Histone H2A Variant Research Articles

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.

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.

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.

The involvement of histones and post‐translational modifications during DNA repair in bdelloid rotifers

Histones are architectural proteins that package eukaryotic DNA into nucleosomes and regulate the expression and replication of the genome. Four canonical histones (H2A, H2B, H3, and H4) are present in duplicates to form an octamer within a nucleosome. Histone N‐and C‐terminal tails are often targets for post‐translational modifications (PTMs). For example, C‐terminal phosphorylation of the histone variant H2AX serves as an epigenetic marker for DNA damage and links damaged chromatin to DNA repair proteins. Bdelloid rotifers are aquatic microinvertebrates that have the ability to effectively repair DNA double‐strand breaks (DSBs) induced by high doses (&gt;1000 Gray) of ionizing radiation. The bdelloid Adineta vaga possesses five histone H2A variants, three of which (H2Abd, H2Abd1, H2Abd2) have C‐terminal tails that are significantly longer than those in other eukaryotes. These tails have the potential to act as targets for PTMs. The objective of this project was to investigate the role of bdelloid H2A variants in DNA repair by characterizing PTMs associated with these proteins and to identify their binding partners. We induced DSBs by irradiating three species of bdelloids with 280 Gray of ionizing radiation. Histones were then isolated at different time points post‐irradiation and high‐resolution mass spectrometry was used to characterize the histones present. We identified each of the five bdelloid‐specific H2A variants along with histones H2B, H3 and H4. Several PTMs (acetylations, methylations and phosphorylations) were identified on the N‐and C‐terminal tails of all histones. Of the identified PTMs, many were only present during DNA repair; among these, several were located in the long C‐terminal tails of H2A variants. To determine the significance of the H2A variants and associated PTMS in bdelloids, we want to identify binding partners to these proteins during DNA repair. To accomplish this, we produced 6×histidine‐tagged proteins representing each of the five variants in A. vaga. Western blots with custom polyclonal antibodies specific for each variant were used to verify the identity of the tagged proteins. Recombinant proteins and peptides (+/− PTMs) will be used in pulldown assays to identify binding partners of histones during DNA repair. In sum, we have identified several novel histone PTMs associated with DNA repair in bdelloid rotifers. These findings will improve our understanding of the role of histones and epigenetics in the specialized DNA repair system in bdelloids.Support or Funding InformationThis work was supported by grants from the NCRR (5P20RR016460) &amp; NIGMS (8P20GM103429) from the NIH and from the Arkansas Science and Technology Authority.

The role of histone variant H2A.Z in co‐transcriptional splicing in Saccharomyces cerevisiae

In eukaryotes, a dynamic ribonucleic protein machine known as the spliceosome catalyzes the removal of introns from pre‐messenger RNA (pre‐mRNA). Although RNA‐synthesis and RNA‐processing have typically been studied as biochemically distinct reactions, recent studies have shown these two processes 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 S. cerevisiae, H2A.Z is deposited in to the chromatin by the SWR1‐complex and is typically found near the 5′ ends of protein‐coding genes. H2A.Z has been implicated in transcription elongation and polymerase processivity. Here we show that H2A.Z, encoded by HTZ1 in yeast, is important for efficient splicing of intron‐containing genes, and splicing in cells lacking H2A.Z is impaired under suboptimal conditions, such as mutant splicing complexes or suboptimal splice sites. HTZ1, is particularly dependent on an intact U2 snRNP, as it shows extensive genetic interactions with components of the U2 snRNP and U2 snRNP associated proteins. Consistent with this, H2A.Z is required for the splicing of introns containing a mutated splice site. RNA‐seq reveals that intron‐containing genes with non‐consensus branchpoint sequences are particularly sensitive to loss of H2A.Z. We find that deletion of HTZ1 results in persistent association of the U2 snRNP with nascent RNA, suggesting altered spliceosomal rearrangements in the absence of H2A.Z. Together, these results demonstrate that histone variant H2A.Z is required for efficient splicing of a subset of intron‐containing genes and indicate a role for H2A.Z in co‐transcriptional spliceosome assembly.Support or Funding InformationRuth L. Kirschstein National Research Service Award GM007185.

The histone variant H2A.X is a regulator of the epithelial-mesenchymal transition.

The epithelial–mesenchymal transition (EMT), considered essential for metastatic cancer, has been a focus of much research, but important questions remain. Here, we show that silencing or removing H2A.X, a histone H2A variant involved in cellular DNA repair and robust growth, induces mesenchymal-like characteristics including activation of EMT transcription factors, Slug and ZEB1, in HCT116 human colon cancer cells. Ectopic H2A.X re-expression partially reverses these changes, as does silencing Slug and ZEB1. In an experimental metastasis model, the HCT116 parental and H2A.X-null cells exhibit a similar metastatic behaviour, but the cells with re-expressed H2A.X are substantially more metastatic. We surmise that H2A.X re-expression leads to partial EMT reversal and increases robustness in the HCT116 cells, permitting them to both form tumours and to metastasize. In a human adenocarcinoma panel, H2A.X levels correlate inversely with Slug and ZEB1 levels. Together, these results point to H2A.X as a regulator of EMT.

Effects of H2A Histone Variants on DNA Sequence and Nucleosome Structure using Coarse Grain Simulations

Coarse grain molecular dynamics (CG-MD) simulations are seeing a rising tendency in a wide range of applications because their potential to enhance the sampling of all-atomistic simulations (AA-MD) and the capacity to make near-atomistic millisecond-timescale simulations practical, putting the second threshold on the horizon. In this field, the coarse-grained Martini force field has a prominent position and has recently extended its already range of applications to DNA-containing biomolecules [1].Eukaryotic genomic DNA exists as highly compacted nucleosome arrays called chromatin. Each nucleosome contains a 147-base-pair (bp) stretch of DNA, which is sharply bent and tightly wrapped around a histone protein octamer [2]. Several mechanisms regulate DNA accessibility, including replacement of canonical histones with specialized histone variants. Among the core histone variants, the H2A family is the biggest one -to date 5 histones variants are documented- showing also the highest sequence divergence among histone families. H2A variants show striking differences in three sites that are critical in intra- and inter-nucleosome interactions: the docking domain, which is close to the DNA entry/exit region, the L1 loop and the acidic patch, which is involved in nucleosome-nucleosome interactions. We have studied the structural and dynamic differences in those regions among H2A histone variants-containing nucleosome structures.Furthermore, it has been shown that DNA sequence effects vary between nucleosomes resulting in a significant factor in their stability. For this reason, we have also analyzed the differences in the associated DNA flexibility along the wrapped DNA sequence derived from MD simulations.

DNA Hypomethylation and Histone Variant macroH2A1 Synergistically Attenuate Chemotherapy-Induced Senescence to Promote Hepatocellular Carcinoma Progression.

Aging is a major risk factor for progression of liver diseases to hepatocellular carcinoma (HCC). Cellular senescence contributes to age-related tissue dysfunction, but the epigenetic basis underlying drug-induced senescence remains unclear. macroH2A1, a variant of histone H2A, is a marker of senescence-associated heterochromatic foci that synergizes with DNA methylation to silence tumor-suppressor genes in human fibroblasts. In this study, we investigated the relationship between macroH2A1 splice variants, macroH2A1.1 and macroH2A1.2, and liver carcinogenesis. We found that protein levels of both macroH2A1 isoforms were increased in the livers of very elderly rodents and humans, and were robust immunohistochemical markers of human cirrhosis and HCC. In response to the chemotherapeutic and DNA-demethylating agent 5-aza-deoxycytidine (5-aza-dC), transgenic expression of macroH2A1 isoforms in HCC cell lines prevented the emergence of a senescent-like phenotype and induced synergistic global DNA hypomethylation. Conversely, macroH2A1 depletion amplified the antiproliferative effects of 5-aza-dC in HCC cells, but failed to enhance senescence. Senescence-associated secretory phenotype and whole-transcriptome analyses implicated the p38 MAPK/IL8 pathway in mediating macroH2A1-dependent escape of HCC cells from chemotherapy-induced senescence. Furthermore, chromatin immunoprecipitation sequencing revealed that this hepatic antisenescence state also required active transcription that could not be attributed to genomic occupancy of these histones. Collectively, our findings reveal a new mechanism by which drug-induced senescence is epigenetically regulated by macroH2A1 and DNA methylation and suggest macroH2A1 as a novel biomarker of hepatic senescence that could potentially predict prognosis and disease progression.

Leukemia-Associated MLL-AF10 Fusion Maintains an Active State of Chromatin on Its Target Genes By Recruiting Tip60

Chromosome translocation involving the mixed lineage leukemia (MLL) gene which generates an in-frame fusion gene of the MLL 5′-region and partner genes, is a common rearrangement in acute myeloid and lymphoid leukemia that is associated with poor prognosis. Knock-in and retroviral transduction studies show that MLL-fusion results in constitutive activation of the transcription of target genes such as Hoxa9 and Meis1 during the development of leukemia. Recent studies show that several transcription regulators, such as Dot1L, Cbx8, PAF1, and AEP/EAP complexes, are required for the leukemogenic activity of MLL-fusion; however, the underlying mechanisms remain elusive. To clarify the mechanism of epigenetic regulation by MLL-fusions, we established a novel leukemia model by generating a conditional MLL-AF10 fusion gene, MLL-AF10 flox, in which the 3′-AF10 region is deleted by 4-OHT-activated Cre-ERT recombinase, resulting in inactivation of MLL-AF10 flox (Figure 1A). Mouse hematopoietic stem/progenitor cells (c-kit+) were immortalized by retroviral transduction of MLL-AF10 flox and cultured in vitro or transplanted into irradiated recipient mice to induce AML in vivo. Treatment of MLL-AF10 flox cells with 4-OHT in vitro to inactivate MLL-AF10 flox downregulated Hoxa9 expression and markedly decreased colony-forming ability. In addition, the inactivation of MLL-AF10 flox rapidly decreased the acetylation level of the histone H2A variant H2A.Z on the Hoxa9 locus. These results suggest that MLL-AF10, possibly together with a histone acetyltransferase (HAT), regulates the acetylation of H2A.Z on the Hoxa9 locus. To identify the HAT responsible for H2A.Z acetylation induced by MLL-AF10, protein complexes associated with H2A.Z-containing nucleosomes were purified, resulting in the identification of Tip60, a MYST-type HAT in a complex with H2A.Z. MLL-AF10 physically interacted with Tip60 via the AF10 C-terminal portion of MLL-AF10 (Figure 1B). ChIP analysis showed that MLL-AF10 and Tip60 co-localize on the Hoxa9 locus in MLL-AF10-transformed cells (MLL-AF10 cells). Furthermore, conditional deletion of Tip60 in MLL-AF10 (Tip60 Flox/Flox, Cre-ERT2) cells dramatically downregulated Hoxa9 expression and resulted in the accumulation of unacetylated H2A.Z on the Hoxa9 locus. Consistent with these data, in vitro acetylation analysis showed that Tip60 directly acetylates H2A.Z. To assess the role of Tip60 in leukemia development in vivo, MLL-AF10 (Tip60 Flox/Flox, Cre-ERT2) leukemia cells were injected into recipient mice. Animals receiving intraperitoneal injection of tamoxifen to delete Tip60 failed to develop MLL-AF10 leukemia (Figure 1C). These data indicate that Tip60 is required for the development of MLL-AF10 leukemia and suggest that MLL-AF10 recruits Tip60 to acetylate H2A.Z on the Hoxa9 locus. The effect of H2A.Z acetylation on Hoxa9 expression was examined by purifying nucleosomes containing acetylation-deficient 3KR H2A.Z (which mimics unacetylated H2A.Z), in which lysines 4, 7, and 11 were substituted by arginine. 3KR H2A.Z preferentially formed nucleosomes with histone H3 trimethylation at lysine 27, which is catalyzed by polycomb repressive complex 2 (PRC2). This finding suggests that nucleosomes including unacetylated H2A.Z are the preferential targets of PRC2. Loss of Tip60 in MLL-AF10 cells resulted in decreased levels of acetylated H2A.Z on the Hoxa9 locus and the recruitment of Ezh2 (a catalytic subunit of PRC2) and increased histone H3 K27 trimethylation. Taken together, these data indicate that Tip60 is a critical factor in the development of MLL-AF10 leukemia. MLL-AF10 may maintain an active chromatin state on its target genesby recruiting Tip60, which acetylates H2A.Z to prevent PRC2 recruitment and gene silencing. On the other hand, unacetylated H2A.Z may be a signal for PRC2 recruitment, which would be induced as a result of Tip60 loss or inactivation of MLL-AF10. [Display omitted] DisclosuresKitabayashi:Daiichi Sankyo Co., Ltd.: Research Funding.

The Structural Determinants behind the Epigenetic Role of Histone Variants.

Histone variants are an important part of the histone contribution to chromatin epigenetics. In this review, we describe how the known structural differences of these variants from their canonical histone counterparts impart a chromatin signature ultimately responsible for their epigenetic contribution. In terms of the core histones, H2A histone variants are major players while H3 variant CenH3, with a controversial role in the nucleosome conformation, remains the genuine epigenetic histone variant. Linker histone variants (histone H1 family) haven’t often been studied for their role in epigenetics. However, the micro-heterogeneity of the somatic canonical forms of linker histones appears to play an important role in maintaining the cell-differentiated states, while the cell cycle independent linker histone variants are involved in development. A picture starts to emerge in which histone H2A variants, in addition to their individual specific contributions to the nucleosome structure and dynamics, globally impair the accessibility of linker histones to defined chromatin locations and may have important consequences for determining different states of chromatin metabolism.

Diversification of histone H2A variants during plant evolution.

Among eukaryotes, the four core histones show an extremely high conservation of their structure and form nucleosomes that compact, protect, and regulate access to genetic information. Nevertheless, in multicellular eukaryotes the two families, histone H2A and histone H3, have diversified significantly in key residues. We present a phylogenetic analysis across the green plant lineage that reveals an early diversification of the H2A family in unicellular green algae and remarkable expansions of H2A variants in flowering plants. We define motifs and domains that differentiate plant H2A proteins into distinct variant classes. In non-flowering land plants, we identify a new class of H2A variants and propose their possible role in the emergence of the H2A.W variant class in flowering plants.

Expression of Non-acetylatable H2A.Z in Myoblast Cells Blocks Myoblast Differentiation through Disruption of MyoD Expression

H2A.Z is a histone H2A variant that is essential for viability in Tetrahymena and Drosophila and also during embryonic development of mice. Although implicated in diverse cellular processes, including transcriptional regulation, chromosome segregation, and heterochromatin formation, its essential function in cells remains unknown. Cellular differentiation is part of the developmental process of multicellular organisms. To elucidate the roles of H2A.Z and H2A.Z acetylation in cellular differentiation, we examined the effects of expressing wild type (WT) or a non-acetylatable form of H2A.Z in the growth and differentiation of the myoblast C2C12 cell line. Ectopic expression of wild type or mutant H2A.Z resulted in distinct phenotypes in the differentiation of the C2C12 cells and the formation of myotubes. Most strikingly, expression of the H2A.Z non-acetylatable mutant (H2A.Z-Ac-mut) resulted in a complete block of myoblast differentiation. We determined that this phenotype is caused by a loss of MyoD expression in the Ac-mut-expressing cells prior to and after induction of differentiation. Moreover, chromatin accessibility assays showed that the promoter region of MyoD is less accessible in the differentiation-defective cells. Altogether, these new findings show that expression of the Ac-mut form of H2A.Z resulted in a dominant phenotype that blocked differentiation due to chromatin changes at the MyoD promoter.

H2A.Z helps genes remember their history so we can remember ours.

Histone variant exchange is a novel epigenetic regulator of cognition. We speculate that H2A.Z, a variant of canonical histone H2A, exerts unique effects on transcription during distinct stages of memory formation, ultimately acting to maintain memory of previous transcriptional states and poise genes for re-activation. Hippocampus-dependent memory formation is initiated by transient expression of memory-related genes, which support the storage of recently acquired memories. Soon after, memories undergo systems consolidation, which transfers memories from the hippocampus to the cortex for long-term storage, and requires ongoing re-activation of memory-related genes. We speculate that learning-induced H2A.Z eviction from nucleosomes initially contributes to stimulus-induced transcriptional induction needed for the initial process of memory consolidation. During systems consolidation, we speculate that delayed incorporation of H2A.Z into nucleosomes of memory-related genes in the cortex is needed to poise genes for rapid re-activation, thus supporting the long-term process of memory stabilization.

Histone H2A/H2B chaperones: from molecules to chromatin-based functions in plant growth and development.

Nucleosomal core histones (H2A, H2B, H3 and H4) must be assembled, replaced or exchanged to preserve or modify chromatin organization and function according to cellular needs. Histone chaperones escort histones, and play key functions during nucleosome assembly/disassembly and in nucleosome structure configuration. Because of their location at the periphery of nucleosome, histone H2A-H2B dimers are remarkably dynamic. Here we focus on plant histone H2A/H2B chaperones, particularly members of the NUCLEOSOME ASSEMBLY PROTEIN-1 (NAP1) and FACILITATES CHROMATIN TRANSCRIPTION (FACT) families, discussing their molecular features, properties, regulation and function. Covalent histone modifications (e.g. ubiquitination, phosphorylation, methylation, acetylation) and H2A variants (H2A.Z, H2A.X and H2A.W) are also discussed in view of their crucial importance in modulating nucleosome organization and function. We further discuss roles of NAP1 and FACT in chromatin-based processes, such as transcription, DNA replication and repair. Specific functions of NAP1 and FACT are evident when their roles are considered with respect to regulation of plant growth and development and in plant responses to environmental stresses. Future major challenges remain in order to define in more detail the overlapping and specific roles of various members of the NAP1 family as well as differences and similarities between NAP1 and FACT family members, and to identify and characterize their partners as well as new families of chaperones to understand histone variant incorporation and chromatin target specificity.

DNA Damage Initiated by the Sulfur Mustard Analog Mechlorethamine (methylbis(2‐chloroethyl)amine) in Mouse Keratinocytes is Associated with an Oxidative Stress Response

Sulfur mustard and nitrogen mustard (HN2, mechlorethamine) are bifunctional alkylating agent that cause dermal inflammation, edema and blistering. Skin keratinocytes are known to be important targets for mustards, which form both monofunctional DNA adducts and inter- and intrastrand DNA cross-links in the cells leading to cytotoxicity. In these studies, we used PAM212 mouse keratinocytes to analyze mechanisms underlying HN2 toxicity. Treatment of PAM212 cells with HN2 (3-30 μM) caused cytostasis; this was associated with a cell cycle block in late S phase and G2M phase. HN2 also caused DNA double strand breaks as shown by increased expression of phospho-H2A.X, a H2A histone variant that binds to double stranded DNA breaks creating a repair recognition domain. HN2-induced phosphorylation of H2A.X was both time- and concentration-dependent. Maximal increases were evident in cells at 24 h post HN2. HN2 induced double strand breaks were correlated with increases in the cellular antioxidants heme oxygenase-1 (HO-1) and heat shock protein27 (hsp27), which peaked 3 h and 6 h post HN2-treatment. These findings are consistent with the idea that HN2 initiates a DNA damage response in mouse keratinocytes that leads to induction of an adaptive stress response. Interfering with DNA strand breaks may be an effective strategy to mitigate mustard-induced damage in keratinocytes. Support: AR055073 and ES005022