Histone Methylation Marks Research Articles

ASXL1 Mutations Are Associated with Widespread and Distinct DNA Methylation Alterations

Background: ASXL1 is frequently mutated in a range of myeloid malignancies, including MDS, CMML, and AML, and is strongly associated with a poor prognosis in these myeloid disorders. ASXL1 is known to play a central role in epigenetic regulation, activating or repressing the transcription of genes involved in either differentiation or proliferation through its effects on histone methylation marks. In particular, ASXL1 is thought to be involved in recruiting PRC2 for its role in epigenetic modification. Even though DNA methylation is critically important in regulating gene expression in hematopoiesis, the effect of ASXL1 mutations on cytosine methylation is not well elucidated. Methods: We have previously studied the effects of the ASXL1 mutation through the use of the KBM5 CML cell line, which carries a homozygous nonsense ASXL1G710X mutation, resulting in loss of ASXL1 protein expression (Valetta et al, Oncotarget, 2015) . In particular, we saw that the ASXL1 mutation results in failure of PRC2 to recruit the HOXA cluster, resulting in increased expression of HOXA genes and resultant myeloid transformation. We used CRISPR/Cas9 mediated correction of the ASXL1 mutation in the leukemic KBM5 cell line to generate isogenic cell lines for methylome, expression, and chromatin accessibility analysis. Results: We have previously shown that correction of the ASXL1G710X mutation in KBM5 cells by CRISPR gene editing restored ASXL1 protein expression, with restored function of PRC2, including HOXA gene down-regulation with resultant reduced cell growth and increased myeloid differentiation, as evidenced by increased expression of CD11b, CD14 and CD15 by flow cytometry. In this current study, RNA-seq performed on the isogenic KBM5 cells confirmed the expression changes and CRISPR mediated correction of the ASXL1 mutation. Methylation DNA immunoprecipitation demonstrated widespread increased cytosine methylation in the KBM5 ASXL1 mutated cell line when compared to the corrected wild type cells. Evaluating this data alongside the RNA sequencing data, a clear difference in expression was noted between the genes that have methylation peaks in the gene promoter versus those that have peaks in the gene bodies. Specifically, increased methylation of the promoter region of genes was associated with decreased gene expression, while increased methylation of the gene body was associated with increased gene expression. Promoter methylation was found to be associated with down-regulation of differentiation associated CD14 . Gene body methylation was seen in widely over-expressed HOXA cluster transcripts and CD15. ATAC-seq demonstrated corresponding changes in chromatin accessibility. Due to the increased methylation in the mutant cells, we next treated the isogenic KBM5 cells with the DNMT inhibitor 5-Azacytidine. Proliferation assays of the ASXL1 mutated and corrected cells revealed significantly decreased cell viability of the KBM5 mutated cells with concordant increase in differentiation. To validate the results in primary samples, we assessed DNA methylation in a cohort of MDS ASXL1 mutant and wild type peripheral blood mononuclear samples. The HELP assay revealed increased methylation in ASXL1 mutant samples particularly in the gene bodies, consistent with the results seen in the isogenic cell line. Conclusions: It has been hypothesized that the ASXL1 mutation results in hypomethylation with resultant increased expression of genes, as previously seen with increased expression of the HOXA gene cluster in ASXL1 mutated cells. Our current study demonstrates increased methylation of the ASXL1 mutant versus the corrected cell line and reveals a significant amount of gene body methylation in the mutant cells. Specifically, we demonstrate that aberrant methylation in mutant cells provides a therapeutic window for DNMT inhibitors and is associated with site specific associations with chromatin accessibility and gene expression. Our study reinforces that high resolution whole genome methylation studies are needed to dissect the specific effects of mutations in epigenetic regulators such as ASXL1 in MDS and AML. Disclosures Verma: Janssen: Research Funding; BMS: Research Funding; Celgene: Honoraria; Stelexis: Equity Ownership, Honoraria; Acceleron: Honoraria.

Low doses of carbendazim and chlorothalonil synergized to impair mouse spermatogenesis through epigenetic pathways.

Pesticides have been extensively produced and used to help the agricultural production which leads to the contamination of the environment, soil, groundwater sources, and even foodstuffs. Fungicides carbendazim (CBZ) and chlorothalonil (Chl) are widely applied in agriculture and other aspects. CBZ or Chl have been reported to disrupt spermatogenesis and decrease semen quality. However, it is not understood the effects of pubertal exposure to low doses of CBZ and Chl together, and the underlying mechanisms. Therefore, the aim of current investigation was to explore the negative impacts of pubertal exposure to low doses of CBZ and Chl together on spermatogenesis and the role of epigenetic modifications in the process. We demonstrated that CBZ and Chl together synergize to decrease sperm motility in vitro (CBZ 1.0+Chl 0.1, CBZ 10.0+CHl 1.0, CBZ 100.0+Chl 10μM in incubation medium for 24h) and sperm concentration and motility in vivo with ICR mice (CBZ 0.1+Chl 0.1, CBZ 1.0+CHl 1.0, CBZ 10.0+Chl 10mg/kg body weight; oral gavage for five weeks). CBZ+Chl significantly increase reactive oxygen species (ROS) and apoptosis by the increase in the protein level of caspase 8 in vitro. Moreover, CBZ+Chl synergized to disrupt mouse spermatogenesis with the disturbance in sperm production proteins and sperm proteins (VASA, A-Myb, STK31, AR, Acrosin). CBZ+Chl synergized to decrease the protein level of estrogen receptor alpha and the protein level of DNA methylation marker 5mC in Leydig cells, and to increase the protein levels of histone methylation marker H3K9 and the methylation enzyme G9a in germ cells. Therefore, greater attention should be paid to the use of CBZ and Chl as pesticides to minimise their adverse impacts on spermatogenesis.

Histone demethylase KDM4A regulates adipogenic and osteogenic differentiation via epigenetic regulation of C/EBPα and canonical Wnt signaling.

Epigenetic modifications play a central role in cell differentiation and development. In the current study, we have recognized lysine demethylase 4A (KDM4A) as a novel epigenetic regulator of osteoblast and adipocyte differentiation. Kdm4a expression was upregulated during osteogenesis and adipogenesis of primary marrow stromal cells and established stromal ST2 line. Overexpression of wild-type Kdm4a promoted adipogenic differentiation and blocked osteogenic differentiation of the progenitor cells. This effect was largely alleviated when the catalytically dead mutation was made. Conversely, depletion or inactivation of Kdm4a in undifferentiated progenitor cells inhibited the formation of adipocytes and promoted the differentiation of osteoblasts. Mechanism explorations showed that overexpression of Kdm4a upregulated the expression of secreted frizzled-related protein 4 (Sfrp4) and CCAAT/enhancer-binding protein α (C/ebpα). Chromatin immunoprecipitation assay demonstrated that KDM4A directly bound the promoters of Sfrp4 and C/ebpα, removed the histone methylation mark H3K9me3, and reduced DNA methylation levels of CpG in promoter regions of C/ebpα and Sfrp4. Furthermore, overexpression of Kdm4a inactivated canonical Wnt signaling. Moreover, activation of canonical Wnt signaling through silencing of Sfrp4 in ST2 attenuated the inhibition of osteogenic differentiation and the enhancement of adipogenic differentiation by KDM4A. These data have identified KDM4A as a novel regulator of osteoblast and adipocyte differentiation and suggest KDM4A inhibition as a potential therapeutic target for treating metabolic disorders such as osteoporosis.

Regulation of PLPP3 gene expression by NF-κB family transcription factors

Lipid phosphate phosphatase 3 (LPP3), encoded by the PLPP3 gene, is an integral membrane enzyme that dephosphorylates phosphate esters of glycero- and sphingophospholipids. Cell surface LPP3 can terminate the signaling actions of bioactive lysophosphatidic acid (LPA) and sphingosine 1 phosphate, which likely explains its role in developmental angiogenesis, vascular injury responses, and cell migration. Heritable variants in the final intron PLPP3 associate with interindividual variability in coronary artery disease risk that may result from disruption of enhancer sequences that normally act in cis to increase expression of the gene. However, the mechanisms regulating PLPP3 expression are not well understood. We show that the human PLPP3 promoter contains three functional NF-κB response elements. All of these are required for maximal induction of PLPP3 promoter activity in reporter assays. The identified sequences recruit RelA and RelB components of the NF-κB transcription complex to chromatin, and these transcription factors bind to the identified target sequences in two different cell types. LPA promotes binding of Rel family transcription factors to the PLPP3 promoter and increases PLPP3 gene expression through mechanisms that are attenuated by an NF-κB inhibitor, LPA receptor antagonists, and inhibitors of phosphoinositide 3 kinase. These findings indicate that up-regulation of PLPP3 during inflammation and atherosclerosis results from canonical activation of the NF-κB signaling cascade to increase PLPP3 expression through nuclear import and binding of RelA and RelB transcription factors to the PLPP3 promoter and suggest a mechanism by which the LPP3 substrate, LPA, can regulate PLPP3 expression.

Pubertal exposure to low doses of zearalenone disrupting spermatogenesis through ERα related genetic and epigenetic pathways

Endocrine disruptor zearalenone (ZEA) has been found to damage the reproductive system especially spermatogenesis. In our previous report, we have found that low dose (lower than No-Observed Effect Level, NOEL) ZEA exposure disturbed mouse spermatogenesis and diminished mouse semen quality. The purpose of current investigation was to explore the underlying mechanisms of pubertal low dose ZEA exposure upsetting spermatogenesis. And it was demonstrated that pubertal low dose ZEA exposure disrupted the meiosis process and the important genetic pathways to inhibit the spermatogenesis and even to diminish the semen quality with the decrease in spermatozoa motility and concentration. The DNA methylation markers 5mC and 5hmC were decreased, the histone methylation marker H3K27 was increased, at the same time estrogen receptor alpha was diminished in mouse testis after pubertal low dose ZEA exposure. The data indicate that the disruption in spermatogenesis by pubertal low dose ZEA exposure may be through the alterations in genetic and epigenetic pathways, and the interactions with estrogen receptor signaling pathway. Therefore, we should pay great attention on ZEA exposure to reduce its adverse impacts on male reproductive health.

Abstract 2991: Targeting glutamine metabolism leads to terminal differentiation in acute myeloid leukemia (AML)

Abstract The ability of All Trans Retinoic Acid (ATRA) to induce terminal differentiation in Acute Promyelocytic Leukemia (APL) has revolutionized treatment for this deadly subset of AML. Unfortunately, identifying targets that induce differentiation in non-APL cases of AML has met with limited success. Our lab has been interested in the role of glutamine metabolism in cellular differentiation programs. As such, we have investigated the degree to which glutamine contributes to the maintenance of an undifferentiated state in AML. Herein, we report that in vitro incubation with the glutamine blocking agent, 6-diazo-5-oxo-L-norleucine (DON) robustly induces differentiation in the human AML cell line, U937. DON treatment induces phenotypic and molecular changes indicative of differentiation, including increased granularity and vacuolization, and upregulation of differentiation markers CD11b and CD14. The differentiation of these cells was confirmed by their highly abrogated growth in methylcellulose-based colony-forming assays. Differentiated AML cells were not exposed to DON during colony-forming assays, suggesting an irreversible change in cellular differentiation triggered by short-term pretreatment. We have demonstrated that DON-induced cellular differentiation is active in other AML cell lines as well. Importantly, the differentiating effect of DON can be abrogated by the addition of a cell permeable form of α-ketoglutarate (αKG), dimethyl-2-ketoglutarate (DMK). αKG is a key metabolite in glutamine metabolism and is also a critical cofactor for a range of demethylase enzymes involved in epigenetic remodeling. Indeed, we have noted a robust increase in histone methylation marks, such as H3K27Me3, during DON-induced differentiation. Further, we have observed a dramatic increase in the transcription of critical transcription factors (CEBPA, FLT3, MYB, SOX4) twenty-four hours after DON treatment, a time point immediately preceding observed phenotypic differentiation changes. In light of these findings, experiments are currently underway to use DON differentiation therapy as a means of treating mouse models of AML. Taken as a whole, these data support the use of glutamine blockade as a novel differentiating therapy in AML. Given the remarkable curative potential of differentiation therapy and the poor survival rates of adults with AML (particularly older patients), our findings represent a particularly novel approach to treating this deadly disease. Citation Format: Robert Leone, Im-Meng Sun, Roshan Chikarmane, Radhika Viswanathan, Sanjeda Patwari, Matthew Arwood, Gabriel Ghiaur, Jonathan Powell. Targeting glutamine metabolism leads to terminal differentiation in acute myeloid leukemia (AML) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2991.

Abstract 1864: Verticillin A causes DNA damage and apoptosis in high grade serous ovarian cancer

Abstract High grade serous ovarian cancer (HGSOC) is the most lethal gynecological malignancy affecting women worldwide and the fifth most common cause of cancer related deaths among U.S. women. New targeted therapies are needed to prevent HGSOC progression and metastasis related lethality from the disease. The goal of this study was to test the novel natural compound, Verticillin A, for its anticancer properties and mode of action in HGSOC cells. Verticillin A is an epipolythiodioxopiperazine (ETP) alkaloid that is isolated from several terrestrial and marine filamentous fungi and has been shown to be cytotoxic in several cancer cell lines including OVCAR8, OVCAR4 and Kuramochi. Our data indicated that Verticillin A treatment caused cytotoxicity in HGSOC cell lines in a dose-dependent manner. Furthermore, treatment with Verticillin A in HGSOC cell line OVCAR8 and OVCAR4 enhanced apoptosis, which was demonstrated by PARP cleavage and Annexin V/ Propidium iodide staining. To determine whether Verticillin A caused in vivo tumor growth inhibition, OVCAR8-RFP cells were xenografted in mice to form tumors and the mice were treated with Verticillin A. Encapsulated nanoparticles of Verticillin A decreased tumor growth in vivo and had low cytotoxicity compared to the naked drug. RNA-Seq analysis was performed with OVCAR8 cells treated with Verticillin A and the data found an upregulation of apoptosis signaling pathway and oxidative stress response and downregulation of cancer stemness signaling pathways. A proteomic histone profiling performed in OVCAR8 cells indicated that Verticillin A caused epigenetic modifications with global changes in histone methylation and acetylation marks. Thus, our study identifies Verticillin A as a novel epigenetic modifier in ovarian cancer cells and indicates therapeutic potential for treatment of HGSOC. Note: This abstract was not presented at the meeting. Citation Format: Amrita Salvi, Julia Austin, Daniel Lantvit, Joanna Burdette. Verticillin A causes DNA damage and apoptosis in high grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1864.

Effect of Dietary Manipulation of B Vitamins During Pregnancy and Its Impact on Neurobehavior Development and Epigenetic Regulation of Imprinted Genes (P15-026-19)

ObjectivesTill date there have been studies to check the individual effect of maternal folate and vitamin B12 during pregnancy but less is known regarding the combined effect of both vitamins on growth and neuro-behaviour of the off-springs. So, the present study was designed with an aim to check the combined effect of both vitamins on: Neurobehaviour development of off-springs and epigenetic regulation of imprinted (H19, IGF2, IGF2R) genes. MethodsFour week old C57BL/6 female mice were randomized into nine groups BNFN, BNFO, BNFD, BDFN, BDFD, BDFO, BOFN, BOFO, BOFD and were fed diet for six weeks while male mice were on normal diet. Mating was performed and pregnant dams gave birth to the F1 off-springs. Both male and female fetuses in F1 generation were fed diets as per the dietary groups of their mothers. After six weeks males were used for neurobehavior studies and mating was carried out, on day 20 of gestation maternal and fetal tissues (F2 generation) were isolated. Folate, homocysteine and B12 levels were analyzed along with mRNA expression, promoter DNA methylation and histone modification of imprinted genes. ResultsFolate, vitamin B12 and homocysteine levels were altered in different folate and B12 dietary groups (P < 0.01). Adult mice exposed to folate and vitamin B12 conditions expressed delayed locomoter functions in BDFD (25.74%), BNFD (20.75%) and BOFD (20.16%) groups, impaired spatial learning and memory ability and increased GSH/GSSG levels in brain under deficient conditions. In maternal and fetal tissues, H19 was found to be over-expressed under deficient conditions which was co-related with decreased promoter methylation and decrease suppressive histone methylation marks H3K9me3 and H3K27me3 (P < 0.01) and increase in H3K4me2. Genomic imprinting patterns were found to be reversed in H19 in both male and female fetuses. IGF2 along with IGF2R showed loss of expression with increased methylation in promoter region (P < 0.05) and suppressive histone methylation marks in deficient conditions. AbbreviationsBN: B12 Normal FN: Folate Normal FD: Folate deficient FO: Folate over supplementation BD: B12 deficient BO: B12 over supplementation ConclusionsMaternal dietary ratio of folate and B12 during pregnancy influence neurobehaviour development and alters the establishment of proper imprints during development. Funding SourcesDepartment of Biotechnology.

Gestational exposure to low-dose zearalenone disrupting offspring spermatogenesis might be through epigenetic modifications.

Zearalenone (ZEA), a F-2 mycotoxin produced by Fusarium, has been found to be an endocrine disruptor through oestrogen receptor signalling pathway to impair spermatogenesis. The disruption on reproductive systems by ZEA exposure might be transgenerational. In our previous report, we have found that low dose (lower than no-observed effect level, NOEL) of ZEA impaired mouse spermatogenesis and decreased mouse semen quality. The purpose of the current investigation was to explore the impacts of low-dose ZEA on spermatogenesis in the offspring after prenatal exposure and the underlying mechanisms. And it demonstrated that prenatal low-dose ZEA exposure disrupted the meiosis process to inhibit the spermatogenesis in offspring and even to diminish the semen quality by the decrease in spermatozoa motility and concentration. The DNA methylation marker 5hmC was decreased, the histone methylation markers H3K9 and H3K27 were elevated, and oestrogen receptor alpha was reduced in the offspring testis after prenatal low-dose ZEA exposure. The data suggest that the disruption in spermatogenesis by prenatal low-dose ZEA exposure may be through the modifications on epigenetic pathways (DNA methylation and histone methylation) and the interactions with oestrogen receptor signalling pathway. Moreover, in the current study, the male offspring were indirectly exposed to low-dose ZEA through placenta and the spermatogenesis in offspring was disrupted which suggested that the toxicity of ZEA on reproductive systems was very severe. Therefore, we strongly recommend that greater attention should be paid to this mycotoxin to minimize its adverse impact on human spermatogenesis.

Destabilization of chromosome structure by histone H3 lysine 27 methylation.

Chromosome and genome stability are important for normal cell function as instability often correlates with disease and dysfunction of DNA repair mechanisms. Many organisms maintain supernumerary or accessory chromosomes that deviate from standard chromosomes. The pathogenic fungus Zymoseptoria tritici has as many as eight accessory chromosomes, which are highly unstable during meiosis and mitosis, transcriptionally repressed, show enrichment of repetitive elements, and enrichment with heterochromatic histone methylation marks, e.g., trimethylation of H3 lysine 9 or lysine 27 (H3K9me3, H3K27me3). To elucidate the role of heterochromatin on genome stability in Z. tritici, we deleted the genes encoding the methyltransferases responsible for H3K9me3 and H3K27me3, kmt1 and kmt6, respectively, and generated a double mutant. We combined experimental evolution and genomic analyses to determine the impact of these deletions on chromosome and genome stability, both in vitro and in planta. We used whole genome sequencing, ChIP-seq, and RNA-seq to compare changes in genome and chromatin structure, and differences in gene expression between mutant and wildtype strains. Analyses of genome and ChIP-seq data in H3K9me3-deficient strains revealed dramatic chromatin reorganization, where H3K27me3 is mostly relocalized into regions that are enriched with H3K9me3 in wild type. Many genome rearrangements and formation of new chromosomes were found in the absence of H3K9me3, accompanied by activation of transposable elements. In stark contrast, loss of H3K27me3 actually increased the stability of accessory chromosomes under normal growth conditions in vitro, even without large scale changes in gene activity. We conclude that H3K9me3 is important for the maintenance of genome stability because it disallows H3K27me3 in regions considered constitutive heterochromatin. In this system, H3K27me3 reduces the overall stability of accessory chromosomes, generating a “metastable” state for these quasi-essential regions of the genome.

Learning the Formation Mechanism of Domain-Level Chromatin States with Epigenomics Data

Epigenetic modifications can extend over long genomic regions to form domain-level chromatin states that play critical roles in gene regulation. The molecular mechanism for the establishment and maintenance of these states is not fully understood and remains challenging to study with existing experimental techniques. Here, we took a data-driven approach and parameterized an information-theoretic model to infer the formation mechanism of domain-level chromatin states from genome-wide epigenetic modification profiles. This model reproduces statistical correlations among histone modifications and identifies well-known states. Importantly, it predicts drastically different mechanisms and kinetic pathways for the formation of euchromatin and heterochromatin. In particular, long, strong enhancer and promoter states grow gradually from short but stable regulatory elements via a multistep process. On the other hand, the formation of heterochromatin states is highly cooperative, and no intermediate states are found along the transition path. This cooperativity can arise from a chromatin looping-mediated spreading of histone methylation mark and supports collapsed, globular three-dimensional conformations rather than regular fibril structures for heterochromatin. We further validated these predictions using changes of epigenetic profiles along cell differentiation. Our study demonstrates that information-theoretic models can go beyond statistical analysis to derive insightful kinetic information that is otherwise difficult to access.

Perinatal Inflammation Results in Altered Histone 3 and Histone 4 Methylation Patterns

BackgroundBronchopulmonary dysplasia (BPD) continues to be a significant pulmonary complication for preterm infants, but the mechanisms associated with altered lung development remain poorly understood. Our mouse model of perinatal inflammation mimics the pathophysiology observed in infants with severe BPD. We have previously reported decreased pulmonary miR‐29b and miR~17–92 cluster expression and increased global DNA methylation. These studies tested the hypothesis that histone methylation may also be responsible for the lung pathophysiology observed in this model. First, we investigated protein arginine methyltransferase (PRMT)‐mediated changes in H4R3me2 (symmetrical and asymmetrical). Second, we explored lysine methylation, specifically H3K4me3, H3K9me2, and H3K27me3.MethodsPregnant C3H/HeN mice received an intraperitoneal LPS injection on E16 and newborn pups were exposed in 85% oxygen from birth to 14 days of life. On postnatal day 14 or 28, mouse lung tissues were harvested. Tissue homogenates were analyzed by western blot for PRMT‐1, PRMT‐5, and H4R3me2 symmetrical and asymmetrical and by ELISA for H3K4me3, H3K9me2, and H3K27me3 (Epigentek).ResultsPerinatal inflammation resulted in decreased expression of PRMT‐1 (1.0 v 0.45 arbitrary units, p&lt;0.0001) and PRMT‐5 (1.0 v 0.48 arbitrary units, p=0.02) which corresponded to decreases in H4R3me2 symmetrical methylation (1.3 v 0.39 arbitrary units, p=0.04). H3K9me2 (39.8 vs 25.0 ug/mL, p=0.03) and H3K4me3 (41.1 vs 30.3 ug/mL, p&lt;0.05) were also decreased in the exposed group while H3K27me3 was not significant.ConclusionOur data suggest that maternal inflammation combined with neonatal hyperoxia influences expression of proteins involved in histone methylation and alters specific histone methylation marks. Further investigations will explore expression of histone lysine methyltransferases and demethylases. Overall the changes observed suggest a disruption in chromatin structure which may be responsible for altered gene expression in this model.Support or Funding InformationNIH NICHD R01HD088033This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

SMARCAD1 ATPase activity is required to silence endogenous retroviruses in embryonic stem cells

Endogenous retroviruses (ERVs) can confer benefits to their host but present a threat to genome integrity if not regulated correctly. Here we identify the SWI/SNF-like remodeler SMARCAD1 as a key factor in the control of ERVs in embryonic stem cells. SMARCAD1 is enriched at ERV subfamilies class I and II, particularly at active intracisternal A-type particles (IAPs), where it preserves repressive histone methylation marks. Depletion of SMARCAD1 results in de-repression of IAPs and adjacent genes. Recruitment of SMARCAD1 to ERVs is dependent on KAP1, a central component of the silencing machinery. SMARCAD1 and KAP1 occupancy at ERVs is co-dependent and requires the ATPase function of SMARCAD1. Our findings uncover a role for the enzymatic activity of SMARCAD1 in cooperating with KAP1 to silence ERVs. This reveals ATP-dependent chromatin remodeling as an integral step in retrotransposon regulation in stem cells and advances our understanding of the mechanisms driving heterochromatin establishment.

Extensive allele-level remodeling of histone methylation modification in reciprocal F1 hybrids of rice subspecies.

Epigenetic mechanisms play a major role in heterosis, partly as a result of the remodeling of epigenetic modifications in F1 hybrids. Based on chromatin immunoprecipitation-sequencing (ChIP-Seq) analyses, we show that at the allele level extensive histone methylation remodeling occurred for a subset of genomic loci in reciprocal F1 hybrids of Oryza sativa (rice) cultivars Nipponbare and 93-11, representing the two subspecies japonica and indica. Globally, the allele modification-altered loci in leaf or root of the reciprocal F1 hybrids involved ˜12-43% or more of the genomic regions carrying either of two typical histone methylation markers, H3K4me3 (>21000 genomic regions) and H3K27me3 (>11000 genomic regions). Nevertheless, at the total modification level, the majority (from ˜43 to >90%) of the modification-altered alleles lay within the range of parental additivity in the hybrids because of concerted alteration in opposite directions, consistent with an overall attenuation of allelic differences in the modifications. Importantly, of the genomic regions that did show non-additivity in total modification level by either marker in the two tissues of hybrids, >80% manifested transgressivity, which involved genes enriched in specific functional categories. Extensive allele-level alteration of H3K4me3 alone was positively correlated with genome-wide changes in allele-level gene expression, whereas at the total level, both H3K4me3 and H3K27me3 remodeling, although affecting just a small number of genes, contributes to the overall non-additive gene expression to variable extents, depending on tissue/marker combinations. Our results emphasize the importance of allele-level analysis in hybrids to assess the remodeling of epigenetic modifications and their relation to changes in gene expression.

Homozygous Loss of a Critical CTCF Recognition Sequence in the HOXA gene Cluster Eliminates CTCF Binding but Does Not Appreciably Reduce HOXA9 expression in NPM1 Mutated AML Cells

HOX genes encode homeodomain transcription factors that play a central role in hematopoietic stem/progenitor cell (HSPCs) self-renewal and the development of acute myeloid leukemia (AML). HOXA and HOXB genes display canonical expression patterns in primary AML samples that correlate with specific recurrent mutations, including expression of only HOXA genes in AMLs with MLL rearrangements and both HOXA and HOXB gene expression in normal karyotype AMLs with mutations in NPM1 (i.e., the NPMc mutation). Although regulation of HOXA genes in MLL-rearranged AML cells has been studied extensively, the regulatory mechanisms that govern HOXA expression in other AML types remains unclear. The chromatin organizing factor CTCF contributes to HOXA gene regulation by controlling chromatin looping and three-dimensional structure in a variety of cell types, including AML cells, and a recent study demonstrated that expression of HOXA9 in MLL-rearranged AML cell lines is dependent on a critical CTCF-binding site between HOXA7 and HOXA9 (CBS7/9). However, whether this site is required for HOXA gene expression in AMLs with NPMc remains unknown.The AML cell line OCI-AML3 contains the NPMc mutation (in addition to a DNMT3AR882C mutant allele) and expresses both HOXA and HOXB genes. We therefore used this cell line to investigate the role of CTCF in regulating HOXA expression in AML cells with this canonical stem cell expression pattern. ChIP-seq for CTCF via ‘ChIPmentation’ identified eight CTCF binding sites across the HOXA cluster in OCI-AML3 cells, including CBS7/9, which is consistent with previous data in AML cells. These sites were identical to those identified in MLL-rearranged AML cell lines, suggesting that CTCF binding is conserved between these AML types. Similar to previous work, ChIP-seq for histone methylation marks associated with active (H3K4 trimethylation) and repressed (H3K27 trimeylation) chromatin demonstrated distinct chromatin domains at the HOXA locus on either side of CBS7/9, indicating that CTCF acts as a barrier between these domains. Using a computational prediction tool for CTCF binding, we identified two potential CTCF-binding sequences at the CBS7/9 locus, including one bidirectional major site (CBS7/9-A; 20 bp) with a high binding score, separated by only 6 bp from a secondary unidirectional site (CBS7/9-B; 9 bp).To define the precise sequence important for CTCF binding in OCI-AML3 cells, we used CRISPR-Cas9 to generate a library of mutant cells with deletions of different sizes at the CBS7/9 locus, and then performed ChIP-seq on the mutant cells to measure CTCF binding. We found that mutants with deletions at CBS7/9-A were depleted relative to wild-type cells. Further analysis demonstrated that deletions as small as 10 bp affecting the CBS7/9-A site were sufficient to reduce CTCF binding to background levels. CRISPR-Cas9 generated deletions in the MLL-rearranged THP-1 cell line that spanned the same sequence also eliminated CTCF ChIP-seq signal, indicating that it is equally important for CTCF binding in MLL-rearranged and NPM1-mutant AML cells.Our collection of mutant OCI-AML3 cell lines included five with biallelic deletions of the CBS7/9-A site, which completely eliminated all CTCF binding at this locus, but did not affect cellular proliferation or survival. Surprisingly, preliminary analysis of expression in the five homozygous mutant OCI-AML3 cells demonstrated that HOXA9 gene expression by RT-qPCR remained largely intact compared to wild-type OCI-AML3 cells, and was not statistically different from three OCI-AML3 cell lines with a heterozygous deletion of the CBS7/9 site.In summary, we have mapped the precise recognition sequence for a critical CTCF binding site in the HOXA locus in human AML cells. Deletion of both alleles of this regulatory sequence in NPM1-mutated OCI-AML3 cells does not affect cell viability and appears to have only modest effects on expression of the adjacent HOXA9 gene. This is in contrast to a recent report suggesting that homozygous deletion of this CTCF binding site is critical for maintaining HOXA gene expression and may be required for cell viability in MLL-rearranged AML cell lines. These differences suggest that maintenance of HOXA expression in NPM1-mutated vs. MLL-rearranged AML cells may occur through distinct regulatory mechanisms. DisclosuresNo relevant conflicts of interest to declare.

Novel KDM5B splice variants identified in patients with developmental disorders: Functional consequences

Histone lysine methylation influences processes such as gene expression and DNA repair. Thirty Jumonji C (JmjC) domain-containing proteins have been identified and phylogenetically clustered into seven subfamilies. Most JmjC domain-containing proteins have been shown to possess histone demethylase activity toward specific histone methylation marks. One of these subfamilies, the KDM5 family, is characterized by five conserved domains and includes four members. Interestingly, de novo loss-of-function and missense variants in KDM5B were identified in patients with intellectual disability (ID) and autism spectrum disorder (ASD) but also in unaffected individuals. Here, we report two novel de novo splice variants in the KDM5B gene in three patients with ID and ASD. The c.808 + 1G > A variant was identified in a boy with mild ID and autism traits and is associated with a significant reduced KDM5B mRNA expression without alteration of its H3K4me3 pattern. In contrast, the c.576 + 2T > C variant was found in twins with global delay in developmental milestones, poor language and ASD. This variant causes the production of an abnormal transcript which may produce an altered protein with the loss of the ARID1B domain with an increase in global gene H3K4me3. Our data reinforces the recent observation that the KDM5B haploinsufficiency is not a mechanism involved in intellectual disability and that KDM5B disorder associated with LOF variants is a recessive disorder. However, some variants may also cause gain of function, and need to be interpreted with caution, and functional studies should be performed to identify the molecular consequences of these different rare variants.

Super-Resolution Imaging of Higher-Order Chromatin Structures at Different Epigenomic States in Single Mammalian Cells

SUMMARYHistone modifications influence higher-order chromatin structures at individual epigenomic states and chromatin environments to regulate gene expression. However, genome-wide higher-order chromatin structures shaped by different histone modifications remain poorly characterized. With stochastic optical reconstruction microscopy (STORM), we characterized the higher-order chromatin structures at their epigenomic states, categorized into three major types in interphase: histone acetylation marks form spatially segregated nanoclusters, active histone methylation marks form spatially dispersed larger nanodomains, and repressive histone methylation marks form condensed large aggregates. These distinct structural characteristics are also observed in mitotic chromosomes. Furthermore, active histone marks coincide with less compact chromatin and exhibit a higher degree of co-localization with other active marks and RNA polymerase II (RNAP II), while repressive marks coincide with densely packed chromatin and spatially distant from repressive marks and active RNAP II. Taken together, super-resolution imaging reveals three distinct chromatin structures at various epigenomic states, which may be spatially coordinated to impact transcription.

ATR inhibition controls aggressive prostate tumors deficient in Y-linked histone demethylase KDM5D.

Epigenetic modifications control cancer development and clonal evolution in various cancer types. Here, we show that loss of the male-specific histone demethylase lysine-specific demethylase 5D (KDM5D) encoded on the Y chromosome epigenetically modifies histone methylation marks and alters gene expression, resulting in aggressive prostate cancer. Fluorescent in situ hybridization demonstrated that segmental or total deletion of the Y chromosome in prostate cancer cells is one of the causes of decreased KDM5D mRNA expression. The result of ChIP-sequencing analysis revealed that KDM5D preferably binds to promoter regions with coenrichment of the motifs of crucial transcription factors that regulate the cell cycle. Loss of KDM5D expression with dysregulated H3K4me3 transcriptional marks was associated with acceleration of the cell cycle and mitotic entry, leading to increased DNA-replication stress. Analysis of multiple clinical data sets reproducibly showed that loss of expression of KDM5D confers a poorer prognosis. Notably, we also found stress-induced DNA damage on the serine/threonine protein kinase ATR with loss of KDM5D. In KDM5D-deficient cells, blocking ATR activity with an ATR inhibitor enhanced DNA damage, which led to subsequent apoptosis. These data start to elucidate the biological characteristics resulting from loss of KDM5D and also provide clues for a potential novel therapeutic approach for this subset of aggressive prostate cancer.

Reading between the Lines: "ADD"-ing Histone and DNA Methylation Marks toward a New Epigenetic "Sum".

Reading between the Lines: "ADD"-ing Histone and DNA Methylation Marks toward a New Epigenetic "Sum".

Dynamics of promoter bivalency and RNAP II pausing in mouse stem and differentiated cells

BackgroundMammalian embryonic stem cells display a unique epigenetic and transcriptional state to facilitate pluripotency by maintaining lineage-specification genes in a poised state. Two epigenetic and transcription processes involved in maintaining poised state are bivalent chromatin, characterized by the simultaneous presence of activating and repressive histone methylation marks, and RNA polymerase II (RNAPII) promoter proximal pausing. However, the dynamics of histone modifications and RNAPII at promoters in diverse cellular contexts remains underexplored.ResultsWe collected genome wide data for bivalent chromatin marks H3K4me3 and H3K27me3, and RNAPII (8WG16) occupancy together with expression profiling in eight different cell types, including ESCs, in mouse. The epigenetic and transcription profiles at promoters grouped in over thirty clusters with distinct functional identities and transcription control.ConclusionThe clustering analysis identified distinct bivalent clusters where genes in one cluster retained bivalency across cell types while in the other were mostly cell type specific, but neither showed a high RNAPII pausing. We noted that RNAPII pausing is more associated with active genes than bivalent genes in a cell type, and was globally reduced in differentiated cell types compared to multipotent.