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Background: Epigenetic mechanisms such as chromatin accessibility impact transcription factor binding to DNA and transcriptional specificity. The androgen receptor (AR), a master regulator of the male phenotype and prostate cancer pathogenesis, acts primar

Background: Crohn's disease (CD) is a chronic inflammatory condition affecting the gastrointestinal tract, characterized by complications such as strictures, fistulas, and neoplasia. Despite medical advancements, a significant number of patients with Crohn's disease require surgery, and many experience post-operative complications and recurrence. Previous studies have analyzed gene expression to study recurrence and post-operative complications independently. This study aims to identify overlapping differentially expressed genes and pathways for recurrence and post-operative complications., Methods: A dataset including 45 patients with Crohn's disease, including gene expression from ileum and colon tissue, endoscopic recurrence, and intra-abdominal septic complications was analyzed. Gene set enrichment analysis was used to identify gene pathways associated with the outcomes. Finally, a multi-variable logistic regression model was created to assess whether gene pathways were independently associated with both outcomes., Results: In ileum tissue, several inflammatory pathways, including interferon alpha and gamma response were upregulated in patients with endoscopic recurrence and intra-abdominal septic complications. In addition, there was upregulation of the epithelial mesenchymal transition pathway. In colon tissue, metabolic processes, such as myogenesis and oxidative phosphorylation were downregulated in both outcomes. In a multivariate model, downregulation of myogenesis in colon tissue was significantly associated with both endoscopic recurrence and intra-abdominal septic complications., Conclusion: These findings shed light on the underlying biology of these outcomes and suggest potential biomarkers or therapeutic targets to reduce their occurrence. Further validation and multi-institutional studies are warranted to confirm these results and improve post-operative outcomes for patients with Crohn's disease., (© 2024. The Author(s).)

Polymerized β-actin may provide a structural basis for chromatin accessibility and actin transport into the nucleus can guide mesenchymal stem cell (MSC) differentiation. Using MSC, we show that using CK666 to inhibit Arp2/3 directed secondary actin branching results in decreased nuclear actin structure, and significantly alters chromatin access measured with ATACseq at 24 h. The ATAC-seq results due to CK666 are distinct from those caused by cytochalasin D (CytoD), which enhances nuclear actin structure. In addition, nuclear visualization shows Arp2/3 inhibition decreases pericentric H3K9me3 marks. CytoD, alternatively, induces redistribution of H3K27me3 marks centrally. Such alterations in chromatin landscape are consistent with differential gene expression associated with distinctive differentiation patterns. Further, knockdown of the non-enzymatic monomeric actin binding protein, Arp4, leads to extensive chromatin unpacking, but only a modest increase in transcription, indicating an active role for actin-Arp4 in transcription. These data indicate that dynamic actin remodeling can regulate chromatin interactions., (© 2024. The Author(s).)

Network inference is used to model transcriptional, signaling, and metabolic interactions among genes, proteins, and metabolites that identify biological pathways influencing disease pathogenesis. Advances in machine learning (ML)-based inference models exhibit the predictive capabilities of capturing latent patterns in genomic data. Such models are emerging as an alternative to the statistical models identifying causative factors driving complex diseases. We present CoVar, an ML-based framework that builds upon the properties of existing inference models, to find the central genes driving perturbed gene expression across biological states. Unlike differentially expressed genes (DEGs) that capture changes in individual gene expression across conditions, CoVar focuses on identifying variational genes that undergo changes in their expression network interaction profiles, providing insights into changes in the regulatory dynamics, such as in disease pathogenesis. Subsequently, it finds core genes from among the nearest neighbors of these variational genes, which are central to the variational activity and influence the coordinated regulatory processes underlying the observed changes in gene expression. Through the analysis of simulated as well as yeast expression data perturbed by the deletion of the mitochondrial genome, we show that CoVar captures the intrinsic variationality and modularity in the expression data, identifying key driver genes not found through existing differential analysis methodologies., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Roy et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Crohn's disease (CD) is a chronic inflammatory gut disorder. Molecular mechanisms underlying the clinical heterogeneity of CD remain poorly understood. MicroRNAs (miRNAs) are important regulators of gut physiology, and several have been implicated in the pathogenesis of adult CD. However, there is a dearth of large-scale miRNA studies for pediatric CD. We hypothesized that specific miRNAs uniquely mark pediatric CD. We performed small RNA-Seq of patient-matched colon and ileum biopsies from treatment-naive pediatric patients with CD (n = 169) and a control cohort (n = 108). Comprehensive miRNA analysis revealed 58 miRNAs altered in pediatric CD. Notably, multinomial logistic regression analysis revealed that index levels of ileal miR-29 are strongly predictive of severe inflammation and stricturing. Transcriptomic analyses of transgenic mice overexpressing miR-29 show a significant reduction of the tight junction protein gene Pmp22 and classic Paneth cell markers. The dramatic loss of Paneth cells was confirmed by histologic assays. Moreover, we found that pediatric patients with CD with elevated miR-29 exhibit significantly lower Paneth cell counts, increased inflammation scores, and reduced levels of PMP22. These findings strongly indicate that miR-29 upregulation is a distinguishing feature of pediatric CD, highly predictive of severe phenotypes, and associated with inflammation and Paneth cell loss.

Pediatric Crohn's disease (CD) is characterized by a severe disease course with frequent complications. We sought to apply machine learning-based models to predict risk of developing future complications in pediatric CD using ileal and colonic gene expression. Gene expression data was generated from 101 formalin-fixed, paraffin-embedded (FFPE) ileal and colonic biopsies obtained from treatment-naïve CD patients and controls. Clinical outcomes including development of strictures or fistulas and progression to surgery were analyzed using differential expression and modeled using machine learning. Differential expression analysis revealed downregulation of pathways related to inflammation and extra-cellular matrix production in patients with strictures. Machine learning-based models were able to incorporate colonic gene expression and clinical characteristics to predict outcomes with high accuracy. Models showed an area under the receiver operating characteristic curve (AUROC) of 0.84 for strictures, 0.83 for remission, and 0.75 for surgery. Genes with potential prognostic importance for strictures (REG1A, MMP3, and DUOX2) were not identified in single gene differential analysis but were found to have strong contributions to predictive models. Our findings in FFPE tissue support the importance of colonic gene expression and the potential for machine learning-based models in predicting outcomes for pediatric CD., (© 2024. The Author(s).)

Motivation: Analysis of open chromatin regions across multiple samples from two or more distinct conditions can determine altered gene regulatory patterns associated with biological phenotypes and complex traits. The ATAC-seq assay allows for tractable genome-wide open chromatin profiling of large numbers of samples. Stable, broadly applicable genomic annotations of open chromatin regions are not available. Thus, most studies first identify open regions using peak calling methods for each sample independently. These are then heuristically combined to obtain a consensus peak set. Reconciling sample-specific peak results post hoc from larger cohorts is particularly challenging, and informative spatial features specific to open chromatin signals are not leveraged effectively., Results: We propose a novel method, ROCCO, that determines consensus open chromatin regions across multiple samples simultaneously. ROCCO employs robust summary statistics and solves a constrained optimization problem formulated to account for both enrichment and spatial dependence of open chromatin signal data. We show this formulation admits attractive theoretical and conceptual properties as well as superior empirical performance compared to current methodology., Availability and Implementation: Source code, documentation, and usage demos for ROCCO are available on GitHub at: https://github.com/nolan-h-hamilton/ROCCO. ROCCO can also be installed as a stand-alone binary utility using pip/PyPI., (© The Author(s) 2023. Published by Oxford University Press.)

The purpose of this study was to determine the toxicological and pharmacokinetic properties of sucralose-6-acetate, a structural analog of the artificial sweetener sucralose. Sucralose-6-acetate is an intermediate and impurity in the manufacture of sucralose, and recent commercial sucralose samples were found to contain up to 0.67% sucralose-6-acetate. Studies in a rodent model found that sucralose-6-acetate is also present in fecal samples with levels up to 10% relative to sucralose which suggest that sucralose is also acetylated in the intestines. A MultiFlow® assay, a high-throughput genotoxicity screening tool, and a micronucleus (MN) test that detects cytogenetic damage both indicated that sucralose-6-acetate is genotoxic. The mechanism of action was classified as clastogenic (produces DNA strand breaks) using the MultiFlow® assay. The amount of sucralose-6-acetate in a single daily sucralose-sweetened drink might far exceed the threshold of toxicological concern for genotoxicity (TTC genotox ) of 0.15 µg/person/day. The RepliGut® System was employed to expose human intestinal epithelium to sucralose-6-acetate and sucralose, and an RNA-seq analysis was performed to determine gene expression induced by these exposures. Sucralose-6-acetate significantly increased the expression of genes associated with inflammation, oxidative stress, and cancer with greatest expression for the metallothionein 1 G gene (MT1G). Measurements of transepithelial electrical resistance (TEER) and permeability in human transverse colon epithelium indicated that sucralose-6-acetate and sucralose both impaired intestinal barrier integrity. Sucralose-6-acetate also inhibited two members of the cytochrome P450 family (CYP1A2 and CYP2C19). Overall, the toxicological and pharmacokinetic findings for sucralose-6-acetate raise significant health concerns regarding the safety and regulatory status of sucralose itself.

Based upon our demonstration that the smooth muscle cell-selective (SMC-selective) putative methyltransferase, Prdm6, interacts with myocardin-related transcription factor-A, we examined Prdm6's role in SMCs in vivo using cell type-specific knockout mouse models. Although SMC-specific depletion of Prdm6 in adult mice was well tolerated, Prdm6 depletion in Wnt1-expressing cells during development resulted in perinatal lethality and a completely penetrant patent ductus arteriosus (DA) phenotype. Lineage tracing experiments in Wnt1Cre2 Prdm6fl/fl ROSA26LacZ mice revealed normal neural crest-derived SMC investment of the outflow tract. In contrast, myography measurements on DA segments isolated from E18.5 embryos indicated that Prdm6 depletion significantly reduced DA tone and contractility. RNA-Seq analyses on DA and ascending aorta samples at E18.5 identified a DA-enriched gene program that included many SMC-selective contractile associated proteins that was downregulated by Prdm6 depletion. Chromatin immunoprecipitation-sequencing experiments in outflow tract SMCs demonstrated that 50% of the genes Prdm6 depletion altered contained Prdm6 binding sites. Finally, using several genome-wide data sets, we identified an SMC-selective enhancer within the Prdm6 third intron that exhibited allele-specific activity, providing evidence that rs17149944 may be the causal SNP for a cardiovascular disease GWAS locus identified within the human PRDM6 gene.

Introduction: In colitis, macrophage functionality is altered compared to normal homeostatic conditions. Loss of IL-10 signaling results in an inappropriate chronic inflammatory response to bacterial stimulation. It remains unknown if inhibition of bromodomain and extra-terminal domain (BET) proteins alters usage of DNA regulatory elements responsible for driving inflammatory gene expression. We determined if the BET inhibitor, (+)-JQ1, could suppress inflammatory activation of macrophages in Il10 -/- mice., Methods: We performed ATAC-seq and RNA-seq on Il10 -/- bone marrow-derived macrophages (BMDMs) cultured in the presence and absence of lipopolysaccharide (LPS) with and without treatment with (+)-JQ1 and evaluated changes in chromatin accessibility and gene expression. Germ-free Il10 -/- mice were treated with (+)-JQ1, colonized with fecal slurries and underwent histological and molecular evaluation 14-days post colonization., Results: Treatment with (+)-JQ1 suppressed LPS-induced changes in chromatin at distal regulatory elements associated with inflammatory genes, particularly in regions that contain motifs for AP-1 and IRF transcription factors. This resulted in attenuation of inflammatory gene expression. Treatment with (+)-JQ1 in vivo resulted in a mild reduction in colitis severity as compared with vehicle-treated mice., Conclusion: We identified the mechanism of action associated with a new class of compounds that may mitigate aberrant macrophage responses to bacteria in colitis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Hoffner O’Connor, Berglind, Kennedy Ng, Keith, Lynch, Schaner, Steinbach, Herzog, Trad, Jeck, Arthur, Simon, Sartor, Furey and Sheikh.)

Alternate splicing events can create isoforms that alter gene function, and genetic variants associated with alternate gene isoforms may reveal molecular mechanisms of disease. We used subcutaneous adipose tissue of 426 Finnish men from the METSIM study and identified splice junction quantitative trait loci (sQTLs) for 6,077 splice junctions (FDR < 1%). In the same individuals, we detected expression QTLs (eQTLs) for 59,443 exons and 15,397 genes (FDR < 1%). We identified 595 genes with an sQTL and exon eQTL but no gene eQTL, which could indicate potential isoform differences. Of the significant sQTL signals, 2,114 (39.8%) included at least one proxy variant (linkage disequilibrium r 2 > 0.8) located within an intron spanned by the splice junction. We identified 203 sQTLs that colocalized with 141 genome-wide association study (GWAS) signals for cardiometabolic traits, including 25 signals for lipid traits, 24 signals for body mass index (BMI), and 12 signals for waist-hip ratio adjusted for BMI. Among all 141 GWAS signals colocalized with an sQTL, we detected 26 that also colocalized with an exon eQTL for an exon skipped by the sQTL splice junction. At a GWAS signal for high-density lipoprotein cholesterol colocalized with an NR1H3 sQTL splice junction, we show that the alternative splice product encodes an NR1H3 transcription factor that lacks a DNA binding domain and fails to activate transcription. Together, these results detect splicing events and candidate mechanisms that may contribute to gene function at GWAS loci., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 American Society of Human Genetics. All rights reserved.)

Background & Aims: The intestinal barrier comprises a monolayer of specialized intestinal epithelial cells (IECs) that are critical in maintaining mucosal homeostasis. Dysfunction within various IEC fractions can alter intestinal permeability in a genetically susceptible host, resulting in a chronic and debilitating condition known as Crohn's disease (CD). Defining the molecular changes in each IEC type in CD will contribute to an improved understanding of the pathogenic processes and the identification of cell type-specific therapeutic targets. We performed, at single-cell resolution, a direct comparison of the colonic epithelial cellular and molecular landscape between treatment-naïve adult CD and non-inflammatory bowel disease control patients., Methods: Colonic epithelial-enriched, single-cell sequencing from treatment-naïve adult CD and non-inflammatory bowel disease patients was investigated to identify disease-induced differences in IEC types., Results: Our analysis showed that in CD patients there is a significant skew in the colonic epithelial cellular distribution away from canonical LGR5+ stem cells, located at the crypt bottom, and toward one specific subtype of mature colonocytes, located at the crypt top. Further analysis showed unique changes to gene expression programs in every major cell type, including a previously undescribed suppression in CD of most enteroendocrine driver genes as well as L-cell markers including GCG. We also dissect an incompletely understood SPIB+ cell cluster, revealing at least 4 subclusters that likely represent different stages of a maturational trajectory. One of these SPIB+ subclusters expresses crypt-top colonocyte markers and is up-regulated significantly in CD, whereas another subcluster strongly expresses and stains positive for lysozyme (albeit no other canonical Paneth cell marker), which surprisingly is greatly reduced in expression in CD. In addition, we also discovered transposable element markers of colonic epithelial cell types as well as transposable element families that are altered significantly in CD in a cell type-specific manner. Finally, through integration with data from genome-wide association studies, we show that genes implicated in CD risk show heretofore unknown cell type-specific patterns of aberrant expression in CD, providing unprecedented insight into the potential biological functions of these genes., Conclusions: Single-cell analysis shows a number of unexpected cellular and molecular features, including transposable element expression signatures, in the colonic epithelium of treatment-naïve adult CD., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Acute ozone (O 3 ) exposure is associated with multiple adverse cardiorespiratory outcomes, the severity of which varies across individuals in human populations and inbred mouse strains. However, molecular determinants of response, including susceptibility biomarkers that distinguish who will develop severe injury and inflammation, are not well characterized. We and others have demonstrated that airway macrophages (AMs) are an important resident immune cell type that are functionally and transcriptionally responsive to O 3 inhalation. Here, we sought to explore influences of strain, exposure, and strain-by-O 3 exposure interactions on AM gene expression and identify transcriptional correlates of O 3 -induced inflammation and injury across six mouse strains, including five Collaborative Cross (CC) strains. We exposed adult mice of both sexes to filtered air (FA) or 2 ppm O 3 for 3 h and measured inflammatory and injury parameters 21 h later. Mice exposed to O 3 developed airway neutrophilia and lung injury with strain-dependent severity. In AMs, we identified a common core O 3 transcriptional response signature across all strains, as well as a set of genes exhibiting strain-by-O 3 exposure interactions. In particular, a prominent gene expression contrast emerged between a low- (CC017/Unc) and high-responding (CC003/Unc) strain, as reflected by cellular inflammation and injury. Further inspection indicated that differences in their baseline gene expression and chromatin accessibility profiles likely contribute to their divergent post-O 3 exposure transcriptional responses. Together, these results suggest that aspects of O 3 -induced respiratory responses are mediated through altered AM transcriptional signatures and further confirm the importance of gene-environment interactions in mediating differential responsiveness to environmental agents.

Inflammatory bowel diseases (IBD), namely Crohn's disease (CD) and ulcerative colitis (UC) are chronic inflammation within the gastrointestinal tract. IBD patient conditions and treatments, such as with immunosuppressants, may result in a higher risk of viral and bacterial infection and more severe outcomes of infections. The effect of the clinical and demographic factors on the prognosis of COVID-19 among IBD patients is still a significant area of investigation. The lack of available data on a large set of COVID-19 infected IBD patients has hindered progress. To circumvent this lack of large patient data, we present a random sampling approach to generate clinical COVID-19 outcomes (outpatient management, hospitalized and recovered, and hospitalized and deceased) on 20,000 IBD patients modeled on reported summary statistics obtained from the Surveillance Epidemiology of Coronavirus Under Research Exclusion (SECURE-IBD), an international database to monitor and report on outcomes of COVID-19 occurring in IBD patients. We apply machine learning approaches to perform a comprehensive analysis of the primary and secondary covariates to predict COVID-19 outcome in IBD patients. Our analysis reveals that age, medication usage and the number of comorbidities are the primary covariates, while IBD severity, smoking history, gender and IBD subtype (CD or UC) are key secondary features. In particular, elderly male patients with ulcerative colitis, several preexisting conditions, and who smoke comprise a highly vulnerable IBD population. Moreover, treatment with 5-ASAs (sulfasalazine/mesalamine) shows a high association with COVID-19/IBD mortality. Supervised machine learning that considers age, number of comorbidities and medication usage can predict COVID-19/IBD outcomes with approximately 70% accuracy. We explore the challenge of drawing demographic inferences from existing COVID-19/IBD data. Overall, there are fewer IBD case reports from US states with poor health ranking hindering these analyses. Generation of patient characteristics based on known summary statistics allows for increased power to detect IBD factors leading to variable COVID-19 outcomes. There is under-reporting of COVID-19 in IBD patients from US states with poor health ranking, underpinning the perils of using the repository to derive demographic information., (© 2021. The Author(s).)

The host receptor for SARS-CoV-2, angiotensin-converting enzyme 2 (ACE2), is highly expressed in small intestine. Our aim was to study colonic ACE2 expression in Crohn's disease (CD) and non-inflammatory bowel disease (non-IBD) controls. We hypothesized that the colonic expression levels of ACE2 impacts CD course. We examined the expression of colonic ACE2 in 67 adult CD and 14 NIBD control patients using RNA-seq and quantitative (q) RT-PCR. We validated ACE2 protein expression and localization in formalin-fixed, paraffin-embedded matched colon and ileal tissues using immunohistochemistry. The impact of increased ACE2 expression in CD for the risk of surgery was evaluated by a multivariate regression analysis and a Kaplan-Meier estimator. To provide critical support for the generality of our findings, we analyzed previously published RNA-seq data from two large independent cohorts of CD patients. Colonic ACE2 expression was significantly higher in a subset of adult CD patients which was defined as the ACE2-high CD subset. IHC in a sampling of ACE2-high CD patients confirmed high ACE2 protein expression in the colon and ileum compared to ACE2-low CD and NIBD patients. Notably, we found that ACE2-high CD patients are significantly more likely to undergo surgery within 5 years of CD diagnosis, and a Cox regression analysis found that high ACE2 levels is an independent risk factor for surgery (OR 2.17; 95% CI, 1.10-4.26; p = 0.025). Increased intestinal expression of ACE2 is associated with deteriorated clinical outcomes in CD patients. These data point to the need for molecular stratification that can impact CD disease-related outcomes.

Background: The intestinal microbiota play a key role in the onset, progression, and recurrence of Crohn disease (CD). Most microbiome studies assay fecal material, which does not provide region-specific information on mucosally adherent bacteria that directly interact with host systems. Changes in luminal oxygen have been proposed as a contributor to CD dybiosis., Methods: The authors generated 16S rRNA data using colonic and ileal mucosal bacteria from patients with CD and without inflammatory bowel disease. We developed profiles reflecting bacterial abundance within defined aerotolerance categories. Bacterial diversity, composition, and aerotolerance profiles were compared across intestinal regions and disease phenotypes., Results: Bacterial diversity decreased in CD in both the ileum and the colon. Aerotolerance profiles significantly differed between intestinal segments in patients without inflammatory bowel disease, although both were dominated by obligate anaerobes, as expected. In CD, high relative levels of obligate anaerobes were maintained in the colon and increased in the ileum. Relative abundances of similar and distinct taxa were altered in colon and ileum. Notably, several obligate anaerobes, such as Bacteroides fragilis, dramatically increased in CD in one or both intestinal segments, although specific increasing taxa varied across patients. Increased abundance of taxa from the Proteobacteria phylum was found only in the ileum. Bacterial diversity was significantly reduced in resected tissues of patients who developed postoperative disease recurrence across 2 independent cohorts, with common lower abundance of bacteria from the Bacteroides, Streptococcus, and Blautia genera., Conclusions: Mucosally adherent bacteria in the colon and ileum show distinct alterations in CD that provide additional insights not revealed in fecal material., (© 2020 Crohn’s & Colitis Foundation. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Gene transcription profiles across tissues are largely defined by the activity of regulatory elements, most of which correspond to regions of accessible chromatin. Regulatory element activity is in turn modulated by genetic variation, resulting in variable transcription rates across individuals. The interplay of these factors, however, is poorly understood. Here we characterize expression and chromatin state dynamics across three tissues-liver, lung, and kidney-in 47 strains of the Collaborative Cross (CC) mouse population, examining the regulation of these dynamics by expression quantitative trait loci (eQTL) and chromatin QTL (cQTL). QTL whose allelic effects were consistent across tissues were detected for 1,101 genes and 133 chromatin regions. Also detected were eQTL and cQTL whose allelic effects differed across tissues, including local-eQTL for Pik3c2g detected in all three tissues but with distinct allelic effects. Leveraging overlapping measurements of gene expression and chromatin accessibility on the same mice from multiple tissues, we used mediation analysis to identify chromatin and gene expression intermediates of eQTL effects. Based on QTL and mediation analyses over multiple tissues, we propose a causal model for the distal genetic regulation of Akr1e1, a gene involved in glycogen metabolism, through the zinc finger transcription factor Zfp985 and chromatin intermediates. This analysis demonstrates the complexity of transcriptional and chromatin dynamics and their regulation over multiple tissues, as well as the value of the CC and related genetic resource populations for identifying specific regulatory mechanisms within cells and tissues., Competing Interests: The authors have declared that no competing interests exist.

Background & Aims: Intestinal epithelial cell (IEC) barrier dysfunction is critical to the development of Crohn's disease (CD). However, the mechanism is understudied. We recently reported increased microRNA-31-5p (miR-31-5p) expression in colonic IECs of CD patients, but downstream targets and functional consequences are unknown., Methods: microRNA-31-5p target genes were identified by integrative analysis of RNA- and small RNA-sequencing data from colonic mucosa and confirmed by quantitative polymerase chain reaction in colonic IECs. Functional characterization of activin receptor-like kinase 1 (ACVRL1 or ALK1) in IECs was performed ex vivo using 2-dimensional cultured human primary colonic IECs. The impact of altered colonic ALK1 signaling in CD for the risk of surgery and endoscopic relapse was evaluated by a multivariate regression analysis and a Kaplan-Meier estimator., Results: ALK1 was identified as a target of miR-31-5p in colonic IECs of CD patients and confirmed using a 3'-untranslated region reporter assay. Activation of ALK1 restricted the proliferation of colonic IECs in a 5-ethynyl-2-deoxyuridine proliferation assay and down-regulated the expression of stemness-related genes. Activated ALK1 signaling increased colonic IEC differentiation toward colonocytes. Down-regulated ALK1 signaling was associated with increased stemness and decreased colonocyte-specific marker expression in colonic IECs of CD patients compared with healthy controls. Activation of ALK1 enhanced epithelial barrier integrity in a transepithelial electrical resistance permeability assay. Lower colonic ALK1 expression was identified as an independent risk factor for surgery and was associated with a higher risk of endoscopic relapse in CD patients., Conclusions: Decreased colonic ALK1 disrupted colonic IEC barrier integrity and was associated with poor clinical outcomes in CD patients., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Genome-wide association studies (GWASs) have identified thousands of genetic loci associated with cardiometabolic traits including type 2 diabetes (T2D), lipid levels, body fat distribution, and adiposity, although most causal genes remain unknown. We used subcutaneous adipose tissue RNA-seq data from 434 Finnish men from the METSIM study to identify 9,687 primary and 2,785 secondary cis-expression quantitative trait loci (eQTL; <1 Mb from TSS, FDR < 1%). Compared to primary eQTL signals, secondary eQTL signals were located further from transcription start sites, had smaller effect sizes, and were less enriched in adipose tissue regulatory elements compared to primary signals. Among 2,843 cardiometabolic GWAS signals, 262 colocalized by LD and conditional analysis with 318 transcripts as primary and conditionally distinct secondary cis-eQTLs, including some across ancestries. Of cardiometabolic traits examined for adipose tissue eQTL colocalizations, waist-hip ratio (WHR) and circulating lipid traits had the highest percentage of colocalized eQTLs (15% and 14%, respectively). Among alleles associated with increased cardiometabolic GWAS risk, approximately half (53%) were associated with decreased gene expression level. Mediation analyses of colocalized genes and cardiometabolic traits within the 434 individuals provided further evidence that gene expression influences variant-trait associations. These results identify hundreds of candidate genes that may act in adipose tissue to influence cardiometabolic traits., (Copyright © 2019 American Society of Human Genetics. All rights reserved.)

The IBDs, Crohn's disease and ulcerative colitis, are chronic inflammatory conditions of the gastrointestinal tract resulting from an aberrant immune response to enteric microbiota in genetically susceptible individuals. Disease presentation and progression within and across IBDs, especially Crohn's disease, are highly heterogeneous in location, severity of inflammation and other phenotypes. Current clinical classifications fail to accurately predict disease course and response to therapies. Genome-wide association studies have identified >240 loci that confer risk of IBD, but the clinical utility of these findings remains unclear, and mechanisms by which the genetic variants contribute to disease are largely unknown. In the past 5 years, the profiling of genome-wide gene expression, epigenomic features and gut microbiota composition in intestinal tissue and faecal samples has uncovered distinct molecular signatures that define IBD subtypes, including within Crohn's disease and ulcerative colitis. In this Review, we summarize studies in both adult and paediatric patients that have identified different IBD subtypes, which in some cases have been associated with distinct clinical phenotypes. We posit that genome-scale molecular phenotyping in large cohorts holds great promise not only to further our understanding of the diverse molecular causes of IBD but also for improving clinical trial design to develop more personalized disease management and treatment.

Background: Crohn's disease (CD) is highly heterogeneous, due in large part to variability in cellular processes that underlie the natural history of CD, thereby confounding effective therapy. There is a critical need to advance understanding of the cellular mechanisms that drive CD heterogeneity., Methods: We performed small RNA sequencing of adult colon tissue from CD and NIBD controls. Colonic epithelial cells and immune cells were isolated from colonic tissues, and microRNA-31 (miR-31) expression was measured. miR-31 expression was measured in colonoid cultures generated from controls and patients with CD. We performed small RNA-sequencing of formalin-fixed paraffin-embedded colon and ileum biopsies from treatment-naive pediatric patients with CD and controls and collected data on disease features and outcomes., Results: Small RNA-sequencing and microRNA profiling in the colon revealed 2 distinct molecular subtypes, each with different clinical associations. Notably, we found that miR-31 expression was a driver of these 2 subtypes and, further, that miR-31 expression was particularly pronounced in epithelial cells. Colonoids revealed that miR-31 expression differences are preserved in this ex vivo system. In adult patients, low colonic miR-31 expression levels at the time of surgery were associated with worse disease outcome as measured by need for an end ileostomy and recurrence of disease in the neoterminal ileum. In pediatric patients, lower miR-31 expression at the time of diagnosis was associated with future development of fibrostenotic ileal CD requiring surgeryCONCLUSIONS. These findings represent an important step forward in designing more effective clinical trials and developing personalized CD therapies., Funding: This work was supported by CCF Career Development Award (SZS), R01-ES024983 from NIEHS (SZS and TSF), 1R01DK104828-01A1 from NIDDK (SZS and TSF), P01-DK094779-01A1 from NIDDK (SZS), P30-DK034987 from NIDDK (SZS), 1-16-ACE-47 ADA Pathway Award (PS), UNC Nutrition Obesity Research Center Pilot & Feasibility Grant P30DK056350 (PS), CCF PRO-KIIDS NETWORK (SZS and PS), UNC CGIBD T32 Training Grant from NIDDK (JBB), T32 Training Grant (5T32GM007092-42) from NIGMS (MH), and SHARE from the Helmsley Trust (SZS). The UNC Translational Pathology Laboratory is supported, in part, by grants from the National Cancer Institute (3P30CA016086) and the UNC University Cancer Research Fund (UCRF) (PS).

Epigenetic effects of environmental chemicals are under intense investigation to fill existing knowledge gaps between environmental/occupational exposures and adverse health outcomes. Chromatin accessibility is one prominent mechanism of epigenetic control of transcription, and understanding of the chemical effects on both could inform the causal role of epigenetic alterations in disease mechanisms. In this study, we hypothesized that baseline variability in chromatin organization and transcription profiles among various tissues and mouse strains influence the outcome of exposure to the DNA damaging chemical 1,3-butadiene. To test this hypothesis, we evaluated DNA damage along with comprehensive quantification of RNA transcripts (RNA-seq), identification of accessible chromatin (ATAC-seq), and characterization of regions with histone modifications associated with active transcription (ChIP-seq for acetylation at histone 3 lysine 27, H3K27ac). We collected these data in the lung, liver, and kidney of mice from two genetically divergent strains, C57BL/6J and CAST/EiJ, that were exposed to clean air or to 1,3-butadiene (~600 ppm) for 2 weeks. We found that tissue effects dominate differences in both gene expression and chromatin states, followed by strain effects. At baseline, xenobiotic metabolism was consistently more active in CAST/EiJ, while immune system pathways were more active in C57BL/6J across tissues. Surprisingly, even though all three tissues in both strains harbored butadiene-induced DNA damage, little transcriptional effect of butadiene was observed in liver and kidney. Toxicologically relevant effects of butadiene in the lung were on the pathways of xenobiotic metabolism and inflammation. We also found that variability in chromatin accessibility across individuals (i.e., strains) only partially explains the variability in transcription. This study showed that variation in the basal states of epigenome and transcriptome may be useful indicators for individuals or tissues susceptible to genotoxic environmental chemicals.

Objective: The clinical presentation and course of Crohn's disease (CD) is highly variable. We sought to better understand the cellular and molecular mechanisms that guide this heterogeneity, and characterise the cellular processes associated with disease phenotypes., Design: We examined both gene expression and gene regulation (chromatin accessibility) in non-inflamed colon tissue from a cohort of adult patients with CD and control patients. To support the generality of our findings, we analysed previously published expression data from a large cohort of treatment-naïve paediatric CD and control ileum., Results: We found that adult patients with CD clearly segregated into two classes based on colon tissue gene expression-one that largely resembled the normal colon and one where certain genes showed expression patterns normally specific to the ileum. These classes were supported by changes in gene regulatory profiles observed at the level of chromatin accessibility, reflective of a fundamental shift in underlying molecular phenotypes. Furthermore, gene expression from the ilea of a treatment-naïve cohort of paediatric patients with CD could be similarly subdivided into colon-like and ileum-like classes. Finally, expression patterns within these CD subclasses highlight large-scale differences in the immune response and aspects of cellular metabolism, and were associated with multiple clinical phenotypes describing disease behaviour, including rectal disease and need for colectomy., Conclusions: Our results strongly suggest that these molecular signatures define two clinically relevant forms of CD irrespective of tissue sampling location, patient age or treatment status., Competing Interests: Competing interests: None declared., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.)

Background: The damaging effects of exposure to environmental toxicants differentially affect genetically distinct individuals, but the mechanisms contributing to these differences are poorly understood. Genetic variation affects the establishment of the gene regulatory landscape and thus gene expression, and we hypothesized that this contributes to the observed heterogeneity in individual responses to exogenous cellular insults., Objectives: We performed an in vivo study of how genetic variation and chromatin organization may dictate susceptibility to DNA damage, and influence the cellular response to such damage, caused by an environmental toxicant., Materials and Methods: We measured DNA damage, messenger RNA (mRNA) and microRNA (miRNA) expression, and genome-wide chromatin accessibility in lung tissue from two genetically divergent inbred mouse strains, C57BL/6J and CAST/EiJ, both in unexposed mice and in mice exposed to a model DNA-damaging chemical, 1,3-butadiene., Results: Our results showed that unexposed CAST/EiJ and C57BL/6J mice have very different chromatin organization and transcription profiles in the lung. Importantly, in unexposed CAST/EiJ mice, which acquired relatively less 1,3-butadiene-induced DNA damage, we observed increased transcription and a more accessible chromatin landscape around genes involved in detoxification pathways. Upon chemical exposure, chromatin was significantly remodeled in the lung of C57BL/6J mice, a strain that acquired higher levels of 1,3-butadiene-induced DNA damage, around the same genes, ultimately resembling the molecular profile of CAST/EiJ., Conclusions: These results suggest that strain-specific changes in chromatin and transcription in response to chemical exposure lead to a "compensation" for underlying genetic-driven interindividual differences in the baseline chromatin and transcriptional state. This work represents an example of how chemical and environmental exposures can be evaluated to better understand gene-by-environment interactions, and it demonstrates the important role of chromatin response in transcriptomic changes and, potentially, in deleterious effects of exposure. https://doi.org/10.1289/EHP1937.

It is well established that genetic variability has a major impact on susceptibility to common diseases, responses to drugs and toxicants, and influences disease-related outcomes. The appreciation that epigenetic marks also vary across the population is growing with more data becoming available from studies in humans and model organisms. In addition, the links between genetic variability, toxicity outcomes and epigenetics are being actively explored. Recent studies demonstrate that gene-by-environment interactions involve both chromatin states and transcriptional regulation, and that epigenetics provides important mechanistic clues to connect expression-related quantitative trait loci (QTL) and disease outcomes. However, studies of Gene×Environment×Epigenetics further extend the complexity of the experimental designs and create a challenge for selecting the most informative epigenetic readouts that can be feasibly performed to interrogate multiple individuals, exposures, tissue types and toxicity phenotypes. We propose that among the many possible epigenetic experimental methodologies, assessment of chromatin accessibility coupled with total RNA levels provides a cost-effective and comprehensive option to sufficiently characterize the complexity of epigenetic and regulatory activity in the context of understanding the inter-individual variability in responses to toxicants.

The intestinal epithelium serves critical physiologic functions that are shared among all vertebrates. However, it is unknown how the transcriptional regulatory mechanisms underlying these functions have changed over the course of vertebrate evolution. We generated genome-wide mRNA and accessible chromatin data from adult intestinal epithelial cells (IECs) in zebrafish, stickleback, mouse, and human species to determine if conserved IEC functions are achieved through common transcriptional regulation. We found evidence for substantial common regulation and conservation of gene expression regionally along the length of the intestine from fish to mammals and identified a core set of genes comprising a vertebrate IEC signature. We also identified transcriptional start sites and other putative regulatory regions that are differentially accessible in IECs in all 4 species. Although these sites rarely showed sequence conservation from fish to mammals, surprisingly, they drove highly conserved IEC expression in a zebrafish reporter assay. Common putative transcription factor binding sites (TFBS) found at these sites in multiple species indicate that sequence conservation alone is insufficient to identify much of the functionally conserved IEC regulatory information. Among the rare, highly sequence-conserved, IEC-specific regulatory regions, we discovered an ancient enhancer upstream from her6/HES1 that is active in a distinct population of Notch-positive cells in the intestinal epithelium. Together, these results show how combining accessible chromatin and mRNA datasets with TFBS prediction and in vivo reporter assays can reveal tissue-specific regulatory information conserved across 420 million years of vertebrate evolution. We define an IEC transcriptional regulatory network that is shared between fish and mammals and establish an experimental platform for studying how evolutionarily distilled regulatory information commonly controls IEC development and physiology.

Background: High-throughput sequence (HTS) data exhibit position-specific nucleotide biases that obscure the intended signal and reduce the effectiveness of these data for downstream analyses. These biases are particularly evident in HTS assays for identifying regulatory regions in DNA (DNase-seq, ChIP-seq, FAIRE-seq, ATAC-seq). Biases may result from many experiment-specific factors, including selectivity of DNA restriction enzymes and fragmentation method, as well as sequencing technology-specific factors, such as choice of adapters/primers and sample amplification methods., Results: We present a novel method to detect and correct position-specific nucleotide biases in HTS short read data. Our method calculates read-specific weights based on aligned reads to correct the over- or underrepresentation of position-specific nucleotide subsequences, both within and adjacent to the aligned read, relative to a baseline calculated in assay-specific enriched regions. Using HTS data from a variety of ChIP-seq, DNase-seq, FAIRE-seq, and ATAC-seq experiments, we show that our weight-adjusted reads reduce the position-specific nucleotide imbalance across reads and improve the utility of these data for downstream analyses, including identification and characterization of open chromatin peaks and transcription-factor binding sites., Conclusions: A general-purpose method to characterize and correct position-specific nucleotide sequence biases fills the need to recognize and deal with, in a systematic manner, binding-site preference for the growing number of HTS-based epigenetic assays. As the breadth and impact of these biases are better understood, the availability of a standard toolkit to correct them will be important.

Motivation: Identifying the locations of transcription factor binding sites is critical for understanding how gene transcription is regulated across different cell types and conditions. Chromatin accessibility experiments such as DNaseI sequencing (DNase-seq) and Assay for Transposase Accessible Chromatin sequencing (ATAC-seq) produce genome-wide data that include distinct 'footprint' patterns at binding sites. Nearly all existing computational methods to detect footprints from these data assume that footprint signals are highly homogeneous across footprint sites. Additionally, a comprehensive and systematic comparison of footprinting methods for specifically identifying which motif sites for a specific factor are bound has not been performed., Results: Using DNase-seq data from the ENCODE project, we show that a large degree of previously uncharacterized site-to-site variability exists in footprint signal across motif sites for a transcription factor. To model this heterogeneity in the data, we introduce a novel, supervised learning footprinter called Detecting Footprints Containing Motifs (DeFCoM). We compare DeFCoM to nine existing methods using evaluation sets from four human cell-lines and eighteen transcription factors and show that DeFCoM outperforms current methods in determining bound and unbound motif sites. We also analyze the impact of several biological and technical factors on the quality of footprint predictions to highlight important considerations when conducting footprint analyses and assessing the performance of footprint prediction methods. Finally, we show that DeFCoM can detect footprints using ATAC-seq data with similar accuracy as when using DNase-seq data., Availability and Implementation: Python code available at https://bitbucket.org/bryancquach/defcom., Contact: bquach@email.unc.edu or tsfurey@email.unc.edu., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com)

Intestinal macrophages (IMs) are uniquely programmed to tolerate exposure to bacteria without mounting potent inflammatory responses. The cytokine IL-10 maintains the macrophage anti-inflammatory response such that loss of IL-10 results in chronic intestinal inflammation. To investigate how IL-10-deficiency alters IM programming and bacterial tolerance, we studied changes in chromatin accessibility in response to bacteria in macrophages from two distinct niches, the intestine and bone-marrow, from both wild-type and IL-10-deficient (Il10(-/-) ) mice. We identified chromatin accessibility changes associated with bacterial exposure and IL-10 deficiency in both bone marrow derived macrophages and IMs. Surprisingly, Il10(-/-) IMs adopted chromatin and gene expression patterns characteristic of an inflammatory response, even in the absence of bacteria. Further, when recombinant IL-10 was added to Il10(-/-) cells, it could not revert the chromatin landscape to a normal state. Our results demonstrate that IL-10 deficiency results in stable chromatin alterations in macrophages, even in the absence of bacteria. This supports a model in which IL-10-deficiency leads to chromatin alterations that contribute to a loss of IM tolerance to bacteria, which is a primary initiating event in chronic intestinal inflammation., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Motivation: Advances in chromosome conformation capture and next-generation sequencing technologies are enabling genome-wide investigation of dynamic chromatin interactions. For example, Hi-C experiments generate genome-wide contact frequencies between pairs of loci by sequencing DNA segments ligated from loci in close spatial proximity. One essential task in such studies is peak calling, that is, detecting non-random interactions between loci from the two-dimensional contact frequency matrix. Successful fulfillment of this task has many important implications including identifying long-range interactions that assist interpreting a sizable fraction of the results from genome-wide association studies. The task - distinguishing biologically meaningful chromatin interactions from massive numbers of random interactions - poses great challenges both statistically and computationally. Model-based methods to address this challenge are still lacking. In particular, no statistical model exists that takes the underlying dependency structure into consideration., Results: In this paper, we propose a hidden Markov random field (HMRF) based Bayesian method to rigorously model interaction probabilities in the two-dimensional space based on the contact frequency matrix. By borrowing information from neighboring loci pairs, our method demonstrates superior reproducibility and statistical power in both simulation studies and real data analysis., Availability and Implementation: The Source codes can be downloaded at: http://www.unc.edu/∼yunmli/HMRFBayesHiC CONTACT: ming.hu@nyumc.org or yunli@med.unc.edu, Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

To increase our understanding of the genetic basis of adiposity and its links to cardiometabolic disease risk, we conducted a genome-wide association meta-analysis of body fat percentage (BF%) in up to 100,716 individuals. Twelve loci reached genome-wide significance (P<5 × 10(-8)), of which eight were previously associated with increased overall adiposity (BMI, BF%) and four (in or near COBLL1/GRB14, IGF2BP1, PLA2G6, CRTC1) were novel associations with BF%. Seven loci showed a larger effect on BF% than on BMI, suggestive of a primary association with adiposity, while five loci showed larger effects on BMI than on BF%, suggesting association with both fat and lean mass. In particular, the loci more strongly associated with BF% showed distinct cross-phenotype association signatures with a range of cardiometabolic traits revealing new insights in the link between adiposity and disease risk.

Background: There is a dire need for reliable prognostic markers that can guide effective therapeutic intervention in Crohn's disease (CD). We examined whether different phenotypes in CD can be classified based on colonic microRNA (miRNA) expression and whether miRNAs have prognostic utility for CD., Methods: High-throughput sequencing of small and total RNA isolated from colon tissue from patients with CD and controls without Inflammatory Bowel Disease (non-IBD) was performed. To identify miRNAs associated with specific phenotypes of CD, patients were stratified according to disease behavior (nonstricturing, nonpenetrating; stricturing; penetrating), and miRNA profiles in each subset were compared with those of the non-IBD group. Validation assays were performed using quantitative reverse transcription polymerase chain reaction. These miRNAs were further evaluated by quantitative reverse transcriptase polymerase chain reaction on formalin-fixed, paraffin-embedded tissue (index biopsies) of patients with nonpenetrating CD at the time of diagnosis that either retained the nonpenetrating phenotype or progressed to penetrating/fistulizing CD., Results: We found a suite of miRNAs, including miR-31-5p, miR-215, miR-223-3p, miR-196b-5p, and miR-203 that stratify patients with CD according to disease behavior independent of the effect of inflammation. Furthermore, we also demonstrated that expression levels of miR-215 in index biopsies of patients with CD might predict the likelihood of progression to penetrating/fistulizing CD. Finally, using a novel statistical simulation approach applied to colonic RNA-sequencing data for patients with CD and non-IBD controls, we identified miR-31-5p and miR-203 as candidate master regulators of gene expression profiles associated with CD., Conclusions: miRNAs may serve as clinically useful prognostic markers guiding initial therapy and identifying patients who would benefit most from effective intervention.

Background: Genetic variation can alter transcriptional regulatory activity contributing to variation in complex traits and risk of disease, but identifying individual variants that affect regulatory activity has been challenging. Quantitative sequence-based experiments such as ChIP-seq and DNase-seq can detect sites of allelic imbalance where alleles contribute disproportionately to the overall signal suggesting allelic differences in regulatory activity., Methods: We created an allelic imbalance detection pipeline, AA-ALIGNER, to remove reference mapping biases influencing allelic imbalance detection and evaluate accuracy of allelic imbalance predictions in the absence of complete genotype data. Using the sequence aligner, GSNAP, and varying amounts of genotype information to remove mapping biases we investigated the accuracy of allelic imbalance detection (binomial test) in CREB1 ChIP-seq reads from the GM12878 cell line. Additionally we thoroughly evaluated the influence of experimental and analytical parameters on imbalance detection., Results: Compared to imbalances identified using complete genotypes, using imputed partial sample genotypes, AA-ALIGNER detected >95 % of imbalances with >90 % accuracy. AA-ALIGNER performed nearly as well using common variants when genotypes were unknown. In contrast, predicting additional heterozygous sites and imbalances using the sequence data led to >50 % false positive rates. We evaluated effects of experimental data characteristics and key analytical parameter settings on imbalance detection. Overall, total base coverage and signal dispersion across the genome most affected our ability to detect imbalances, while parameters such as imbalance significance, imputation quality thresholds, and alignment mismatches had little effect. To assess the biological relevance of imbalance predictions, we used electrophoretic mobility shift assays to functionally test for predicted allelic differences in CREB1 binding in the GM12878 lymphoblast cell line. Six of nine tested variants exhibited allelic differences in binding. Two of these variants, rs2382818 and rs713875, are located within inflammatory bowel disease-associated loci., Conclusions: AA-ALIGNER accurately detects allelic imbalance in quantitative sequence data using partial genotypes or common variants filling a critical methodological gap in these analyses, as full genotypes are rarely available. Importantly, we demonstrate how experimental and analytical features impact imbalance detection providing guidance for similar future studies.

Mapping expression quantitative trait loci (eQTL) has identified genetic variants associated with transcription rates and has provided insight into genotype-phenotype associations obtained from genome-wide association studies (GWAS). Traditional eQTL mapping methods present significant challenges for the multiple-testing burden, resulting in a limited ability to detect eQTL that reside distal to the affected gene. To overcome this, we developed a novel eQTL testing approach, " NET: work-based, L: arge-scale I: dentification o F: dis T: al eQTL" (NetLIFT), which performs eQTL testing based on the pairwise conditional dependencies between genes' expression levels. When applied to existing data from yeast segregants, NetLIFT replicated most previously identified distal eQTL and identified 46% more genes with distal effects compared to local effects. In liver data from mouse lines derived through the Collaborative Cross project, NetLIFT detected 5744 genes with local eQTL while 3322 genes had distal eQTL. This analysis revealed founder-of-origin effects for a subset of local eQTL that may contribute to previously described phenotypic differences in metabolic traits. In human lymphoblastoid cell lines, NetLIFT was able to detect 1274 transcripts with distal eQTL that had not been reported in previous studies, while 2483 transcripts with local eQTL were identified. In all species, we found no enrichment for transcription factors facilitating eQTL associations; instead, we found that most trans-acting factors were annotated for metabolic function, suggesting that genetic variation may indirectly regulate multigene pathways by targeting key components of feedback processes within regulatory networks. Furthermore, the unique genetic history of each population appears to influence the detection of genes with local and distal eQTL., (Copyright © 2014 by the Genetics Society of America.)

RNA-Seq is quickly becoming the preferred method for comprehensively characterizing whole transcriptome activity, and the analysis of count data from RNA-Seq requires new computational tools. We developed GSAASeqSP, a novel toolset for genome-wide gene set association analysis of sequence count data. This toolset offers a variety of statistical procedures via combinations of multiple gene-level and gene set-level statistics, each having their own strengths under different sample and experimental conditions. These methods can be employed independently, or results generated from multiple or all methods can be integrated to determine more robust profiles of significantly altered biological pathways. Using simulations, we demonstrate the ability of these methods to identify association signals and to measure the strength of the association. We show that GSAASeqSP analyses of RNA-Seq data from diverse tissue samples provide meaningful insights into the biological mechanisms that differentiate these samples. GSAASeqSP is a powerful platform for investigating molecular underpinnings of complex traits and diseases arising from differential activity within the biological pathways. GSAASeqSP is available at http://gsaa.unc.edu.

Despite an emerging understanding of the genetic alterations giving rise to various tumors, the mechanisms whereby most oncogenes are overexpressed remain unclear. Here we have utilized an integrated approach of genomewide regulatory element mapping via DNase-seq followed by conventional reporter assays and transcription factor binding site discovery to characterize the transcriptional regulation of the medulloblastoma oncogene Orthodenticle Homeobox 2 (OTX2). Through these studies we have revealed that OTX2 is differentially regulated in medulloblastoma at the level of chromatin accessibility, which is in part mediated by DNA methylation. In cell lines exhibiting chromatin accessibility of OTX2 regulatory regions, we found that autoregulation maintains OTX2 expression. Comparison of medulloblastoma regulatory elements with those of the developing brain reveals that these tumors engage a developmental regulatory program to drive OTX2 transcription. Finally, we have identified a transcriptional regulatory element mediating retinoid-induced OTX2 repression in these tumors. This work characterizes for the first time the mechanisms of OTX2 overexpression in medulloblastoma. Furthermore, this study establishes proof of principle for applying ENCODE datasets towards the characterization of upstream trans-acting factors mediating expression of individual genes.

Comprehensive sequencing of human cancers has identified recurrent mutations in genes encoding chromatin regulatory proteins. For clear cell renal cell carcinoma (ccRCC), three of the five commonly mutated genes encode the chromatin regulators PBRM1, SETD2, and BAP1. How these mutations alter the chromatin landscape and transcriptional program in ccRCC or other cancers is not understood. Here, we identified alterations in chromatin organization and transcript profiles associated with mutations in chromatin regulators in a large cohort of primary human kidney tumors. By associating variation in chromatin organization with mutations in SETD2, which encodes the enzyme responsible for H3K36 trimethylation, we found that changes in chromatin accessibility occurred primarily within actively transcribed genes. This increase in chromatin accessibility was linked with widespread alterations in RNA processing, including intron retention and aberrant splicing, affecting ∼25% of all expressed genes. Furthermore, decreased nucleosome occupancy proximal to misspliced exons was observed in tumors lacking H3K36me3. These results directly link mutations in SETD2 to chromatin accessibility changes and RNA processing defects in cancer. Detecting the functional consequences of specific mutations in chromatin regulatory proteins in primary human samples could ultimately inform the therapeutic application of an emerging class of chromatin-targeted compounds.

The epithelium lining the epididymis has a pivotal role in ensuring a luminal environment that can support normal sperm maturation. Many of the individual genes that encode proteins involved in establishing the epididymal luminal fluid are well characterized. They include ion channels, ion exchangers, transporters, and solute carriers. However, the molecular mechanisms that coordinate expression of these genes and modulate their activities in response to biological stimuli are less well understood. To identify cis-regulatory elements for genes expressed in human epididymis epithelial cells, we generated genome-wide maps of open chromatin by DNase-seq. This analysis identified 33,542 epididymis-selective DNase I hypersensitive sites (DHS), which were not evident in five cell types of different lineages. Identification of genes with epididymis-selective DHS at their promoters revealed gene pathways that are active in immature epididymis epithelial cells. These include processes correlating with epithelial function and also others with specific roles in the epididymis, including retinol metabolism and ascorbate and aldarate metabolism. Peaks of epididymis-selective chromatin were seen in the androgen receptor gene and the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which has a critical role in regulating ion transport across the epididymis epithelium. In silico prediction of transcription factor binding sites that were overrepresented in epididymis-selective DHS identified epithelial transcription factors, including ELF5 and ELF3, the androgen receptor, Pax2, and Sox9, as components of epididymis transcriptional networks. Active genes, which are targets of each transcription factor, reveal important biological processes in the epididymis epithelium.

DNase-seq is primarily used to identify nucleosome-depleted DNase I hypersensitive (DHS) sites genome-wide that correspond to active regulatory elements. However, ≈ 40 yr ago it was demonstrated that DNase I also digests with a ≈ 10-bp periodicity around nucleosomes matching the exposure of the DNA minor groove as it wraps around histones. Here, we use DNase-seq data from 49 samples representing diverse cell types to reveal this digestion pattern at individual loci and predict genomic locations where nucleosome rotational positioning, the orientation of DNA with respect to the histone surface, is stably maintained. We call these regions DNase I annotated regions of nucleosome stability (DARNS). Compared to MNase-seq experiments, we show DARNS correspond well to annotated nucleosomes. Interestingly, many DARNS are positioned over only one side of annotated nucleosomes, suggesting that the periodic digestion pattern attenuates over the nucleosome dyad. DARNS reproduce the arrangement of nucleosomes around transcription start sites and are depleted at ubiquitous DHS sites. We also generated DARNS from multiple lymphoblast cell line (LCL) samples. We found that LCL DARNS were enriched at DHS sites present in most of the original 49 samples but absent in LCLs, while multi-cell-type DARNS were enriched at LCL-specific DHS sites. This indicates that variably open DHS sites are often occupied by rotationally stable nucleosomes in cell types where the DHS site is closed. DARNS provide additional information about precise DNA orientation within individual nucleosomes not available from other nucleosome positioning assays and contribute to understanding the role of chromatin in gene regulation.

Regulatory elements recruit transcription factors that modulate gene expression distinctly across cell types, but the relationships among these remains elusive. To address this, we analyzed matched DNase-seq and gene expression data for 112 human samples representing 72 cell types. We first defined more than 1800 clusters of DNase I hypersensitive sites (DHSs) with similar tissue specificity of DNase-seq signal patterns. We then used these to uncover distinct associations between DHSs and promoters, CpG islands, conserved elements, and transcription factor motif enrichment. Motif analysis within clusters identified known and novel motifs in cell-type-specific and ubiquitous regulatory elements and supports a role for AP-1 regulating open chromatin. We developed a classifier that accurately predicts cell-type lineage based on only 43 DHSs and evaluated the tissue of origin for cancer cell types. A similar classifier identified three sex-specific loci on the X chromosome, including the XIST lincRNA locus. By correlating DNase I signal and gene expression, we predicted regulated genes for more than 500K DHSs. Finally, we introduce a web resource to enable researchers to use these results to explore these regulatory patterns and better understand how expression is modulated within and across human cell types.