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1
O'Neill, Laura P., Hugh T. Spotswood, Milan Fernando, and Bryan M. Turner. "Differential loss of histone H3 isoforms mono-, di- and tri-methylated at lysine 4 during X-inactivation in female embryonic stem cells." Biological Chemistry 389, no. 4 (April 1, 2008): 365–70. http://dx.doi.org/10.1515/bc.2008.046.
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Abstract Silencing of genes on one of the two female X chromosomes early in development helps balance expression of X-linked genes between XX females and XY males and involves chromosome-wide changes in histone variants and modifications. Mouse female embryonic stem (ES) cells have two active Xs, one of which is silenced on differentiation, and provide a powerful model for studying the dynamics of X inactivation. Here, we use immunofluorescence microscopy of metaphase chromosomes to study changes in H3 mono-, di- or tri-methylated at lysine 4 (H3K4me1, -2 or -3) on the inactivating X (Xi) in female ES cells. H3K4me3 is absent from Xi in approximately 25% of chromosome spreads by day 2 of differentiation and in 40–50% of spreads by days 4–6, making it one of the earliest detectable changes on Xi. In contrast, loss of H3K4me2 occurs 1–2 days later, when histone acetylation also diminishes. Remarkably, H3K4me1 is depleted on both (active) X chromosomes in undifferentiated female ES cells, and on the single X in males, and remains depleted on Xi. Consistent with this, chromatin immunoprecipitation reveals differentiation-related reductions in H3K4me2 and H3K4me3 at the promoter regions of genes undergoing X-inactivation in female ES cells, but no comparable change in H3K4me1.
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Guo, Qiaoyan, Xiaoxia Li, Hongbo Han, Chaoyuan Li, Shujun Liu, Wenhui Gao, and Guangdong Sun. "Histone Lysine Methylation in TGF-β1 Mediated p21 Gene Expression in Rat Mesangial Cells." BioMed Research International 2016 (2016): 1–9. http://dx.doi.org/10.1155/2016/6927234.
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Transforming growth factor beta1- (TGF-β1-) induced p21-dependent mesangial cell (MC) hypertrophy plays a key role in the pathogenesis of chronic renal diseases including diabetic nephropathy (DN). Increasing evidence demonstrated the role of posttranscriptional modifications (PTMs) in the event; however, the precise regulatory mechanism of histone lysine methylation remains largely unknown. Here, we examined the roles of both histone H3 lysine 4 and lysine 9 methylations (H3K4me/H3K9me) in TGF-β1 induced p21 gene expression in rat mesangial cells (RMCs). Our results indicated that TGF-β1 upregulated the expression of p21 gene in RMCs, which was positively correlated with the increased chromatin marks associated with active genes (H3K4me1/H3K4me2/H3K4me3) and negatively correlated with the decreased levels of repressive marks (H3K9me2/H3K9me3) at p21 gene promoter. TGF-β1 also elevated the recruitment of the H3K4 methyltransferase (HMT) SET7/9 to the p21 gene promoter. SET7/9 gene silencing with small interfering RNAs (siRNAs) significantly abolished the TGF-β1 induced p21 gene expression. Taken together, these results reveal the key role of histone H3Kme in TGF-β1 mediated p21 gene expression in RMC, partly through HMT SET7/9 occupancy, suggesting H3Kme and SET7/9 may be potential renoprotective agents in managing chronic renal diseases.
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Lichtenberg, Jens, Elisabeth F. Heuston, Cheryl A. Keller, Ross C. Hardison, and David M. Bodine. "Comparison of Expression and Epigenetic Profiles in Human and Mouse Erythropoiesis and Megakaryopoiesis Using a Systems Biology Model." Blood 126, no. 23 (December 3, 2015): 2383. http://dx.doi.org/10.1182/blood.v126.23.2383.2383.
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Abstract To date numerous datasets of gene expression and epigenetic profiles for mouse and human hematopoietic cells have been generated. While individual data sets for a particular cell type have been correlated, no approach exists to harness all expression and epigenetic profiles for the different types of hematopoietic cells. Our goal is to develop a systems biology platform to compare epigenetic profiles of hematopoietic cells towards a better understanding of epigenetic mechanisms governing hematopoiesis. To provide the necessary foundation to support systematic studies of hematopoiesis, we have developed the Systems Biology Repository (SBR, http://sbrblood.nhgri.nih.gov), a data "ranch" for organizing and analyzing transcriptome and epigenome data cells throughout differentiation. To populate SBR, we extracted, curated, annotated, and integrated all human and mouse hematopoietic datasets available through the Encyclopedia of DNA Elements (ENCODE), the Gene Expression Omnibus (GEO) and the Short Read Repository (SRR). These include genome-wide profiles of DNA methylation, histone methylation and acetylation, transcription factor occupancy (ChIPSeq), chromatin accessibility (DNaseISeq, ATACSeq, FAIRESeq), and coding as well as non-coding transcriptional profiles (RNASeq). To demonstrate the utility of SBR, we conducted three different analyses. The first was a vertical study of HistoneSeq (H3K4me1, H3K4me2, H3K4me3, and H3K27ac), DNA methylation and RNASeq profiles during mouse erythroid differentiation. We found a global decrease in DNA methylation from hematopoietic stem and progenitor cells (HSC) through common myeloid progenitors (CMP), erythroid progenitor cells (MEP) and erythroblasts (ERY; 92936 peaks in HSC to 14422 in ERY). The number of expressed genes (using a tags per million cutoff of 10) increased in erythroid progenitors (8901 in HSC to 10778 in CMP and 10670 in MEP) before decreasing in ERY (8654). 62% of histone marks delineating active enhancers (H3K27ac, H3K4me1) are present in both HSC and ERY, while 48% arise de novo during differentiation. In contrast, only 16% of active promoter specific histone marks (H3K4me2, H3K4me3) are present in both HSC and ERY. For a horizontal analysis we compared the DNA methylation, RNASeq, histone modification (H3K4me1, H3K4me2, H3K4me3, and H3K27ac) and transcription factor binding (GATA1 and NFE2) profiles of erythroblasts (ERY) and megakaryocytes (MEG). We found a similar relationship between gene expression and the histone and DNA methylation profiles in each cell type but differences between expression and in transcription factor occupancy. DNA methylation and H3K4me3 was enriched in the gene body of expressed genes (>36%) for both ERY (p ≤ 0.001) and MEG (p ≤ 0.01). In contrast DNA methylation was enriched in the upstream and downstream regions of non-coding RNA genes (p ≤ 0.001). Transcription factor occupancy was cell type specific: 79% of GATA1 sites are in ERY and 72% of NFE2 sites are in MEG. In erythroblasts, DNA methylation and GATA1 binding in the gene body are associated with gene silencing (4 fold difference, p ≤ 0.001), while in megakaryocytes, DNA methylation and NFE2 binding in the gene body are associated with gene activation (8 fold difference, p ≤ 0.001). We used the Mouse Genome Informatics homology map data to perform a cross-species comparison of the expression profiles of mouse and human multipotent progenitors (MPP), proerythroblasts and orthochromatic erythroblasts. We found a total of 5247 genes expressed at significantly different levels (p ≤ 0.001) between human and mouse MPP, while only 2010 genes were expressed at significantly similar levels (p ≤ 0.001). At the proerythroblast and orthochromatic erythroblast stages 7696 genes and 6571 genes were expressed at significantly different levels (p ≤ 0.001) between human and mouse respectively, while 2024 and 2560 genes were expressed at significantly similar levels (p ≤ 0.001). These data are consistent with previous studies showing differences in the transcriptional profiles of mouse and human hematopoietic cells. In summary, SBR provides a foundation to model the genetic and epigenetic landscape in both the mouse and human hematopoietic system, and enables functional correlations to be made between the species. As SBR is expanded to include data from patient cells, it will be possible to model epigenetic changes associated with disease. Disclosures No relevant conflicts of interest to declare.
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Adelman, Emmalee R., Jian Shi, and Maria E. Figueroa. "Aging Human Hematopoietic Stem Cells Manifest Massive Epigenetic Reprogramming and Altered Gene Splicing of Key Hematopoietic Gene Sets." Blood 128, no. 22 (December 2, 2016): 885. http://dx.doi.org/10.1182/blood.v128.22.885.885.
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Abstract Aging leads to impairment of hematopoietic stem cell (HSC) function with decreased self-renewal, imbalanced differentiation potential and an increased risk to develop myeloid malignancies. These malignancies are associated with epigenetic deregulation, which contributes to pathogenesis. Notably, studies in murine models have revealed epigenetic changes in aged HSC. However, it is unknown if this occurs in normal human HSC aging and whether it may contribute to HSC dysfunction. Therefore, we performed comprehensive epigenomic and transcriptional profiling in primary human HSC (Lin-, CD34+, CD38-) isolated from young (18-30 yo), mid (45-55 yo) and old (65-75 yo) healthy donors. Using a micro-ChIP-seq protocol we profiled H3K4me1, H3K4me3, H3K27me3 and H3K27ac in 4-7 donors per age group, as well as genome-wide DNA methylation (5mC), hydroxymethylation (5hmC) and RNA-seq. Analysis of enhancer-associated marks revealed that with age there is marked reduction in both H3K4me1 and H3K27ac (20,783 and 15,625 peaks lost, respectively; log10likelihood ratio >3). Gene ontology analysis of these lost peaks revealed their association with genes involved in hematopoiesis and, RNA splicing and chromatin organization, respectively (ChIPenrich, FDR<0.05). In addition, regions depleted in H3K4me1 are enriched for PU.1, FLI1, ETS, and CTCF binding sites (Homer, q<0.00001). We next asked if aging results in specific remodeling of poised (H3K4me1>H3K4me3, H3K27ac-) and active (H3K4me1>H3K4me3, H3K27ac+) enhancers. We found age-related loss of H3K4me1 enrichment at 10,696 poised enhancers, which are associated with hematopoiesis and T- and B-cell receptor signaling (FDR<0.05). We also identified 17,242 active enhancers in young HSC, 7,057 of which are depleted in old HSC. This loss of active enhancers targets genes associated with hematopoiesis, immune signaling and myeloid malignancies (FDR<0.05). Next we analyzed the impact of aging on promoter-associated marks, H3K4me3 and H3K27me3. Remarkably, while aging leads to loss of 22,689 H3K4me3 peaks, only 1,339 H3K27me3 peaks are lost. Loss of H3K4me3 targets genes involved in inflammatory response, development and WNT signaling (FDR<0.05). Given this uneven change in H3K4me3/H3K27me3 with aging, we hypothesized this may correlate with changes in bivalently marked promoters, which regulate key developmental genes. Out of 3,947 bivalent promoters in young HSC, 842 are lost in aged HSC. This loss of bivalency affects genes involved in WNT, Cadherin and Hedgehog signaling pathways (FDR<0.05). Next we analyzed changes in cytosine modifications. We observe widespread gain of 5hmC (n=14,554 differentially hydroxymethylated regions [DHMR]; FDR <0.005), with specific enrichment at introns and exons (p<2.2e-16), as well as enrichment for GATA and KLF binding sites (Homer, q<0.00001). These DHMR target genes involved in hematopoiesis, proteins regulated by alternative splicing, and pathways associated with cancer (FDR<0.05). In contrast, much more subtle changes are found in 5mC with HSC aging, with only 529 differentially methylated regions (q-value <0.05, meth.diff >20%). However, these subtle changes also target genes associated with cadherin and WNT signaling. Finally, RNA-seq analysis revealed that this age-associated epigenetic reprogramming is accompanied by an overall trend to gene downregulation. Amongst the genes most affected are the nuclear lamin gene LMNA (mutated in progeria syndrome), splicing factors SRSF7 and U2AF1 and, the transcription factors KLF3/6 and HIF1α (FDR <0.05, fold change >1.5). Notably, changes in expression also include significant differential exon usage, which may be mediated by DHMR at intron-exon boundaries: 575 genes show altered exon usage (FDR <0.05, fold change>1.5) including LMNA and the epigenetic modifiers BRD9, CITED2, KDM6A and SETD6. In summary, we have completed the first comprehensive epigenomic profiling of aging in human HSC. Our findings show massive epigenetic remodeling in aged HSC, consisting of loss of activating histone marks primarily targeting enhancers and bivalent promoters at genes involved in hematopoiesis and developmental pathways. Cytosine modifications show widespread changes in 5hmC, targeting intron-exon boundaries. Globally, this epigenetic reprogramming results in overall gene downregulation and altered splicing of genes important for HSC regulation. Disclosures No relevant conflicts of interest to declare.
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Deshpande, Neha, Rachel Jordan, Michelle Henderson Pozzi, and Mary Bryk. "Histone 3 lysine 4 monomethylation supports activation of transcription in S. cerevisiae during nutrient stress." Current Genetics 68, no. 2 (January 18, 2022): 181–94. http://dx.doi.org/10.1007/s00294-022-01226-2.
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AbstractMono-methylation of the fourth lysine on the N-terminal tail of histone H3 was found to support the induction of RNA polymerase II transcription in S. cerevisiae during nutrient stress. In S. cerevisiae, the mono-, di- and tri-methylation of lysine 4 on histone H3 (H3K4) is catalyzed by the protein methyltransferase, Set1. The three distinct methyl marks on H3K4 act in discrete ways to regulate transcription. Nucleosomes enriched with tri-methylated H3K4 are usually associated with active transcription whereas di-methylated H3K4 is associated with gene repression. Mono-methylated H3K4 has been shown to repress gene expression in S. cerevisiae and is detected at enhancers and promoters in eukaryotes. S. cerevisiae set1Δ mutants unable to methylate H3K4 exhibit growth defects during histidine starvation. The growth defects are rescued by either a wild-type allele of SET1 or partial-function alleles of set1, including a mutant that predominantly generates H3K4me1 and not H3K4me3. Rescue of the growth defect is associated with induction of the HIS3 gene. Growth defects observed when set1Δ cultures were starved for isoleucine and valine were also rescued by wild-type SET1 or partial-function set1 alleles. The results show that H3K4me1, in the absence of H3K4me3, supports transcription of the HIS3 gene and expression of one or more of the genes required for biosynthesis of isoleucine and valine during nutrient stress. Set1-like methyltransferases are evolutionarily conserved, and research has linked their functions to developmental gene regulation and several cancers in higher eukaryotes. Identification of mechanisms of H3K4me1-mediated activation of transcription in budding yeast will provide insight into gene regulation in all eukaryotes.
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Adelman, Emmalee, André Olsson, Tingting Qin, R. Coleman Lindsley, Rafael Bejar, Nathan Salomonis, Lee Grimes, and Maria E. Figueroa. "Integrative Epigenetic and Single-Cell RNA-Seq Profiling of Human Hematopoietic Stem Cells Reveals Epigenetic Reprogramming of Enhancer and Regulatory Elements during Normal Aging." Blood 130, Suppl_1 (December 7, 2017): 770. http://dx.doi.org/10.1182/blood.v130.suppl_1.770.770.
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Abstract Aging is associated with impaired hematopoietic stem cell (HSC) function, increased risk of myeloid malignancies and the acquisition of clonal hematopoiesis of indeterminate potential (CHIP). Little is known about how epigenetic regulation contributes to these age-related changes in human HSC biology. Here we report a comprehensive epigenetic and transcriptomic profiling study of human HSC aging. The HSC enriched (HSCe; Lin- CD34+ CD38-) population was purified from young (18-30 yo) and aged (65-75 yo) healthy donors and used for ChIP-Seq of H3K4me1, H3K27ac, H3K4me3, H3K27me3, DNA methylation, and bulk and single-cell (sc) RNA-seq. 5-hydroxymethylcytosine (hmC) was also profiled in the Lin- CD34+ CD38+ fraction (n=4-7 per modification, per age group). Targeted exon sequencing of 128 genes revealed only 1 out of 24 donors with any mutation (DNMT3A mutation with variant allele frequency of 0.12); thus, we concluded that any observed epigenetic or transcriptional changes with age could not be due to CHIP. Analysis of histone modifications revealed significant changes in aged HSCe compared to young, affecting 21,022 H3K4me1, 15,686 H3K4me3 and 27,071 H3K27ac peaks, with the vast majority of peaks (>98%) losing signal intensity with age (log likelihood ratio >3). In contrast, only 1,748 H3K27me3 peaks changed with age. Genes with age-related loss of H3K4me1, H3K4me3, or H3K27ac tended to lower expression in aged HSCe compared to young, while genes with reduced H3K27me3 tended to higher expression (t-test, p < 0.05). Functional annotation of regions with decreased H3K4me1 and H3K27ac showed they are associated with genes involved in hematopoiesis and chromatin organization, and RNA splicing, respectively; while sites with age-associated decrease in H3K4me3 and H3K27me3 are associated with developmental pathways (ChIP-enrich, FDR <0.05). Given these marked changes in H3K4me1 and H3K27ac, we hypothesized that enhancers may be deregulated with HSCe aging. We found that 35% (n=4,519) of all active enhancers (H3K27ac+, H3K4me1>H3K4me3, > 3 kb from TSS) lost H3K27ac with age, including enhancers regulating numerous hematopoietic transcription factors such as RUNX3, FLI1, GATA2, GFI1, HIF1A, and KLF6, as well as epigenetic modifiers BCOR, DNMT3A, DOT1 L and KMT2A, and the gene mutated in progeria syndromes, LMNA .KEGG pathway analysis of all active enhancers lost with age exhibited enrichment for B- and T-cell signaling, and leukemic and apoptosis pathways (ChIP-enrich, FDR<0.05). In addition, analysis of bivalent promoters revealed that 1,017 out of 3,967 bivalent promoters identified in young HSCe shifted from bivalency towards repression in aged HSCe, due to loss of H3K4me3. These lost bivalent promoters are enriched for WNT, Hedgehog and Cadherin signaling pathways and include several HOXC cluster genes and WNT factors (ChIP-enrich, FDR<0.05). Notably, analysis of DNA methylation showed only focal changes, with 529 differentially methylated regions with aging (q-value < 0.05 and methylation difference ≥20%), which were associated with cell adhesion, cadherins, and WNT-signaling (ChIP-enrich, FDR <0.05). In contrast, global profiling of hmC revealed 14,554 peaks gained (FDR<0.05) at regions enriched for GATA and KLF family transcription factor binding motifs (Homer, q<1.0e-4). At the expression level, 502 genes were differentially expressed with age (FDR < 0.05 and Fold change ≥ 1.5), with downregulation of LMNA, the splicing factors U2AF1 and SREK1, hematopoietic transcription factors HIF1A, BCL6 and KLF factors 3, 6, 7 and 10, and the epigenetic modifiers KDM3A, SETD6, SETD8 and SETD1A . Strikingly, analysis of sc-RNA-seq of young and aged HSCe showed that while 4 out of 208 young HSCe possessed elements of the aged HSCe gene signature, no young HSCe displayed the complete aged HSCe expression profile. In summary, integrative profiling of aged human HSCe reveals widespread epigenetic changes, targeting active enhancers of hematopoietic transcription factors and genes involved in immune function, thus implicating enhancer deregulation in aged HSC loss of function. Importantly, both mutational analysis and single cell RNA-seq suggest that these changes cannot be attributed to clonal hematopoiesis alone, but rather, are due in part to reprogramming of aged HSCs. Disclosures Lindsley: Takeda Pharmaceuticals: Consultancy; Jazz Pharmaceuticals: Consultancy; MedImmune: Research Funding. Bejar: Genoptix: Consultancy, Honoraria, Patents & Royalties; AbbVie/Genetech: Honoraria, Other: Ad-hoc advisory board; Modus Outcomes: Consultancy, Honoraria; Foundation Medicine: Honoraria, Other: Ad-hoc advisory board; Otsuka/Astex: Honoraria, Other: Ad-hoc advisory board; Celgene: Consultancy, Honoraria, Other: DSMB, Steering Committee, Research Funding.
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Ingvarsdottir, Kristin, Chris Edwards, Min Gyu Lee, Jung Shin Lee, David C. Schultz, Ali Shilatifard, Ramin Shiekhattar, and Shelley L. Berger. "Histone H3 K4 Demethylation during Activation and Attenuation of GAL1 Transcription in Saccharomyces cerevisiae." Molecular and Cellular Biology 27, no. 22 (September 17, 2007): 7856–64. http://dx.doi.org/10.1128/mcb.00801-07.
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ABSTRACT In mammalian cells, histone lysine demethylation is carried out by two classes of enzymes, the LSD1/BHC110 class and the jumonji class. The enzymes of the jumonji class in the yeast Saccharomyces cerevisiae have recently also been shown to have lysine demethylation activity. Here we report that the protein encoded by YJR119c (termed KDM5), coding for one of five predicted jumonji domain proteins in yeast, specifically demethylates trimethylated histone H3 lysine 4 (H3K4me3), H3K4me2, and H3K4me1 in vitro. We found that loss of KDM5 increased mono-, di-, and trimethylation of lysine 4 during activation of the GAL1 gene. Interestingly, cells deleted of KDM5 also displayed a delayed reduction of K4me3 upon reestablishment of GAL1 repression. These results indicate that K4 demethylation has two roles at GAL1, first to establish appropriate levels of K4 methylation during gene activation and second to remove K4 trimethylation during the attenuation phase of transcription. Thus, analysis of lysine demethylation in yeast provides new insight into the physiological roles of jumonji demethylase enzymes.
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Ngo, Vu, Zhao Chen, Kai Zhang, John W. Whitaker, Mengchi Wang, and Wei Wang. "Epigenomic analysis reveals DNA motifs regulating histone modifications in human and mouse." Proceedings of the National Academy of Sciences 116, no. 9 (February 12, 2019): 3668–77. http://dx.doi.org/10.1073/pnas.1813565116.
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Histones are modified by enzymes that act in a locus, cell-type, and developmental stage-specific manner. The recruitment of enzymes to chromatin is regulated at multiple levels, including interaction with sequence-specific DNA-binding factors. However, the DNA-binding specificity of the regulatory factors that orchestrate specific histone modifications has not been broadly mapped. We have analyzed 6 histone marks (H3K4me1, H3K4me3, H3K27ac, H3K27me3, K3H9me3, H3K36me3) across 121 human cell types and tissues from the NIH Roadmap Epigenomics Project as well as 8 histone marks (with addition of H3K4me2 and H3K9ac) from the mouse ENCODE Consortium. We have identified 361 and 369 DNA motifs in human and mouse, respectively, that are the most predictive of each histone mark. Interestingly, 107 human motifs are conserved between the two species. In human embryonic cell line H1, we mutated only the found DNA motifs at particular loci and the significant reduction of H3K27ac levels validated the regulatory roles of the perturbed motifs. The functionality of these motifs was also supported by the evidence that histone-associated motifs, especially H3K4me3 motifs, significantly overlap with the expression of quantitative trait loci SNPs in cancer patients more than the known and random motifs. Furthermore, we observed possible feedbacks to control chromatin dynamics as the found motifs appear in the promoters or enhancers associated with various histone modification enzymes. These results pave the way toward revealing the molecular mechanisms of epigenetic events, such as histone modification dynamics and epigenetic priming.
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Schulz, Vincent P., Kimberly Lezon-Geyda, Yelena Maksimova, and Patrick G. Gallagher. "Enhancers and Super Enhancers Are Associated With Genes That Control Phenotypic Traits In Primary Human Erythroid Cells." Blood 122, no. 21 (November 15, 2013): 1200. http://dx.doi.org/10.1182/blood.v122.21.1200.1200.
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Abstract Identification of cell-type specific enhancers is important for understanding the regulation of programs controlling cellular development and differentiation. Recent studies have shown that enhancers are frequently associated with biologically relevant and disease-associated genetic variants. We hypothesized that unique sets of enhancers and super enhancers regulate gene expression in erythroid cells, a specialized cell type evolved to carry oxygen, and associated variants influence erythroid phenotypic variability. Active enhancers are part of a chromatin landscape marked by histone H3 lysine 4 monomethylation (H3K4me1) and histone H3 lysine 27 acetylation (H3K27Ac). A subset of enhancers, called super enhancers, important for regulating genes critical for cell-type specific identify, have been described. Super enhancers span large regions of chromatin, have domains of transcription factors (TF), significant amounts of H3K4me1 and H3K27Ac modification, and significant amounts of Mediator (MED1) occupancy, frequently with the transcriptional activator BRD4. Using ChIP-seq, genome wide maps of enhancers were constructed for H3K4me1, H3K27Ac, MED1, and BRD4 using primary human erythroid cell chromatin. These data were combined with parallel gene expression analyses determined via RNA-seq and enhancers and super enhancers identified. Cell and tissue-type specific enhancers act over distances of tens to hundreds of kilobases, thus bona fide erythroid enhancers are expected to be enriched in the genomic vicinity of genes expressed and functional in erythroid cells. Sites of occupancy of H3K4me1 were correlated with levels of gene expression in erythroid cells. To exclude gene promoters, H3K4me1 within 1kb of annotated transcriptional start sites (TSS) were excluded from analyses. Consistent with their predicted function, there was significantly higher levels of erythroid transcription for genes with H3K4me1 occupancy within 1-50kb of the TSS of genes cf. genes with H3K4me1 occupancy >50kb of a TSS (p value<2.2e-16). There was also significantly higher expression of genes with H3K4me1 occupancy within 1-50kb of the TSS in erythroid cells cf. non-erythroid cells (T lymphocyte). The top over represented TF motifs at sites of H3K4me1 were GATA1, AP1/NFE2, and KLF1. To explore whether candidate erythroid enhancers are enriched in regions associated with biologically relevant erythroid cell traits, candidate enhancers were mapped to a data set of erythroid-associated SNPs from the NHGRI GWAS catalog. 32 enhancers mapped to sites previously associated with biologically relevant erythroid traits. SNPs changed TF binding motifs at numerous enhancers including GATA1 motifs in the BCL11A, TFRC and ATP24 loci, an NFE2 motif in the ATP2B4 locus, and a TAL1 motif in the BCL11A locus. Super enhancers were identified as described (Cell 153:307, 2013) by finding regions with the highest levels of clustered chromatin modification/occupancy. Super enhancers defined by H3K4me1 and H3K27Ac modifications yielded 231 regions, BRD4 occupancy yielded 166 regions, and MED1 occupancy yielded 52 regions. H3K4me1/H3K27Ac-marked SE regions were found near the FOXO3, GATA2, STAT5A, TAL1, and ZFPM1 gene loci. BRD4- and MED1-marked super enhancers were found near the critical erythroid volume regulatory gene PIEZO1. The top over represented TF motifs at super enhancer sites defined by H3K4me1 were TAL1/RUNX1, GATA1, KLF1, defined by BRD4 were TAL1, KLF1, and MYC, and defined by MED1 were GATA1, MYC and CTCF. Mapping of super enhancers to erythroid-associated SNPs from the GWAS catalog of the NHGRI revealed many super enhancers mapped to regions associated with biologically relevant erythroid cell traits. For example, super enhancers identified by H3K4me1 mapped to loci for BCL11A, TFRC, KIT, HBS1L, MYB, ANK1, HK1, and the alpha-globin gene cluster; super enhancers identified by BRD4 localized to the alpha-globin cluster and the PIEZO1 gene locus. Perturbation of enhancer function during erythroid development and differentiation may lead to dysregulation of gene expression with concomitant phenotypic consequences. Insights into regulation of programs of gene expression in obtained from study of erythroid enhancers will provide insights into the functional significance of sequence variation associated with quantitative traits and inherited and acquired hematologic disease. Disclosures: No relevant conflicts of interest to declare.
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Mantsoki, Anna, Karla Parussel, and Anagha Joshi. "Identification and Characterisation of Putative Enhancer Elements in Mouse Embryonic Stem Cells." Bioinformatics and Biology Insights 15 (January 2021): 117793222097462. http://dx.doi.org/10.1177/1177932220974623.
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Enhancer elements control mammalian transcription largely in a cell-type-specific manner. The genome-wide identification of enhancer elements and their activity status in a cellular context is therefore fundamental to understanding cell identity and function. We determined enhancer activity in mouse embryonic stem (ES) cells using chromatin modifications and characterised their global properties. Specifically, we first grouped enhancers into 5 groups using multiple H3K4me1, H3K27ac, and H3K27me3 modification data sets. Active enhancers (simultaneous presence of H3K4me1 and H3K27ac) were enriched for binding of pluripotency factors and were found near pluripotency-related genes. Although both H3K4me1-only and active enhancers were enriched for super-enhancers and a TATA box like motif, active enhancers were preferentially bound by RNA polII (s2) and were enriched for bidirectional transcription, while H3K4me1-only enhancers were enriched for RNA polII (8WG16) suggesting they were likely poised. Bivalent enhancers (simultaneous presence of H3K4me1 and H3K27me3) were preferentially in the vicinity of bivalent genes. They were enriched for binding of components of polycomb complex as well as Tcf3 and Oct4. Moreover, a ‘CTTTCTC’ de-novo motif was enriched at bivalent enhancers, previously identified at bivalent promoters in ES cells. Taken together, 3 histone modifications successfully demarcated active, bivalent, and poised enhancers with distinct sequence and binding features.
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Nikitin, Kolosov, Murzina, Pats, Zamyatin, Tkachev, Sorokin, Kopylov, and Buzdin. "Retroelement-Linked H3K4me1 Histone Tags Uncover Regulatory Evolution Trends of Gene Enhancers and Feature Quickly Evolving Molecular Processes in Human Physiology." Cells 8, no. 10 (October 8, 2019): 1219. http://dx.doi.org/10.3390/cells8101219.
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Background: Retroelements (REs) are mobile genetic elements comprising ~40% of human DNA. They can reshape expression patterns of nearby genes by providing various regulatory sequences. The proportion of regulatory sequences held by REs can serve a measure of regulatory evolution rate of the respective genes and molecular pathways. Methods: We calculated RE-linked enrichment scores for individual genes and molecular pathways based on ENCODE project epigenome data for enhancer-specific histone modification H3K4me1 in five human cell lines. We identified consensus groups of molecular processes that are enriched and deficient in RE-linked H3K4me1 regulation. Results: We calculated H3K4me1 RE-linked enrichment scores for 24,070 human genes and 3095 molecular pathways. We ranked genes and pathways and identified those statistically significantly enriched and deficient in H3K4me1 RE-linked regulation. Conclusion: Non-coding RNA genes were statistically significantly enriched by RE-linked H3K4me1 regulatory modules, thus suggesting their high regulatory evolution rate. The processes of gene silencing by small RNAs, DNA metabolism/chromatin structure, sensory perception/neurotransmission and lipids metabolism showed signs of the fastest regulatory evolution, while the slowest processes were connected with immunity, protein ubiquitination/degradation, cell adhesion, migration and interaction, metals metabolism/ion transport, cell death, intracellular signaling pathways.
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Cattoglio, Claudia, Danilo Pellin, Ermanno Rizzi, Giulietta Maruggi, Giorgio Corti, Francesca Miselli, Daniela Sartori, et al. "High-definition mapping of retroviral integration sites identifies active regulatory elements in human multipotent hematopoietic progenitors." Blood 116, no. 25 (December 16, 2010): 5507–17. http://dx.doi.org/10.1182/blood-2010-05-283523.
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Abstract Integration of retroviral vectors in the human genome follows nonrandom patterns that favor insertional deregulation of gene expression and increase the risk of their use in clinical gene therapy. The molecular basis of retroviral target site selection is still poorly understood. We used deep sequencing technology to build genomewide, high-definition maps of > 60 000 integration sites of Moloney murine leukemia virus (MLV)– and HIV-based retroviral vectors in the genome of human CD34+ multipotent hematopoietic progenitor cells (HPCs) and used gene expression profiling, chromatin immunoprecipitation, and bioinformatics to associate integration to genetic and epigenetic features of the HPC genome. Clusters of recurrent MLV integrations identify regulatory elements (alternative promoters, enhancers, evolutionarily conserved noncoding regions) within or around protein-coding genes and microRNAs with crucial functions in HPC growth and differentiation, bearing epigenetic marks of active or poised transcription (H3K4me1, H3K4me2, H3K4me3, H3K9Ac, Pol II) and specialized chromatin configurations (H2A.Z). Overall, we mapped 3500 high-frequency integration clusters, which represent a new resource for the identification of transcriptionally active regulatory elements. High-definition MLV integration maps provide a rational basis for predicting genotoxic risks in gene therapy and a new tool for genomewide identification of promoters and regulatory elements controlling hematopoietic stem and progenitor cell functions.
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Turner, Stephen, Moshe Olshansky, Jasmine Li, Michelle Nguyen, Eddie Chen, T. H. Nguyen, Sudha Rao, and Brendan Russ. "Genome wide mapping of epigenetic signatures identify novel enhancer elements that underpin virus-specific CD8+ T cell differentiation (IRM14P.445)." Journal of Immunology 194, no. 1_Supplement (May 1, 2015): 198.5. http://dx.doi.org/10.4049/jimmunol.194.supp.198.5.
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Abstract The molecular mechanisms that underpin acquisition and maintenance of lineage specific gene expression by virus-specific CD8+ T cells are not fully delineated. Post-translational modifications (PTMs) of genome associated histone proteins are a key mechanism for regulating gene expression. We mapped genome wide changes in histone PTM deposition during virus-specific CD8+ T cell differentiation. By comparing the location and level of deposition of a combination of H3K4me1, H3K4me2, H3K4me3, H3K27me3, H3K27Ac, chromatin accessibility and binding of the histone acetyltransferase, p300, we could map both known and putative enhancer elements that are differentially regulated between naive, effector and memory virus-specific CD8+ T cells. Using this epigenetic blueprint, we identified putative transcriptional enhancers located upstream of the Ccl5 gene, a key CD8+ T cell effector gene. We demonstrate that acquisition of Ccl5 transcription upon T cell activation is regulated via temporal and transcription factor (TF) dependent chromatin remodelling. Using chromatin conformation capture (3C) to measure enhancer-promoter interactions, we show that that TF dependent looping of these enhancer elements onto the Ccl5 promoter correlated with transcriptional activation and potential. These studies form a platform for more in depth analysis of key non-coding regulatory elements that regulate acquisition of virus-specific T cell gene expression and maintenance into memory.
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Wang, Jingjing, Yuriy L. Orlov, Xue Li, Yincong Zhou, Yongjing Liu, Chunhui Yuan, and Ming Chen. "In situ dissecting the evolution of gene duplication with different histone modification patterns based on high-throughput data analysis in Arabidopsis thaliana." PeerJ 9 (January 5, 2021): e10426. http://dx.doi.org/10.7717/peerj.10426.
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Background Genetic regulation is known to contribute to the divergent expression of duplicate genes; however, little is known about how epigenetic modifications regulate the expression of duplicate genes in plants. Methods The histone modification (HM) profile patterns of different modes of gene duplication, including the whole genome duplication, proximal duplication, tandem duplication and transposed duplication were characterized based on ChIP-chip or ChIP-seq datasets. In this study, 10 distinct HM marks including H2Bub, H3K4me1, H3K4me2, H3K4me3, H3K9ac, H3K9me2, H3K27me1, H3K27me3, H3K36me3 and H3K14ac were analyzed. Moreover, the features of gene duplication with different HM patterns were characterized based on 88 RNA-seq datasets of Arabidopsis thaliana. Results This study showed that duplicate genes in Arabidopsis have a more similar HM pattern than single-copy genes in both their promoters and protein-coding regions. The evolution of HM marks is found to be coupled with coding sequence divergence and expression divergence after gene duplication. We found that functionally selective constraints may impose on epigenetic evolution after gene duplication. Furthermore, duplicate genes with distinct functions have more divergence in histone modification compared with the ones with the same function, while higher expression divergence is found with mutations of chromatin modifiers. This study shows the role of epigenetic marks in regulating gene expression and functional divergence after gene duplication in plants based on sequencing data.
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Placek, Katarzyna, Kairong Cui, Gangqing Hu, Ji-Eun Lee, Chaochen Wang, Joanne Konkel, Dunfang Zhang, WanJun Chen, Kai Ge, and Keji Zhao. "KMT2D histone methyltransferase modulates chromatin accessibility during regulatory T cell development (LYM6P.714)." Journal of Immunology 194, no. 1_Supplement (May 1, 2015): 135.2. http://dx.doi.org/10.4049/jimmunol.194.supp.135.2.
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Abstract Gene regulatory elements are associated with histone H3 lysine 4 (H3K4) methylation and H3 acetylation and DNase I hypersensitivity. Whilst much is known about changes in histone modification patterns and chromatin accessibility during T cell differentiation, little is known about the role of histone modifying enzymes in this process. KMT2D is a histone methyltransferase that shapes enhancer landscape by regulating H3K4me and H3K27ac. To determine whether KMT2D plays a role in T cell development, we deleted KMT2D in T cells. Our data show that KMT2D deficiency results in reduced number of T cells in periphery. Moreover in the absence of KMT2D, differentiation of regulatory T (Treg) cells is compromised. KMT2D co-localizes with active chromatin marks. We showed that DNaseI hypersensitive sites (HS) bound by KMT2D tend to decrease in accessibility upon KMT2D deletion in CD4+ T cells that leads to reduced expression of nearby genes. The decreased HS are linked to genes involved in Treg differentiation and are enriched in transcription factor (TF) binding motif important for CD4+ T cell development. We found that KMT2D co-localizes and interacts with TF in CD4+ T cells. KMT2D binds to Treg master regulator: Foxp3 upstream enhancer and imposes H3K4me1 and H3K4me2 at Foxp3 regulatory elements. Our data suggest that KMT2D modulates CD4+ T cell development by maintaining chromatin accessibility and thus enabling TFs binding at genes involved in Treg cell differentiation.
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Chang, Chu-Yuan, Jui-Hung Hung, Liang-Wei Huang, Joye Li, Ka Shing Fung, Cheng-Fu Kao, and Linyi Chen. "Epigenetic Regulation of WNT3A Enhancer during Regeneration of Injured Cortical Neurons." International Journal of Molecular Sciences 21, no. 5 (March 10, 2020): 1891. http://dx.doi.org/10.3390/ijms21051891.
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Traumatic brain injury is known to reprogram the epigenome. Chromatin immunoprecipitation-sequencing of histone H3 lysine 27 acetylation (H3K27ac) and tri-methylation of histone H3 at lysine 4 (H3K4me3) marks was performed to address the transcriptional regulation of candidate regeneration-associated genes. In this study, we identify a novel enhancer region for induced WNT3A transcription during regeneration of injured cortical neurons. We further demonstrated an increased mono-methylation of histone H3 at lysine 4 (H3K4me1) modification at this enhancer concomitant with a topological interaction between sub-regions of this enhancer and with promoter of WNT3A gene. Together, this study reports a novel mechanism for WNT3A gene transcription and reveals a potential therapeutic intervention for neuronal regeneration.
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Rada-Iglesias, Alvaro. "Is H3K4me1 at enhancers correlative or causative?" Nature Genetics 50, no. 1 (December 22, 2017): 4–5. http://dx.doi.org/10.1038/s41588-017-0018-3.
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Tutino, Vincent M., Cathleen C. Kuo, Naval Avasthi, Hamid H. Rai, Muhammad Waqas, Adnan H. Siddiqui, James N. Jarvis, and Kerry E. Poppenberg. "Chromatin architecture around stroke haplotypes provides evidence that genetic risk is conferred through vascular cells." Epigenomics 14, no. 5 (March 2022): 243–59. http://dx.doi.org/10.2217/epi-2021-0307.
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Introduction: Genome-wide association studies (GWAS) have identified numerous stroke-associated SNPs. To understand how SNPs affect gene expression related to increased stroke risk, we studied epigenetic landscapes surrounding 26 common, validated stroke-associated loci. Methods: We mapped the SNPs to linkage disequilibrium (LD) blocks and examined H3K27ac, H3K4me1, H3K9ac, and H3K4me3 histone marks and transcription-factor binding-sites in pathologically relevant cell types (hematopoietic and vascular cells). Hi-C data were used to identify topologically associated domains (TADs) encompassing the LD blocks and overlapping genes. Results: Fibroblasts, smooth muscle, and endothelial cells showed significant enrichment for enhancer-associated marks within stroke-associated LD blocks. Genes within encompassing TADs reflected vessel homeostasis, cellular turnover, and enzymatic activity. Conclusions: Stroke-associated genetic variants confer risk predominantly through vascular cells rather than hematopoietic cell types.
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Alajem, Adi, Hava Roth, Sofia Ratgauzer, Danny Bavli, Alex Motzik, Shlomtzion Lahav, Itay Peled, and Oren Ram. "DNA methylation patterns expose variations in enhancer-chromatin modifications during embryonic stem cell differentiation." PLOS Genetics 17, no. 4 (April 12, 2021): e1009498. http://dx.doi.org/10.1371/journal.pgen.1009498.
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In mammals, cellular identity is defined through strict regulation of chromatin modifications and DNA methylation that control gene expression. Methylation of cytosines at CpG sites in the genome is mainly associated with suppression; however, the reason for enhancer-specific methylation is not fully understood. We used sequential ChIP-bisulfite-sequencing for H3K4me1 and H3K27ac histone marks. By collecting data from the same genomic region, we identified enhancers differentially methylated between these two marks. We observed a global gain of CpG methylation primarily in H3K4me1-marked nucleosomes during mouse embryonic stem cell differentiation. This gain occurred largely in enhancer regions that regulate genes critical for differentiation. The higher levels of DNA methylation in H3K4me1- versus H3K27ac-marked enhancers, despite it being the same genomic region, indicates cellular heterogeneity of enhancer states. Analysis of single-cell RNA-seq profiles demonstrated that this heterogeneity correlates with gene expression during differentiation. Furthermore, heterogeneity of enhancer methylation correlates with transcription start site methylation. Our results provide insights into enhancer-based functional variation in complex biological systems.
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Chen, Jigang, Yanhong Guo, Wei Zeng, Li Huang, Qi Pang, Ling Nie, Jiao Mu, Fahuan Yuan, and Bing Feng. "ER stress triggers MCP-1 expression through SET7/9-induced histone methylation in the kidneys of db/db mice." American Journal of Physiology-Renal Physiology 306, no. 8 (April 15, 2014): F916—F925. http://dx.doi.org/10.1152/ajprenal.00697.2012.
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Epigenetics plays a key role in the pathogenesis of diabetic nephropathy (DN), although the precise regulatory mechanism is still unclear. Here, we examined the role of endoplasmic reticulum (ER) stress in histone H3 lysine 4 (H3K4) methyltransferase SET7/9-induced monocyte chemoattractant protein-1 (MCP-1) expression in the kidneys of db/db mice. Our results indicate that the expression of MCP-1 significantly increased in the kidneys of db/db mice in a time-dependent manner. An increased chromatin mark associated with an active gene (H3K4me1) at MCP-1 promoters accompanied this change in expression. The expression of SET7/9 and the recruitment to these promoters were also elevated. SET7/9 gene silencing with small interfering (si) RNAs significantly attenuated the expression of H3K4me1 and MCP-1. Furthermore, expression of signaling regulator glucose-regulated protein 78 (GRP78), a monitor of ER stress, significantly increased in the kidneys of db/db mice. The expression of spliced X-box binding protein 1 (XBP1s), an ER stress-inducible transcription factor, and recruitment to the SET7/9 promoters were also increased. XBP1s gene silencing with siRNAs significantly attenuated the expression of SET7/9, H3K4me1, and MCP-1. The chaperone betaine not only effectively downregulated the GRP78 and XBP1s expression levels but also markedly decreased the SET7/9, H3K4me1, and MCP-1 levels. Luciferase reporter assay demonstrated that XBP1s participated in ER stress-induced SET7/9 transcription, Taken together, these results reveal that ER stress can trigger the expression of MCP-1, in part through the XBP1s-mediated induction of SET7/9.
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Local, Andrea, Hui Huang, Claudio P. Albuquerque, Namit Singh, Ah Young Lee, Wei Wang, Chaochen Wang, et al. "Identification of H3K4me1-associated proteins at mammalian enhancers." Nature Genetics 50, no. 1 (December 18, 2017): 73–82. http://dx.doi.org/10.1038/s41588-017-0015-6.
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Carr, Ryan M., Terra Lasho, David Marks, Ezequiel Tolosa, Luciana L. Almada, Bonnie Alver, Moritz Binder, et al. "Clinical Categorization of Chronic Myelomonocytic Leukemia into Proliferative and Dysplastic Subtypes Correlates with Distinct Genomic, Transcriptomic and Epigenomic Signatures." Blood 134, Supplement_1 (November 13, 2019): 1710. http://dx.doi.org/10.1182/blood-2019-123877.
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Introduction: Chronic myelomonocytic leukemia (CMML), an aggressive myeloid malignancy, can be categorized into two subtypes, proliferative CMML (pCMML) and dysplastic (dCMML), based on a white blood cell (WBC) count of ≥ 13 x 109/L for the former (Arber et al. Blood 2016). While this WBC cut off is somewhat arbitrary, patients with pCMML have unique phenotypic features and a shorter survival. We carried out this study to assess the genomic, transcriptomic and epigenetic landscapes of these two CMML subtypes. Methods: Peripheral blood (PB) and bone marrow (BM) mononuclear cells (MNC) were obtained from WHO-defined CMML patients. Next generation sequencing (NGS) using a 36-gene panel was performed on 350 patients with Illumina HiSeq4000 platform with median read depth of 400X. RNA sequencing (RNA-seq) was performed on 25 patients by bulk whole transcriptome sequencing using Illumina TruSeq. DNA immunoprecipitation sequencing (DIP-seq) was performed on 18 patients using 5-methylcystocine (5mC), 5-hydroxymethylcytosine (5hmC) and bridging monoclonal antibodies with subsequent paired-end sequencing using HiSeq4000. Chromatin immunoprecipitation sequencing (ChIP-seq) was performed on 30 patients with Illumina HiSeq2500 to a depth of 25 million for histone 3 lysine 4 monomethylation (H3K4me1) and histone 3 lysine 4 trimethylation (H3K4me3) and 50 million reads for histone 3 lysine 27 trimethylation (H3K27me3) and Input per sample. Results: Five hundred and seventy-three patients with WHO defined CMML were included; median age 71 years (range 18-95 years), 67% males. 282 patients had pCMML (49%), while 291 (51%) had dCMML. As pre-defined, patients with pCMML were more likely to have higher absolute monocyte counts (p<0.0001), circulating immature myeloid cells (p<0.0001), PB blasts (p<0.0001), and higher lactate dehydrogenase levels (p=0.03). At last follow up 234 (41%) deaths and 70 (20%) leukemic transformations were documented. The median OS for pCMML vs dCMML in this cohort was 19 months vs 30 months (p<0.0001, Figure 1A) and validated in an independent Austrian cohort (p=0.02). Genomic profiling: NGS performed on 350 patients (BM MNC) revealed a higher frequency of NRAS (35 vs 17%, p=0.004), cumulative RAS pathway (NRAS, KRAS, CBL and PTPN11) (73 vs 47%, p=0.001), ASXL1 (p=0.003) and JAK2V617F (p=0.04) mutations in pCMML relative to dCMML (Figure 1B); while dCMML had a higher frequency of SF3B1 mutations (p=0.02). There were no differences in distribution of TET2 and SRSF2Transcriptomic analysis: RNA-seq was performed on PB MNC from RAS pathway mutant pCMML patients (n=12) and RAS pathway wildtype dCMML patients (n=13). Unsupervised clustering analysis resulted in appropriate segregation revealing distinct expression profiles between disease subtypes (Figure 1C). Compared to dCMML, 3729 genes were significantly differentially upregulated and 2658 genes were differentially downregulated in pCMML. Among genes most highly upregulated were mitotic checkpoint kinases including AURBK, PLK1, PLK2, PLK4 andEpigenetic profiling: ChIP-seq of PB and BM MNC from pCMML (n=18) and dCMML (n=12) patients and healthy, age-matched controls (n=10) revealed a global increase in H3K4me1, without significant differences in H3K4me3 or H3K27me3 occupancies (regardless of stratification by ASXL1 mutational status; 40% ASXL1mt in pCMML, 30% dCMML) in pCMML vs dCMML (Figure 1D). H3K4me1 occupancy was also increased in a sequence-specific manner at the transcription start sites of the aforementioned mitotic kinases (PLK1 and WEE1). DIP-seq was performed on PB MNC to assess global differences in 5-mC and 5-hmC levels, between pCMML (n=9) and dCMML (n=9), with no differences seen between the two subtypes (regardless of TET2 mutational status, 40% TET2mt in each subtype) (Figure 1E). Conclusions: Despite the somewhat arbitrary WBC distinction between pCMML and dCMML, clear phenotypic, genetic, transcriptomic, epigenetic and survival differences exist between the two subtypes, providing strong biological rationale for this distinction. pCMML patients have a higher frequency of oncogenic RAS pathway mutations, a unique transcriptomic profile enriched in mitotic check point kinases and a unique chromatin configuration with global and sequence specific enrichment in H3K4me1, with no significant global differences in 5mC, 5hmC, or H3K4me3 and H3K27me3 occupancy. Figure 1 Disclosures Geissler: AOP: Honoraria; Pfizer: Honoraria; AstraZeneca: Honoraria; Novartis: Honoraria; Celgene: Honoraria; Roche: Honoraria; Abbvie: Honoraria; Ratiopharm: Honoraria; Amgen: Honoraria. Al-Kali:Astex Pharmaceuticals, Inc.: Research Funding. Patnaik:Stem Line Pharmaceuticals.: Membership on an entity's Board of Directors or advisory committees.
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Hatzi, Katerina, Calvo-Vidal Maria Nieves, Leandro Cerchietti, and Ari M. Melnick. "The Histone Demethylase LSD1 Acts As a BCL6 Corepressor In Germinal Center B Cells." Blood 122, no. 21 (November 15, 2013): 781. http://dx.doi.org/10.1182/blood.v122.21.781.781.
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Abstract The BCL6 transcription repressor (B-Cell Lymphoma 6) is required for B cells to form germinal centers (GC) and undergo antibody affinity maturation. BCL6 mediates its functions mainly through the recruitment of corepressor complexes with distinct enzymatic activities including the SMRT/NCOR, BCOR and MTA3 corepressors. Preliminary chromatin immunoprecipitation (ChIP) studies designed to dissect the biochemical actions of BCL6 on chromatin, showed that BCL6 inhibition results in considerable induction of H3K4me2 levels at selected BCL6 target loci. Consistent with these observations a BCL6 expressing lymphoma cell line had lower levels of H3K4me2 compared to BCL6 negative lymphoma cells. Hence, we hypothesized that in addition to HDAC and PRC1-like complexes BCL6 associates with distinct complexes bearing H3K4me2 demethylase activity. We observed that one of the few H3K4 demethylases known to target H3K4me1/2 residues, Lysine Specific Demethylase-1 (LSD1) is highly expressed in GC B cells. More specifically both LSD1 transcript and protein levels were induced in the transition from naïve to GC B cell state, similar to BCL6 levels. Collectively these results indicate that BCL6 might functionally interact with LSD1 in GC B cells to mediate transcriptional repression of key gene networks. We initially asked if BCL6 and LSD1 proteins interact in GC-derived B cells using coimmunoprecipitation and GST pull down experiments. We found that BCL6 and LSD1 proteins endogenously interact in these cells. Additional GST pull-down experiments using full length GST-LSD1 and in-vitro translated S35 BCL6 confirmed this interaction suggesting it is possibly direct. Moreover, immunoprecitated BCL6 complexes could demethylate H3K4me1 but not H3K4me3 in in vitro assays using histone substrates. To further evaluate the role of LSD1 in the physiological GC response we challenged C57BL/6 mice with T-cell dependent antigen (sheep red blood cells) and we examined GC formation in mice treated with a specific LSD1 inhibitor compared to untreated control mice. LSD1 inhibition resulted in GC formation deficiency indicating that LSD1 enzymatic activity plays key roles in normal immune response. To study the functional role of BCL6-LSD1 complexes we performed Q-ChIP assays for LSD1 at BCL6 target loci and showed that several BCL6 target loci, including MLL2, CDKN1A, BRAF and CCR6 are also bound by LSD1. To extend these findings we performed LSD1 ChIP-seq in a GC-derived lymphoma cell line. These genomic studies revealed an extensive overlap of BCL6 and LSD1 binding sites (1509 common loci). Roughly 560 gene promoters were bound by both BCL6 and LSD1 at the same site. Moreover, about 1,000 BCL6-LSD1 binding sites were outside promoters and might play a role in the regulation of inter/intragenic elements similar to the role of LSD1 in enhancer regulation in embryonic stem cells. Loss of BCL6 at several targeted loci resulted in LSD1 release from the chromatin suggesting that BCL6 mediates tethering of LSD1 at these loci. Genes bound or associated with BCL6-LSD1 complexes were significantly enriched in pathways such as B cell activation, chromatin organization and cell cycle control. Taken together these results suggest that LSD1 acts as a BCL6 corepressor in GC B cells and is an essential transcriptional regulator for the formation of normal GC response. BCL6 and LSD1 possibly cooperate to establish a transcriptional program crucial for the GC stage of B cell differentiation. Because LSD1 is expressed at high levels in lymphoma cells, targeting the LSD1 enzymatic activity could potentially be exploited as a lymphoma targeted therapy. Disclosures: No relevant conflicts of interest to declare.
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Wang, Xiaokang, Wojciech Rosikiewicz, Yurii Sedkov, Baisakhi Mondal, Tanner Martinez, Satish Kallappagoudar, Andrey Tvardovskiy, et al. "The MLL3/4 complexes and MiDAC co-regulate H4K20ac to control a specific gene expression program." Life Science Alliance 5, no. 11 (July 12, 2022): e202201572. http://dx.doi.org/10.26508/lsa.202201572.
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The mitotic deacetylase complex MiDAC has recently been shown to play a vital physiological role in embryonic development and neurite outgrowth. However, how MiDAC functionally intersects with other chromatin-modifying regulators is poorly understood. Here, we describe a physical interaction between the histone H3K27 demethylase UTX, a complex-specific subunit of the enhancer-associated MLL3/4 complexes, and MiDAC. We demonstrate that UTX bridges the association of the MLL3/4 complexes and MiDAC by interacting with ELMSAN1, a scaffolding subunit of MiDAC. Our data suggest that MiDAC constitutes a negative genome-wide regulator of H4K20ac, an activity which is counteracted by the MLL3/4 complexes. MiDAC and the MLL3/4 complexes co-localize at many genomic regions, which are enriched for H4K20ac and the enhancer marks H3K4me1, H3K4me2, and H3K27ac. We find that MiDAC antagonizes the recruitment of UTX and MLL4 and negatively regulates H4K20ac, and to a lesser extent H3K4me2 and H3K27ac, resulting in transcriptional attenuation of associated genes. In summary, our findings provide a paradigm how the opposing roles of chromatin-modifying components, such as MiDAC and the MLL3/4 complexes, balance the transcriptional output of specific gene expression programs.
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Sokolov, Alexey, Svetlana Zhenilo, Sergey Rastorguev, Alexander Mazur, and Egor Prokhortchouk. "Analysis of distribution of chromatin marks across "divergence islands" in three-spined stickleback (Gasterosteus aculeatus)." F1000Research 5 (December 20, 2016): 2880. http://dx.doi.org/10.12688/f1000research.10428.1.
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The three-spined stickleback (Gasterosteus aculeatus) is a well-known model organism for studying adaptations to water salinity. In this work, we investigate the dynamics of an epigenetic landscape of water salinity adaptation using three chromatin marks: H3K27ac, H3K4me1 and H3K4me3. The choice of marks was determined by the fact that some adaptive genomic loci are situated in gene-free regions, suggesting their regulatory role as enhancers. Histone modifications seem to be a promising mechanism that could regulate such regions. Difference between histone modifications in sea and freshwater - both in genes and intergenic enhancers - may contribute to epigenetic plasticity of stickleback adaptation. As a result of this study, we found differential chromatin peaks in "divergence islands" at enhancer elements and promoters of genes, which are responsible for stress adaptation and homeostasis. However, a full genome study analysis is required to fully understand mechanism of adaptation to water salinity.
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Tian, Yi, Zhengcai Jia, Jun Wang, Zemin Huang, Jun Tang, Yanhua Zheng, Yan Tang, et al. "Global Mapping of H3K4me1 and H3K4me3 Reveals the Chromatin State-Based Cell Type-Specific Gene Regulation in Human Treg Cells." PLoS ONE 6, no. 11 (November 23, 2011): e27770. http://dx.doi.org/10.1371/journal.pone.0027770.
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Aday, Aaron W., Lihua Julie Zhu, Abirami Lakshmanan, Jie Wang, and Nathan D. Lawson. "Identification of cis regulatory features in the embryonic zebrafish genome through large-scale profiling of H3K4me1 and H3K4me3 binding sites." Developmental Biology 357, no. 2 (September 2011): 450–62. http://dx.doi.org/10.1016/j.ydbio.2011.03.007.
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Sakabe, Noboru J., Ivy Aneas, Nicholas Knoblauch, Debora R. Sobreira, Nicole Clark, Cristina Paz, Cynthia Horth, et al. "Transcriptome and regulatory maps of decidua-derived stromal cells inform gene discovery in preterm birth." Science Advances 6, no. 49 (December 2020): eabc8696. http://dx.doi.org/10.1126/sciadv.abc8696.
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While a genetic component of preterm birth (PTB) has long been recognized and recently mapped by genome-wide association studies (GWASs), the molecular determinants underlying PTB remain elusive. This stems in part from an incomplete availability of functional genomic annotations in human cell types relevant to pregnancy and PTB. We generated transcriptome (RNA-seq), epigenome (ChIP-seq of H3K27ac, H3K4me1, and H3K4me3 histone modifications), open chromatin (ATAC-seq), and chromatin interaction (promoter capture Hi-C) annotations of cultured primary decidua-derived mesenchymal stromal/stem cells and in vitro differentiated decidual stromal cells and developed a computational framework to integrate these functional annotations with results from a GWAS of gestational duration in 56,384 women. Using these resources, we uncovered additional loci associated with gestational duration and target genes of associated loci. Our strategy illustrates how functional annotations in pregnancy-relevant cell types aid in the experimental follow-up of GWAS for PTB and, likely, other pregnancy-related conditions.
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Greenfield, Graeme, Suzanne McPherson, James Smith, Adam Mead, Claire Harrison, Ken Mills, and Mary Frances McMullin. "Modification of the Histone Landscape with JAK Inhibition in Myeloproliferative Neoplasms." Cancers 12, no. 9 (September 18, 2020): 2669. http://dx.doi.org/10.3390/cancers12092669.
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Dysregulation of epigenetic processes is increasingly understood to play a role in the pathogenesis of myeloproliferative neoplasms (MPNs). Ruxolitinib, a JAK/STAT inhibitor, has proved a useful addition to the therapeutic arsenal for these disorders, but has limited disease modifying activity. We determined the effect of JAK inhibition on the histone landscape of MPN cells in cell line models of MPNs and validated using samples from the MAJIC randomised clinical trial of ruxolitinib in polycythaemia vera and essential thrombocythaemia. We demonstrated an epigenetic modifying effect of ruxolitinib using a histone modification assay. The majority of 21 histone H3 modifications were upregulated, with H3K27me3 and H3K36me2 significant in the combined cell line results. Chromatin immunoprecipitation and sequencing (CHIP-seq) for three marks of interest, H3K4me1, H3K4me3 and H3K27ac, was consistent with the histone modification assay showing a significant increase in H3K4me3 and H3K27ac peaks at promoter regions, both marks of active transcription. In contrast, RNA sequencing demonstrates a coordinated reduction in gene expression in a number of cell pathways including PI3K-AKT signalling, transcriptional misregulation in cancer and JAK-STAT signalling in spite of these histone changes. This highlights the complex mechanisms of transcriptional control within the cells which was reflected in analysis of the histone landscape in patient samples following ruxolitinib treatment.
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Kwon, Minjung, Kihyun Park, Kwangbeom Hyun, Jeong-Heon Lee, Linjiao Zhou, Young-Wook Cho, Kai Ge, David G. Skalnik, Tom W. Muir, and Jaehoon Kim. "H2B ubiquitylation enhances H3K4 methylation activities of human KMT2 family complexes." Nucleic Acids Research 48, no. 10 (May 4, 2020): 5442–56. http://dx.doi.org/10.1093/nar/gkaa317.
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Abstract In mammalian cells, distinct H3K4 methylation states are created by deposition of methyl groups by multiple complexes of histone lysine methyltransferase 2 (KMT2) family proteins. For comprehensive analyses that directly compare the catalytic properties of all six human KMT2 complexes, we employed a biochemically defined system reconstituted with recombinant KMT2 core complexes (KMT2CoreCs) containing minimal components required for nucleosomal H3K4 methylation activity. We found that each KMT2CoreC generates distinct states and different levels of H3K4 methylation, and except for MLL3 all are stimulated by H2Bub. Notably, SET1BCoreC exhibited the strongest H3K4 methylation activity and, to our surprise, did not require H2B ubiquitylation (H2Bub); in contrast, H2Bub was required for the H3K4me2/3 activity of the paralog SET1ACoreC. We also found that WDR5, RbBP5, ASH2L and DPY30 are required for efficient H3K4 methyltransferase activities of all KMT2CoreCs except MLL3, which could produce H3K4me1 in the absence of WDR5. Importantly, deletion of the PHD2 domain of CFP1 led to complete loss of the H3K4me2/3 activities of SET1A/BCoreCs in the presence of H2Bub, indicating a critical role for this domain in the H2Bub-stimulated H3K4 methylation. Collectively, our results suggest that each KMT2 complex methylates H3K4 through distinct mechanisms in which individual subunits differentially participate.
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Cao, Peng, Fan Li, Yajie Xiao, Shan Hu, Kangle Kong, Peng Han, Jiaqi Yue, et al. "Identification and Validation of 7-lncRNA Signature of Epigenetic Disorders by Comprehensive Epigenetic Analysis." Disease Markers 2022 (February 21, 2022): 1–14. http://dx.doi.org/10.1155/2022/5118444.
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The survival rate of patients with lung adenocarcinoma (LUAD) is low. This study analyzed the correlation between the expression of long noncoding RNA (lncRNA) and epigenetic alterations along with the investigation of the prognostic value of these outcomes for LUAD. Differentially expressed lncRNAs were identified based on multiomic data and positively related genes using DESeq2 in R, differentially histone-modifying genes specific to LUAD based on histone modification data, gene enhancers from information collected from the FANTOM5 (Function Annotation Of The Mammalian Genome-5) (fantom.gsc.riken.jp/5) human enhancer database, gene promoters using the ChIPseeker and the human lincRNAs Transcripts database in R, and differentially methylated regions (DMRs) using Bumphunter in R. Overall survival was estimated by Kaplan-Meier, comparisons were performed among groups using log-rank tests to derive differences between sample subclasses, and epigenetic lncRNAs (epi-lncRNAs) potentially relevant to LUAD prognosis were identified. A total of seven dysregulated epi-lncRNAs in LUAD were identified by comparing histone modifications and alterations in histone methylation regions on lncRNA promoter and enhancer elements, including H3K4me2, H3K27me3, H3K4me1, H3K9me3, H4K20me1, H3K9ac, H3K79me2, H3K27ac, H3K4me3, and H3K36me3. Furthermore, 69 LUAD-specific dysregulated epi-lncRNAs were identified. Moreover, lncRNAs-based prognostic analysis of LUAD samples was performed and explored that seven of these lncRNAs, including A2M-AS1, AL161431.1, DDX11-AS1, FAM83A-AS1, MHENCR, MNX1-AS1, and NKILA (7-EpiLncRNA), showed the potential to serve as markers for LUAD prognosis. Additionally, patients having a high 7-EpiLncRNA score showed a generally more unfavorable prognosis compared with those which scored lower. Seven lncRNAs were identified as markers of prognosis in patients with LUAD. The outcomes of this research will help us understand epigenetically aberrant regulation of lncRNA expression in LUAD in a better way and have implications for research advances in the regulatory role of lncRNAs in LUAD.
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Alaterre, Elina, Laurie Herviou, Hugues De Boussac, Giorgio Papadopoulos, Stéphanie Boireau, Nicolas Robert, Guilhem Requirand, et al. "Comprehensive Characterization of the Epigenetic Landscape in Multiple Myeloma." Blood 136, Supplement 1 (November 5, 2020): 2–3. http://dx.doi.org/10.1182/blood-2020-138801.
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Multiple myeloma (MM) is a B cell neoplasia characterized by the accumulation of clonal plasma cells within the bone marrow. Epigenetic modifications, including DNA methylation and histone post-translational changes, are involved in MM development and progression, but also in drug resistance. We have derived a large cohort of patient-derived HMCLs that remain dependent on the addition of exogenous MM bone marrow growth factors, reflecting primary tumor conditions. We have described their molecular diversity by analyzing the gene expression profile and mutational landscape and have showed that HMCL molecular diversity reflects part of the molecular heterogeneity of primary MM cells. However, the epigenetic landscape of HMCLs has never been described. A comprehensive characterization of the epigenetic landscape of HMCLs would advance our understanding of MM pathophysiology and may attempt to identify new therapeutic targets. In our study, we presented the epigenetic landscape of HMCLs. We performed chromatin immunoprecipitation sequencing (ChIP-seq) to analyze changes of the histone marks (H3K9me3 to follow heterochromatin, H3K4me1 and H3K27ac to follow enhancer activity, H3K4me3 and H3K36me3 to follow active transcription and H3K27me3 to follow Polycomb-silenced chromatin) on 16 HMCLs, representative of the molecular heterogeneity of MM. The differential analysis of histone modification profiles of HMCLs highlighted links between histone modifications and cytogenetic abnormalities or recurrent mutations. H3K4me3 and H3K27me3 profile analysis revealed specific clusters of HMCL related to 1q gain and t(4;14) translocation, respectively. These two cytogenetic abnormalities lead to deregulation of epigenetic player expression (e.g. SETDB1 and MMSET) and thus, could alter histone modification profile. Using histone modifications associated to enhancer regions (H3K4me1 and H3K27ac), we identified super-enhancers (SE) associated with genes involved in the biology of MM. 607 to 2510 predicted super-enhancers per HMCL were identified, including MAF, MYC, CCND1, CCND2, TRAF3 or NSD2. These super-enhancers differ from typical enhancers in both size and H3K4me1 and H3K27ac levels. The SE-associated genes identified in HMCLs with a prognostic value in two independent cohorts of newly diagnosed patients (CoMMpass cohort; N = 674 and Montpellier cohort; N = 69 with RNA-seq data) were used to build a score predicting MM patient outcome (Figure 1). Moreover, among the 28 genes that compose the risk-score (BSG, HK2, HNRNPC, HSPA9, IL10, ILF3, LDHB, MDH1, MYBPC2, NCL, NUDC, PARP1, PDIA6, PRPS1, RPL8, RPL13A, RPL27A, RPL35, SF3B2, SLC7A5, SLC25A39, SMARCA4, SPN, STC2, THY1, TNPO2, TPR), public datasets of RNAi and CRISPR/Cas9 screening revealed four genes (YWHAQ, IL10, HK2 and THY1) identified as significant essential myeloma genes, suggesting that they could represent potential therapeutic targets. We also identified promoters of genes characterized by a co-localization of H3K9me3 and H3K27me3 repressive marks in HMCLs. We evaluated the prognostic value of these genes in the CoMMpass and Montpellier cohorts, and selected genes associated with poor outcome when their expression is low in MM cells of patients (ARHGEF5, BIVM, DEF8, GRID2IP, HDAC9, HSPA1L, KDM4C, NLRP2, P4HA3, PAG1, PM20D1, RMND5A, SEMA6A, SFMBT2, THEMIS2, TPRKB, ZFP2 and ZNF5188B) underlining potential new tumor suppressor genes. These potential tumor suppressor genes associated with repressive histone marks were used to build a second risk score splitting MM patients in low- and high-risk groups in CoMMpass and Montpellier cohorts. Finally, we explored H3K4me3 marks comparing drug-resistant and -sensitive HMCLs (N = 16) to identify regions involved in drug resistance. From these data, we developed epigenetic biomarkers based on this H3K4me3 modification predicting lenalidomide and romidepsin HDCAi response. This study provides a comprehensive characterization of the MM epigenetic landscape representing unique resources for future biological studies and could help to identify novel critical epigenetic modifications involved in MM progression and drug resistance. Furthermore, risk-scores based on super enhancers and repressive regions together with epigenetic biomarkers of drug response could represent new tools for precision oncology in MM. Disclosures De Boussac: Diag2Tec: Current Employment. Bruyer:Diag2Tec: Current Employment. Vincent:janssen: Membership on an entity's Board of Directors or advisory committees, Other: Congress support; Celgene: Membership on an entity's Board of Directors or advisory committees, Other: Congress support; takeda: Membership on an entity's Board of Directors or advisory committees, Other: Congress support. Moreaux:Diag2Tec: Consultancy.
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Wang, Hongfang, Chongzhi Zang, Len Taing, Hoifung Wong, Yumi Yashiro-Ohtani, Stephen Blacklow, Warren S. Pear, X. Shirley Liu, and Jon C. Aster. "Genome-Wide Analysis of NOTCH1, ETS Family Factors, and RUNX1 Binding in Human T Lymphoblastic Leukemia Cells Reveals Distinct Regulatory Elements." Blood 120, no. 21 (November 16, 2012): 1277. http://dx.doi.org/10.1182/blood.v120.21.1277.1277.
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Abstract Abstract 1277 NOTCH1 regulates gene expression by forming transcription activation complexes with the DNA-binding factor RBPJ and gain-of-function NOTCH1 mutations are common in human and murine T lymphoblastic leukemia/lymphoma (T-LL). Via ChIP-seq studies of T-LL cells with constitutive Notch activation, we previously showed that NOTCH1/RBPJ binding sites in T-LL genomes are highly enriched for motifs corresponding to Ets factors and Runx factors. In this study, we determined the relationship of NOTCH1, RBPJ, ETS1, GABPA and RUNX1 binding sites in human T-LL cells by performing ChIP-Seq for each of these factors, as well as the chromatin marks H3K4me1, H3K4me3, and H3K27me3, and aligning the resulting sequences to human genome reference hg19 using programs available through Cistrome. Peak calling was performed with MACS2, and motif analysis was performed using SeqPos, which relies on JASPAR, TRANSFAC, Protein Binding Microarray (PBM), Yeast-1-hybrid (y1h), and human protein-DNA interaction (hPDI) databases to find known motifs and can also perform de novo motif discovery. Our analysis showed even more pervasive overlap of NOTCH1/RBPJ binding with ETS1/GABPA and RUNX1 factor binding than was predicted by motif analysis, in part due to binding of Ets factors and RUNX1 to non-canonical sequences. Heat-map analysis with K-means clustering on NOTCH1 binding regions identified three major classes of RBPJ/NOTCH1: class 1, characterized by high NOTCH/RBPJ signals, binding of the cofactors ZNF143, ETS1 and GABPA, high H3K4me3 signals, localization to promoters, and binding motifs for ZNF143; class 2, characterized by low NOTCH/RBPJ signals, binding of the cofactors ETS1, GABPA and RUNX1, high H3K4me3 signals, and Ets factor and CREB binding motifs; and class 3, characterized by high NOTCH/RBPJ signals, binding of RUNX1 and ETS1 cofactors, high H3K4me1 signals, intergenic localization (consistent with enhancers), and motifs for RUNX factors, ETS factors, and RBPJ. Of note, the nearest binding sites to the most responsive NOTCH1 target genes (defined as >2 fold stimulation when NOTCH1 was activated following release of gamma-secretase inhibitor (GSI) blockade by drug washout) were preferentially associated with Class 3 sites. Furthermore, shRNA knockdown of Ets factors and RUNX1 in T-LL cell lines induced apoptosis and reduced cell proliferation, implicating these factors in maintenance of T-LL growth and survival. Combination of knockdown of either Ets factors or RUNX1 with GSI treatment resulted in more severe phenotype in terms of apoptosis and cell growth compared to the knockdown or GSI treatment alone. In summary, our studies represent a step forward towards genome-wide understanding of how Notch works in concerts with other transcription factors to regulate the transcriptome of T-LL cells. Disclosures: No relevant conflicts of interest to declare.
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Kurotaki, Daisuke, Naoki Osato, Akira Nishiyama, Michio Yamamoto, Tatsuma Ban, Hideaki Sato, Jun Nakabayashi, et al. "Essential role of the IRF8-KLF4 transcription factor cascade in murine monocyte differentiation." Blood 121, no. 10 (March 7, 2013): 1839–49. http://dx.doi.org/10.1182/blood-2012-06-437863.
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Key Points IRF8 induces the Klf4 gene in myeloid progenitors; this transcription factor cascade is essential for Ly6C+ monocyte development. IRF8 binding to genomic targets promotes H3K4me1, a chromatin signature for promoter-distal enhancers, thereby inducing gene expression.
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Rodier, Julie-Anne, and Catherine Pena. "H3K4me1 in VTA Mediates Early Life Stress-Induced Stress Sensitivity." Biological Psychiatry 89, no. 9 (May 2021): S75. http://dx.doi.org/10.1016/j.biopsych.2021.02.201.
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Rasid, Orhan, Christine Chevalier, Tiphaine Marie-Noelle Camarasa, Catherine Fitting, Jean-Marc Cavaillon, and Melanie Anne Hamon. "H3K4me1 Supports Memory-like NK Cells Induced by Systemic Inflammation." Cell Reports 29, no. 12 (December 2019): 3933–45. http://dx.doi.org/10.1016/j.celrep.2019.11.043.
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Drucker, Kristen, Connor Yanchus, Thomas Kollmeyer, Asma Ali, Decker Paul, Matthew Kosel, Arijit Panda, et al. "EPCO-02. GERMLINE SINGLE NUCLEOTIDE POLYMORPHISM rs55705857 AT 8q24 INTERACTS WITH SOMATIC IDH1 MUTATION TO ENHANCE HUMAN GLIOMA FORMATION." Neuro-Oncology 23, Supplement_6 (November 2, 2021): vi1. http://dx.doi.org/10.1093/neuonc/noab196.001.
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Abstract BACKGROUND Determination of the causation of germline single nucleotide polymorphisms (SNPs) located in non-coding regions of the genome is challenging. The genomic region of 8q24 has been identified as important in many kinds of cancer, linked to a topologically associated domain (TAD) encompassing MYC; this TAD contains a GWAS SNP (rs55705857) associated with IDH-mutant glioma. METHODS Germline genotyping data from 622 IDH-mutant glioma and 668 controls were used to fine map the rs55705857 locus by detailed haplotype analysis. Chromatin immunoprecipitation sequencing (ChIP-seq) of histone markers H3K4me1, H3K4me3, H3K27ac and H3K36me3 was performed on normal brain samples (n=8) and human glioma samples (n=11 IDH-wt and 52 IDH-mut). RNAseq from 9 normal and 83 brain tumors (n=26 IDH-wt and 55 IDH-mut) were used to assess differential gene expression. RESULTS Fine-mapping identified rs55705857 SNP as the most likely causative allele (OR=8.69; p<0.001) within 8q24 for the development of IDH-mutant glioma. At rs55705857, both H3K27ac and H3K4me1 in IDH-mutant vs IDH-wt tumors were increased 3.05- and 1.58-fold, respectively (DiffBind; p=5.81×10-7 and p=2.31×10-3). ChromHMM analysis of the marks indicated that promoter and enhancer functions were significantly increased, and the activity broadened at rs55705857 in IDH-mut gliomas compared to IDH-wt tumors and normal brain samples. This enhancement correlated with significant increased MYC expression in IDH-mut gliomas (p=3.1×10-13), as well as alterations of Myc signaling targets. Publicly available ATACseq, ChIPseq and long-range DNA interaction data demonstrated that the rs55705857 locus is open and interacts with the MYC promoter. CONCLUSIONS Fine-mapping of the 8q24 locus provided strong evidence that rs55705857 is the causative 8q24 locus associated with IDH-mut glioma. Functional experiments suggest that IDH mutation facilitates rs55705857 interaction with MYC to alter downstream MYC targets.
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Bendayan, Marion, Liliana Caceres, Emine Saïs, Nelly Swierkowski-Blanchard, Laura Alter, Amélie Bonnet-Garnier, and Florence Boitrelle. "Human Sperm Morphology as a Marker of Its Nuclear Quality and Epigenetic Pattern." Cells 11, no. 11 (May 30, 2022): 1788. http://dx.doi.org/10.3390/cells11111788.
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Background: Human sperm chromatin condensation is a sum of epigenetic events that allows for the near-complete replacement of histones with protamines. Under high-magnification microscopy, nuclear vacuoles have been described as thumbprints with poor chromatin condensation. The objective of this study is to examine whether vacuolated spermatozoa carry specific epigenetic marks, which may influence embryo development. Methods: The presence and three-dimensional distribution of ten epigenetic marks (protamine-P2, histone-H3, H3K4me1/me2/me3, H3K9me1/me2/me3, H3K27me3, H4k20me2) were evaluated and compared in morphometrically normal spermatozoa according to the presence or absence of a large vacuole occupying more than 15% of the head surface (n = 4193). Results: Vacuolated spermatozoa were significantly more frequently labelled with H3 and H3K4me3 than normal spermatozoa (88.1% ± 2.7 and 78.5% ± 5.2 vs. 74.8% ± 4.8 and 49.1% ± 7.4, respectively; p = 0.009 and p < 0.001) and significantly less marked by P2 and H3K27me3 (50.2% ± 6.2 and 63.9% ± 6.3 vs. 82.1% ± 4.4 and 73.6% ± 5.1, respectively; p < 0.001 and p = 0.028). In three dimensions, vacuoles are nuclear concavities filled with DNA carrying the H3K4me3 marker. Conclusion: High-magnification microscopy is a simple tool to estimate in real time the sperm epigenetic profile. The selection of normal spermatozoa without vacuoles and the deselection of spermatozoa with vacuoles appear to be epigenetically favorable to embryo development and safe offspring.
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Kingsley, N. B., Colin Kern, Catherine Creppe, Erin N. Hales, Huaijun Zhou, T. S. Kalbfleisch, James N. MacLeod, Jessica L. Petersen, Carrie J. Finno, and Rebecca R. Bellone. "Functionally Annotating Regulatory Elements in the Equine Genome Using Histone Mark ChIP-Seq." Genes 11, no. 1 (December 18, 2019): 3. http://dx.doi.org/10.3390/genes11010003.
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One of the primary aims of the Functional Annotation of ANimal Genomes (FAANG) initiative is to characterize tissue-specific regulation within animal genomes. To this end, we used chromatin immunoprecipitation followed by sequencing (ChIP-Seq) to map four histone modifications (H3K4me1, H3K4me3, H3K27ac, and H3K27me3) in eight prioritized tissues collected as part of the FAANG equine biobank from two thoroughbred mares. Data were generated according to optimized experimental parameters developed during quality control testing. To ensure that we obtained sufficient ChIP and successful peak-calling, data and peak-calls were assessed using six quality metrics, replicate comparisons, and site-specific evaluations. Tissue specificity was explored by identifying binding motifs within unique active regions, and motifs were further characterized by gene ontology (GO) and protein–protein interaction analyses. The histone marks identified in this study represent some of the first resources for tissue-specific regulation within the equine genome. As such, these publicly available annotation data can be used to advance equine studies investigating health, performance, reproduction, and other traits of economic interest in the horse.
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Hu, Qiwen, Casey S. Greene, and Elizabeth A. Heller. "Specific histone modifications associate with alternative exon selection during mammalian development." Nucleic Acids Research 48, no. 9 (April 22, 2020): 4709–24. http://dx.doi.org/10.1093/nar/gkaa248.
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Abstract Alternative splicing (AS) is frequent during early mouse embryonic development. Specific histone post-translational modifications (hPTMs) have been shown to regulate exon splicing by either directly recruiting splice machinery or indirectly modulating transcriptional elongation. In this study, we hypothesized that hPTMs regulate expression of alternatively spliced genes for specific processes during differentiation. To address this notion, we applied an innovative machine learning approach to relate global hPTM enrichment to AS regulation during mammalian tissue development. We found that specific hPTMs, H3K36me3 and H3K4me1, play a role in skipped exon selection among all the tissues and developmental time points examined. In addition, we used iterative random forest model and found that interactions of multiple hPTMs most strongly predicted splicing when they included H3K36me3 and H3K4me1. Collectively, our data demonstrated a link between hPTMs and alternative splicing which will drive further experimental studies on the functional relevance of these modifications to alternative splicing.
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Nanan, Kyster, and David P. LeBrun. "Identification and Characterization By ChIP-Seq Of Genomic Sites Bound By E2A-PBX1 In Acute Lymphoblastic Leukemia Demonstrates Associations With p300 Recruitment, Transcriptionally Active Chromatin and Abundant Transcription." Blood 122, no. 21 (November 15, 2013): 2501. http://dx.doi.org/10.1182/blood.v122.21.2501.2501.
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Abstract E2A-PBX1 (EP1) is a chimeric oncogenic transcription factor expressed consequent to the 1;19 chromosomal translocation in cases of acute lymphoblastic leukemia (ALL). EP1 can induce transcription of reporter genes and EP1-driven oncogenesis requires direct binding of EP1 with the transcriptional co-activator and histone acetyltransferase p300. Therefore, we hypothesized that EP1 recruits p300 and other co-activators to cis-acting regulatory elements throughout the genome thereby inducing or maintaining transcription of target genes some of which contribute to the neoplastic phenotype. Here we have used chromatin immunoprecipitation followed by next generation DNA sequencing (ChIP-seq) to identify and characterize EP1-bound sites across the genome of the t(1;19)-associated, ALL-derived cell line RCH-ACV. ChIP was performed with an anti-FLAG antibody using sheared chromatin prepared from RCH-ACV cells that stably expressed FLAG-tagged EP1; ChIP from parent RCH-ACV cells not expressing FLAG-EP1 served as a negative control for peak calling. Parallel immunoprecipitations were performed with antibodies for p300 and the chromatin marks H3K4me3, H3K4me1 and H3K27me3. Sequencing of DNA purified from the immunoprecipitated material and of total RNA (RNA-seq) was carried out commercially by BGI whereas bioinformatic analyses were performed in-house. Bioinformatic analysis of data from replicate samples identified 3166 EP1 binding peaks across the RCH-ACV genome (irreproducible discovery rate threshold <0.01). Most EP1 binding sites were located in intronic (1408 sites) or intergenic (1346 sites) regions. Binding site consensus analysis showed overrepresentation of binding motifs for REST, CTCF, MYC, PAX5 and other transcription factors suggesting indirect recruitment of EP1 to DNA mediated by protein-protein interactions. EP1-bound regions were enriched for p300 binding (Figure 1), consistent with the documented importance of p300 recruitment in EP1 oncogenesis. A particular association with H3K4me3 relative to H3K4me1 or H3K27me3 (Figure 2) suggested association with active promoters. Three hundred and forty-two genes had EP1 binding sites within 1000 bp of their transcriptional start sites and these genes were associated with differentially abundant transcription (Figure 3, P<0.001). Querying the online Mammalian Phenotype Ontology tool with genes associated with EP1 binding generated terms that were obviously rich in phenotypes pertaining to B-lymphopoiesis. In summary, our results suggest that EP1 recruits p300 and other co-activators to transcriptionally active chromatin in ALL cells. Results from studies currently underway to confirm the dependency of target gene expression and p300 recruitment upon binding of EP1 at specific binding sites will be presented. Disclosures: No relevant conflicts of interest to declare.
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Okitsu, Cindy Yen, John Cheng Feng Hsieh, and Chih-Lin Hsieh. "Transcriptional Activity Affects the H3K4me3 Level and Distribution in the Coding Region." Molecular and Cellular Biology 30, no. 12 (April 19, 2010): 2933–46. http://dx.doi.org/10.1128/mcb.01478-09.
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ABSTRACT Histone lysine methylation and CpG DNA methylation contribute to transcriptional regulation. We have shown previously that dimethylated and trimethylated forms of histone H3 at lysine 4 (H3K4me2 and H3K4me3) are primarily depleted from CpG-methylated DNA regions by using patch-methylated stable episomes (minichromosomes) in human cells. This effect on H3K4me2 is clearly not linked to the transcriptional activity in the methylated DNA region; however, transcriptional activity may play a role in the presence of H3K4me3. Here, we present clear evidence of the impact of transcriptional activity on the overall level of H3K4me3 in the coding region and the lack of impact on H3K4me2. Our data also demonstrate the influence of transcriptional activity on the distribution of H3K4me3 and H3K4me2, but not that of total H3, in the 5′ end of the coding region relative to the 3′ end. The nature of the promoter (viral or endogenous) affects H3K4me3 much more than it affects H3K4me2, suggesting a potential fundamental difference in the recruitment of methyltransferase for H3K4 trimethylation.
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Bogliotti, Y. S., L. B. Ferré, D. J. Humpal, and P. J. Ross. "68 EPIGENETIC REMODELING OF HISTONE 3 MARKS DURING BOVINE PRE-IMPLANTATION DEVELOPMENT." Reproduction, Fertility and Development 26, no. 1 (2014): 148. http://dx.doi.org/10.1071/rdv26n1ab68.
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During pre-implantation development, substantial epigenetic changes occur that are thought to play key roles in achieving embryonic genome activation and totipotency. Embryonic genome activation occurs at the 8- to 16-cell stage in cattle and, although it is a crucial step of development, the specific mechanisms involved are still poorly understood. The aim of this study was to determine whether 4 histone 3 marks associated with active genes are remodelled during oocyte and early embryo development in cattle. The dynamics of acetylation of lysine 27 (H3K27ac), di-methylation of lysine 79 (H3K79me2), and mono- and tri-methylation of lysine 4 (H3K4me1, H3K4me3) were assessed by immunofluorescence and confocal microscopy. Ovaries were obtained from an abattoir. Immature germinal vesicle stage oocytes were aspirated from small antral follicles and matured for 24 h to the metaphase II stage (MII). Embryos were produced by in vitro fertilization and collected at different stages of development: pronuclear [PN; 18 h post-fertilization (hpf)], 2-cell (30 hpf), 4-cell (44 hpf), 8-cell (56 hpf), 16-cell (72 hpf), morula (120 hpf), and blastocyst (180 hpf). Three to 4 biological replicates were done per antibody and a total of 197 oocytes per embryo were imaged (8 to 16 per stage/antibody). The images were analysed using Fiji (Schindelin et al. 2012 Nat. Methods 9, 676–682). The average nuclear intensity per oocyte per embryo was adjusted by the average of 2 cytoplasmic areas (background). An ANOVA mixed model was used for statistical analysis using SAS (SAS Institute Inc., Cary, NC, USA). The least squares means of the different stages were compared (within each antibody group) using a Tukey-Kramer adjustment and were considered to be significantly different at P < 0.05. The H3K79me2 marks showed a significant increase from germinal vesicle to MII, a change opposite that of H3K27ac, which experienced a significant decrease between these two stages. The H3K4me1/me3 marks showed no significant changes during oocyte maturation. All 3 methylation marks presented a significant reduction in nuclear intensity from MII to PN, indicating that these marks are actively removed right after fertilization. The opposite effect was observed for the acetylation mark, in which the levels increased significantly from MII to PN. The H3K4me1/me3 marks showed a gradual decrease in intensity levels from the 2-cell stage onward, reaching a minimum at the 16-cell per morula stages. The H3K79me2 levels were low from PN to 16-cell stage, at which point its intensity levels began to increase, reaching statistical significance at the blastocyst stage. The H3K27ac marks showed a slow decrease in intensity levels from the PN stage, achieving statistical significance as it dropped to a minimum at the 16-cell stage. These results show that the global levels of the assayed epigenetic marks undergo dynamic changes during oocyte maturation and embryo development, suggesting that their remodelling may be important for early development. The authors thank Alta Genetics for providing the semen.
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Heuston, Elisabeth F., Cheryl A. Keller, Jens Lichtenberg, Stacie M. Anderson, NIH Intramural Sequencing Center, Ross C. Hardison, and David M. Bodine. "Establishment of Enhancer Elements during Erythro-Megakaryopoiesis." Blood 128, no. 22 (December 2, 2016): 1486. http://dx.doi.org/10.1182/blood.v128.22.1486.1486.
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Abstract Enhancers are cis acting regulatory modules associated with lineage-specific gene expression. The Encyclopedia of DNA Elements project (ENCODE) showed that enhancers are in open chromatin regions identified by the Assay for Transposable-Accessible Chromatin (ATAC) and bound with histone H3 is mono-methylated at lysine 4 (H3K4me1). Chromatin regions marked by H3K4me1 alone identifies "poised" enhancers (not active), while the additional presence and histone H3 acetylated at lysine 27 (H3K27ac) identifies "active" enhancers. To establish a genome-wide enhancer map in the erythro-megakaryocytic lineage, we performed ChIPSeq of H3K4me1 and H3K27ac in primary erythroblasts (EB) and megakaryocytes (MEG) isolated from mouse bone marrow. We also assayed primary mouse EB, MEG, hematopoietic stem and progenitor cells (LSK), and common myeloid progenitor cells (CMP) for open chromatin regions with ATAC and transcriptome profiling by RNASeq. Finally, we compared histone-defined enhancers in mature cells with the corresponding ATAC regions in progenitor cells to identify the preservation of poised and active enhancers through hematopoiesis. We identified 6565 and 3543 active enhancers in EB and MEG respectively; approximately 10% (434) were shared. We further refined our enhancer set to the ~90% of EB and MEG active enhancers that overlap with ATAC regions (AER, histone-marked active enhancer within an ATAC region). To identify enhancers in the open chromatin of progenitor cells, we overlaid EB and MEG AER with CMP ATAC sites. This revealed that 82% (5226/6399) of EB AER and 87% (1437/3302) of MEG AER were present in CMP. Overlaying the EB and MEG AER onto LSK ATAC showed that 67% (4278/6399) of EB-specific AER and 79% (2594/3302) of MEG-specific AER overlapped with LSK ATAC sites. To identify the EB and MEG AER in LSK-accessible chromatin that are active (not poised), we compared our LSK enhancer set with the indexing-first ChIP (iChIP) histone marks identified by Lara-Astiaso et al., (Science, 2014). 1840 of the 4278 (43%) LSK-accessible EB AER overlapped with LSK iChIP H3K4me1 marks; 632 of these (15% overall) also had the active H3K27ac mark. 1083 of the 2594 (42%) MEG AER that were present in LSK overlapped with LSK iChIP H3K4me1 marks; 241 of these (9% overall) had the H3K27ac mark. For both EB and MEG, AER not marked by iChIP K4me1 were within gene bodies. To further characterize enhancer roles in lineage commitment, we profiled super enhancers (SE), which have highly lineage-specific activity. We defined SE as the 2% of AER with the highest H3K27ac levels (Hnisz et al., Cell, 2013) and identified 101 EB and 98 MEG SE; all of these were cell-specific. We found that 65% (66/101) of EB SE and 87% (85/98) of MEG SE overlapped with LSK ATAC sites. 30 of the 66 (45%) LSK-accessible EB SE overlapped with LSK iChIP H3K4me1 marks; 9 of these (14% overall) also had the active H3K27ac mark. In comparison, 15 of the 85 (18%) LSK-accessible MEG SE overlapped with LSK iChIP H3K4me1 marks; 4 of these (5% overall) also had the active H3K27ac mark. We correlated our LSK-accessible, iChIP-marked active AER with gene expression by assigning each AER to the nearest gene. We then used RNASeq data to perform gene set enrichment analysis via Ingenuity Pathway Analysis. We found that the LSK-accessible EB-specific AER gene set included erythropoietin-regulated genes (p £ 9x10-5) and genes associated with Fanconi anemia (6x10-4). Conversely, LSK-accessible and iChIP-active MEG AER were associated an increase of progenitor cell populations and proliferation activities for several hematopoietic lineages (p £ 2x10-5). However, the genes in the non-megakaryocyte pathways were significantly down-regulated as LSK committed to the megakaryocyte lineage. In summary, our results demonstrate the establishment of poised and active enhancers in hematopoietic progenitors and their preservation through erythro-megakaryopoiesis. We show that >40% of EB and MEG enhancers were also enhancers in LSK and CMP; the EB and MEG enhancers that were not LSK enhancers were primarily within gene bodies. We also found that MEG, but not EB, super enhancers were less likely than conventional enhancers to be established in LSK. Finally, our data show that, while LSK-established EB enhancers target EB-specific functions, LSK-established MEG enhancers have more universal hematopoietic functions that are down-regulated during megakaryocytic lineage commitment. Disclosures No relevant conflicts of interest to declare.
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REN, GANG, KAIRONG CUI, Chengyu LIU, Jinfang (Jeff) Zhu, and KEJI ZHAO. "Negative regulation of Ifng expression by MLL4 through CNS-28." Journal of Immunology 204, no. 1_Supplement (May 1, 2020): 76.10. http://dx.doi.org/10.4049/jimmunol.204.supp.76.10.
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Abstract Interferon-g (IFN-g) is a key cytokine produced by type 1 T helper (Th1) cells in response to viral or intracellular bacterial infections in mammals. While a number of positive regulatory elements have been well studied, no negative regulatory elements at the Ifng locus for its expression have been identified. Here we report a negative regulatory mechanism of Ifng expression by MLL4, a histone methyltransferase responsible for H3K4me1. Deletion of Mll4 resulted in loss of H3K4me1 signals at the previously unrecognized CNS-28 site and led to enhanced IFN-g expression during Th1 differentiation. GATA3 bound to the CNS-28 site and deletion of Gata3 resulted in a similar upregulation of IFN-g with Mll4 deletion. Furthermore, deletion of Mll4 compromised GATA3 binding to CNS-28 and led to a substantial reorganization of the 3D chromatin structure at the Ifng genomic locus in both naïve CD4+ T cells and Th1 cells. Thus, we conclude that MLL4 negatively regulates Ifng expression by facilitating GATA3 binding to the CNS-28 site and maintaining a special local chromatin organization.
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Wang, Chaochen, Ji-Eun Lee, Binbin Lai, Todd S. Macfarlan, Shiliyang Xu, Lenan Zhuang, Chengyu Liu, Weiqun Peng, and Kai Ge. "Enhancer priming by H3K4 methyltransferase MLL4 controls cell fate transition." Proceedings of the National Academy of Sciences 113, no. 42 (October 3, 2016): 11871–76. http://dx.doi.org/10.1073/pnas.1606857113.
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Transcriptional enhancers control cell-type–specific gene expression. Primed enhancers are marked by histone H3 lysine 4 (H3K4) mono/di-methylation (H3K4me1/2). Active enhancers are further marked by H3K27 acetylation (H3K27ac). Mixed-lineage leukemia 4 (MLL4/KMT2D) is a major enhancer H3K4me1/2 methyltransferase with functional redundancy with MLL3 (KMT2C). However, its role in cell fate maintenance and transition is poorly understood. Here, we show in mouse embryonic stem cells (ESCs) that MLL4 associates with, but is surprisingly dispensable for the maintenance of, active enhancers of cell-identity genes. As a result, MLL4 is dispensable for cell-identity gene expression and self-renewal in ESCs. In contrast, MLL4 is required for enhancer-binding of H3K27 acetyltransferase p300, enhancer activation, and induction of cell-identity genes during ESC differentiation. MLL4 protein, rather than MLL4-mediated H3K4 methylation, controls p300 recruitment to enhancers. We also show that, in somatic cells, MLL4 is dispensable for maintaining cell identity but essential for reprogramming into induced pluripotent stem cells. These results indicate that, although enhancer priming by MLL4 is dispensable for cell-identity maintenance, it controls cell fate transition by orchestrating p300-mediated enhancer activation.
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Stephens, Kimberly E., Weiqiang Zhou, Zhicheng Ji, Hongkai Ji, Yun Guan, and Sean D. Taverna. "63438 Differential chromatin accessibility at dorsal root ganglia enhancers is associated with nerve injury." Journal of Clinical and Translational Science 5, s1 (March 2021): 5. http://dx.doi.org/10.1017/cts.2021.414.
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ABSTRACT IMPACT: Our improved understanding of the changes in chromatin accessibility that occur in persistent pain states may identify regulatory genomic elements that play essential roles in modulating gene expression in the DRG. OBJECTIVES/GOALS: Efforts to understand genetic variability involved in an individual’s susceptibility to persistent pain support a role for upstream regulation by epigenetic mechanisms. Our objective was to examine the transcriptomic and epigenetic basis of persistent pain following nerve injury. METHODS/STUDY POPULATION: We used a multiomic approach to identify novel molecular pathways associated with nerve injury-induced pain hypersensitivity. Adult Sprague Dawley rats were randomized to Chronic Constriction Injury (CCI) to the sciatic nerve or no treatment (naive). The ipsilateral L4-L6 dorsal root ganglia (DRG)s were removed on Day 14 and used for ChIP-seq for H3K4me1, ATAC-seq, and RNA-seq. We assessed for differential chromatin accessibility, transcription factor motifs, and enrichment for biological processes in chromatin accessible regions associated with cis-regulatory regions identified by ATAC-seq and H3K4me1 enrichment. Luciferase assays determined the functional significance of these sequences. RESULTS/ANTICIPATED RESULTS: We identified 58,446 genomic regions where H3K4me1 enrichment overlapped with chromatin accessibility. Differential analysis identified 2145 of these 58,446 regions that had changes in accessibility after CCI. The majority of these regions were located in introns or intergenic regions. Functional annotation of the differentially accessible regions identified disparate molecular functions enriched following nerve injury which suggests that altered chromatin structure plays a role in the development of mechanical hypersensitivity. Motif analysis identified specific transcription factor families whose binding sequences were enriched in regions of increased or decreased accessibility. Luciferase assays showed significant enhancement or repression of gene transcription. DISCUSSION/SIGNIFICANCE OF FINDINGS: Our data provides a comprehensive map of chromatin accessibility changes in the DRG after CCI and emphasizes the importance of chromatin structure in the development and maintenance of chronic pain.
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Pham, Thu-Hang, Monika Lichtinger, Chris Benner, Sabine Pape, Lucia Schwarzfischer, Maja Klug, Andreas Gogol-Doering, et al. "Genome-Wide Profiling of Epigenetic and Transcription Factor Regulation In Human Macrophage Differentiation." Blood 116, no. 21 (November 19, 2010): 3875. http://dx.doi.org/10.1182/blood.v116.21.3875.3875.
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Abstract Abstract 3875 The differentiation of human macrophages is accompanied by distinctive phenotypical changes and generally proceeds in the absence of proliferation. The molecular events governing this process are still poorly understood. Using ChIP-Seq technology we studied epigenetic changes as well as alterations in transcription factor occupancy during human monocyte differentiation and correlated these events with gene expression levels in hematopoietic cell types. We show that putative enhancer regions marked by histone H3 lysine4 monomethylation (H3K4me1) at different developmental stages (human progenitor cells, peripheral blood monocytes and in vitro differentiated macrophages) are enriched in distinct sets of transcription factor motifs corresponding to lineage-determining factors. Cell stage-specific histone methylation at promoter-distal sites corresponds with increased mRNA expression levels of neighboring genes. We generated global DNA-binding maps in monocytes and macrophages for two transcription factors (PU.1 and C/EBPβ) with a well established role in monocyte/macrophage differentiation. Comparison of human binding sites with corresponding mouse data revealed a surprisingly low level of conservation (∼10-15%) of PU.1-or C/EBPβ -bound sites between man and mouse, despite a highly conserved binding preference for both transcription factors. During monocytic differentiation, human macrophages primarily gained additional binding sites for both transcription factors (as well as promoter-distal H3K4me1). Interestingly, only neighboring genes with multiple binding events showed significantly increased, macrophage-specific mRNA expression as compared to monocytic as well as lymphocytic cell types. Human macrophage-specific H3K4me1-marked regions as well as macrophage-specific PU.1- and C/EBP-bound sites were characterized by overlapping sets of novel sequence motifs, suggesting that the combinatorial interaction of corresponding DNA-binding factors with PU.1 and C/EBPβ may be required for the establishment of human macrophage-specific enhancers. These data provide novel insights into PU.1 and C/EBPβ mediated gene regulation during human macrophage differentiation. Disclosures: No relevant conflicts of interest to declare.
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Xie, Lin, Ning Ding, Honghong Zhang, Kun Liu, Jiantuan Xiong, Shengchao Ma, Anning Yang, Huiping Zhang, and Yideng Jiang. "SNF5 promotes IL-1β expression via H3K4me1 in atherosclerosis induced by homocysteine." International Journal of Biochemistry & Cell Biology 135 (June 2021): 105974. http://dx.doi.org/10.1016/j.biocel.2021.105974.
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Binder, Moritz, Alexandre Gaspar Maia, Ryan M. Carr, Terra Lasho, Thomas E. Witzig, Christopher Pin, Kurt Berger, et al. "Distal Enhancer Elements in ASXL1-Mutant Chronic Myelomonocytic Leukemia." Blood 134, Supplement_1 (November 13, 2019): 2981. http://dx.doi.org/10.1182/blood-2019-124917.
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Introduction: Additional Sex Combs-Like 1 (ASXL1) is a chromatin modifier frequently affected by truncating mutations in myeloid malignancies. These mutations are associated with poor survival outcomes and increased rates of acute leukemic transformation. In chronic myelomonocytic leukemia (CMML), ASXL1 mutations are thought to affect transcriptional activity mainly by modifying histone marks, however additional epigenomic mechanisms have not been fully explored. We interrogated the epigenome of patients with ASXL1-mutant (MT) and -wildtype (WT) CMML using a multiomics approach to define cis-regulatory elements (CREs) such as distal enhancers (DEs). Methods: Bone marrow mononuclear cells from patients with CMML were subjected to targeted NGS of DNA, whole transcriptome shotgun sequencing (RNA-seq), immunoprecipitation (IP) of DNA (hydroxy-)methyl residues (DIP-seq), IP of the histone modifications H3K4me1, H3K4me3, and H3K27me3 (ChIP-seq), and DNA transposase accessibility studies (ATAC-seq). After quality control all samples were sequenced on an Illumina HiSeq 4000 before further processing and data analysis. Global assessments of DNA (hydroxy-)methylation, DNA accessibility, and histone modifications between ASXL1 MT and WT CMML were performed. The samples in the two groups were treated as biological replicates and subjected to a consensus peak calling strategy requiring an overlap of at least 30% between samples and an adjusted p-value < 5x10-5 for a signal peak to be considered statistically significant. Differential gene expression was estimated to define the up-regulated genes in ASXL1 MT CMML. Potential CREs were defined as sites with statistically significant signal peaks overlapping in at least two of the three epigenomic marks: H3K4me1, 5hmC, and ATAC. Potential DEs were defined as CREs in non-coding regions outside promoter regions (defined as transcription start site ±3kb) that were annotated in GeneHancer. Annotated DEs only present in ASXL1 MT but not WT CMML (specific DEs) were intersected with the list of up-regulated genes and the ReMap atlas. Results: Sixteen WHO-defined CMML patients were included, median age 69 years (48 - 77), 63% male; of which 8 patients (50%, all truncating frame shift mutations) were ASXL1 MT and 8 (50%) WT. The burden and spectrum of co-mutations was similar between ASXL1 WT and MT CMML (21 versus 23 per group; p = 0.684; heatmap). There was a predominant up-regulation of gene expression in ASXL1 MT CMML: 707 genes up- and 124 down-regulated (volcano plot, FDR < 0.050 for all genes). There were 64336 potential CREs, the vast majority (97%) being present in both ASXL1 MT and WT CMML (left Venn diagram). These CREs were most commonly located in introns, promoter regions, and distal non-coding regions (bar graph and pie chart). There were 1303 CREs unique to ASXL1 MT CMML (specific DEs), 1161 (90%) of which were annotated in GeneHancer (left Euler diagram). Of these 1161 annotated specific DEs 859 (74%) were located outside promoter regions and 34 (4%) of them were known to be associated with genes up-regulated in ASXL1 MT CMML (Euler diagrams). These specific DEs were characterized by an increase in H3K4me1 occupancy and DNA accessibility (average signal tracks, purple bars indicating annotated DEs, thin bars below peaks indicating statistical significance). We previously observed epigenomic modification of promoter regions in 519 of the 707 up-regulated genes (73%) facilitating transcriptional activity in ASXL1 MT CMML. For 13 of the up-regulated genes (right Venn diagram, blue genes in volcano plot) the specific DEs were the sole identified mechanism, while for the other 21 genes there were additional mechanisms noted in the promoter region. The top five transcription factor candidates binding the 34 specific DEs included JMJD1C, MYC, KDM5B, RCOR1, and HDAC2 (-log10(E) > 40 for all candidates). Conclusions: Using a multiomics approach based on H3K4me1, 5hmC, and ATAC data we identified potential CREs in ASXL1 MT CMML and characterized potential DEs using publicly available annotation data. Specific DEs were associated with up-regulated genes serving as a possible explanation for the observed transcriptional activity, shedding further light on the adverse prognostic impact associated with ASXL1 mutations. Figure 1 Disclosures Patnaik: Stem Line Pharmaceuticals.: Membership on an entity's Board of Directors or advisory committees.