DNase I Hypersensitive Sites Research Articles

Histone H3K4me1 strongly activates the DNase I hypersensitive sites in super-enhancers than those in typical enhancers.

Super-enhancers (SEs), which consist of multiple enhancer elements, are occupied by master transcription factors and co-activators, such as Mediator, and are highly acetylated at histone H3K27. Here, we have characterized the SEs in terms of DNase I hypersensitive sites (DHSs) by analyzing publicly available chromatin immunoprecipitation (ChIP)-seq and DNase-seq data of K562 cells and compared with the DHSs in typical enhancers (TEs). DHSs in the SEs were highly marked by histone H3K4me1 than DHSs in TEs. Loss of H3K4me1 by the deletion of catalytic domains in histone methyltransferases MLL3 and MLL4 remarkably decreased histone H3K27ac and histone H3 depletion at SE DHSs than at TE DHSs. The levels of enhancer RNA (eRNA) transcripts and mRNA transcripts from the putative target genes were notably reduced at and near SE DHSs than TE DHSs following H3K4me1 loss. These results indicate that histone H3K4me1 is a marker for DHSs in SEs and that this modification has a more significant impact on the activation of SE DHSs than TE DHSs.

CHIPS: A Snakemake pipeline for quality control and reproducible processing of chromatin profiling data

Motivation: The chromatin profile measured by ATAC-seq, ChIP-seq, or DNase-seq experiments can identify genomic regions critical in regulating gene expression and provide insights on biological processes such as diseases and development. However, quality control and processing chromatin profiling data involves many steps, and different bioinformatics tools are used at each step. It can be challenging to manage the analysis. Results: We developed a Snakemake pipeline called CHIPS (CHromatin enrIchment ProcesSor) to streamline the processing of ChIP-seq, ATAC-seq, and DNase-seq data. The pipeline supports single- and paired-end data and is flexible to start with FASTQ or BAM files. It includes basic steps such as read trimming, mapping, and peak calling. In addition, it calculates quality control metrics such as contamination profiles, polymerase chain reaction bottleneck coefficient, the fraction of reads in peaks, percentage of peaks overlapping with the union of public DNaseI hypersensitivity sites, and conservation profile of the peaks. For downstream analysis, it carries out peak annotations, motif finding, and regulatory potential calculation for all genes. The pipeline ensures that the processing is robust and reproducible. Availability: CHIPS is available at https://github.com/liulab-dfci/CHIPS.

Defining the Role of a GATA-2 Enhancer during Hematopoietic Differentiation of Human iPSCs

Division of Cellular and Gene Therapies, Center for Biologics Evaluation and Research, US Food and Drug Administration, USARationale : Differentiation of hematopoietic stem cells from human induced pluripotent stem cells (iPSCs) has the potential to provide novel therapeutic options for a wide range of blood diseases; however, despite significant progress that has been made in driving iPSC differentiation, issues with limited differentiation potential and engraftment capability in vivo still persist. A better understanding of the key regulators of hematopoiesis is essential for the development of improved differentiation protocols. GATA-2, a transcription factor shown to be essential for hematopoiesis, has been examined to determine its role in both hematopoiesis and endothelial cell development. Mouse studies have described an early developmental Gata-2 hematopoietic-specific enhancer located 5′ to its distal first exon. Based on homology within this regulatory domain between mouse and human sequences, we hypothesize that this cis-regulatory region plays an essential role in human hematopoietic expression of GATA-2 and in endothelial-to-hematopoietic transition.Methods : In this study, we use a genome editing approach and an in vitro human iPSC differentiation system to examine our hypothesis. A pair of CRISPR/Cas9 guide RNA was designed to recognize sequences of a DNase I hypersensitive site (DHS) within the putative enhancer region immediately upstream of the GATA-2 distal first exon. Human iPSCs were treated with CRISPR/Cas9 to create cell lines that have a deletion at this DHS. These genetically modified iPSC lines were compared to their isogenic parental controls, to examine functional consequences of the deletion. Both adherent culture-based and spin-embryoid body formation-based protocols were used for mesodermal differentiation, endothelial specification and hematopoietic induction.Results : Screening of human iPSC clones after co-transfection of Cas9 and guide RNA-expression vectors has identified clones with ~400 bp deletion of the DNase I hypersensitive site that is conserved among vertebrates. When maintained in iPSC expansion condition, these iPSC clones display typical undifferentiated human iPSC morphology and expression of pluripotency-related markers such as TRA-1-60. Upon treatment with a Wnt signaling activator, BMP4, bFGF and VEGF, the genetically modified cells showed similar differentiation efficiency, compared to parental controls, to mesoderm and endothelial lineages. However, they exhibit a lower proliferation rate in endothelial expansion conditions. Preliminary studies also showed a deficiency of the modified cells in their ability to form capillary-like structures in tube formation assays. In hematopoietic differentiation experiments using the spin-EB method, these cells showed very low viability upon hematopoietic induction and failed to produce hematopoietic progenitor cells. These results suggest that the DNase I hypersensitive site upstream of GATA2 distal first exon plays an important role in human endothelial cell function including transition from hemogenic endothelium to hematopoietic cells upon stimulation.Currently, experiments are being conducted to characterize the functionality of endothelial-like cells and to confirm previous observations in additional iPSC lines. Differential expression of GATA-2 between parental and genome edited cells at various developmental stages is being determined. Hematopoietic differentiation capacity of these iPSCs by stromal cell co-culture methods is being used to confirm observations from the spin-EB protocol. Defining the functional outcome of this targeted deletion, across several iPSC lines, will provide a system for understanding molecular regulations in the first steps of human hematopoietic stem cell generation. DisclosuresNo relevant conflicts of interest to declare.

Controlling Hematopoietic Stem Cell Quiescence By Ablating the TGFβ-Responsive, Hematopoietic-Hpecific Enhancer of CDKN1C (p57)

Introduction: The cyclin-dependent kinase inhibitor (CDKI) CDKN1C (p57) is a putative tumor suppressor gene with strong differential expression in both human and murine hematopoietic stem cells (HSCs). The most immature, quiescent HSCs are marked by p57 expression and p57 is rapidly downregulated as HSCs enter cell cycle and differentiate. Genetic deletion of p57 leads to abnormal HSC cell cycle entry and, in murine models, a propensity for HSC exhaustion indicating that p57 is an essential gatekeeper of HSC function. TGFβ signaling is an important mediator of HSC quiescence and mandates the withdrawal of HSCs from cell cycle following hematopoietic stress. We have found that p57 is a direct transcriptional target of TGFβ in HSCs where it serves as an essential downstream mediator of TGFβ signaling. In HSCs, genetic deletion of p57 phenocopies the effects of TGFβ blockade in vivo. The regulation of p57 is complex and interesting. In almost all epithelial cell types, p57 is poorly expressed and is not a transcriptional target of TGFβ. This is also true for most differentiated hematopoietic lineages. The mechanism for the tissue-type and differentiation stage-specific expression of p57 and its control by TGFβ in HSCs is not known. We hypothesized that a hematopoietic enhancer for p57 must be responsible for TGFβ induction in HSCs.Methods: We performed chromatin immunoprecipitation (ChIP/ChIP-chip/ChIP-seq) in human, AML-derived M091 cells that faithfully upregulate p57 in response to TGFβ signaling in a manner indistinguishable from human cord blood derived CD34+ cells. Cultured cells were studied while proliferating and following treatment with TGFβ for various times. ChIP was performed for histone modifications that mark enhancer elements (histone H3 lysine 4 mono-methylation, H3K4me1, and H3K27-acetlylation, H3K27ac) and promoters (histone H3 lysine 4 tri-methylation, H3K4me2, and H3K27-acetlylation, H3K27ac). Genomic localization of receptor activated phospho-Smad2 was also identified before and after TGFβ stimulation. Putative enhancers were cloned into reporter constructs that were then studied after stable incorporation into the genome of M091 and K562 cells.Results: We identified putative enhancer elements more than 100 KB from the p57 gene body (Fig. A). A cluster of three small elements fulfilled our biochemical criterion for a hematopoietic-specific enhancer: enhancer histone marks (H3K4me1/H3K27ac); hyper-acetylation and phospho-Smad2 binding in response to TGFβ (Fig. B, D). When subcloned and tested in reporter assays, the elements had enhancer function and two of the three were induced by TGFβ (Fig. C). Interestingly, the ENCODE consortium identified the genomic region of our putative enhancers as overlapping with a DNAseI hypersensitive site and harboring the chromatin signature of a “Strong Enhancer” only in hematopoietic cells (K562) but not in other cell type tested. Nonetheless, unequivocal classification of this element as a p57 enhancer required in vivo analysis of HSCs with targeted deletion of the enhancer element.We designed and cloned, ten guide-RNAs (gRNAs) targeting regions upstream and downstream of this putative enhancer cluster. These gRNAs were then tested in HeLa cells by transient transfection with wild-type Cas9 nuclease. Using this approach, we selected a pair of gRNAs with the highest efficiency in deleting the entire enhancer cluster as assessed by ddPCR (~30% alleles). These gRNAs or non-targeting control gRNAs and Cas9 were then transfected into M091 cells along with a GFP reporter. Transfected GFP+ cells were then sorted 36 hrs later. A second lot of unsorted cells were grown in bulk cultures. Allelic ratios of this enhancer element were tracked by ddPCR to assess the relative outgrowth of genomically unmodified cells compared to those that had this putative p57 enhancer deleted. The targeted MO91 population has exhibited resistance to TGFβ as assessed using a growth curve assay.Conclusions: A hematopoietic-specific enhancer lying ~100 kb from the p57 gene body is important for TGFβ-mediated upregulation of p57 and consequent cell cycle withdrawal in immature hematopoietic cells. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Characterization of a HBB -Locus Fetal Chromatin Domain Region during Adult Erythropoiesis

The hemoglobinopathies including sickle cell disease (SCD) and β-thalassemia are the most common genetic disorders in the world producing significant morbidity and mortality. Drug-mediated fetal hemoglobin (HbF) induction ameliorates the clinical severity of SCD and improves long-term survival. To define mechanisms of γ-globin regulation, previously we demonstrated the role of the Gγ-globin cAMP response element, in drug-mediated HbF induction (Sangerman et al., Blood 2006). Subsequently, data generated by the ENCODE project led us to investigate a region 4 kb upstream of Gγ-globin, which harbors a DNase hypersensitive site and enrichment of the histone 3 lysine 4, mono-methylated (H3K4Me1) enhancer mark, that we named the Fetal Chromatin Domain (FCD). Recently, a synthetic zinc finger DNA-binding domain targeting this region was shown to reduce γ-globin gene expression in K562 cells (Shen et al., Blood 128:320, 2016). Initially, we performed multispecies sequence alignments which revealed the FCD is located in a genomic region shared by Old World monkeys (humans and Baboons) with a delayed fetal to adult globin switch that occurs after birth. By contrast, the FCD region is absent in New World monkeys where the switch occurs during the fetal developmental stage. These findings support involvement of the FCD region in γ-globin regulation during hemoglobin switching.Subsequent studies were performed to define the functional role of the FCD during erythropoiesis. ENCODE long RNA-sequencing assay detected PolyA RNA species throughout the FCD region in K562 cells. Based on these findings, we designed two small-interference RNA molecules targeting the core FCD (siFCD). Both siFCD molecules were transfected into KU812 cells and individual γ-globin gene expression measured using RT-qPCR with gene-specific primers. The level of Gγ-globin mRNA increased by 64%, while Aγ-globin transcription was reduced 38%. These results suggest the FCD facilitates differential regulation of the γ-globin genes. To further characterize FCD function, a series of γ-globin promoter luciferase reporters were constructed with the sense or antisense 500-bp core FCD region, cloned downstream of the luciferase gene. In KU812 stable cell lines, the sense FCD construct enhanced Aγ-globin promoter activity 10-fold, whereas the antisense construct repressed promoter activity. By contrast, the FCD in both orientations silenced Gγ-globin promoter activity. These results support unidirectional gene-specific enhancer properties of the FCD. Subsequent pulldown assay with a 34-bp biotinylated FCD probe confirmed NFE2, BHLHE40 and cMyc binding, which was abolished by mutation of the E-box sequence (CACGTG) in the FCD core.To build on the KU812 cell data, we performed μChIP assay using primary erythroid progenitors generated from adult CD34+ stem cells (Zhu et al., Haematologica 2017). In our system, the γ-globin to β-globin gene switch occurred on day 4 in culture. Initially, we measured levels of acetylated histone H3 (AcH3), H3K4Me1, and TFIID binding in the β-globin locus control region hypersensitivity site 2 (HS2) and FCD. As expected, high AcH3 and H3K4Me1 marks occurred in HS2 consistent with its enhancer function. Similar patterns of histone marks and binding of TFIID, NFE2 and BHLHE40 occurred in HS2 and FCD region. It is known that HS2 participates in DNA looping to accomplish developmentally regulated globin gene expression during hemoglobin switching therefore chromosome conformation capture (3C) assay was performed in primary erythroid progenitors to determine if the FCD facilitates changes in chromatin structure. We observed strong long-range interactions between the FCD and HS2 with 4.4-fold maximal chromatin enrichment on day 5; likewise the FCD showed interactions with Aγ-globin (4.7-fold) and Gγ-globin (3.5-fold) by day 3 before switching occurred. Of note, low level interactions occurred between the FCD and β-globin gene at all time points tested.Collectively, these data support the FCD as a unidirectional enhancer element involved in γ-globin regulation during erythropoiesis. To further define mechanisms of FCD function, the effects of CrisprCas9-mediated deletions in the FCD on globin gene expression will be explored. DisclosuresNo relevant conflicts of interest to declare.

Genome-Wide Identification of DNase I Hypersensitive Sites in Plants.

DNase I hypersensitive site (DHS) mapping combined with high-throughput sequencing (DNase-seq) enables the identification of cis-regulatory DNA elements (CREs) genome wide. However, despite the wide applications of DNase-seq in plants, its application to the highly repetitive genomes of plants has lagged. Here, we describe a modified DNase-seq method, making it more practical for application to plants with genomes enriched with repetitive DNA. This approach adopts a double-hit-based strategy, in which small (<250-bp) DNA fragments digested by DNase I are selected and used for sequencing library construction. Using these protocols, we have conducted DNase-seq in plants with high content of repetitive DNA, including maize, sugarcane, and tetraploid cotton. Genome-wide maps of DHS and CREs have been created using these DNase-seq datasets. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Nuclei isolation Basic Protocol 2: DNase I digestion Basic Protocol 3: Target DNA isolation Basic Protocol 4: Library construction and validation.

Purifying selection on noncoding deletions of human regulatory loci detected using their cellular pleiotropy.

Genomic deletions provide a powerful loss-of-function model in noncoding regions to assess the role of purifying selection on genetic variation. Regulatory element function is characterized by nonuniform tissue and cell type activity, necessarily linking the study of fitness consequences from regulatory variants to their corresponding cellular activity. We generated a callset of deletions from genomes in the Alzheimer's Disease Neuroimaging Initiative (ADNI) and used deletions from The 1000 Genomes Project Consortium (1000GP) in order to examine whether purifying selection preserves noncoding sites of chromatin accessibility marked by DNase I hypersensitivity (DHS), histone modification (enhancer, transcribed, Polycomb-repressed, heterochromatin), and chromatin loop anchors. To examine this in a cellular activity-aware manner, we developed a statistical method, pleiotropy ratio score (PlyRS), which calculates a correlation-adjusted count of “cellular pleiotropy” for each noncoding base pair by analyzing shared regulatory annotations across tissues and cell types. By comparing real deletion PlyRS values to simulations in a length-matched framework and by using genomic covariates in analyses, we found that purifying selection acts to preserve both DHS and enhancer noncoding sites. However, we did not find evidence of purifying selection for noncoding transcribed, Polycomb-repressed, or heterochromatin sites beyond that of the noncoding background. Additionally, we found evidence that purifying selection is acting on chromatin loop integrity by preserving colocalized CTCF binding sites. At regions of DHS, enhancer, and CTCF within chromatin loop anchors, we found evidence that both sites of activity specific to a particular tissue or cell type and sites of cellularly pleiotropic activity are preserved by selection.

CEPZ: A Novel Predictor for Identification of DNase I Hypersensitive Sites.

DNase I hypersensitive sites (DHSs) have proven to be tightly associated with cis-regulatory elements, commonly indicating specific function on the chromatin structure. Thus, identifying DHSs plays a fundamental role in decoding gene regulatory behavior. While traditional experimental methods turn to be time-consuming and expensive, computational techniques promise to be practical to discovering and analyzing regulatory factors. In this study, we applied an efficient model that considered composition information and physicochemical properties and effectively selected features with a boosting algorithm. CEPZ, our predictor, greatly improved a Matthews correlation coefficient and accuracy of 0.7740 and 0.9113 respectively, more competitive than any predictor before. This result suggests that it may become a useful tool for DHSs research in the human and other complex genomes. Our research was anchored on the properties of dinucleotides and we identified several dinucleotides with significant differences in the distribution of DHS and non-DHS samples, which are likely to have a special meaning in the chromatin structure. The datasets, feature sets and the relevant algorithm are available at https://github.com/YanZheng-16/CEPZ_DHS/.

Genomic Footprinting Analyses from DNase-seq Data to Construct Gene Regulatory Networks.

Chromatin accessibility is directly linked with transcription in eukaryotes. Accessible regions associated with regulatory proteins are highly sensitive to DNase I digestion and are termed DNase I hypersensitive sites (DHSs). DHSs can be identified by DNase I digestion, followed by high-throughput DNA sequencing (DNase-seq). The single-base-pair resolution digestion patterns from DNase-seq allows identifying transcription factor (TF) footprints of local DNA protection that predict TF-DNA binding. The identification of differential footprinting between two conditions allows mapping relevant TF regulatory interactions. Here, we provide step-by-step instructions to build gene regulatory networks from DNase-seq data. Our pipeline includes steps for DHSs calling, identification of differential TF footprints between treatment and control conditions, and construction of gene regulatory networks. Even though the data we used in this example was obtained from Arabidopsis thaliana, the workflow developed in this guide can be adapted to work with DNase-seq data from any organism with a sequenced genome.

Exposure to extracellular vesicles from Pseudomonas aeruginosa result in loss of DNA methylation at enhancer and DNase hypersensitive site regions in lung macrophages

ABSTRACT Various pathogens use differing strategies to evade host immune response including modulating the host’s epigenome. Here, we investigate if EVs secreted from P. aeruginosa alter DNA methylation in human lung macrophages, thereby potentially contributing to a dysfunctional innate immune response. Using a genome-wide DNA methylation approach, we demonstrate that P. aeruginosa EVs alter certain host cell DNA methylation patterns. We identified 1,185 differentially methylated CpGs (FDR < 0.05), which were significantly enriched for distal DNA regulatory elements including enhancer regions and DNase hypersensitive sites. Notably, all but one of the 1,185 differentially methylated CpGs were hypomethylated in association with EV exposure. Significantly hypomethylated CpGs tracked to genes including AXL, CFB and CCL23. Gene expression analysis identified 310 genes exhibiting significantly altered expression 48 hours post P. aeruginosa EV treatment, with 75 different genes upregulated and 235 genes downregulated. Some CpGs associated with cytokines such as CSF3 displayed strong negative correlations between DNA methylation and gene expression. Our infection model illustrates how secreted products (EVs) from bacteria can alter DNA methylation of the host epigenome. Changes in DNA methylation in distal DNA regulatory regions in turn can modulate cellular gene expression and potential downstream cellular processes.

Regulatory mechanism of chicken lysozyme gene expression in oviducts examined using transgenic technology

The use of promoters that strongly express target genes in the chicken oviduct is beneficial for the production of proteinaceous materials into egg white by transgenic chickens. To examine the regulatory mechanisms of chicken lysozyme gene expression invivo, genetically manipulated chickens that express human erythropoietin under the control of a lysozyme promoter-enhancer were established. By using several deletion mutants of the promoter-flanking region, we found that a-1.9kb DNase I hypersensitive site (DHS) was essential for oviduct-specific expression in genetically manipulated chickens. The concentration of human erythropoietin in egg white was 14-75μg/ml, suggesting that the chicken lysozyme promoter containing-1.9kb DHS is sufficient for the production of pharmaceuticals using transgenic chickens.

The prediction of human DNase I hypersensitive sites based on DNA sequence information

DNase I hypersensitive sites (DHSs) are special regions of the chromosome with loose structure that can be recognized, bound, and cleaved by DNase I enzyme. In these specific regions, the chromatin lacks condensed structure, resulting in increased accessibility. DHSs are hallmarks of gene expression regulation, and the characterization of DHSs is important to understand transcriptional regulatory mechanism and also to facilitate localization of cis-regulatory elements such as promoters, enhancers, insulators, silencers, and locus control regions. Although many experimental methods have been proposed to identify DHSs, these methods are time-consuming and expensive, making it urgent to develop computational methods to predict DHSs. In this study, we described a sequence-based predictor to identity DHSs in the human genome. In the predictor, optimal features were selected from a large feature set including various k-mer nucleotide compositions and correlation information of physicochemical properties of dinucleotides by using a two-step feature selection algorithm. Using 5-fold cross-validation, the proposed method achieved a Matthews correlation coefficient and accuracy of 0.66 and 0.87, respectively, which are higher than those of published DHS predictors, indicating the good performance of our method. The benchmark datasets and trained DHS model are available at https://github.com/Jackie-Suv/iDHS-SVM.

LincSNP 3.0: an updated database for linking functional variants to human long non-coding RNAs, circular RNAs and their regulatory elements.

We describe an updated comprehensive database, LincSNP 3.0 (http://bioinfo.hrbmu.edu.cn/LincSNP), which aims to document and annotate disease or phenotype-associated variants in human long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs) or their regulatory elements. LincSNP 3.0 has updated with several novel features, including (i) more types of variants including single nucleotide polymorphisms (SNPs), linkage disequilibrium SNPs (LD SNPs), somatic mutations and RNA editing sites have been expanded; (ii) more regulatory elements including transcription factor binding sites (TFBSs), enhancers, DNase I hypersensitive sites (DHSs), topologically associated domains (TADs), footprintss, methylations and open chromatin regions have been added; (iii) the associations among circRNAs, regulatory elements and variants have been identified; (iv) more experimentally supported variant-lncRNA/circRNA-disease/phenotype associations have been manually collected; (v) the sources of lncRNAs, circRNAs, SNPs, somatic mutations and RNA editing sites have been updated. Moreover, four flexible online tools including Genome Browser, Variant Mapper, Circos Plotter and Functional Annotation have been developed to retrieve, visualize and analyze the data. Collectively, LincSNP 3.0 provides associations among functional variants, regulatory elements, lncRNAs and circRNAs in diseases. It will serve as an important and continually updated resource for investigating functions and mechanisms of lncRNAs and circRNAs in diseases.

Systematic Evaluation of Somatic Cis-Regulatory Mutations in Follicular Lymphoma

Background: Follicular lymphoma (FL) is an incurable indolent B cell malignancy characterized in the majority of cases by the t(14;18) translocation. While the mutational landscape of the coding genome is nearing completion, less is known about the characteristics of its noncoding genome. Our expectation is that the distribution of noncoding mutations will be non-random, determined by epigenomics features, such as chromatin modification and accessibility. Our strategy is therefore to integrate whole genome epigenomic marks and mutations in order to enrich for variants with regulatory potential and identify unique mutational processes specific to thesecis-regulatory elements (CREs).Allele-specific expression (ASE) patterns allow further refinement to resolve functional CREs and bona fide mutations associated with changes in gene expression. Methods: The H3K27Ac consensus CREs were determined using the ChIP-Seq data of 9 FL patient primary cells (Koues et al., Immunity2015). DNase I-hypersensitive sites (DHSs) and Hi-C of the B-lymphocyte cell line GM12878 was downloaded from ENCODE. Whole genome sequencing and RNA-Seq of 70 FL patients with relative high tumour cellularity (≥30% for DNA tumour purity and ≥25% for RNA B-cell content) were obtained from the International Cancer Genome Consortium project ICGC MMML-Seq. The mutation rate within CREs and DHSs in comparison to their flanking sequences was estimated using a previous pipeline (Sabarinathan et al., Nature2016). We further developed cis-ASE, an integrated analytic pipeline for the identification of recurrent ASE genes, and significantly associated CREs and mutations (Fig1.A). Results: In total 1.04 million noncoding mutations, corresponding to 14.8K mutations per sample with a median of 9,991 noncoding mutations were identified in our series of 70 FL samples. 62K (6.0%) mutations were located within H3K27Ac bound CREs and there was an elevated mutation rate in H3K27Ac CREs compared to the corresponding flanking regions of 1kb up and downstream (χ² test p = 1.95e-19, Fig1.B). For DHSs, we observed the opposite pattern, with a lower mutation rate in DHSs than in the flanking regions (p = 0.04, Fig1.C), most likely reflecting the higher accessibility to global genome repair machinery in DHSs in relation to flanking sequences. Dividing mutations into high (≥0.3) and low (&amp;lt;0.3) adjusted variant allele frequency (VAF) groups (accounting for tumour purity), we observed significantly higher mutation rate in CREs than in the flanking sequences, that was specific for mutations with higher VAFs (p= 6.95e−38), as the difference was much weaker for low VAF mutations (p= 0.02). Mutation signatures 6 and 20, linked to defective DNA mismatch repair, were highly enriched for mutations within H3K27Ac CREs (44%) versus other regions outside (27%) (p&amp;lt; 0.001). ASE was assessed using 45.6k informative SNPs (1.7%) per sample wherein matched genotype data was available from WGS and RNA-Seq profiles. Cis-ASE identified on average 480 ASE genes per sample (binomial test, adjusted p&amp;lt;0.05), corresponding to 1,943 recurrent ASE genes with a minimum threshold of ≥5 samples. These ASE events were not significantly influenced by local copy number changes or promoter methylation. KEGG pathway analysis of recurrent ASE genes identified adherens junction, B cell receptor signaling pathway and Fc gamma R-mediated phagocytosis as the most overrepresented pathways. Cis-ASE further identified 18 ASE-CRE interactions where CRE mutations were significantly correlated with an imbalance in elevated alternative allele ratios. These ASE genes included recognized lymphoma related genes including BCL2, STAT6, MAF, and additional novel targets. The pattern of aberrant somatic hypermutation (aSHM) was assessed for these 18 CREs, and we narrowed down to 15 significant ASE-CRE interactions not strongly affected by aSHM, consisting of 92 bona fide mutation candidates present in 37 FL samples of our cohort (52.9%). Conclusion: Our study identified unique mutation processes operating in H3K27Ac CREs. Using an integrated genomic approach of whole genome mutations, chromatin marks and RNA-Seq, we explored ASE-CRE interactions, and identified 15 H3K27Ac bound CREs enriched for cis-regulatory mutations significantly associated with ASE and total expression of targeted genes, deserving for further exploration to enrich our understanding of FL noncoding genome. Disclosures Okosun: BeiGene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead Sciences: Research Funding.

NIMBus: a negative binomial regression based Integrative Method for mutation Burden Analysis

BackgroundIdentifying frequently mutated regions is a key approach to discover DNA elements influencing cancer progression.However, it is challenging to identify these burdened regions due to mutation rate heterogeneity across the genome and across different individuals. Moreover, it is known that this heterogeneity partially stems from genomic confounding factors, such as replication timing and chromatin organization. The increasing availability of cancer whole genome sequences and functional genomics data from the Encyclopedia of DNA Elements (ENCODE) may help address these issues.ResultsWe developed a negative binomial regression-based Integrative Method for mutation Burden analysiS (NIMBus). Our approach addresses the over-dispersion of mutation count statistics by (1) using a Gamma–Poisson mixture model to capture the mutation-rate heterogeneity across different individuals and (2) estimating regional background mutation rates by regressing the varying local mutation counts against genomic features extracted from ENCODE. We applied NIMBus to whole-genome cancer sequences from the PanCancer Analysis of Whole Genomes project (PCAWG) and other cohorts. It successfully identified well-known coding and noncoding drivers, such as TP53 and the TERT promoter. To further characterize the burdening of non-coding regions, we used NIMBus to screen transcription factor binding sites in promoter regions that intersect DNase I hypersensitive sites (DHSs). This analysis identified mutational hotspots that potentially disrupt gene regulatory networks in cancer. We also compare this method to other mutation burden analysis methods.ConclusionNIMBus is a powerful tool to identify mutational hotspots. The NIMBus software and results are available as an online resource at github.gersteinlab.org/nimbus.

A cell-type deconvolution meta-analysis of whole blood EWAS reveals lineage-specific smoking-associated DNA methylation changes

Highly reproducible smoking-associated DNA methylation changes in whole blood have been reported by many Epigenome-Wide-Association Studies (EWAS). These epigenetic alterations could have important implications for understanding and predicting the risk of smoking-related diseases. To this end, it is important to establish if these DNA methylation changes happen in all blood cell subtypes or if they are cell-type specific. Here, we apply a cell-type deconvolution algorithm to identify cell-type specific DNA methylation signals in seven large EWAS. We find that most of the highly reproducible smoking-associated hypomethylation signatures are more prominent in the myeloid lineage. A meta-analysis further identifies a myeloid-specific smoking-associated hypermethylation signature enriched for DNase Hypersensitive Sites in acute myeloid leukemia. These results may guide the design of future smoking EWAS and have important implications for our understanding of how smoking affects immune-cell subtypes and how this may influence the risk of smoking related diseases.

Trajectories of body mass index and adolescent hair cortisol: importance of persistence, variability, and developmental timing

Longevity gene klotho (KL) is associated with age-related phenotypes including lifespan, cardiometabolic disorders, cognition, and brain morphology, in part, by conferring protection against inflammation. We hypothesized that the KL/inflammation association might be altered in the presence of psychiatric stress and operate via epigenetic pathways. We examined KL polymorphisms, and their interaction with posttraumatic stress disorder (PTSD) symptoms, in association with KL DNA methylation in blood. We further examined KL DNA methylation as a predictor of longitudinal changes in a peripheral biomarker of inflammation (C-reactive protein; CRP).The sample comprised 309 white non-Hispanic military veterans (93.5 % male; mean age: 32 years, range: 19–65; 30 % PTSD per structured diagnostic interview); 111 were reassessed approximately two years later.Analyses revealed a methylation quantitative trait locus at rs9527025 (C370S, previously implicated in numerous studies of aging) in association with a Cytosine-phosphate-Guanine site (cg00129557; B = −.65, p = 1.29 X 10−20), located within a DNase hypersensitivity site in the body of KL. There was also a rs9527025 x PTSD severity interaction (B = .004, p = .035) on methylation at this locus such that the minor allele was associated with reduced cg00129557 methylation in individuals with few or no PTSD symptoms while this effect was attenuated in those with elevated levels of PTSD. Path models revealed that methylation at cg00129557 was inversely associated with CRP over time (B = −.14, p = .005), controlling for baseline CRP. There was also an indirect effect of rs9527025 X PTSD on subsequent CRP via cg00129557 methylation (indirect B = −.002, p = .033).Results contribute to our understanding of the epigenetic correlates of inflammation in PTSD and suggest that KL methylation may be a mechanism by which KL genotype confers risk vs. resilience to accelerated aging in those experiencing traumatic stress.

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

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

Index and biological spectrum of human DNase I hypersensitive sites

DNase I hypersensitive sites (DHSs) are generic markers of regulatory DNA1–5 and contain genetic variations associated with diseases and phenotypic traits6–8. We created high-resolution maps of DHSs from 733 human biosamples encompassing 438 cell and tissue types and states, and integrated these to delineate and numerically index approximately 3.6 million DHSs within the human genome sequence, providing a common coordinate system for regulatory DNA. Here we show that these maps highly resolve the cis-regulatory compartment of the human genome, which encodes unexpectedly diverse cell- and tissue-selective regulatory programs at very high density. These programs can be captured comprehensively by a simple vocabulary that enables the assignment to each DHS of a regulatory barcode that encapsulates its tissue manifestations, and global annotation of protein-coding and non-coding RNA genes in a manner orthogonal to gene expression. Finally, we show that sharply resolved DHSs markedly enhance the genetic association and heritability signals of diseases and traits. Rather than being confined to a small number of distal elements or promoters, we find that genetic signals converge on congruently regulated sets of DHSs that decorate entire gene bodies. Together, our results create a universal, extensible coordinate system and vocabulary for human regulatory DNA marked by DHSs, and provide a new global perspective on the architecture of human gene regulation.

Global reference mapping of human transcription factor footprints

Combinatorial binding of transcription factors to regulatory DNA underpins gene regulation in all organisms. Genetic variation in regulatory regions has been connected with diseases and diverse phenotypic traits1, but it remains challenging to distinguish variants that affect regulatory function2. Genomic DNase I footprinting enables the quantitative, nucleotide-resolution delineation of sites of transcription factor occupancy within native chromatin3–6. However, only a small fraction of such sites have been precisely resolved on the human genome sequence6. Here, to enable comprehensive mapping of transcription factor footprints, we produced high-density DNase I cleavage maps from 243 human cell and tissue types and states and integrated these data to delineate about 4.5 million compact genomic elements that encode transcription factor occupancy at nucleotide resolution. We map the fine-scale structure within about 1.6 million DNase I-hypersensitive sites and show that the overwhelming majority are populated by well-spaced sites of single transcription factor–DNA interaction. Cell-context-dependent cis-regulation is chiefly executed by wholesale modulation of accessibility at regulatory DNA rather than by differential transcription factor occupancy within accessible elements. We also show that the enrichment of genetic variants associated with diseases or phenotypic traits in regulatory regions1,7 is almost entirely attributable to variants within footprints, and that functional variants that affect transcription factor occupancy are nearly evenly partitioned between loss- and gain-of-function alleles. Unexpectedly, we find increased density of human genetic variation within transcription factor footprints, revealing an unappreciated driver of cis-regulatory evolution. Our results provide a framework for both global and nucleotide-precision analyses of gene regulatory mechanisms and functional genetic variation.