription PCR showed that the H2A‐HDX gene transcribes the full‐length C‐terminal tail. We also identified PTMs specific to irradiated samples in the N‐ and C‐terminal tails of the H2A variants, suggesting a potential role for epigenetics and histone H2A variants during DNA repair. Future pulldown assays using proteins and N‐ and C‐terminal tail peptides (+/− PTMs) will identify binding partners to H2A‐HDX and shed light on the role of histone H2A variants in DNA repair or other cellular processes in bdelloid rotifers.Support or Funding InformationThis work was supported by grants from the National Center for Research Resources (5P20RR016460) & National Institute of General Medical Sciences (8P20GM103429) from the NIH.

Mild Hypothermia Attenuates the Anesthetic Isoflurane-Induced Cytotoxicity.

The commonly used inhalation anesthetic isoflurane has been reported to induce DNA damage and cytotoxicity. However, the methods to attenuate these effects remain largely to be determined. Mild hypothermia has neuroprotective effects. We therefore set out to assess whether mild hypothermia could protect the isoflurane-induced DNA damage and cytotoxicity. Moreover, we investigated the underlying mechanisms by assessing the effects of mild hypothermia on the isoflurane-induced changes in ATP levels. H4 human neuroglioma cells were treated with 2% isoflurane for 3 or 6 h with and without mild hypothermia (35°C). We assessed the cell viability by using 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) and lactate dehydrogenase (LDH) assay. We determined DNA damage by measuring levels of phosphorylation of the histone protein H2A variant X at Ser139 (γH2A.X), the marker of DNA damage. We also measured ATP levels in the cells. Here we showed that the treatment with 2% isoflurane for 6 h induced cytotoxicity and DNA damage in the cells. Moreover, the treatment with 2% isoflurane for 3 h decreased ATP levels without inducing cytotoxicity. Mild hypothermia attenuated the isoflurane-induced cytotoxicity, DNA damage, and ATP reduction in the cells. Taken together, these data suggest that the isoflurane-induced reduction in ATP levels occurred before the isoflurane-induced cytotoxicity. Isoflurane may induce DNA damage and cause cytotoxicity through reducing ATP levels. Mild hypothermia would ameliorate isoflurane-induced DNA damage and cytotoxicity by attenuating the isoflurane-induced reduction in ATP levels. These pilot studies have established a system and will promote the future investigations of anesthesia neurotoxicity.

Genomic characterization and dynamic methylation of promoter facilitates transcriptional regulation of H2A variants, H2A.1 and H2A.2 in various pathophysiological states of hepatocyte

Differential expression of homomorphous variants of H2A family of histone H2A.1 and H2A.2 have been associated with hepatocellular carcinoma and maintenance of undifferentiated state of hepatocyte. However, not much is known about the transcriptional regulation of these H2A variants. The current study revealed the presence of 43bp 5′-regulatory region upstream of translation start site and a 26bp 3′ stem loop conserved region for both the H2A.1 and H2A.2 variants. However, alignment of both H2A.1 and H2A.2 5′-untranslated region (UTR) sequences revealed no significant degree of homology between them despite the coding exon being very similar amongst the variants. Further, transient transfection coupled with dual luciferase assay of cloned 5′ upstream sequences of H2A.1 and H2A.2 of length 1.2 (−1056 to +144) and 1.379kb (−1160 to +219) from experimentally identified 5′UTR in rat liver cell line (CL38) confirmed their promoter activity. Moreover, in silico analysis revealed a presence of multiple CpG sites interspersed in the cloned promoter of H2A.1 and a CpG island near TSS for H2A.2, suggesting that histone variants transcription might be regulated epigenetically. Indeed, treatment with DNMT and HDAC inhibitors increased the expression of H2A.2 with no significant change in H2A.1 levels. Further, methyl DNA immunoprecipitation coupled with quantitative analysis of DNA methylation using real-time PCR revealed hypo-methylation and hyper-methylation of H2A.1 and H2A.2 respectively in embryonic and HCC compared to control adult liver tissue. Collectively, the data suggests that differential DNA methylation on histone promoters is a dynamic player regulating their expression status in different pathophysiological stages of liver.

ZMYM3 regulates BRCA1 localization at damaged chromatin to promote DNA repair.

Chromatin connects DNA damage response factors to sites of damaged DNA to promote the signaling and repair of DNA lesions. The histone H2A variants H2AX, H2AZ, and macroH2A represent key chromatin constituents that facilitate DNA repair. Through proteomic screening of these variants, we identified ZMYM3 (zinc finger, myeloproliferative, and mental retardation-type 3) as a chromatin-interacting protein that promotes DNA repair by homologous recombination (HR). ZMYM3 is recruited to DNA double-strand breaks through bivalent interactions with both histone and DNA components of the nucleosome. We show that ZMYM3 links the HR factor BRCA1 to damaged chromatin through specific interactions with components of the BRCA1-A subcomplex, including ABRA1 and RAP80. By regulating ABRA1 recruitment to damaged chromatin, ZMYM3 facilitates the fine-tuning of BRCA1 interactions with DNA damage sites and chromatin. Consistent with a role in regulating BRCA1 function, ZMYM3 deficiency results in impaired HR repair and genome instability. Thus, our work identifies a critical chromatin-binding DNA damage response factor, ZMYM3, which modulates BRCA1 functions within chromatin to ensure the maintenance of genome integrity.

The role of variant histone H2AV in Drosophila melanogaster larval hematopoiesis.

Replication-independent histone variants can replace the canonical replication-dependent histones. Vertebrates have multiple H2A variant histones, including H2AZ and H2AX that are present in most eukaryotes. H2AZ regulates transcriptional activation as well as the maintenance of gene silencing, while H2AX is important in DNA damage repair. The fruit fly Drosophila melanogaster has only one histone H2A variant (H2AV), which is a chimera of H2AZ and H2AX. In this study we found that lack of H2AV led to the formation of black melanotic masses in Drosophila third instar larvae. The formation of these masses was found in conjunction with a loss of the majority of the primary lymph gland lobes. Interestingly, the cells of the posterior signaling center were preserved in these mutants. Reduction of H2AV levels by RNAi knockdown caused a milder phenotype that preserved the lymph gland structure but that included precocious differentiation of the prohemocytes located within the medullary zone and the secondary lobes of the lymph gland. Mutant rescue experiments suggest that the H2AZ-like rather than the H2AX-like function of H2AV is primarily required for normal hematopoiesis.

ANKHD1 Silencing Delays S Phase Progression in Multiple Myeloma Cells Via Activation of ATM/ATR -CDC25a Pathway

Ankyrin repeat and KH domain-containing protein 1, ANKHD1, is highly expressed in myeloma cells and plays an important role in multiple myeloma (MM) progression and growth. ANKHD1 is found to be overexpressed in S phase of cell cycle in MM cells and silencing of ANKHD1 expression leads to accumulation of cells in S phase, suggesting a role in S phase progression (1). Earlier studies by our group reported that ANKHD1 silencing downregulates all replication dependent histones and that this downregulation may be associated with replication stress and DNA damage (2). We observed increased expression of γH2AX protein (phosphorylated histone H2A variant, H2AX, at Serine 139), a marker for DNA double strand breaks (DSBs) and an early sign of DNA damage induced by replication stress, in ANKHD1 silenced MM cells. In the present study we further sought to investigate the mechanisms underlying the induction of DNA damage on ANKHD1 silencing. We first confirmed the increased expression of γH2AX by flow cytometry analysis and observed that both the mean fluorescence intensity as well as percentage of γH2AX positive cells were higher in ANKHD1 silenced MM cells as compared to control cells. Phosphorylation of histone 2AX requires activation of the phosphatidylinositol-3-OH-kinase-like family of protein kinases, DNA-PKcs (DNA-dependent protein kinase), ATM (ataxia telangiectasia mutated)andATR (ATM-Rad3-related) that serves as central components of the signaling cascade initiated by DSBs. Hence, we checked for the expression of these kinases and observed increased phosphorylation of both ATM and ATR kinases in ANKHD1 silenced MM cells. There was no difference in the expressions of DNA-PKcs in control and ANKHD1 silenced cells by western blot. We next checked for the expression of CHK1 (checkpoint kinase 1) and CHK2 (checkpoint kinase 2), essential serine threonine kinases downstream of ATM and ATR. We observed a decrease in pCHK2 (phosphorylated CHK2 at Thr 68), with no change in expression of pCHK1 (phosphorylated CHK1 at Ser 345) total CHK1 or total CHK2. We also checked for expression of CDC25a (a member of the CDC25 family of dual-specificity phosphatases), that is specifically degraded in response to DNA damage (DSBs) and delays S phase progression via activation of ATM /ATR-CHK2 signaling pathway. Expression of CDC25a was significantly decreased in ANKHD1 silencing cells, confirming the induction of DSBs, and probably accounting for S phase delay on ANKHD1 silencing. Since there was decrease in active CHK2 (pCHK2) and no change in CHK1 required for degradation of CDC25a, we assume that decrease in CDC25a in ANKHD1 silenced MM cells may be via activation of ATM/ ATR pathway independent of CHK2/CHK1. Expression of several other downstream factors of DSBs induced DNA damage response and repair such as BRCA1, PTEN, DNMT1, SP1, HDAC2 were also found to be modulated in ANKHD1 silenced MM cells.In conclusion, ANKHD1 silencing in MM cells leads to DNA damage and modulates expression of several genes implicated in DNA damage and repair. DNA damage induced after ANKHD1 silencing in MM cells activates ATM/ ATR-CDC25a pathway which may lead to the activation of S phase checkpoint in MM cells. Results however are preliminary and further studies are required to understand the role of ANKHD1 in intra S phase check point.

The Role of H2AFY in U2AF1 Mutant Cells and Normal Hematopoiesis

Mutations in genes that are involved in pre-mRNA splicing (i.e., spliceosome genes; U2AF1, SF3B1, SRSF2 and ZRSR2) are particularly frequent in MDS, affecting ~50% of MDS patients. Our group showed that expression of U2AF1(S34F), the most common U2AF1 mutant, causes altered splicing in human and mouse cells and altered hematopoiesis in mice. How U2AF1(S34F) expression contributes to altered hematopoiesis is unclear. We hypothesized that U2AF1(S34F) alters splicing of target genes that affect hematopoiesis. Using three independent sets of RNA-seq data from human acute myeloid leukemia (AML) patients, human CD34+ hematopoietic cells, and mouse common myeloid progenitor cells that express either wild-type or mutant U2AF1, we identified consistent alternative splicing of H2AFY. H2AFY, also known as macroH2A1, is a histone H2A variant implicated in transcription, development, and tumorigenesis. Alternative splicing of H2AFY generates two isoforms, H2AFY1.1 and H2AFY1.2. In many solid cancers, alternative splicing of H2AFY is observed, resulting in reductions in H2AFY1.1 isoform expression. Similarly, in all three RNA-seq datasets and RT-PCR of primary MDS samples, mutant U2AF1 samples express H2AFY1.1 at 30% of the levels observed in samples without a spliceosome mutation (p<0.01). In order to determine whether expression of H2AFY1.1 could rescue a cellular phenotype in U2AF1 mutant cells, we exogenously expressed H2AFY1.1 in doxycycline-inducible U2AF1(S34F) mouse bone marrow cells ex vivo using a lentivirus co-expressing GFP. We observed an attenuation of cell death induced by U2AF1(S34F) expression (%GFP+ live cells relative to uninduced cells: U2AF1(S34F) cells-45% [control virus] vs. 89% [H2AFY1.1 virus], p<0.001; wild-type cells-94% [control virus] vs. 97% [H2AFY1.1 virus], ns). Next, we asked whether loss of H2afy1.1 expression phenocopies any of the hematopoietic alterations observed in U2AF1(S34F) mice (reductions in white blood cell counts [WBC], B-cells and monocytes, or expansion of progenitor cells). To address this question, we first investigated the role of H2afy in normal mouse hematopoiesis. H2afy-/- mice (lacking both H2afy1.1 and H2afy1.2 isoforms) are viable and born at the expected Mendelian frequency. H2afy-/- and H2afy+/-mice exhibited reduced WBC compared to H2afy+/+ littermate mice (7.7, 8.1, and 9.9 K cells/µL, respectively, p<0.01 compared to wild-type mice, N=15 each genotype, 8-12 weeks old), whereas the red blood cell and platelet counts were no different. Flow cytometric analysis of the peripheral blood cell lineages revealed reduced B-cell numbers in H2afy-/- and H2afy+/-mice compared to H2afy+/+ mice (mean 4.0, 4.5, and 5.6 K cells/µL, respectively, p<=0.003). Stem (KLS and KLS-SLAM) and progenitor cells (CMP, GMP, MEP) were not different between genotypes. There was a 2-fold reduction in CFU-pre-B colonies in the H2afy-/- bone marrow cells compared to wild-type controls (p= 0.01, N=10), but CFU-C colonies were no different. To determine whether the reduction in B-cells is hematopoietic cell-intrinsic, we transplanted mouse bone marrow cells collected from H2afy+/+ or H2afy-/- mice into lethally irradiated congenic recipients. At 6 weeks post-transplant, mice who received the H2afy-/- bone marrow cells compared to mice who received H2afy+/+ cells exhibited a marked reduction in WBC (5.0, 9.1 K cells/µL, respectively, p<0.001, N=10) and peripheral blood B-cells (1.9, 5.3 K cells/µL, respectively, p<0.001), but not in myeloid cells. This suggests that hematopoietic expression of H2afy contributes to B-cell development in mice. To assess stem/progenitor cell function in vivo, we performed a competitive repopulation assay. As early as 1 month post-transplant (and persistent to 4 months), there was a reduction in H2afy-/- donor-derived B-cells in the peripheral blood compared to mice receiving H2afy+/+ cells (14%, 58%, respectively, p<0.001, N=14-15). The reduction in peripheral white blood cell counts and B-cells in H2afy-/- mice mirrors the phenotypic changes observed in U2AF1(S34F) mice, suggesting that alterations in H2afy may contribute to altered hematopoiesis in U2AF1 mutant mice. Ongoing studies are designed to address whether the B-cell phenotype in H2afy-/- and U2AF1 mutant mice is due to a specific loss of H2afy1.1 or 1.2 isoform expression in hematopoietic cells. DisclosuresNo relevant conflicts of interest to declare.

Loss of H3K9me3 Correlates with ATM Activation and Histone H2AX Phosphorylation Deficiencies in Hutchinson-Gilford Progeria Syndrome.

Compelling evidence suggests that defective DNA damage response (DDR) plays a key role in the premature aging phenotypes in Hutchinson-Gilford progeria syndrome (HGPS). Studies document widespread alterations in histone modifications in HGPS cells, especially, the global loss of histone H3 trimethylated on lysine 9 (H3K9me3). In this study, we explore the potential connection(s) between H3K9me3 loss and the impaired DDR in HGPS. When cells are exposed to a DNA-damaging agent Doxorubicin (Dox), double strand breaks (DSBs) are generated that result in the phosphorylation of histone H2A variant H2AX (gammaH2AX) within an hour. We find that the intensities of gammaH2AX foci appear significantly weaker in the G0/G1 phase HGPS cells compared to control cells. This reduction is associated with a delay in the recruitment of essential DDR factors. We further demonstrate that ataxia-telangiectasia mutated (ATM) is responsible for the amplification of gammaH2AX signals at DSBs during G0/G1 phase, and its activation is inhibited in the HGPS cells that display significant loss of H3K9me3. Moreover, methylene (MB) blue treatment, which is known to save heterochromatin loss in HGPS, restores H3K9me3, stimulates ATM activity, increases gammaH2AX signals and rescues deficient DDR. In summary, this study demonstrates an early DDR defect of attenuated gammaH2AX signals in G0/G1 phase HGPS cells and provides a plausible connection between H3K9me3 loss and DDR deficiency.

Sequence analysis and expression patterns of histone H2A variants in Phytophthora infestans

In eukaryotic cells, DNA is packaged inside the nucleus together with histones to form nucleosomes. Each histone molecule contains two of each core histone subunits H2A, H2B, H3 and H4. Among core histones, the H2A family is of great interest due to the high diversity of specialized variants. These variants have shown important role in critical cellular processes. Epigenetic mechanism in oomycetes is barely known. Phytophthora infestans is a severe pathogen and a model species in oomycetes. In this study, we studied the sequence and expression pattern of H2A variants of P. infestans through genome search, sequences alignment, phylogenetic analysis and realtime qPCR detection. P. infestans contains conserved genes encoding histone variants H2A.X.1, H2A.X.2 and H2A.Z, and these genes have specific expression patterns during development and infection stages. Our datasets provide useful inputs to help explore the epigenetic mechanisms of oomycetes.

An Assay for Measuring Histone Variant Exchange within Nucleosomes In Vitro.

The incorporation of histone variants into specific chromatin regions is a mechanism by which cells can regulate many important biological processes. One such example is H2A.Z, a highly conserved variant of H2A that is incorporated in genomic regulatory regions and contributes to control gene expression. H2A.Z variant exchange involves the removal of H2A-H2B dimers from a preassembled nucleosome and their replacement with H2A.Z-H2B dimers. A specific family of chromatin remodeling complexes, homologous to the yeast Swr1 complex, have been shown to be capable of this histone exchange activity both in vivo and in vitro. Here, we describe an assay to measure the histone H2A.Z exchange activity of recombinant human p400 on immobilized mononucleosomes in vitro. The assay can be adapted to other histone exchange complexes/catalytic subunits purified from any species.

Histone macroH2A1.2 promotes metabolic health and leanness by inhibiting adipogenesis.

BackgroundObesity has tremendous impact on the health systems. Its epigenetic bases are unclear. MacroH2A1 is a variant of histone H2A, present in two alternatively exon-spliced isoforms macroH2A1.1 and macroH2A1.2, regulating cell plasticity and proliferation, during pluripotency and tumorigenesis. Their role in adipose tissue plasticity is unknown.ResultsHere, we show evidence that macroH2A1.1 protein levels in the visceral adipose tissue of obese humans positively correlate with BMI, while macroH2A1.2 is nearly absent. We thus introduced a constitutive GFP-tagged transgene for macroH2A1.2 in mice, and we characterized their metabolic health upon being fed a standard chow diet or a high fat diet. Despite unchanged food intake, these mice exhibit lower adipose mass and improved glucose metabolism both under a chow and an obesogenic diet. In the latter regimen, transgenic mice display smaller pancreatic islets and significantly less inflammation. MacroH2A1.2 overexpression in the mouse adipose tissue induced dramatic changes in the transcript levels of key adipogenic genes; genomic analyses comparing pre-adipocytes to mature adipocytes uncovered only minor changes in macroH2A1.2 genomic distribution upon adipogenic differentiation and suggested differential cooperation with transcription factors. MacroH2A1.2 overexpression markedly inhibited adipogenesis, while overexpression of macroH2A1.1 had opposite effects.ConclusionsMacroH2A1.2 is an unprecedented chromatin component powerfully promoting metabolic health by modulating anti-adipogenic transcriptional networks in the differentiating adipose tissue. Strategies aiming at enhancing macroH2A1.2 expression might counteract excessive adiposity in humans.Electronic supplementary materialThe online version of this article (doi:10.1186/s13072-016-0098-9) contains supplementary material, which is available to authorized users.

Albendazole as a promising molecule for tumor control

This work evaluated the antitumor effects of albendazole (ABZ) and its relationship with modulation of oxidative stress and induction of DNA damage. The present results showed that ABZ causes oxidative cleavage on calf-thymus DNA suggesting that this compound can break DNA. ABZ treatment decreased MCF-7 cell viability (EC50=44.9 for 24h) and inhibited MCF-7 colony formation (~67.5% at 5μM). Intracellular ROS levels increased with ABZ treatment (~123%). The antioxidant NAC is able to revert the cytotoxic effects, ROS generation and loss of mitochondrial membrane potential of MCF-7 cells treated with ABZ. Ehrlich carcinoma growth was inhibited (~32%) and survival time was elongated (~50%) in animals treated with ABZ. Oxidative biomarkers (TBARS and protein carbonyl levels) and activity of antioxidant enzymes (CAT, SOD and GR) increased, and reduced glutathione (GSH) was depleted in animals treated with ABZ, indicating an oxidative stress condition, leading to a DNA damage causing phosphorylation of histone H2A variant, H2AX, and triggering apoptosis signaling, which was confirmed by increasing Bax/Bcl-xL rate, p53 and Bax expression. We propose that ABZ induces oxidative stress promoting DNA fragmentation and triggering apoptosis and inducing cell death, making this drug a promising leader molecule for development of new antitumor drugs.

Efficacy and epigenetic interactions of novel DNA hypomethylating agent guadecitabine (SGI-110) in preclinical models of hepatocellular carcinoma

ABSTRACTHepatocellular carcinoma (HCC) is a deadly malignancy characterized at the epigenetic level by global DNA hypomethylation and focal hypermethylation on the promoter of tumor suppressor genes. In most cases it develops on a background of liver steatohepatitis, fibrosis, and cirrhosis. Guadecitabine (SGI-110) is a second-generation hypomethylating agent, which inhibits DNA methyltransferases. Guadecitabine is formulated as a dinucleotide of decitabine and deoxyguanosine that is resistant to cytidine deaminase (CDA) degradation and results in prolonged in vivo exposure to decitabine following small volume subcutaneous administration of guadecitabine. Here we found that guadecitabine is an effective demethylating agent and is able to prevent HCC progression in pre-clinical models. In a xenograft HCC HepG2 model, guadecitabine impeded tumor growth and inhibited angiogenesis, while it could not prevent liver fibrosis and inflammation in a mouse model of steatohepatitis. Demethylating efficacy of guadecitabine on LINE-1 elements was found to be the highest 8 d post-infusion in blood samples of mice. Analysis of a panel of human HCC vs. normal tissue revealed a signature of hypermethylated tumor suppressor genes (CDKN1A, CDKN2A, DLEC1, E2F1, GSTP1, OPCML, E2F1, RASSF1, RUNX3, and SOCS1) as detected by methylation-specific PCR. A pronounced demethylating effect of guadecitabine was obtained also in the promoters of a subset of tumor suppressors genes (CDKN2A, DLEC1, and RUNX3) in HepG2 and Huh-7 HCC cells. Finally, we analyzed the role of macroH2A1, a variant of histone H2A, an oncogene upregulated in human cirrhosis/HCC that synergizes with DNA methylation in suppressing tumor suppressor genes, and it prevents the inhibition of cell growth triggered by decitabine in HCC cells. Guadecitabine, in contrast to decitabine, blocked growth in HCC cells overexpressing macroH2A1 histones and with high CDA levels, despite being unable to fully demethylate CDKN2A, RUNX3, and DLEC1 promoters altered by macroH2A1. Collectively, our findings in human and mice models reveal novel epigenetic anti-HCC effects of guadecitabine, which might be effective specifically in advanced states of the disease.

An Essential Viral Transcription Activator Modulates Chromatin Dynamics.

Although ICP4 is the only essential transcription activator of herpes simplex virus 1 (HSV-1), its mechanisms of action are still only partially understood. We and others propose a model in which HSV-1 genomes are chromatinized as a cellular defense to inhibit HSV-1 transcription. To counteract silencing, HSV-1 would have evolved proteins that prevent or destabilize chromatinization to activate transcription. These proteins should act as HSV-1 transcription activators. We have shown that HSV-1 genomes are organized in highly dynamic nucleosomes and that histone dynamics increase in cells infected with wild type HSV-1. We now show that whereas HSV-1 mutants encoding no functional ICP0 or VP16 partially enhanced histone dynamics, mutants encoding no functional ICP4 did so only minimally. Transient expression of ICP4 was sufficient to enhance histone dynamics in the absence of other HSV-1 proteins or HSV-1 DNA. The dynamics of H3.1 were increased in cells expressing ICP4 to a greater extent than those of H3.3. The dynamics of H2B were increased in cells expressing ICP4, whereas those of canonical H2A were not. ICP4 preferentially targets silencing H3.1 and may also target the silencing H2A variants. In infected cells, histone dynamics were increased in the viral replication compartments, where ICP4 localizes. These results suggest a mechanism whereby ICP4 activates transcription by disrupting, or preventing the formation of, stable silencing nucleosomes on HSV-1 genomes.

Splice variants of the SWR1-type nucleosome remodeling factor Domino have distinct functions during Drosophila melanogaster oogenesis

SWR1-type nucleosome remodeling factors replace histone H2A by variants to endow chromatin locally with specialized functionality. In Drosophila melanogaster a single H2A variant, H2A.V, combines functions of mammalian H2A.Z and H2A.X in transcription regulation and the DNA damage response. A major role in H2A.V incorporation for the only SWR1-like enzyme in flies, Domino, is assumed but not well documented in vivo. It is also unclear whether the two alternatively spliced isoforms, DOM-A and DOM-B, have redundant or specialized functions. Loss of both DOM isoforms compromises oogenesis, causing female sterility. We systematically explored roles of the two DOM isoforms during oogenesis using a cell type-specific knockdown approach. Despite their ubiquitous expression, DOM-A and DOM-B have non-redundant functions in germline and soma for egg formation. We show that chromatin incorporation of H2A.V in germline and somatic cells depends on DOM-B, whereas global incorporation in endoreplicating germline nurse cells appears to be independent of DOM. By contrast, DOM-A promotes the removal of H2A.V from stage 5 nurse cells. Remarkably, therefore, the two DOM isoforms have distinct functions in cell type-specific development and H2A.V exchange.

Label-Free Relative Quantitation of Isobaric and Isomeric Human Histone H2A and H2B Variants by Fourier Transform Ion Cyclotron Resonance Top-Down MS/MS.

Histone variants are known to play a central role in genome regulation and maintenance. However, many variants are inaccessible by antibody-based methods or bottom-up tandem mass spectrometry due to their highly similar sequences. For many, the only tractable approach is with intact protein top-down tandem mass spectrometry. Here, ultra-high-resolution FT-ICR MS and MS/MS yield quantitative relative abundances of all detected HeLa H2A and H2B isobaric and isomeric variants with a label-free approach. We extend the analysis to identify and relatively quantitate 16 proteoforms from 12 sequence variants of histone H2A and 10 proteoforms of histone H2B from three other cell lines: human embryonic stem cells (WA09), U937, and a prostate cancer cell line LaZ. The top-down MS/MS approach provides a path forward for more extensive elucidation of the biological role of many previously unstudied histone variants and post-translational modifications.