Histone Modifications H3K4me3 Research Articles

Allele-specific binding variants causing ChIP-seq peak height of histone modification are not enriched in expression QTL annotations

BackgroundGenome sequence variants affecting complex traits (quantitative trait loci, QTL) are enriched in functional regions of the genome, such as those marked by certain histone modifications. These variants are believed to influence gene expression. However, due to the linkage disequilibrium among nearby variants, pinpointing the precise location of QTL is challenging. We aimed to identify allele-specific binding (ASB) QTL (asbQTL) that cause variation in the level of histone modification, as measured by the height of peaks assayed by ChIP-seq (chromatin immunoprecipitation sequencing). We identified DNA sequences that predict the difference between alleles in ChIP-seq peak height in H3K4me3 and H3K27ac histone modifications in the mammary glands of cows.ResultsWe used a gapped k-mer support vector machine, a novel best linear unbiased prediction model, and a multiple linear regression model that combines the other two approaches to predict variant impacts on peak height. For each method, a subset of 1000 sites with the highest magnitude of predicted ASB was considered as candidate asbQTL. The accuracy of this prediction was measured by the proportion where the predicted direction matched the observed direction. Prediction accuracy ranged between 0.59 and 0.74, suggesting that these 1000 sites are enriched for asbQTL. Using independent data, we investigated functional enrichment in the candidate asbQTL set and three control groups, including non-causal ASB sites, non-ASB variants under a peak, and SNPs (single nucleotide polymorphisms) not under a peak. For H3K4me3, a higher proportion of the candidate asbQTL were confirmed as ASB when compared to the non-causal ASB sites (P < 0.01). However, these candidate asbQTL did not enrich for the other annotations, including expression QTL (eQTL), allele-specific expression QTL (aseQTL) and sites conserved across mammals (P > 0.05).ConclusionsWe identified putatively causal sites for asbQTL using the DNA sequence surrounding these sites. Our results suggest that many sites influencing histone modifications may not directly affect gene expression. However, it is important to acknowledge that distinguishing between putative causal ASB sites and other non-causal ASB sites in high linkage disequilibrium with the causal sites regarding their impact on gene expression may be challenging due to limitations in statistical power.

EZH2 inhibition reduces macrophage inflammatory responses in atherosclerosis

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): Dutch Heart Foundation (Hartstichting) Aim Epigenetic processes are essential modulators of macrophage inflammatory responses. We postulate that interference in the epigenetic machinery of macrophages might offer novel approaches to combat atherosclerosis. Here, we investigate the repressive histone modification H3K27Me3 deposited by the polycomb repressive complex 2 (PRC2) with its catalytic component EZH2. We studied the therapeutic potential of macrophage EZH2 inhibition in the context of atherosclerosis. Methods Human monocyte-derived macrophages and murine peritoneal and bone-marrow derived macrophages were treated with the EZH2-specific inhibitor GSK126 and subsequently activated with LPS to mimic TLR4-inflammatory responses. The impact of Ezh2 inhibition on macrophage differentiation and activation compared to vehicle (DMSO) was assessed by RNA-seq, ChIP-seq, flow cytometry, western blot, and ELISA. To study its impact on atherosclerosis, female Ldlr-/- mice on a high-cholesterol diet (9weeks) were treated the last four weeks with GSK126 or control. Results Ezh2 inhibition by GSK126 in vitro lowered global H3K27Me3 levels without altering macrophage viability and differentiation, showcasing effective EZH2 inhibition. RNA-seq revealed that of more than one-third of the LPS-induced genes were significantly downregulated by EZH2 inhibition. Subsequent pathway analysis identified cytokine and interferon signaling, co-stimulation, and cell migration as the top down-regulated pathways (padj&amp;lt;0.05). Furthermore, ChIP-seq revealed considerably altered regulation of H3K27Me3 deposition. Indeed, we confirmed that gene and cytokine expression of the inflammatory mediators IL-6, IL-12, and TNF were reduced. Furthermore, membrane marker expression of co-stimulatory CD40, CD80, and CD86 were significantly decreased. In murine atherosclerosis, we observed a reduction in the Virmani plaque severity score and are currently assessing lesion size and composition. Conclusion Overall, we show that EZH2 inhibition reduces inflammatory responses in human and murine macrophages in vitro. We are currently assessing the impact of EZH2 inhibition on lesion size and composition in murine atherosclerosis. Additionally, we are performing ex vivo experiments on human endarterectomy plaques to assess the therapeutic potential of EZH2 inhibition on human atherosclerosis.

Unraveling genetic underpinnings of purine content in pork

The significance of purine base content as an important nutrition indicator in foods arises from its potential to trigger hyperuricemia or gout via high-purine diet. Livestock meats, including pork, generally contain moderate to high total purine content (TP). Recent research revealed substantial variations within and across pig breeds, implying genetic factors influencing this trait. Thus, this study aimed to unravel the genetic underpinnings governing purine base content in pork. The heritability estimates (h2) for the four purine traits ranged from 0.14 to 0.35. A total of 14, 36, 19 and 25 quantitative trait loci (QTLs) were identified for guanine, adenine, hypoxanthine, and TP, respectively. Our comprehensive gene set enrichment analysis and gene network analysis revealed 15 promising candidate genes intricately interwoven within diverse purine metabolism pathways, such as purine ribonucleotide metabolic process, purine nucleotide metabolism and transport, and purine salvage pathways, all contributing to TP. Strikingly, most genetic variants significantly associated with TP displayed analogous effects on multiple purine bases. Two distinct and highly significant QTLs (P < 10-12) emerged on Sus Scrofa chromosome (SSC) 12: one impacting guanine content and the other concurrently influencing adenine and hypoxanthine levels. The peak of the guanine QTL on SSC12 resided 1.1 kb downstream of the transmembrane protein 238 like (TMEM238L) gene and is encapsulated within a genomic segment characterized by the histone modification H3K27me3. Focused fine-mapping for the SSC12 QTL associated with adenine and hypoxanthine levels narrowed its scope to around 172 kb, encompassing the growth arrest specific 7 (GAS7) and myosin heavy chain 13 (MYH13) genes. However, the observed QTL effect was not attributed to any missense mutations within the two genes. This pioneering study unveils the genetic variations and candidate genes associated with purine content in livestock, laying a robust foundation for the selective breeding of pig lines with reduced purine base content.

Dissecting gene activation and chromatin remodeling dynamics in single human cells undergoing reprogramming

During cell fate transitions, cells remodel their transcriptome, chromatin, and epigenome; however, it has been difficult to determine the temporal dynamics and cause-effect relationship between these changes at the single-cell level. Here, we employ the heterokaryon-mediated reprogramming system as a single-cell model to dissect key temporal events during early stages of pluripotency conversion using super-resolution imaging. We reveal that, following heterokaryon formation, the somatic nucleus undergoes global chromatin decompaction and removal of repressive histone modifications H3K9me3 and H3K27me3 without acquisition of active modifications H3K4me3 and H3K9ac. The pluripotency gene OCT4 (POU5F1) shows nascent and mature RNA transcription within the first 24h after cell fusion without requiring an initial open chromatin configuration at its locus. NANOG, conversely, has significant nascent RNA transcription only at 48h after cell fusion but, strikingly, exhibits genomic reopening early on. These findings suggest that the temporal relationship between chromatin compaction and gene activation during cellular reprogramming is gene context dependent.

Extensive long-range polycomb interactions and weak compartmentalization are hallmarks of human neuronal 3D genome.

Chromatin architecture regulates gene expression and shapes cellular identity, particularly in neuronal cells. Specifically, polycomb group (PcG) proteins enable establishment and maintenance of neuronal cell type by reorganizing chromatin into repressive domains that limit the expression of fate-determining genes and sustain distinct gene expression patterns in neurons. Here, we map the 3D genome architecture in neuronal and non-neuronal cells isolated from the Wernicke's area of four human brains and comprehensively analyze neuron-specific aspects of chromatin organization. We find that genome segregation into active and inactive compartments is greatly reduced in neurons compared to other brain cells. Furthermore, neuronal Hi-C maps reveal strong long-range interactions, forming a specific network of PcG-mediated contacts in neurons that is nearly absent in other brain cells. These interacting loci contain developmental transcription factors with repressed expression in neurons and other mature brain cells. But only in neurons, they are rich in bivalent promoters occupied by H3K4me3 histone modification together with H3K27me3, which points to a possible functional role of PcG contacts in neurons. Importantly, other layers of chromatin organization also exhibit a distinct structure in neurons, characterized by an increase in short-range interactions and a decrease in long-range ones.

LKRSDH-dependent histone modifications of insulin-like peptide sites contribute to age-related circadian rhythm changes.

To understand aging impact on the circadian rhythm, we screened for factors influencing circadian changes during aging. Our findings reveal that LKRSDH mutation significantly reduces rhythmicity in aged flies. RNA-seq identifies a significant increase in insulin-like peptides (dilps) in LKRSDH mutants due to the combined effects of H3R17me2 and H3K27me3 on transcription. Genetic evidence suggests that LKRSDH regulates age-related circadian rhythm changes through art4 and dilps. ChIP-seq analyzes whole genome changes in H3R17me2 and H3K27me3 histone modifications in young and old flies with LKRSDH mutation and controls. The results reveal a correlation between H3R17me2 and H3K27me3, underscoring the role of LKRSDH in regulating gene expression and modification levels during aging. Overall, our study demonstrates that LKRSDH-dependent histone modifications at dilps sites contribute to age-related circadian rhythm changes. This data offers insights and a foundational reference for aging research by unveiling the relationship between LKRSDH and H3R17me2/H3K27me3 histone modifications in aging.

Transcriptionally active chromatin loops contain both ‘active’ and ‘inactive’ histone modifications that exhibit exclusivity at the level of nucleosome clusters

Chromatin state is thought to impart regulatory function to the underlying DNA sequence. This can be established through histone modifications and chromatin organisation, but exactly how these factors relate to one another to regulate gene expression is unclear. In this study, we have used super-resolution microscopy to image the Y loops of Drosophila melanogaster primary spermatocytes, which are enormous transcriptionally active chromatin fibres, each representing single transcription units that are individually resolvable in the nuclear interior. We previously found that the Y loops consist of regular clusters of nucleosomes, with an estimated median of 54 nucleosomes per cluster with wide variation.In this study, we report that the histone modifications H3K4me3, H3K27me3, and H3K36me3 are also clustered along the Y loops, with H3K4me3 more associated with diffuse chromatin compared to H3K27me3. These histone modifications form domains that can be stretches of Y loop chromatin micrometres long, or can be in short alternating domains. The different histone modifications are associated with different sizes of chromatin clusters and unique morphologies. Strikingly, a single chromatin cluster almost always only contains only one type of the histone modifications that were labelled, suggesting exclusivity, and therefore regulation at the level of individual chromatin clusters. The active mark H3K36me3 is more associated with actively elongating RNA polymerase II than H3K27me3, with polymerase often appearing on what are assumed to be looping regions on the periphery of chromatin clusters.These results provide a foundation for understanding the relationship between chromatin state, chromatin organisation, and transcription regulation – with potential implications for pause-release dynamics, splicing complex organisation and chromatin dynamics during polymerase progression along a gene.

Abstract 6146: Characterizing underlying molecular mechanisms of prostate cancer risk variants identified from multi-ethnic GWAS

Abstract Prostate cancer (PCa) is a complex disease with the second leading cause of cancer death in US males. Multi-ethnic genome-wide association studies (GWAS) identified a thousand genetic variants associated with PCa risk in different races. However, the majority of risk variants are located in non-coding regions, making it challenging to understand their contribution to PCa risk. Non-coding regions include functional regulatory elements (REs) such as promoters and enhancers, whose activity can be evaluated by histone modification H3K4me3 and H3K27ac, respectively. In this study, we integrated PCa risk variants with hundreds of H3K4me3 and H3K27ac ChIP-seq data from normal and cancer prostate cell lines and tissues, finding 122 variants in promoters and 301 variants in enhancers. Enhancers are distal to promoters, and they form loops to regulate gene expression, mediated by transcription factors (TFs). Enhancer activities are tightly associated with cell fate. Genetic variants can affect the activity of enhancers by changing the binding preference of TFs and such changes can affect chromatin interactions and dysregulate gene expression. We hypothesize that allele changes of enhancer variants contribute to PCa risk differently among individuals from different ancestries, leading to the development of heterogeneous PCa molecular subgroups, characterized by genetic alterations including TMPRSS2-ERG fusion. By integrating H3K27ac ChIP-seq data from prostate cell lines derived from European (EUR) and African (AFR) ancestries, we found 185 risk variants in enhancers in EUR cell lines and 69 risk variants in enhancers in AFR cell lines. Next, we integrated patient tissue-derived H3K27ac ChIP-seq data and performed chromatin quantitative trait loci (cQTL) and allelic imbalance (AI) analyses to determine risk variants associated with enhancer activity. Among the 185 risk enhancer variants in EUR and 69 in AFR cell lines, we found 130 and 46 with significant association, respectively. To determine TFs that change binding upon allele change of enhancer risk variants, we integrated hundreds of TF ChIP-seq data from prostate cell lines and tissues. Among the 185 risk enhancer variants in EUR and 69 in AFR cell lines, we found 142 and 44 variants bound by TFs, respectively. When we further performed cQTL and AI analyses for FOXA1, HOXB13, and AR, we found 87 and 29 variants significantly associated with TF binding affinity, respectively. Moreover, we found that these associations differ between TMPRSS2-ERG fusion positive and negative tumors. To identify the target genes of enhancer risk variants, we are currently integrating chromatin interaction data including Hi-C and Micro-C. Overall, this study provides valuable insights into the underlying molecular mechanisms of risk variants in various populations and individuals which lead to the development of heterogeneous PCa subgroups. Citation Format: Zexun Wu, Andrew Vu, Ethan S. Nelson-Moore, Huan Cao, Suhn K. Rhie. Characterizing underlying molecular mechanisms of prostate cancer risk variants identified from multi-ethnic GWAS [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6146.

Abstract 561: Targeting Y box binding protein 1 (YBX1) in MYC-amplified medulloblastoma

Abstract Medulloblastoma is the most common malignant pediatric brain tumor. Patients diagnosed with MYC-amplified group 3 medulloblastoma (G3 MB) have a devastating prognosis with a 5-year survival rate of &amp;lt;50%. Standard of care (SOC) consists of maximal resection surgery followed by chemoradiotherapy, but these extensive treatments leave patients with neurocognitive and developmental deficits. Therefore, the identification of novel targets that can be used alone or in combination with SOC is of utmost importance. Using a batch-normalized bulk RNA-seq dataset (GSE124814) comprised of 1350 MB samples and 291 normal cerebella, we reveal upregulation of Y-box binding protein 1 (YBX1) in MB compared to normal cerebellum. Further analysis of this dataset and others demonstrates G3 tumors that display high MYC levels also have the highest YBX1 expression. Analysis of ChIP-seq data shows strong binding of MYC to the YBX1 promoter in G3 MB, concurrent with H3K27ac and H3K4me3 histone modifications. Using a well-established ex vivo model, we demonstrate increased YBX1 expression as normal cerebellar progenitor cells transition to tumorigenic G3 MB stem-like cells. Recent work in leukemia reveals a reciprocal regulation of MYC by YBX1, however, this mechanism is not well established in G3 MB. We utilized an orthogonal pharmacogenetic approach to inhibit YBX1 function and determine its effects on G3 MB tumor biology. Suppression of YBX1 significantly reduced the proliferation and decreased MYC protein expression of G3 MB cells in vitro. These findings support a reciprocal MYC-YBX1 axis, which could influence tumor growth in vivo. Orthotopic implantation of YBX1 KO G3 MB cells leads to increased animal survival. We next evaluated the therapeutic potential of two recently characterized YBX1 inhibitors which display single agent efficacy in vitro and in vivo. YBX1 inhibitors synergized with SOC in vitro lowering the IC50 for both chemo and radiotherapy, which could reduce adverse effects experienced by patients. In silico molecular docking using PyMOL predicts YBX1 inhibitors interact with amino acids within the cold shock domain of YBX1, likely impairing its ability to bind RNA. We performed formaldehyde RNA immunoprecipitation (fRIP)-sequencing to identify changes in RNA transcript levels following YBX1 pharmacological inhibition. Disruption of YBX1-RNA binding impacts the stability of known oncogenic transcripts which may be required for tumorigenesis. Our findings impart an alluring prospect of targeting YBX1 in combination with standard-of-care, and possibly other compounds to improve patients’ outcomes. Citation Format: Stephen Carney, John Hemenway, Ashmitha Rajendran, Matt Hathaway, Eric Nealy, Christian Bonatto, Dhanir Tailor, Sanjay Malhotra, Myron Evans. Targeting Y box binding protein 1 (YBX1) in MYC-amplified medulloblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 561.

Chromatin profiling reveals TFAP4 as a critical transcriptional regulator of bovine satellite cell differentiation

BackgroundSatellite cells are myogenic precursor cells in adult skeletal muscle and play a crucial role in skeletal muscle regeneration, maintenance, and growth. Like embryonic myoblasts, satellite cells have the ability to proliferate, differentiate, and fuse to form multinucleated myofibers. In this study, we aimed to identify additional transcription factors that control gene expression during bovine satellite cell proliferation and differentiation.ResultsUsing chromatin immunoprecipitation followed by sequencing, we identified 56,973 and 54,470 genomic regions marked with both the histone modifications H3K4me1 and H3K27ac, which were considered active enhancers, and 50,956 and 59,174 genomic regions marked with H3K27me3, which were considered repressed enhancers, in proliferating and differentiating bovine satellite cells, respectively. In addition, we identified 1,216 and 1,171 super-enhancers in proliferating and differentiating bovine satellite cells, respectively. Analyzing these enhancers showed that in proliferating bovine satellite cells, active enhancers were associated with genes stimulating cell proliferation or inhibiting myoblast differentiation whereas repressed enhancers were associated with genes essential for myoblast differentiation, and that in differentiating satellite cells, active enhancers were associated with genes essential for myoblast differentiation or muscle contraction whereas repressed enhancers were associated with genes stimulating cell proliferation or inhibiting myoblast differentiation. Active enhancers in proliferating bovine satellite cells were enriched with binding sites for many transcription factors such as MYF5 and the AP-1 family transcription factors; active enhancers in differentiating bovine satellite cells were enriched with binding sites for many transcription factors such as MYOG and TFAP4; and repressed enhancers in both proliferating and differentiating bovine satellite cells were enriched with binding sites for NF-kB, ZEB-1, and several other transcription factors. The role of TFAP4 in satellite cell or myoblast differentiation was previously unknown, and through gene knockdown and overexpression, we experimentally validated a critical role for TFAP4 in the differentiation and fusion of bovine satellite cells into myofibers.ConclusionsSatellite cell proliferation and differentiation are controlled by many transcription factors such as AP-1, TFAP4, NF-kB, and ZEB-1 whose roles in these processes were previously unknown in addition to those transcription factors such as MYF5 and MYOG whose roles in these processes are widely known.

Three-dimensional chromatin analysis reveals Sp1 as a mediator to program and reprogram HPV-host epigenetic architecture in cervical cancer

Human papillomavirus (HPV) is predominantly associated with HPV-related cancers, however, the precise mechanisms underlying the HPV-host epigenetic architectures in HPV carcinogenesis remain elusive. Here, we employed high-throughput chromosome conformation capture (Hi-C) to comprehensively map HPV16/18-host chromatin interactions. Our study identified the transcription factor Sp1 as a pivotal mediator in programming HPV-host interactions. By targeting Sp1, the active histone modifications (H3K27ac, H3K4me1, and H3K4me3) and the HPV-host chromatin interactions are reprogrammed, which leads to the downregulation of oncogenes located near the integration sites in both HPV (E6/E7) and the host genome (KLF5/MYC). Additionally, Sp1 inhibition led to the upregulation of immune checkpoint genes by reprogramming histone modifications in host cells. Notably, humanized patient-derived xenograft (PDX-HuHSC-NSG) models demonstrated that Sp1 inhibition promoted anti-PD-1 immunotherapy via remodeling the tumor immune microenvironment in cervical cancer. Moreover, single-cell transcriptomic analysis validated the enrichment of transcription factor Sp1 in epithelial cells of cervical cancer. In summary, our findings elucidate Sp1 as a key mediator involved in the programming and reprogramming of HPV-host epigenetic architecture. Inhibiting Sp1 with plicamycin may represent a promising therapeutic option for HPV-related carcinoma.

Integrative epigenome-transcriptome analysis unravels cancer-specific over-expressed genes potentially regulating immune microenvironment in clear cell renal cell carcinoma

BackgroundClear cell Renal Cell Carcinoma (ccRCC) is the frequently diagnosed histological life-threatening tumor subtype in the urinary system. Integrating multi-omics data is emerging as a tool to provide a comprehensive view of biology and disease for better therapeutic interventions. MethodWe have integrated freely available ccRCC data sets of genome-wide DNA methylome, transcriptome, and active histone modification marks, H3K27ac, H3K4me1, and H3K4me3 specific ChIP-seq data to screen genes with higher expression. Further, these genes were filtered based on their effect on survival upon alteration in expression. ResultsThe six multi-omics-based identified genes, RUNX1, MSC, ADA, TREML1, TGFA, and VWF, showed higher expression with enrichment of active histone marks and hypomethylated CpG in ccRCC. In continuation, the identified genes were validated by an independent dataset and showed a correlation with nodal and metastatic status. Furthermore, gene ontology and pathway analysis revealed that immune-related pathways are activated in ccRCC patients. ConclusionsThe network analysis of six overexpressed genes suggests their potential role in an immunosuppressive environment, leading to tumor progression and poor prognosis. Our study shows that the multi-omics approach helps unravel complex biology for patient subtyping and proposes combination strategies with epi-drugs for more precise immunotherapy in ccRCC.

Histone modification-dependent production of peptide hormones facilitates acquisition of pluripotency during leaf-to-callus transition in Arabidopsis.

Chromatin configuration is critical for establishing tissue identity and changes substantially during tissue identity transitions. The crucial scientific and agricultural technology of in vitro tissue culture exploits callus formation from diverse tissue explants and tissue regeneration via de novo organogenesis. We investigated the dynamic changes in H3ac and H3K4me3 histone modifications during leaf-to-callus transition in Arabidopsis thaliana. We analyzed changes in the global distribution of H3ac and H3K4me3 during the leaf-to-callus transition, focusing on transcriptionally active regions in calli relative to leaf explants, defined by increased accumulation of both H3ac and H3K4me3. Peptide signaling was particularly activated during callus formation; the peptide hormones RGF3, RGF8, PIP1 and PIPL3 were upregulated, promoting callus proliferation and conferring competence for de novo shoot organogenesis. The corresponding peptide receptors were also implicated in peptide-regulated callus proliferation and regeneration capacity. The effect of peptide hormones in plant regeneration is likely at least partly conserved in crop plants. Our results indicate that chromatin-dependent regulation of peptide hormone production not only stimulates callus proliferation but also establishes pluripotency, improving the overall efficiency of two-step regeneration in plant systems.

Regulation of effector gene expression as concerted waves in Leptosphaeria maculans: a two-player game.

Effector genes, encoding molecules involved in disease establishment, are concertedly expressed throughout the lifecycle of plant-pathogenic fungi. However, little is known about how effector gene expression is regulated. Since many effector genes are located in repeat-rich regions, the role of chromatin remodeling in their regulation was recently investigated, notably establishing that the repressive histone modification H3K9me3, deposited by KMT1, was involved in several fungal species including Leptosphaeria maculans. Nevertheless, previous data suggest that a second regulatory layer, probably involving a specific transcription factor (TF), might be required. In L. maculans, a Dothideomycete causing stem canker of oilseed rape, we identified the ortholog of Pf2, a TF belonging to the Zn2Cys6 fungal-specific family, and described as essential for pathogenicity and effector gene expression. We investigated its role together with KMT1, by inactivating and over-expressing LmPf2 in a wild-type strain and a ∆kmt1 mutant. Functional analyses of the corresponding transformants highlighted an essential role of LmPf2 in the establishment of pathogenesis and we found a major effect of LmPf2 on the induction of effector gene expression once KMT1 repression is lifted. Our results show, for the first time, a dual control of effector gene expression.

Leveraging chromatin state transitions for the identification of regulatory networks orchestrating heart regeneration.

The limited regenerative capacity of the human heart contributes to high morbidity and mortality worldwide. In contrast, zebrafish exhibit robust regenerative capacity, providing a powerful model for studying how to overcome intrinsic epigenetic barriers maintaining cardiac homeostasis and initiate regeneration. Here, we present a comprehensive analysis of the histone modifications H3K4me1, H3K4me3, H3K27me3and H3K27ac during various stages of zebrafish heart regeneration. We found a vast gain of repressive chromatin marks one day after myocardial injury, followed by the acquisition of active chromatin characteristics on day four and a transition to a repressive state on day 14, and identified distinct transcription factor ensembles associated with these events. The rapid transcriptional response involves the engagement of super-enhancers at genes implicated in extracellular matrix reorganization and TOR signaling, while H3K4me3 breadth highly correlates with transcriptional activity and dynamic changes at genes involved in proteolysis, cell cycle activity, and cell differentiation. Using loss- and gain-of-function approaches, we identified transcription factors in cardiomyocytes and endothelial cells influencing cardiomyocyte dedifferentiation or proliferation. Finally, we detected significant evolutionary conservation between regulatory regions that drive zebrafish and neonatal mouse heart regeneration, suggesting that reactivating transcriptional and epigenetic networks converging on these regulatory elements might unlock the regenerative potential of adult human hearts.

Cutting Edge: Hypoxia Sensing by the Histone Demethylase UTX (KDM6A) Limits Colitogenic CD4+ T Cells in Mucosal Inflammation.

Hypoxia is a hallmark of inflammatory conditions (e.g., inflammatory bowel disease [IBD]), and adaptive responses have consequently evolved to protect against hypoxia-associated tissue injury. Because augmenting hypoxia-induced protective responses is a promising therapeutic approach for IBD, a more complete understanding of these pathways is needed. Recent work has demonstrated that the histone demethylase UTX is oxygen-sensitive, but its role in IBD is unclear. In this study, we show that hypoxia-induced deactivation of UTX downregulates T cell responses in mucosal inflammation. Hypoxia results in decreased T cell proinflammatory cytokine production and increased immunosuppressive regulatory T cells, and these findings are recapitulated by UTX deficiency. Hypoxia leads to T cell accumulation of H3K27me3 histone modifications, suggesting that hypoxia impairs UTX's histone demethylase activity to dampen T cell colitogenic activity. Finally, T cell-specific UTX deletion ameliorates colonic inflammation in an IBD mouse model, implicating UTX's oxygen-sensitive demethylase activity in counteracting hypoxic inflammation.

A low-input high resolution sequential chromatin immunoprecipitation method captures genome-wide dynamics of bivalent chromatin

BackgroundBivalent chromatin is an exemplar of epigenetic plasticity. This co-occurrence of active-associated H3K4me3 and inactive-associated H3K27me3 histone modifications on opposite tails of the same nucleosome occurs predominantly at promoters that are poised for future transcriptional upregulation or terminal silencing. We know little of the dynamics, resolution, and regulation of this chromatin state outside of embryonic stem cells where it was first described. This is partly due to the technical challenges distinguishing bone-fide bivalent chromatin, where both marks are on the same nucleosome, from allelic or sample heterogeneity where there is a mix of H3K4me3-only and H3K27me3-only mononucleosomes.ResultsHere, we present a robust and sensitive method to accurately map bivalent chromatin genome-wide, along with controls, from as little as 2 million cells. We optimized and refined the sequential ChIP protocol which uses two sequential overnight immunoprecipitation reactions to robustly purify nucleosomes that are truly bivalent and contain both H3K4me3 and H3K27me3 modifications. Our method generates high quality genome-wide maps with strong peak enrichment and low background, which can be analyzed using standard bioinformatic packages. Using this method, we detect 8,789 bivalent regions in mouse embryonic stem cells corresponding to 3,918 predominantly CpG rich and developmentally regulated gene promoters. Furthermore, profiling Dppa2/4 knockout mouse embryonic stem cells, which lose both H3K4me3 and H3K27me3 at approximately 10% of bivalent promoters, demonstrated the ability of our method to capture bivalent chromatin dynamics.ConclusionsOur optimized sequential reChIP method enables high-resolution genome-wide assessment of bivalent chromatin together with all required controls in as little as 2 million cells. We share a detailed protocol and guidelines that will enable bivalent chromatin landscapes to be generated in a range of cellular contexts, greatly enhancing our understanding of bivalent chromatin and epigenetic plasticity beyond embryonic stem cells.

Evaluating histone modification analysis of individual preimplantation embryos

BackgroundWe previously reported a modification of the CUT&Tag method (NTU-CAT) that allows genome-wide histone modification analysis in individual preimplantation embryos. In the present study, NTU-CAT was further simplified by taking advantage of the Well-of-the-Well (WOW) system, which enables the processing of multiple embryos in a shorter time with less reagent and cell loss during the procedure (WOW-CUT&Tag, WOW-CAT).ResultsWOW-CAT allowed histone modification profiling from not only a single blastocyst but also from a portion of it. WOW-CAT generated similar H3K4me3 profiles as NTU-CAT, but they were closer to the profiles produced by chromatin immunoprecipitation-sequencing, such as a valley-like trend and relatively lower false positive rates, indicating that WOW-CAT may attenuate the bias of Tn5 transposase to cut open chromatin regions. Simultaneous WOW-CAT of two halves of single blastocysts was conducted to analyze two different histone modifications (H3K4me3 and H3K27ac) within the same embryo. Furthermore, trophectoderm cells were biopsied and subjected to WOW-CAT in anticipation of preimplantation diagnosis of histone modifications. WOW-CAT allowed the monitoring of epigenetic modifications in the main body of the embryo. For example, analysis of H3K4me3 modifications of XIST and DDX3Y in trophectoderm biopsies could be used to sex embryos in combination with quantitative PCR, but without the need for deep sequencing.ConclusionsThese results suggest the applicability of WOW-CAT for flexible epigenetic analysis of individual embryos in preimplantation epigenetic diagnosis.

The m6 A reader YTHDC2 regulates UVB-induced DNA damage repair and histone modification.

Ultraviolet B (UVB) radiation represents a major carcinogen for the development of all skin cancer types. Mechanistically, UVB induces damage to DNA in the form of lesions, including cyclobutane pyrimidine dimers (CPDs). Disruption of the functional repair processes, such as nucleotide excision repair (NER), allows persistence of DNA damage and contributes to skin carcinogenesis. Recent work has implicated m6 A RNA methylation and its regulatory proteins as having critical roles in facilitating UVB-induced DNA damage repair. However, the biological functions of the m6 A reader YTHDC2 are unknown in this context. Here, we show that YTHDC2 inhibition enhances the repair of UVB-induced DNA damage. We discovered that YTHDC2 inhibition increased the expression of PTEN while it decreased the expression of the PRC2 component SUZ12 and the levels of the histone modification H3K27me3. However, none of these functions were causally linked to the improvements in DNA repair, suggesting that the mechanism utilized by YTHDC2 may be unconventional. Moreover, inhibition of the m6 A writer METTL14 reversed the effect of YTHDC2 inhibition on DNA repair while inhibition of the m6 A eraser FTO mimicked the effect of YTHDC2 inhibition, indicating that YTHDC2 may regulate DNA repair through the m6 A pathway. Finally, compared to normal human skin, YTHDC2 expression was upregulated in human cutaneous squamous cell carcinomas (cSCC), suggesting that it may function as a tumor-promoting factor in skin cancer. Taken together, our findings demonstrate that the m6 A reader YTHDC2 plays a role in regulating UVB-induced DNA damage repair and may serve as a potential biomarker in cSCC.

Single-cell joint profiling of multiple epigenetic proteins and gene transcription.

Sculpting the epigenome with a combination of histone modifications and transcription factor occupancy determines gene transcription and cell fate specification. Here, we first develop uCoTarget, utilizing a split-pool barcoding strategy for realizing ultrahigh-throughput single-cell joint profiling of multiple epigenetic proteins. Through extensive optimization for sensitivity and multimodality resolution, we demonstrate that uCoTarget enables simultaneous detection of five histone modifications (H3K27ac, H3K4me3, H3K4me1, H3K36me3, and H3K27me3) in 19,860 single cells. We applied uCoTarget to the in vitro generation of hematopoietic stem/progenitor cells (HSPCs) from human embryonic stem cells, presenting multimodal epigenomic profiles in 26,418 single cells. uCoTarget reveals establishment of pairing of HSPC enhancers (H3K27ac) and promoters (H3K4me3) and RUNX1 engagement priming for H3K27ac activation along the HSPC path. We then develop uCoTargetX, an expansion of uCoTarget to simultaneously measure transcriptome and multiple epigenome targets. Together, our methods enable generalizable, versatile multimodal profiles for reconstructing comprehensive epigenome and transcriptome landscapes and analyzing the regulatory interplay at single-cell level.