H3K27ac Peaks Research Articles

Comparative Analysis of Promoters and Enhancers in the Pituitary Glands of the Bama Xiang and Large White Pigs.

The epigenetic regulation of gene expression is implicated in complex diseases in humans and various phenotypes in other species. There has been little exploration of regulatory elements in the pig. Here, we performed chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-Seq) to profile histone H3 lysine 4 trimethylation (H3K4me3) and histone H3 lysine 27 acetylation (H3K27ac) in the pituitary gland of adult Bama Xiang and Large White pigs, which have divergent evolutionary histories and large phenotypic differences. We identified a total of 65,044 non-redundant regulatory regions, including 23,680 H3K4me3 peaks and 61,791 H3K27ac peaks (12,318 proximal and 49,473 distal), augmenting the catalog of pituitary regulatory elements in pigs. We found 793 H3K4me3 and 3,602 H3K27ac peaks that show differential activity between the two breeds, overlapping with genes involved in the Notch signaling pathway, response to growth hormone (GH), thyroid hormone signaling pathway, and immune system, and enriched for binding motifs of transcription factors (TFs), including JunB, ATF3, FRA1, and BATF. We further identified 2,025 non-redundant super enhancers from H3K27ac ChIP-seq data, among which 302 were shared in all samples of cover genes enriched for biological processes related to pituitary function. This study generated a valuable dataset of H3K4me3 and H3K27ac regions in porcine pituitary glands and revealed H3K4me3 and H3K27ac peaks with differential activity between Bama Xiang and Large White pigs.

Abstract 2120: Interrogation of regulatory and expression changes in Malignant Rhabdoid Tumours to identify new therapeutic approaches

Abstract Malignant Rhabdoid Tumors (MRTs) are a textbook model of epigenetic dysregulation within cancer. The biallelic inactivation of a single gene SMARCB1 in >95% of MRTs is sufficient to provoke a lethal pediatric cancer. SMARCB1 encodes a core subunit of the SWI/SNF complex and inactivation results in genome-wide deregulation of gene expression/pathways. SMARCB1 loss was previously shown to impair SWI/SNF enhancer targeting at TSS-distal enhancers and a model proposed whereby mutant residual SWI/SNF complexes preferentially binds to super-enhancers over typical-enhancers; thereby inappropriately maintaining progenitor lineage processes and blocking differentiation. This model deemphasizes the action of mutant SWI/SNF complexes upon cell cycle/oncogenic processes. We performed a consensus analysis on a cohort of novel and published ChIP-seq profiles (n=6 +/- SMARCB1 models) to define consensus SMARCB1-dependent epigenetic changes. We further used parallel RNA-seq, methylation and whole-genome CRISPR screen data to emphasize the significance of specific regulatory processes from the perspective of likely biological effects and functional essentiality. We bioinformatically identified consensus super and typical-enhancers from H3K27ac peaks, multiple datasets enabled high confidence regions to be called. SMARCB1 loss did not significantly impact SWI/SNF occupancy of super-enhancers, permutation test revealed significant (p<0.001) overlap of binding across both conditions; in agreement with residual SWI/SNF complex binding theories. However, we noted the same at typical-enhancers implying all enhancers are similarly affected. We hypothesized that the resulting SMARCB1-dependent transcriptional impact, not location of SWI/SNF binding would be paramount, InTAD and RNAseq data were used to assign target genes to enhancers. Of the SMARCB1-dependent genes linked to typical-enhancers 13% showed significantly altered expression compared to 9% for super-enhancers. Ontology analysis identified classic oncogenic pathways such as cell cycle and metabolic genes as enriched among SMARCB1-dependent enhancer targets. Gene Set Enrichment Analysis (GSEA) confirmed significant enrichment of mucopolysaccharide metabolic genes (p<0.002, NES 1.42). Correlating dysregulated gene targets to epigenetically altered enhancers/promoters we ranked loci which drive MRT tumorigenesis. We identified a 40kb upstream region of SMARCB1-dependent methylation and ChIPseq binding strongly correlated to expression of CDK6. We further employed CRISPR activation/repression tools to mimic SMARCB1 re-expression and model their effects. Successful modulation of candidate loci will help define potential future novel targeted therapeutics - e.g use of HDACi - to recapitulate the effects of SMARCB1 re-expression. Citation Format: Emma L. Lishman-Walker, Martina A. Finetti, James P. Hacking, Matthew Selby, Yura Grabovska, Stephen Crosier, Simon Bailey, Steven C. Clifford, Daniel Williamson. Interrogation of regulatory and expression changes in Malignant Rhabdoid Tumours to identify new therapeutic approaches [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2120.

Abstract 2119: Genome-wide epigenetic alterations in T-cell prolymphocytic leukemia

Abstract T-cell prolymphocytic leukemia (T-PLL) is a rare disease showing a rapid clinical course, with a median survival of <1 year. Whole-genome and whole-exome sequencing have identified key genetic lesions in T-PLL, including structural alterations such as inversions, translocations, and large copy number variants, as well as recurrent somatic mutations of genes encoding chromatin regulators and of those in the JAK-STAT signaling pathway. Epigenetic alteration is implicated in the susceptibility and progression of both cancer and non-cancerous diseases. However, genome-wide epigenetic data has not been generated for T-PLL, which prohibits the mechanistic studies of transcriptional misregulation in T-PLL. This study used micrococcal nuclease digestion and sequencing (MNase-seq) to profile the chromatin accessible regions, i.e., gene regulatory regions such as promoters, enhancers and insulators, in both T-PLL patients and healthy individuals. Our analysis revealed the prevalence of lost open chromatin regions in T-PLL. We also generated histone H3 lysine 4 monomethylation (H3K4me1) and lysine 27 acetylation (H3K27ac) chromatin immunoprecipitation and sequencing (ChIP-seq) data from the same samples. We identified 5,462 and 2,833 active enhancers (>=2.5 kb away from transcription start sites) that are lost and gained in T-PLL, respectively, compared to normal. The differential H3K27ac peaks are preferentially associated with the nearest genes differentially expressed between T-PLL and normal (chi-square test p <2.2e-16), indicating a role of epigenetic alteration in transcriptional misregulation. In addition, we identified alterations of active enhancers that target oncogenes with key roles in T-PLL, such as TCL1Aand MYC. Together, our analysis has provided insights into the epigenetic mechanisms that drive oncogenic activation in T-PLL. Citation Format: Shulan Tian, Henan Zhang, Eric Klee, Huihuang Yan, Wei Ding. Genome-wide epigenetic alterations in T-cell prolymphocytic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2119.

Human placental cytotrophoblast epigenome dynamics over gestation and alterations in placental disease.

The human placenta and its specialized cytotrophoblasts rapidly develop, have a compressed lifespan, govern pregnancy outcomes, and program the offspring's health. Understanding the molecular underpinnings of these behaviors informs development and disease. Profiling the extraembryonic epigenome and transcriptome during the 2nd and 3rd trimesters revealed H3K9 trimethylation overlapping deeply DNA hypomethylated domains with reduced gene expression and compartment-specific patterns that illuminated their functions. Cytotrophoblast DNA methylation increased, and several key histone modifications decreased across the genome as pregnancy advanced. Cytotrophoblasts from severe preeclampsia had substantially increased H3K27 acetylation globally and at genes that are normally downregulated at term but upregulated in this syndrome. In addition, some cases had an immature pattern of H3K27ac peaks, and others showed evidence of accelerated aging, suggesting subtype-specific alterations in severe preeclampsia. Thus, the cytotrophoblast epigenome dramatically reprograms during pregnancy, placental disease is associated with failures in this process, and H3K27 hyperacetylation is a feature of severe preeclampsia.

Identification of super enhancer-associated key genes for prognosis of germinal center B-cell type diffuse large B-cell lymphoma by integrated analysis

BackgroundThe pathogenesis of germinal center B-cell type diffuse large B-cell lymphoma (GCB-DLBCL) is not fully elucidated. This study aims to explore the regulation of super enhancers (SEs) on GCB-DLBCL by identifying specific SE-target gene.MethodsWeighted gene co-expression network analysis (WGCNA) was used to screen modules associated with GCB subtype. Functional analysis was performed by gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment. H3K27ac peaks were used to identify SEs. Overall survival analysis was performed using Kaplan–Meier curve with log-rank and Breslow test. The effect of ADNP, ANKRD28 and RTN4IP1 knockdown on Karpas 422 and SUDHL-4 cells proliferation was analyzed by CCK-8. Karpas 422 and SUDHL-4 cells were treated with bromodomain and extra-terminal domain (BET) inhibitor JQ1, and the expression of ADNP, ANKRD28 and RTN4IP1was measured by qRT-PCR.ResultsA total of 26 modules were screened in DLBCL. Turquoise module was closely related to GCB-DLBCL, and its eigengenes were mainly related to autophagy. There were 971 SEs in Karpas 422 cell and 1088 SEs in SUDHL-4 cell. Function of the nearest genes of overall SEs were related to cancer. Six SE-related genes associated with GCB-DLBCL were identified as prognostic markers. Knockdown of ADNP, ANKRD28 and RTN4IP1 inhibited the proliferation of Karpas 422 and SUDHL-4 cells. JQ1 treatment suppressed ADNP, ANKRD28 and RTN4IP1 expression in Karpas 422 and SUDHL-4 cells.ConclusionsA total of 6 SE-related genes associated with GCB-DLBCL overall survival were identified in this study. These results will serve as a theoretical basis for further study of gene regulation and function of GCB-DLBCL.

Dysregulated H3K27 Acetylation Is Implicated in Fatty Liver Hemorrhagic Syndrome in Chickens.

Epigenetic regulation of gene expression has been reported in the pathogenesis of metabolic disorders such as diabetes and liver steatosis in humans. However, the molecular mechanisms of fatty liver hemorrhagic syndrome (FLHS) in chickens have been rarely studied. H3K27ac chromatin immunoprecipitation coupled with high-throughput sequencing and high-throughput RNA sequencing was performed to compare genome-wide H3K27ac profiles and transcriptomes of liver tissue between healthy and FLHS chickens. In total, 1,321 differential H3K27ac regions and 443 differentially expressed genes were identified (| log2Fold change| ≥ 1 and P-value ≤ 0.05) between the two groups. Binding motifs for transcription factors involved in immune processes and metabolic homeostasis were enriched among those differential H3K27ac regions. Differential H3K27ac peaks were associated with multiple known FLHS risk genes, involved in lipid and energy metabolism (PCK1, APOA1, ANGPTL4, and FABP1) and the immune system (FGF7, PDGFRA, and KIT). Previous studies and our current results suggested that the high-energy, low-protein (HELP) diet might have an impact on histone modification and chromatin structure, leading to the dysregulation of candidate genes and the peroxisome proliferator-activated receptor (PPAR) signaling pathway, which causes excessive accumulation of fat in the liver tissue and induces the development of FLHS. These findings highlight that epigenetic modifications contribute to the regulation of gene expression and play a central regulatory role in FLHS. The PPAR signaling pathway and other genes implicated in FLHS are of great importance for the development of novel and specific therapies for FLHS-susceptible commercial laying hens.

HGG-41. STRUCTURAL VARIANT DRIVERS IN PEDIATRIC HIGH-GRADE GLIOMA

BACKGROUNDDriver single nucleotide variants (SNV) and somatic copy number aberrations (SCNA) of pediatric high-grade glioma (pHGGs), including Diffuse Midline Gliomas (DMGs) are characterized. However, structural variants (SVs) in pHGGs and the mechanisms through which they contribute to glioma formation have not been systematically analyzed genome-wide.METHODSUsing SvABA for SVs as well as the latest pipelines for SCNAs and SNVs we analyzed whole-genome sequencing from 174 patients. This includes 60 previously unpublished samples, 43 of which are DMGs. Signature analysis allowed us to define pHGG groups with shared SV characteristics. Significantly recurring SV breakpoints and juxtapositions were identified with algorithms we recently developed and the findings were correlated with RNAseq and H3K27ac ChIPseq.RESULTSThe SV characteristics in pHGG showed three groups defined by either complex, intermediate or simple signature activities. These associated with distinct combinations of known driver oncogenes. Our statistical analysis revealed recurring SVs in the topologically associating domains of MYCN, MYC, EGFR, PDGFRA & MET. These correlated with increased mRNA expression and amplification of H3K27ac peaks. Complex recurring amplifications showed characteristics of extrachromosomal amplicons and were enriched in coding SVs splitting protein regulatory from effector domains. Integrative analysis of all SCNAs, SNVs & SVs revealed patterns of characteristic combinations between potential drivers and signatures. This included two distinct groups of H3K27M DMGs with either complex or simple signatures and different combinations of associated variants.CONCLUSIONRecurrent SVs associate with signatures shaped by an underlying process, which can lead to distinct mechanisms to activate the same oncogene.

Epigenetic Mechanisms Mediating Cell State Transitions in Chondrocytes.

Epigenetic modifications play critical roles in regulating cell lineage differentiation, but the epigenetic mechanisms guiding specific differentiation steps within a cell lineage have rarely been investigated. To decipher such mechanisms, we used the defined transition from proliferating (PC) into hypertrophic chondrocytes (HC) during endochondral ossification as a model. We established a map of activating and repressive histone modifications for each cell type. ChromHMM state transition analysis and Pareto-based integration of differential levels of mRNA and epigenetic marks revealed that differentiation-associated gene repression is initiated by the addition of H3K27me3 to promoters still carrying substantial levels of activating marks. Moreover, the integrative analysis identified genes specifically expressed in cells undergoing the transition into hypertrophy. Investigation of enhancer profiles detected surprising differences in enhancer number, location, and transcription factor binding sites between the two closely related cell types. Furthermore, cell type-specific upregulation of gene expression was associated with increased numbers of H3K27ac peaks. Pathway analysis identified PC-specific enhancers associated with chondrogenic genes, whereas HC-specific enhancers mainly control metabolic pathways linking epigenetic signature to biological functions. Since HC-specific enhancers show a higher conservation in postnatal tissues, the switch to metabolic pathways seems to be a hallmark of differentiated tissues. Surprisingly, the analysis of H3K27ac levels at super-enhancers revealed a rapid adaption of H3K27ac occupancy to changes in gene expression, supporting the importance of enhancer modulation for acute alterations in gene expression. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

CYP2S1 might regulate proliferation and immune response of keratinocyte in psoriasis

ABSTRACT Psoriasis is an autoimmune skin disorder influenced by genetic, epigenetic and environmental factors. We previously found CYP2S1 intragenic DNA methylation cg19430423 site strongly hypomethylated in psoriatic skin tissues. In this study, we performed methylation loci fine-mapping to search the top signals in the entire CYP2S1 gene region, and further carried out gene expression assay, cell proliferation, apoptosis, differentiation and migration in CYP2S1 overexpressed (CYP2S1 over) and silenced (siRNA) human keratinocytes. Target bisulphite conversion sequencing revealed cg19430423 and nearby two loci were the top differentially methylated loci. These three loci located within active enhancer region marked by H3K4Me1 and H3K27AC peaks. Cg19430423 might not bind with ATF1 directly. CYP2S1 over repressed NHEK cell proliferation, but have no confirmed evidence on affecting migration, apoptosis and differentiation. Real-time PCR showed that CYP2S1 inhibited expression of IL1β, IL8, IL33, IL36, LL37, CXCL10 and CCL20 gene. In summary, CYP2S1 might inhibit keratinocyte proliferation, and modulate immune response through IL-8, IL-33, IL-36, CXCL-10, CCL20, thus contribute to the development of psoriasis.

NSD2 overexpression drives clustered chromatin and transcriptional changes in a subset of insulated domains

CTCF and cohesin play a key role in organizing chromatin into topologically associating domain (TAD) structures. Disruption of a single CTCF binding site is sufficient to change chromosomal interactions leading to alterations in chromatin modifications and gene regulation. However, the extent to which alterations in chromatin modifications can disrupt 3D chromosome organization leading to transcriptional changes is unknown. In multiple myeloma, a 4;14 translocation induces overexpression of the histone methyltransferase, NSD2, resulting in expansion of H3K36me2 and shrinkage of antagonistic H3K27me3 domains. Using isogenic cell lines producing high and low levels of NSD2, here we find oncogene activation is linked to alterations in H3K27ac and CTCF within H3K36me2 enriched chromatin. A logistic regression model reveals that differentially expressed genes are significantly enriched within the same insulated domain as altered H3K27ac and CTCF peaks. These results identify a bidirectional relationship between 2D chromatin and 3D genome organization in gene regulation.

Abstract SY25-02: Widespread germline-somatic regulatory differences in renal cell carcinoma prioritize risk mechanisms

Abstract Determining the function of non-coding regulatory variants in cancer remains a key challenge. We investigated genetic (germline and somatic) determinants of regulatory mechanisms in renal cell carcinoma (RCC) using H3K27ac ChIP-seq data in 10 matched tumor/normal samples and RNA-seq data from 496/66 tumor/normal samples from The Cancer Genome Atlas (TCGA). Unsupervised clustering of H3K27ac activity cleanly separated tumor from normal individuals, highlighting extensive epigenetic reprogramming during transformation. We developed a novel method to test each chromatin feature for allele-specific activity (asQTL) and evaluate tumor/normal differences in allele-specificity (d-asQTLs) while modelling local structural variation and read overdispersion. At an FDR of 5%, we identified 1,356 unique asQTL chromatin peaks in normal tissues; 2,868 in tumors; and 1,054 d-asQTLs (primarily imbalanced in tumor). The d-asQTL peaks were significantly enriched for RCC GWAS heritability (32x, P=1.8x10-3), more so than any other functional feature including all H3K27ac peaks (12x), super-enhancers (5x), and regions near asQTL genes (4x). Intersection of asQTLs with RCC genome-wide association study (GWAS) data identified putative functional features for 6/17 known loci including tumor-specific activity at SCARB1, which has recently been implicated as a prognostic biomarker in RCC. d-asQTLs were significantly enriched for tumor-specific binding of transcription factors in the hypoxia pathway, implicating a general mechanism for polygenic germline-somatic interaction. Citation Format: Alexander Gusev, Sandor Spisak, Forough Abbasi, Andre Fay, Hallie Carol, Ji-Heui Seo, Toni Choueiri, Kate Lawrenson, Matthew Freedman. Widespread germline-somatic regulatory differences in renal cell carcinoma prioritize risk mechanisms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr SY25-02.

CTCF is necessary for CD8+ effector T cell differentiation

Abstract Naive T cell differentiation into effector and memory subsets in response to infection requires the coordination of numerous factors that regulate changes in gene expression to support proliferation and cell function. Chromatin accessibility and DNA looping influence transcription factor binding, facilitate enhancer-promoter interactions, and insulate genes. CCCTC-Binding Factor (CTCF) is a highly conserved protein important for organizing the genome and partnering with transcription factors, but its role in CD8+ T cell differentiation has yet to be determined. We show that loss of CTCF in CD8+ T cells alters the expression of genes important for subset differentiation and these changes are reflected during Listeria infection where the formation of memory-precursor cells is favored while the formation of terminal effector cells is impaired. At a memory timepoint, CTCF knockdown enhances intestinal Trm accumulation despite impairing circulating memory maintenance. ChIP-Seq and HiC reveals that terminal effector cells gain CTCF binding within topologically associated domains, often overlapping with H3K27ac peaks which marks active enhancers and promoters. As genes near the overlap of CTCF and H3K27ac peaks have increased expression in terminal effector cells, we propose that one role of CTCF is to promote effector function by stabilizing promoter-enhancer interactions which influences the expression of fate-determining genes. Insights into the role of CTCF in CD8+ T cell differentiation during infection will yield valuable insight into dynamic chromatin changes regulating T cell differentiation and inform the development of therapies which better utilize the T cell response.

Atheroprotective Flow Upregulates ITPR3 (Inositol 1,4,5-Trisphosphate Receptor 3) in Vascular Endothelium via KLF4 (Krüppel-Like Factor 4)-Mediated Histone Modifications.

Objective- The topographical distribution of atherosclerosis in vasculature underscores the importance of shear stress in regulating endothelium. With a systems approach integrating sequencing data, the current study aims to explore the link between shear stress-regulated master transcription factor and its regulation of endothelial cell (EC) function via epigenetic modifications. Approach and Results- H3K27ac (acetylation of histone 3 lysine 27)-ChIP-seq (chromatin immunoprecipitation followed by high throughput sequencing), ATAC-seq (an assay for transposase-accessible chromatin-sequencing), and RNA-seq (RNA-sequencing) were performed to investigate the genome-wide epigenetic regulations in ECs in response to atheroprotective pulsatile shear stress (PS). In silico prediction revealed that KLF4 binding motifs were enriched in the PS-enhanced H3K27ac regions. By integrating PS- and KLF4-modulated H3K27ac, we identified 18 novel PS-upregulated genes. The promoter regions of these genes showed an overlap between the KLF4-enhanced assay for transposase-accessible chromatin signals and the PS-induced H3K27ac peaks. Experiments using ECs isolated from mouse aorta, lung ECs from EC-KLF4-TG versus EC-KLF4-KO mice, and atorvastatin-treated ECs showed that ITPR3 (inositol 1,4,5-trisphosphate receptor 3) was robustly activated by KLF4 and statins. KLF4 ATAC-qPCR (quantitative polymerase chain reaction) and ChIP-qPCR further demonstrated that a specific locus in the promoter region of the ITPR3 gene was essential for KLF4 binding, H3K27ac enrichment, chromatin accessibility, RNA polymerase II recruitment, and ITPR3 transcriptional activation. Deletion of this KLF4 binding locus in ECs by using CRISPR-Cas9 resulted in blunted calcium influx, reduced expression of endothelial nitric oxide synthase, and diminished nitric oxide bioavailability. Conclusions- These results from a novel multiomics study suggest that KLF4 is crucial for PS-modulated H3K27ac that allow the transcriptional activation of ITPR3. This novel mechanism contributes to the Ca2+-dependent eNOS (endothelial nitric oxide synthase) activation and EC homeostasis.

Genetic risk variants for brain disorders are enriched in cortical H3K27ac domains

Most variants associated with complex phenotypes in genome-wide association studies (GWAS) do not directly index coding changes affecting protein structure. Instead they are hypothesized to influence gene regulation, with common variants associated with disease being enriched in regulatory domains including enhancers and regions of open chromatin. There is interest, therefore, in using epigenomic annotation data to identify the specific regulatory mechanisms involved and prioritize risk variants. We quantified lysine H3K27 acetylation (H3K27ac) - a robust mark of active enhancers and promoters that is strongly correlated with gene expression and transcription factor binding – across the genome in entorhinal cortex samples using chromatin immunoprecipitation followed by highly parallel sequencing (ChIP-seq). H3K27ac peaks were called using high quality reads combined across all samples and formed the basis of partitioned heritability analysis using LD score regression along with publicly-available GWAS results for seven psychiatric and neurodegenerative traits. Heritability for all seven brain traits was significantly enriched in these H3K27ac peaks (enrichment ranging from 1.09–2.13) compared to regions of the genome containing other active regulatory and functional elements across multiple cell types and tissues. The strongest enrichments were for amyotrophic lateral sclerosis (ALS) (enrichment = 2.19; 95% CI = 2.12–2.27), autism (enrichment = 2.11; 95% CI = 2.05–2.16) and major depressive disorder (enrichment = 2.04; 95% CI = 1.92–2.16). Much lower enrichments were observed for 14 non-brain disorders, although we identified enrichment in cortical H3K27ac domains for body mass index (enrichment = 1.16; 95% CI = 1.13–1.19), ever smoked (enrichment = 2.07; 95% CI = 2.04–2.10), HDL (enrichment = 1.53; 95% CI = 1.45–1.62) and trigylcerides (enrichment = 1.33; 95% CI = 1.24–1.42). These results indicate that risk alleles for brain disorders are preferentially located in regions of regulatory/enhancer function in the cortex, further supporting the hypothesis that genetic variants for these phenotypes influence gene regulation in the brain.

Young genes have distinct gene structure, epigenetic profiles, and transcriptional regulation.

Species-specific, new, or “orphan” genes account for 10%–30% of eukaryotic genomes. Although initially considered to have limited function, an increasing number of orphan genes have been shown to provide important phenotypic innovation. How new genes acquire regulatory sequences for proper temporal and spatial expression is unknown. Orphan gene regulation may rely in part on origination in open chromatin adjacent to preexisting promoters, although this has not yet been assessed by genome-wide analysis of chromatin states. Here, we combine taxon-rich nematode phylogenies with Iso-Seq, RNA-seq, ChIP-seq, and ATAC-seq to identify the gene structure and epigenetic signature of orphan genes in the satellite model nematode Pristionchus pacificus. Consistent with previous findings, we find young genes are shorter, contain fewer exons, and are on average less strongly expressed than older genes. However, the subset of orphan genes that are expressed exhibit distinct chromatin states from similarly expressed conserved genes. Orphan gene transcription is determined by a lack of repressive histone modifications, confirming long-held hypotheses that open chromatin is important for new gene formation. Yet orphan gene start sites more closely resemble enhancers defined by H3K4me1, H3K27ac, and ATAC-seq peaks, in contrast to conserved genes that exhibit traditional promoters defined by H3K4me3 and H3K27ac. Although the majority of orphan genes are located on chromosome arms that contain high recombination rates and repressive histone marks, strongly expressed orphan genes are more randomly distributed. Our results support a model of new gene origination by rare integration into open chromatin near enhancers.

Differential analysis of chromatin accessibility and histone modifications for predicting mouse developmental enhancers.

Enhancers are distal cis-regulatory elements that modulate gene expression. They are depleted of nucleosomes and enriched in specific histone modifications; thus, calling DNase-seq and histone mark ChIP-seq peaks can predict enhancers. We evaluated nine peak-calling algorithms for predicting enhancers validated by transgenic mouse assays. DNase and H3K27ac peaks were consistently more predictive than H3K4me1/2/3 and H3K9ac peaks. DFilter and Hotspot2 were the best DNase peak callers, while HOMER, MUSIC, MACS2, DFilter and F-seq were the best H3K27ac peak callers. We observed that the differential DNase or H3K27ac signals between two distant tissues increased the area under the precision-recall curve (PR-AUC) of DNase peaks by 17.5–166.7% and that of H3K27ac peaks by 7.1–22.2%. We further improved this differential signal method using multiple contrast tissues. Evaluated using a blind test, the differential H3K27ac signal method substantially improved PR-AUC from 0.48 to 0.75 for predicting heart enhancers. We further validated our approach using postnatal retina and cerebral cortex enhancers identified by massively parallel reporter assays, and observed improvements for both tissues. In summary, we compared nine peak callers and devised a superior method for predicting tissue-specific mouse developmental enhancers by reranking the called peaks.

Abstract NG04: Immune evasion mechanisms in melanoma resistant to targeted- and/or immunotherapy

Abstract NG04: Immune evasion mechanisms in melanoma resistant to targeted- and/or immunotherapy

Integrative Epigenetic and Single-Cell RNA-Seq Profiling of Human Hematopoietic Stem Cells Reveals Epigenetic Reprogramming of Enhancer and Regulatory Elements during Normal Aging

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. DisclosuresLindsley: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.

Recent studies on the evolution of mammalian flight using high-throughput sequencing technology

Bats are the only mammals capable of self-powered flight using wings. Critical adaptations for their flight include a pair of dramatically elongated hands (forelimb digits II–V) with broad wing membranes. Although it has been studied for over ten years, the molecular mechanism of bat wing evolution was still uncertain before 2014. Here we reviewed recently significant studies on this topic, which made a great progress on the knowledge of bat wing formation. Thousands of differentially expressed genes and regulatory elements associated with limb development were found by using advanced high-throughput sequencing technology, including whole genome sequencing, RNA-seq and ChIP-seq. Screening out from RNA-seq data, we identified seven key genes that displayed unique expression patterns in embryonic bat wings and feet, compared with mouse fore- and hindlimbs. The expression of all 5 ′ HoxD genes ( Hoxd9–13 ) and Tbx3 , six known crucial transcription factors for limb and digit development, was extremely high and prolonged in the elongating wing area. The expression of Brinp3 (also called Fam5c ), a tumor suppressor, in bat limbs was bat-specific and significantly high in all short digit regions (the thumb and foot digits). Using RNA-seq analysis, Eckalbar et al. found that more than 7000 genes and a few long noncoding RNAs, including Tbx5-as1 and Hottip, were differentially expressed between forelimb and hindlimb in different stages of bat embryos. They also identified thousands of regions that were differentially modified in forelimb and hindlimb by using ChIP-seq analysis and 2796 bat-accelerated regions (BARs) within H3K27ac peaks by using comparative genomics, several of which cluster near limb-associated genes. Booker et al. discovered 166 BARs that overlap H3K27ac and p300 ChIP-seq peaks in mouse embryonic limbs. They further performed a mouse enhancer assay and showed that five Myotis lucifugus BARs drove gene expression in the embryonic mouse limb, with the majority showing differential enhancer activities compared to the mouse orthologous BAR sequences. Specially, one of them (BAR116), which was located telomeric to the HoxD cluster, had robust forelimb expression for the M. lucifugus sequence and no activity for the mouse sequence at the embryonic corresponding stage. Developing limb expression analysis of Hoxd10 - 13 in another species of bats ( Miniopterus natalensis ) showed that a high-forelimb and weak-hindlimb expression for Hoxd10 - 11 , which was similar to the expression trend observed for M. lucifugus BAR116 in mice. All the above results suggest that multiple changes in gene expression patterns and adaptive evolution of gene expression regulators occurred during the evolution of bat wings. Future studies should focus on functions of key developmental genes and bat-accelerated regulators, interactions between them, and pathways involving these genes.

Unique Long Non-Coding RNA Expression Signature in ETV6/RUNX1-Driven B-Cell Precursor Acute Lymphoblastic Leukemia

Emerging evidence suggests that long non-coding RNAs (lncRNAs) are critically involved in a variety of human tumor entities and the identification of cancer-associated lncRNAs might reveal new prognostic biomarkers or novel therapeutic targets for the treatment of human cancer. In this study, we characterized the lncRNA expression signature associated with ETV6/RUNX1-positive pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL), one of the most prevalent genetic subtypes of childhood leukemia.First, we used primary leukemia patient samples to identify an ETV6/RUNX1 specific expression signature consisting of 596 lncRNA transcripts. Besides using primary BCP-ALL patient samples, we also integrated these lncRNA expression data with RNA sequencing results from a panel of human BCP-ALL leukemia cell lines and identified a unique lncRNA expression profile of 16 lncRNAs exclusively associated with the presence of the ETV6/RUNX1 fusion protein. Notably, lnc-SARDH-1 (also known as DBH-AS1) was the only lncRNA from this list for which an oncogenic role has previously been postulated.Given that previous studies revealed putative overlap between lncRNA expression and the presence of regulatory enhancer elements, we also evaluated the distribution of H3K27ac, a histone modification associated with enhancer activity, at the genomic loci of the 16 ETV6/RUNX1 specific lncRNAs mentioned above. Notably, broad H3K27ac binding was identified for lnc-NKX2-3-1, lnc-RTN4R-1, lnc-GIP-1, lnc-LRP8-3, lnc-TCF12-2, lnc- C8orf4-1, lnc-C8orf4-2, lnc-TINAGL1-1 and lnc-LSM11-4 in ETV6/RUNX1-positive REH cells, whereas more discrete H3K27ac peaks were identified at putative promoter regions for lnc-TIMM21-5 and lnc-CPT2-7. Interestingly, coding genes adjacent to some of these lncRNAs showed unique overexpression in ETV6/RUNX1-positive BCP-ALLs, suggesting a possible cis regulatory relationship between these lncRNAs and their nearby protein coding genes. Finally, we applied shRNA-mediated silencing of endogenous ETV6/RUNX1, integrated these expression profiles with the patient and cell line data, to show that lnc-NKX2-3-1, lnc-TIMM21-5, lnc-ASTN1-1 and lnc-RTN4R-1 were the only lncRNAs that were truly regulated by the oncogenic fusion protein.Interestingly, subsequent lncRNA modulation experiments using LNA GapmeR technology revealed that lnc-TIMM21-5 and lnc-ASTN1-1 modulation has no effect on overall transcription, suggesting that these lncRNAs might act at the translational level or/and at various steps of mRNA processing. In contrast, lnc-NKX2-3-1 and lnc-RTN4R-1 perturbations resulted in severe changes in gene expression, suggesting an alternative mechanism of action for these lncRNAs that is more transcriptionally oriented. Most notably, loss of lnc-RTN4R-1expression significantly affected part of the ETV6/RUNX1-specific mRNA expression signature as exemplified by reduced levels of AK7, PTPRKand GBA3.Altogether, our study identified a panel of ETV6/RUNX1 specific lncRNAs that might be implicated in the biology of human BCP-ALL and could serve as novel biomarkers or novel therapeutic targets for the treatment of this prevalent subtype of human leukemia.TLM and PVV are shared last authors DisclosuresNo relevant conflicts of interest to declare.