Distinct Chromatin Research Articles

High-Density Domain-Focused CRISPR Screens Reveal Epigenetic Regulators of Hox/Meis Gene Expression in Acute Myeloid Leukemia

The aberrant and constitutive activation of the HOXA cluster genes and the their-co-factor MEIS1 (HOX/MEIS) is a recurrent feature in several types of myeloid and lymphoid leukemias. Aberrant HOX/MEIS expression has been shown to drive limitless leukemia stem cell self-renewal and is therefore an attractive target for therapy in acute myeloid leukemia (AML). However, since HOX/MEIS genes encode DNA-binding transcription factors, small molecules targeting these proteins directly are lacking. Furthermore, targeting the HOX/MEIS network is complicated by the fact that these genes are coordinately regulated and have redundant functions in sustaining leukemic self-renewal. One way of therapeutically targeting aberrant HOX/MEIS transcription is the identification and pharmacologic inhibition of upstream chromatin regulators that coordinately modulate their expression. In order to identify such chromatin regulators, we made use of an endogenous GFP reporter knocked-in to the MEIS1 locus in the high HOX/MEIS-expressing U937 human AML cell line. Using this system, we first performed a high-throughput flow-cytometry-based small-molecule inhibitor screen with a library of 261 compounds targeting epigenetic regulators. In our screen, the most potent hits that reproducibly showed >50% MEIS1-GFP inhibition were small molecules that targeted DOT1L, the histone methyltransferase. DOT1L inhibitors have already been well-characterized as HOX/MEIS regulators and most epigenetic regulators are not targeted by existing compound libraries. Therefore, we decided to use a genetic screening approach to more extensively interrogate the landscape of epigenetic regulators of HOX/MEIS expression in AML. For this, we designed a custom computational pipeline and built a CRISPR library of 10,000 sgRNAs targeting functionally conserved protein domains of all catalogued chromatin modulatory proteins (> 600 proteins - 5 sgRNAs per conserved domain). This list of epigenetic regulators included histone modifying enzymes, chromatin readers, nucleosome remodelers, adaptor proteins and proteins involved in DNA and RNA modifications, as well as other transcriptional regulators. Using this comprehensive, domain-focused CRISPR library, we conducted a phenotypic enrichment screen. Specifically, we used flow cytometry to purify the top 20% GFP-MEIS1 (high) and bottom 20% GFP-MEIS1 (low) expressing cells and identified sgRNAs that were enriched particularly in the GFP-MEIS1 -low vs -high fraction using next generation sequencing. Given the extent and complexity of the CRISPR library, our approach uncovered members of six distinct chromatin modifying complexes as MEIS1 regulators (MAGeCKFlute pipeline, 2 SD > mean) and we could validate > 10 of these hits as bonafide regulators of MEIS1 as well as HOXA genes. We also demonstrated their essentiality for the proliferation of HOX-driven AML cells using arrayed sgRNA competition assays. These validated hits included several known as well as novel chromatin readers and writers amenable to small-molecule targeting. We focused our attention on the KAT7/JADE3 complex and the casein kinase 2 (CK2) family that we validated as potent and selective regulators of HOX/MEIS expression in AML cells. Our studies demonstrated that genetic depletion of components of the KAT7 complex or of the CK2 family could reverse HOX/MEIS activation in human AML cells, leading to a progressive loss of proliferative potential. Importantly, the use of the clinical-grade CK2 inhibitor CX4945 (Silmitasertib) caused a concentration-dependent down-regulation of HOX/MEIS expression in models of HOX-driven AML, leading to significant anti-leukemia effects. Our study provides a framework for the multiplexed identification of actionable dependencies targeting therapeutically recalcitrant oncogenic networks in cancer. Specifically for AML, since Silmitasertib is in Phase 2 trials for treatment of other cancers, our studies may solve the long-standing problem of targeting leukemia stem cells in AML potentially overcoming therapy refractoriness in this devastating disease. Disclosures No relevant conflicts of interest to declare.

Computational analysis of TP53 vs. CTNNB1 mutations in hepatocellular carcinoma suggests distinct cancer subtypes with differential gene expression profiles and chromatin states

Genetic variations are important drivers of carcinogenesis. It is extremely important to identify molecular distinctions between patients of the same disease for effective cancer treatment. This study aims to understand cellular and molecular differences between hepatocellular carcinoma patients carrying TP53 or CTNNB1 mutations, which could possess clinical significance. For this purpose, DNA sequencing and mRNA expression data for hepatocellular carcinoma patients were analyzed. Differentially expressed genes and the cellular processes that they are involved in were determined for TP53/CTNNB1-altered patient groups. We found that the mutations of TP53/CTNNB1 genes in the patient cohort was almost mutually exclusive and gene expression profiling in these subgroups were unique. Gene Ontology (GO) enrichment analysis of the differentially expressed genes identified several important cellular processes. In line with this, selected histone variants, histone chaperons, as well as the binding partners of TP53/CTNNB1 showed distinct enrichment levels. TP53/CTNNB1-altered patient groups laso showed different prognostic outcomes and tumor infiltration levels. In conclusion, our results strongly imply differential chromatin states and transcriptional regulation in relation to the mutational status of TP53 vs. CTNNB1, suggesting that these genes might be inducing different cellular pathways involving distinct chromatin environments during the course of carcinogenesis.

USP38 Couples Histone Ubiquitination and Methylation via KDM5B to Resolve Inflammation.

Chromatin modifications, such as histone acetylation, ubiquitination, and methylation, play fundamental roles in maintaining chromatin architecture and regulating gene transcription. Although their crosstalk in chromatin remodeling has been gradually uncovered, the functional relationship between histone ubiquitination and methylation in regulating immunity and inflammation remains unclear. Here, it is reported that USP38 is a novel histone deubiquitinase that works together with the histone H3K4 modifier KDM5B to orchestrate inflammatory responses. USP38 specifically removes the monoubiquitin on H2B at lysine 120, which functions as a prerequisite for the subsequent recruitment of demethylase KDM5B to the promoters of proinflammatory cytokines Il6 and Il23a during LPS stimulation. KDM5B in turn inhibits the binding of NF‐κB transcription factors to the Il6 and Il23a promoters by reducing H3K4 trimethylation. Furthermore, USP38 can bind to KDM5B and prevent it from proteasomal degradation, which further enhances the function of KDM5B in the regulation of inflammation‐related genes. Loss of Usp38 in mice markedly enhances susceptibility to endotoxin shock and acute colitis, and these mice display a more severe inflammatory phenotype compared to wild‐type mice. The studies identify USP38‐KDM5B as a distinct chromatin modification complex that restrains inflammatory responses through manipulating the crosstalk of histone ubiquitination and methylation.

Single-nucleus RNA-seq and FISH identify coordinated transcriptional activity in mammalian myofibers

Skeletal muscle fibers are large syncytia but it is currently unknown whether gene expression is coordinately regulated in their numerous nuclei. Here we show by snRNA-seq and snATAC-seq that slow, fast, myotendinous and neuromuscular junction myonuclei each have different transcriptional programs, associated with distinct chromatin states and combinations of transcription factors. In adult mice, identified myofiber types predominantly express either a slow or one of the three fast isoforms of Myosin heavy chain (MYH) proteins, while a small number of hybrid fibers can express more than one MYH. By snRNA-seq and FISH, we show that the majority of myonuclei within a myofiber are synchronized, coordinately expressing only one fast Myh isoform with a preferential panel of muscle-specific genes. Importantly, this coordination of expression occurs early during post-natal development and depends on innervation. These findings highlight a previously undefined mechanism of coordination of gene expression in a syncytium.

NuA3 HAT antagonizes the Rpd3S and Rpd3L HDACs to optimize mRNA and lncRNA expression dynamics.

In yeast, NuA3 histone acetyltransferase (NuA3 HAT) promotes acetylation of histone H3 lysine 14 (H3K14) and transcription of a subset of genes through interaction between the Yng1 plant homeodomain (PHD) finger and H3K4me3. Although NuA3 HAT has multiple chromatin binding modules with distinct specificities, their interdependence and combinatorial actions in chromatin binding and transcription remain unknown. Modified peptide pulldown assays reveal that the Yng1 N-terminal region is important for the integrity of NuA3 HAT by mediating the interaction between core subunits and two methyl-binding proteins, Yng1 and Pdp3. We further uncover that NuA3 HAT contributes to the regulation of mRNA and lncRNA expression dynamics by antagonizing the histone deacetylases (HDACs) Rpd3S and Rpd3L. The Yng1 N-terminal region, the Nto1 PHD finger and Pdp3 are important for optimal induction of mRNA and lncRNA transcription repressed by the Set2-Rpd3S HDAC pathway, whereas the Yng1 PHD finger–H3K4me3 interaction affects transcriptional repression memory regulated by Rpd3L HDAC. These findings suggest that NuA3 HAT uses distinct chromatin readers to compete with two Rpd3-containing HDACs to optimize mRNA and lncRNA expression dynamics.

TADs enriched in histone H1.2 strongly overlap with the B compartment, inaccessible chromatin, and AT-rich Giemsa bands.

Giemsa staining of metaphase chromosomes results in a characteristic banding useful for identification of chromosomes and its alterations. We have investigated insilico whether Giemsa bands (G bands) correlate with epigenetic and topological features of the interphase genome. Staining of G-positive bands decreases with GC content; nonetheless, G-negative bands are GC heterogeneous. High GC bands are enriched in active histone marks, RNA polymerase II, and SINEs and associate with gene richness, gene expression, and early replication. Low GC bands are enriched in repressive marks, lamina-associated domains, and LINEs. Histone H1 variants distribute heterogeneously among G bands: H1X is enriched at high GC bands and H1.2 is abundant at low GC, compacted bands. According to epigenetic features and H1 content, G bands can be organized in clusters useful to compartmentalize the genome. Indeed, we have obtained Hi-C chromosome interaction maps and compared topologically associating domains (TADs) and A/B compartments to G banding. TADs with high H1.2/H1X ratio strongly overlap with B compartment, late replicating, and inaccessible chromatin and low GC bands. We propose that GC content is a strong driver of chromatin compaction and 3D genome organization, that Giemsa staining recapitulates this organization denoted by high-throughput techniques, and that H1 variants distribute at distinct chromatin domains. DATABASES: Hi-C data on T47D breast cancer cells have been deposited in NCBI's Gene Expression Omnibus and are accessible through GEO Series accession number GSE147627.

Heterochromatin as an Important Driver of Genome Organization.

Heterochromatin is a constituent of eukaryotic genomes with functions spanning from gene expression silencing to constraining DNA replication and repair. Inside the nucleus, heterochromatin segregates spatially from euchromatin and is localized preferentially toward the nuclear periphery and surrounding the nucleolus. Despite being an abundant nuclear compartment, little is known about how heterochromatin regulates and participates in the mechanisms driving genome organization. Here, we review pioneer and recent evidence that explores the functional role of heterochromatin in the formation of distinct chromatin compartments and how failure of the molecular mechanisms forming heterochromatin leads to disarray of genome conformation and disease.

FOXA1 Mutations Reveal Distinct Chromatin Profiles and Influence Therapeutic Response in Breast Cancer

Mutations in the pioneer transcription factor FOXA1 are a hallmark of estrogen receptor-positive (ER+) breast cancers. Examining FOXA1 in ∼5,000 breast cancer patients identifies several hotspot mutations in the Wing2 region and a breast cancer-specific mutation SY242CS, located in the third β strand. Using a clinico-genomically curated cohort, together with breast cancer models, we find that FOXA1 mutations associate with a lower response to aromatase inhibitors. Mechanistically, Wing2 mutations display increased chromatin binding at ER loci upon estrogen stimulation, and an enhanced ER-mediated transcription without changes in chromatin accessibility. In contrast, SY242CS shows neomorphic properties that include the ability to open distinct chromatin regions and activate an alternative cistrome and transcriptome. Structural modeling predicts that SY242CS confers a conformational change that mediates stable binding to a non-canonical DNA motif. Taken together, our results provide insights into how FOXA1 mutations perturb its function to dictate cancer progression and therapeutic response.

A review of epigenetic regulation in wound healing: Implications for the future of wound care.

Epigenetic regulatory mechanisms are essential for maintaining skin homeostasis and aid in the processes of wound healing. The nucleus co-ordinates gene expression using epigenetic regulatory mechanisms based on distinct chromatin structural states and their remodeling. These include DNA methylation and hydroxymethylation, post-translational histone modifications, ATP-dependent chromatin remodeling and higher-order chromatin structure and 3D genome organization. Epigenetic pathways play a key role in co-ordinating the behavior and activity of the multitude of cell types seen during skin repair, and research is now focusing on how wound healing can be modulated by altering the activity of certain reparative genes. Herein, we aim to highlight recent advances in understanding epigenetic regulatory mechanisms, with particular reference to those involved in keratinocyte and fibroblast biology. We also propose future directions for exploration of epigenetic mechanisms, and their potential clinical applications in acute wound care.

Immunochemical Detection of Modified Cytosine Species in Lampbrush Chromatin.

The lampbrush chromosomes found in the giant nucleus or germinal vesicle (GV) of amphibian oocytes provide unique opportunities for discrete closed and open chromatin structural domains to be directly observable by simple light microscopy. Moreover, the method described here for preparing spreads of lampbrush chromatin for immunostaining enables a straightforward approach to establishing the distributions of modified nucleotides within and between structurally and functionally distinctive chromatin domains.

Mapping the Effects of Genetic Variation on Chromatin State and Gene Expression Reveals Loci That Control Ground State Pluripotency.

Mouse embryonic stem cells (mESCs) cultured in the presence of LIF occupy a ground state with highly active pluripotency-associated transcriptional and epigenetic circuitry. However, ground state pluripotency in some inbred strain backgrounds is unstable in the absence of ERK1/2 and GSK3 inhibition. Using an unbiased genetic approach, we dissect the basis of this divergent response to extracellular cues by profiling gene expression and chromatin accessibility in 170 genetically heterogeneous mESCs. We map thousands of loci affecting chromatin accessibility and/or transcript abundance, including 10 QTL hotspots where genetic variation at a single locus coordinates the regulation of genes throughout the genome. For one hotspot, we identify a single enhancer variant ∼10 kb upstream of Lifr associated with chromatin accessibility and mediating a cascade of molecular events affecting pluripotency. We validate causation through reciprocal allele swaps, demonstrating the functional consequences of noncoding variation in gene regulatory networks that stabilize pluripotent states invitro.

Abstract 3439: FOXA1 mutations reveal distinct chromatin profiles and influence therapeutic response in breast cancer

Abstract FOXA1 is an evolutionary conserved pioneer factor that binds to condensed chromatin allowing the recruitment of other transcription factors to the DNA. Although mutations in FOXA1 are a hallmark of estrogen receptor-positive (ER+) breast cancers, it is currently unknown whether and how these mutations affect breast cancer development and response to endocrine therapies. In this work, we studied how the genome-wide chromatin recruitment, accessibility and transcriptional outcomes of recurrent FOXA1 mutations can affect therapeutic response in ER+ breast cancer. By examining the landscape of FOXA1 mutations in a cohort of 4,952 breast cancer patients, we identified several hotspot mutations, some of them present also in other malignancies and some others specific to breast cancer. In particular, we characterized three mutations in the Wing2 region and a breast-cancer specific third β strand mutation, namely SY242CS. We also showed that FOXA1 mutations are enriched in metastatic tumors and mutually exclusive with ESR1 mutations, well-known drivers of resistance to endocrine therapy. Using a clinico-genomicaly curated cohort of patients, together with in vitro and in vivo breast models, we associated FOXA1 missense mutations with a lower response to endocrine therapy. Mechanistically, by means of ChIP-seq, ATAC-seq and RNA-seq analyses, we found that FOXA1 mutations in the Wing2 loop display increased chromatin binding affinity at ER loci upon estrogen stimulation, and an enhanced ER-mediated transcription without changes in chromatin accessibility, decoupling FOXA1 Wing2 mutant binding from their pioneering function. These data correlated with the highly organized 3D conformation conferred by FOXA1 Wing2 mutations we predicted by structural modeling. Thus. the enhanced chromatin binding affinity gained by FOXA1 Wing2 mutations might be a mechanism to sustain active estrogen response even in the presence of therapeutic pressure. In contrast, breast specific SY242CS mutant shows neomorphic properties including the ability to open novel chromatin regions, and activate an alternative cistrome and transcriptome. Using an engineered luciferase reporter system, we validated that a non-canonical motif, shared by both gained accessibility and binding sites, is SY242CS-specific. Structural modeling of the binding of WT or SY242CS FOXA1 to this new motif revealed that SY242CS undergoes conformational change that results in a tight interaction with the new DNA motif, not observed in WT FOXA1. Taken together, our findings provide mechanistic insights into how FOXA1 mutations modulate its function in breast cancer to dictate malignant progression and response to endocrine therapy. More broadly, these results position FOXA1 mutations as potential biomarkers of response and potential targets for the treatment of metastatic ER+ breast cancer. Citation Format: Amaia Arruabarrena-Aristorena, Jesper LV Maag, Srushti Kittane, Yanyan Cai, Jane Park, Pedram Razavi, Lorenzo Ferrando, Pier Selenica, Wouter R. Karthaus, Srinivasaraghavan Kannan, Emiliano Cocco, Sik Y. Ho, Daisylyn Senna Tan, Mirna Sallaku, Charles L. Sawyers, Jorge S. Reis-Fihlo, Chandra S. Verma, Ralf Jauch, Richard Koche, José Baselga, Eneda Toska, Maurizio Scaltriti. FOXA1 mutations reveal distinct chromatin profiles and influence therapeutic response in breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3439.

Cumulative contact frequency of a chromatin region is an intrinsic property linked to its function.

Regulation of gene transcription is a complex process controlled by many factors, including the conformation of chromatin in the nucleus. Insights into chromatin conformation on both local and global scales can be provided by the Hi-C (high-throughput chromosomes conformation capture) method. One of the drawbacks of Hi-C analysis and interpretation is the presence of systematic biases, such as different accessibility to enzymes, amplification, and mappability of DNA regions, which all result in different visibility of the regions. Iterative correction (IC) is one of the most popular techniques developed for the elimination of these systematic biases. IC is based on the assumption that all chromatin regions have an equal number of observed contacts in Hi-C. In other words, the IC procedure is equalizing the experimental visibility approximated by the cumulative contact frequency (CCF) for all genomic regions. However, the differences in experimental visibility might be explained by biological factors such as chromatin openness, which is characteristic of distinct chromatin states. Here we show that CCF is positively correlated with active transcription. It is associated with compartment organization, since compartment A demonstrates higher CCF and gene expression levels than compartment B. Notably, this observation holds for a wide range of species, including human, mouse, and Drosophila. Moreover, we track the CCF state for syntenic blocks between human and mouse and conclude that active state assessed by CCF is an intrinsic property of the DNA region, which is independent of local genomic and epigenomic context. Our findings establish a missing link between Hi-C normalization procedures removing CCF from the data and poorly investigated and possibly relevant biological factors contributing to CCF.

Robust gene expression programs underlie recurrent cell states and phenotype switching in melanoma.

Melanoma cells can switch between a melanocytic and a mesenchymal-like state. Scattered evidence indicates that additional intermediate state(s) may exist. Here, to search for such states and decipher their underlying gene regulatory network (GRN), we studied 10 melanoma cultures using single-cell RNA sequencing (RNA-seq) as well as 26 additional cultures using bulk RNA-seq. Although each culture exhibited a unique transcriptome, we identified shared GRNs that underlie the extreme melanocytic and mesenchymal states and the intermediate state. This intermediate state is corroborated by a distinct chromatin landscape and is governed by the transcription factors SOX6, NFATC2, EGR3, ELF1 and ETV4. Single-cell migration assays confirmed the intermediate migratory phenotype of this state. Using time-series sampling of single cells after knockdown of SOX10, we unravelled the sequential and recurrent arrangement of GRNs during phenotype switching. Taken together, these analyses indicate that an intermediate state exists and is driven by a distinct and stable 'mixed' GRN rather than being a symbiotic heterogeneous mix of cells.

AMPK Enhances Transcription of Selected Nrf2 Target Genes via Negative Regulation of Bach1.

5′-AMP-activated protein kinase (AMPK) and the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) are main players in the cellular adaptive response to metabolic and oxidative/xenobiotic stress, respectively. AMPK does not only balance the rate of fuel catabolism versus anabolism but also emerges as regulator of gene expression. We here examined the influence of AMPK on Nrf2-dependent gene transcription and the potential interplay of the two cellular stress hubs. Using gene expression analyses in wt and AMPKα1 −/− or Nrf2 −/− mouse embryonal fibroblasts, we could show that AMPK only affected a portion of the entire of Nrf2-dependent transcriptome upon exposure to the Nrf2 activator sulforaphane (Sfn). Focusing on selected genes with positive regulation by Nrf2 and either positive or no further regulation by AMPK, we revealed that altered Nrf2 levels could not account for the distinct extent of transactivation of certain Nrf2 targets in wt and AMPK −/− cells (assessed by immunoblot). FAIRE-qPCR largely excluded distinct chromatin accessibility of selected Nrf2-responsive antioxidant response elements (ARE) within the regulatory gene regions in wt and AMPK−/− cells. However, expression analyses and ChIP-qPCR showed that in AMPK−/− cells, levels of BTB and CNC homology 1 (Bach1), a competitor of Nrf2 for ARE sites with predominant repressor function, were higher, and Bach1 also bound to a greater relative extent to the examined ARE sites when compared to Nrf2. The negative influence of AMPK on Bach1 was confirmed by pharmacological and genetic approaches and occurred at the level of mRNA synthesis. Overall, the observed AMPK-mediated boost in transactivation of a subset of Nrf2 target genes involves downregulation of Bach1 and subsequent favored binding of activating Nrf2 over repressing Bach1 to the examined ARE sites.

Inhibition of Glycolysis in Pathogenic TH17 Cells through Targeting a miR -21-Peli1-c-Rel Pathway Prevents Autoimmunity.

It is well known that some pathogenic cells have enhanced glycolysis; the regulatory network leading to increased glycolysis are not well characterized. In this study, we show that CNS-infiltrated pathogenic TH17 cells from diseased mice specifically upregulate glycolytic pathway genes compared with homeostatic intestinal TH17 cells. Bioenergetic assay and metabolomics analyses indicate that in vitro-derived pathogenic TH17 cells are highly glycolytic compared with nonpathogenic TH17 cells. Chromatin landscape analyses demonstrate TH17 cells in vivo that show distinct chromatin states, and pathogenic TH17 cells show enhanced chromatin accessibility at glycolytic genes with NF-κB binding sites. Mechanistic studies reveal that miR-21 targets the E3 ubiquitin ligase Peli1-c-Rel pathway to promote glucose metabolism of pathogenic TH17 cells. Therapeutic targeting c-Rel-mediated glycolysis in pathogenic TH17 cells represses autoimmune diseases. These findings extend our understanding of the regulation TH17 cell glycolysis in vivo and provide insights for future therapeutic intervention to TH17 cell-mediated autoimmune diseases.

Characterization of histone modification patterns and prediction of novel promoters using functional principal component analysis.

Characterization of distinct histone methylation and acetylation binding patterns in promoters and prediction of novel regulatory regions remains an important area of genomic research, as it is hypothesized that distinct chromatin signatures may specify unique genomic functions. However, methods that have been proposed in the literature are either descriptive in nature or are fully parametric and hence more restrictive in pattern discovery. In this article, we propose a two-step non-parametric statistical inference procedure to characterize unique histone modification patterns and apply it to analyzing the binding patterns of four histone marks, H3K4me2, H3K4me3, H3K9ac, and H4K20me1, in human B-lymphoblastoid cells. In the first step, we used a functional principal component analysis method to represent the concatenated binding patterns of these four histone marks around the transcription start sites as smooth curves. In the second step, we clustered these curves to reveal several unique classes of binding patterns. These uncovered patterns were used in turn to scan the whole-genome to predict novel and alternative promoters. Our analyses show that there are three distinct promoter binding patterns of active genes. Further, 19654 regions not within known gene promoters were found to overlap with human ESTs, CpG islands, or common SNPs, indicative of their potential role in gene regulation, including being potential novel promoter regions.

A Chromodomain-Helicase-DNA-Binding Factor Functions in Chromatin Modification and Gene Regulation.

Proteins in the Chromodomain-Helicase/ATPase-DNA-binding domain (CHD) family are divided into three groups. The function of group I CHD proteins in nucleosome positioning is well established, while that of group II members (represented by CHD3/Mi2) remains unclear. Using high-throughput approaches, we investigated the function of the group II rice (Oryza sativa) CHD protein CHR729 in nucleosome positioning, gene expression, histone methylation, and binding. Our data revealed that the chr729 mutation led to increased nucleosome occupancy in the rice genome and altered the expression and histone H3K4me3 modification of many, mainly underexpressed, genes. Further analysis showed that the mutation affected both the deposition and depletion of H3K4me3 in distinct chromatin regions, with concomitant changes in H3K27me3 modification. Genetic and genomic analyses revealed that CHR729 and JMJ703, an H3K4 demethylase, had agonistic, antagonistic, and independent functions in modulating H3K4me3 and the expression of subsets of genes. In addition, CHR729 binding was enriched in H3K4me3-marked genic and H3K27me3-marked intergenic regions. The results indicate that CHR729 has distinct functions in regulating H3K4me3 and H3K27me3 modifications and gene expression at different chromatin domains and provide insight into chromatin regulation of bivalent genes marked by both H3K4me3 and H3K27me3.

Epigenetic Regulation of Kaposi's Sarcoma-Associated Herpesvirus Latency.

Kaposi’s sarcoma-associated herpesvirus (KSHV) is an oncogenic γ-herpesvirus that infects humans and exhibits a biphasic life cycle consisting of latent and lytic phases. Following entry into host cells, the KSHV genome undergoes circularization and chromatinization into an extrachromosomal episome ultimately leading to the establishment of latency. The KSHV episome is organized into distinct chromatin domains marked by variations in repressive or activating epigenetic modifications, including DNA methylation, histone methylation, and histone acetylation. Thus, the development of KSHV latency is believed to be governed by epigenetic regulation. In the past decade, interrogation of the KSHV epitome by genome-wide approaches has revealed a complex epigenetic mark landscape across KSHV genome and has uncovered the important regulatory roles of epigenetic modifications in governing the development of KSHV latency. Here, we highlight many of the findings regarding the role of DNA methylation, histone modification, post-translational modification (PTM) of chromatin remodeling proteins, the contribution of long non-coding RNAs (lncRNAs) in regulating KSHV latency development, and the role of higher-order episomal chromatin architecture in the maintenance of latency and the latent-to-lytic switch.

Sperm Originated Chromatin Imprints and LincRNAs in Organismal Development and Cancer.

SummaryImportance of sperm-derived transcripts and chromatin imprints in organismal development is poorly investigated. Here using an integrative approach, we show that human sperm transcripts are equally important as oocyte. Sperm-specific and sperm-oocyte common transcripts carry distinct chromatin structures at their promoters correlating with corresponding transcript levels in sperm. Interestingly, sperm-specific H3K4me3 patterns at the lincRNA promoters are not maintained in the germ layers and somatic tissues. However, bivalent chromatin at the sperm-specific protein-coding gene promoters is maintained throughout the development. Sperm-specific transcripts reach their peak expression during zygotic genome activation, whereas sperm-oocyte common transcripts are present during early preimplantation development but decline at the onset of zygotic genome activation. Additionally, there is an inverse correlation between sperm-specific and sperm-oocyte lincRNAs throughout the development. Sperm-lincRNAs also show aberrant activation in tumors. Overall, our observations indicate that sperm transcripts carrying chromatin imprints may play an important role in human development and cancer.