An oncogenic JMJD6-DGAT1 axis tunes the epigenetic regulation of lipid droplet formation in clear cell renal cell carcinoma.

Characterized by intracellular lipid droplet accumulation, clear cell renal cell carcinoma (ccRCC) is resistant to cytotoxic chemotherapy and is a lethal disease. Through an unbiased siRNA screen of 2-oxoglutarate (2-OG)-dependent enzymes, which play a critical role in tumorigenesis, we identified Jumonji domain-containing 6 (JMJD6) as an essential gene for ccRCC tumor development. The downregulation of JMJD6 abolished ccRCC colony formation in vitro and inhibited orthotopic tumor growth in vivo. Integrated ChIP-seq and RNA-seq analyses uncovered diacylglycerol O-acyltransferase 1 (DGAT1) as a critical JMJD6 effector. Mechanistically, JMJD6 interacted with RBM39 and co-occupied DGAT1 gene promoter with H3K4me3 to induce DGAT1 expression. JMJD6 silencing reduced DGAT1, leading to decreased lipid droplet formation and tumorigenesis. The pharmacological inhibition (or depletion) of DGAT1 inhibited lipid droplet formation in vitro and ccRCC tumorigenesis in vivo. Thus, the JMJD6-DGAT1 axis represents a potential new therapeutic target for ccRCC.

Genome-wide Screening Identifies SFMBT1 as an Oncogenic Driver in Cancer with VHL Loss.

von Hippel-Lindau (VHL) is a critical tumor suppressor in clear cell renal cell carcinomas (ccRCCs). It is important to identify additional therapeutic targets in ccRCC downstream of VHL loss besides hypoxia-inducible factor 2α (HIF2α). By performing a genome-wide screen, we identified Scm-like with four malignant brain tumor domains 1 (SFMBT1) as a candidate pVHL target. SFMBT1 was considered to be a transcriptional repressor but its role in cancer remains unclear. ccRCC patients with VHL loss-of-function mutations displayed elevated SFMBT1 protein levels. SFMBT1 hydroxylation on Proline residue 651 by EglN1 mediated its ubiquitination and degradation governed by pVHL. Depletion of SFMBT1 abolished ccRCC cell proliferation in vitro and inhibited orthotopic tumor growth in vivo. Integrated analyses of ChIP-seq, RNA-seq, and patient prognosis identified sphingosine kinase 1 (SPHK1) as a key SFMBT1 target gene contributing to its oncogenic phenotype. Therefore, the pVHL-SFMBT1-SPHK1 axis serves as a potential therapeutic avenue for ccRCC.

Septo-dentate gyrus cholinergic circuits modulate function and morphogenesis of adult neural stem cells through granule cell intermediaries.

Cholinergic neurons in the basal forebrain play a crucial role in regulating adult hippocampal neurogenesis (AHN). However, the circuit and molecular mechanisms underlying cholinergic modulation of AHN, especially the initial stages of this process related to the generation of newborn progeny from quiescent radial neural stem cells (rNSCs), remain unclear. Here, we report that stimulation of the cholinergic circuits projected from the diagonal band of Broca (DB) to the dentate gyrus (DG) neurogenic niche promotes proliferation and morphological development of rNSCs, resulting in increased neural stem/progenitor pool and rNSCs with longer radial processes and larger busy heads. Interestingly, DG granule cells (GCs) are required for DB-DG cholinergic circuit-dependent modulation of proliferation and morphogenesis of rNSCs. Furthermore, single-nucleus RNA sequencing of DG reveals cell type-specific transcriptional changes in response to cholinergic circuit stimulation, with GCs (among all the DG niche cells) exhibiting the most extensive transcriptional changes. Our findings shed light on how the DB-DG cholinergic circuits orchestrate the key niche components to support neurogenic function and morphogenesis of rNSCs at the circuit and molecular levels.

Heterogeneity in the progression of retinal pathologies in mice harboring patient mimicking Impg2 mutations.

Biallelic mutations in interphotoreceptor matrix proteoglycan 2 (IMPG2) in humans cause retinitis pigmentosa (RP) with early macular involvement, albeit the disease progression varies widely due to genetic heterogeneity and IMPG2 mutation type. There are currently no treatments for IMPG2-RP. To aid preclinical studies toward eventual treatments, there is a need to better understand the progression of disease pathology in appropriate animal models. Toward this goal, we developed mouse models with patient mimicking homozygous frameshift (T807Ter) or missense (Y250C) Impg2 mutations, as well as mice with a homozygous frameshift mutation (Q244Ter) designed to completely prevent IMPG2 protein expression, and characterized the trajectory of their retinal pathologies across postnatal development until late adulthood. We found that the Impg2T807Ter/T807Ter and Impg2Q244Ter/Q244Ter mice exhibited early onset gliosis, impaired photoreceptor outer segment maintenance, appearance of subretinal deposits near the optic disc, disruption of the outer retina, and neurosensorial detachment, whereas the Impg2Y250C/Y250C mice exhibited minimal retinal pathology. These results demonstrate the importance of mutation type in disease progression in IMPG2-RP and provide a toolkit and preclinical data for advancing therapeutic approaches.

Cas9 gene therapy for Angelman syndrome traps Ube3a-ATS long non-coding RNA.

Angelman syndrome (AS) is a severe neurodevelopmental disorder caused by a mutation or deletion of the maternally inherited UBE3A allele. In neurons, the paternally inherited UBE3A allele is silenced in cis by a long non-coding RNA called UBE3A-ATS. Here, as part of a systematic screen, we found that Cas9 can be used to activate ('unsilence') paternal Ube3a in cultured mouse and human neurons when targeted to Snord115 genes, which are small nucleolar RNAs that are clustered in the 3' region of Ube3a-ATS. A short Cas9 variant and guide RNA that target about 75 Snord115 genes were packaged into an adeno-associated virus and administered to a mouse model of AS during the embryonic and early postnatal stages, when the therapeutic benefit of restoring Ube3a is predicted to be greatest1,2. This early treatment unsilenced paternal Ube3a throughout the brain for at least 17 months and rescued anatomical and behavioural phenotypes in AS mice. Genomic integration of the adeno-associated virus vector into Cas9 target sites caused premature termination of Ube3a-ATS at the vector-derived polyA cassette, or when integrated in the reverse orientation, by transcriptional collision with the vector-derived Cas9 transcript. Our study shows that targeted genomic integration of a gene therapy vector can restore the function of paternally inherited UBE3A throughout life, providing a path towards a disease-modifying treatment for a syndromic neurodevelopmental disorder.

USP13 promotes deubiquitination of ZHX2 and tumorigenesis in kidney cancer.

Clear cell renal cell carcinoma (ccRCC) is characterized by the loss of tumor suppressor Von Hippel Lindau (VHL) function. VHL is the component of an E3 ligase complex that promotes the ubiquitination and degradation of hypoxia inducible factor α (HIF-α) (including HIF1α and HIF2α) and Zinc Fingers And Homeoboxes 2 (ZHX2). Our recent research showed that ZHX2 contributed to ccRCC tumorigenesis in a HIF-independent manner. However, it is still unknown whether ZHX2 could be modified through deubiquitination even in the absence of pVHL. Here, we performed a deubiquitinase (DUB) complementary DNA (cDNA) library binding screen and identified USP13 as a DUB that bound ZHX2 and promoted ZHX2 deubiquitination. As a result, USP13 promoted ZHX2 protein stability in an enzymatically dependent manner, and depletion of USP13 led to ZHX2 down-regulation in ccRCC. Functionally, USP13 depletion led to decreased cell proliferation measured by two-dimensional (2D) colony formation and three-dimensional (3D) anchorage-independent growth. Furthermore, USP13 was essential for ccRCC tumor growth in vivo, and the effect was partially mediated by its regulation on ZHX2. Our findings support that USP13 may be a key effector in ccRCC tumorigenesis.

Novel Porcine Model Reveals Two Distinct LGR5 Cell Types During Lung Development and Homeostasis.

Cells expressing LGR5 play a pivotal role in homeostasis, repair, and regeneration in multiple organs including skin and gastrointestinal tract, yet little is known about their role in the lung. Findings from mice, a widely used animal model, suggest that lung LGR5 expression differs from that of humans. In this work, using a new transgenic pig model, we identify two main populations of LGR5+ cells in the lung that are conserved in human, but not mouse lungs. Using RNA sequencing, 3D imaging and organoid models, we determine that in the fetal lung, epithelial LGR5 expression is transient in a subpopulation of SOX9+/ETV+/SFTPC+ progenitor lung tip cells. In contrast, epithelial LGR5 expression is absent from postnatal lung, but is reactivated in bronchioalveolar organoids derived from basal airway cells. We also describe a separate population of mesenchymal LGR5+ cells that surrounds developing and mature airways, lies adjacent to airway basal cells, and is closely associated with nerve fibers. Transcriptionally, mesenchymal LGR5+ cells include a subset of peribronchial fibroblasts (PBF) that express unique patterns of SHH, FGF, WNT and TGF-ß signaling pathway genes. These results support distinct roles for LGR5+ cells in the lung and describe a physiologically relevant animal model for further studies on the function of these cells in repair and regeneration.

USP37 promotes deubiquitination of HIF2α in kidney cancer.

Clear cell renal cell carcinoma (ccRCC) is characterized by loss of tumor suppressor Von Hippel Lindau (VHL) function, which leads to accumulation of hypoxia inducible factor α (including HIF1α and HIF2α). HIF2α was previously reported to be one of the major oncogenic drivers in ccRCC, however, its therapeutic targets remain challenging. Here we performed a deubiquitinase (DUB) complementary DNA (cDNA) library binding screen and discovered that ubiquitin-specific peptidase 37 (USP37) is a DUB that binds HIF2α and promotes HIF2α deubiquitination. As a result, USP37 promotes HIF2α protein stability in an enzymatically dependent manner, and depletion of USP37 leads to HIF2α down-regulation in ccRCC. Functionally, USP37 depletion causes decreased cell proliferation measured by MTS, two-dimensional (2D) colony formation as well as three-dimensional (3D) anchorage- independent growth. USP37 is also essential for maintaining kidney tumorigenesis in an orthotopic xenograft model and its depletion leads to both decreased primary kidney tumorigenesis and spontaneous lung metastasis. Our results suggest that USP37 is a potential therapeutic target in ccRCC.

Integrative QTL analysis of gene expression and chromatin accessibility identifies multi-tissue patterns of genetic regulation.

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.

A conditional mouse expressing an activating mutation in NRF2 displays hyperplasia of the upper gastrointestinal tract and decreased white adipose tissue.

Activation of the nuclear factor (erythroid-derived 2)-like 2 (NFE2L2 or NRF2) transcription factor is a critical and evolutionarily conserved cellular response to oxidative stress, metabolic stress, and xenobiotic insult. Deficiency of NRF2 results in hypersensitivity to a variety of stressors, whereas its aberrant activation contributes to several cancer types, most commonly squamous cell carcinomas of the esophagus, oral cavity, bladder, and lung. Between 10% and 35% of patients with squamous cell carcinomas display hyperactive NRF2 signaling, harboring activating mutations and copy number amplifications of the NFE2L2 oncogene or inactivating mutations or deletions of KEAP1 or CUL3, the proteins of which co-complex to ubiquitylate and degrade NRF2 protein. To better understand the role of NRF2 in tumorigenesis and more broadly in development, we engineered the endogenous Nfe2l2 genomic locus to create a conditional mutant LSL-Nrf2E79Q mouse model. The E79Q mutation, one of the most commonly observed NRF2-activating mutations in human squamous cancers, codes for a mutant protein that does not undergo KEAP1/CUL3-dependent degradation, resulting in its constitutive activity. Expression of NRF2 E79Q protein in keratin 14 (KRT14)-positive murine tissues resulted in hyperplasia of squamous cell tissues of the tongue, forestomach, and esophagus, a stunted body axis, decreased weight, and decreased visceral adipose depots. RNA-seq profiling and follow-up validation studies of cultured NRF2E79Q murine esophageal epithelial cells revealed known and novel NRF2-regulated transcriptional programs, including genes associated with squamous cell carcinoma (e.g. Myc), lipid and cellular metabolism (Hk2, Ppard), and growth factors (Areg, Bmp6, Vegfa). These data suggest that in addition to decreasing adipogenesis, KRT14-restricted NRF2 activation drives hyperplasia of the esophagus, forestomach, and tongue, but not formation of squamous cell carcinoma. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

COVID-19 and the problem of clinical knowledge.

COVID-19 presents many challenges, both clinical and philosophical. In this paper we discuss a major lacuna that COVID-19 revealed in our philosophy and understanding of medicine. Whereas we have some understanding of how physician-scientists interrogate the world to learn more about medicine, we do not understand the epistemological costs and benefits of the various ways clinicians acquire new knowledge in their fields. We will also identify reasons this topic is important both when the world is facing a pandemic and when it is not.

Social perceptions of warmth and competence influence behavioral intentions and neural processing.

Perceptions of the primary social dimensions, warmth and competence, determine how we view and relate to social targets. To discern how warmth and competence might affect neural processing and its downstream behavioral consequences, we manipulated impressions of targets' warmth and competence and then measured intentions toward the target and motor resonance, a neural process previously linked to social processing. While EEG was recorded, 66 participants watched videos of people performing a simple motor activity and completed a measure of hypothetical intentions to help or harm. Both perceptions of warmth and competence predicted an increase in helping intentions. Moreover, participants showed the least motor resonance with high competence-medium warmth targets, suggesting the importance of both social dimensions in driving neural simulation of targets' actions. Perceptions of a person's warmth and competence can affect not only how others might intend to treat them, but also how they might process their basic experiences on a neural level.

Transcriptome-Wide Regulation of Key Developmental Pathways in the Mouse Neural Tube by Prenatal Alcohol Exposure.

BACKGROUND: Early gestational alcohol exposure is associated with severe craniofacial and CNS dysmorphologies and behavioral abnormalities during adolescence and adulthood. Alcohol exposure during the formation of the neural tube (gestational day [GD] 8 to 10 in mice; equivalent to4th week of human pregnancy) disrupts development of ventral midline brain structures such as the pituitary, septum, and ventricles. This study identifies transcriptomic changes in the rostroventral neural tube (RVNT), the region of the neural tube that gives rise to the midline structures sensitive to alcohol exposure during neurulation. METHODS: Female C57BL/6J mice were administered 2 doses of alcohol (2.9 g/kg) or vehicle 4 hours apart on GD 9.0. The RVNTs of embryos were collected 6 or 24 hours after the first dose and processed for RNA-seq. RESULTS: Six hours following GD 9.0 alcohol exposure (GD 9.25), over 2,300 genes in the RVNT were determined to be differentially regulated by alcohol. Enrichment analysis determined that PAE affected pathways related to cell proliferation, p53 signaling, ribosome biogenesis, and immune activation. In addition, over 100 genes involved in primary cilia formation and function and regulation of morphogenic pathways were altered 6 hours after alcohol exposure. The changes to gene expression were largely transient, as only 91 genes identified as differentially regulated by prenatal alcohol at GD 10 (24 hours postexposure). Functionally, the differentially regulated genes at GD 10 were related to organogenesis and cell migration. CONCLUSIONS: These data give a comprehensive view of the changing landscape of the embryonic transcriptome networks in regions of the neural tube that give rise to brain structures impacted by a neurulation-stage alcohol exposure. Identification of gene networks dysregulated by alcohol will help elucidate the pathogenic mechanisms of alcohol's actions.

Differential rates of intravascular uptake and pain perception during lumbosacral epidural injection among adults using a 22-gauge needle versus 25-gauge needle: a randomized clinical trial.

BACKGROUND: Inadvertent intravascular injection has been suggested as the most probable mechanism behind serious neurological complications during transforaminal epidural steroid injections. Authors believe a smaller gauge needle may lead to less intravascular uptake and less pain. Theoretically, there is less chance for a smaller gauge needle to encounter a blood vessel during an injection compared to a larger gauge needle. Studies have also shown smaller gauge needle to cause less pain. The aim of the study was to quantify the difference between a 22-gauge needle and 25-gauge needle during lumbosacral transforaminal epidural steroid injection in regards to intravascular uptake and pain perception. METHODS: This was a prospective single blind randomized clinical trial performed at outpatient spine practice locations of two academic institutions. One hundred sixty-two consecutive patients undergoing lumbosacral transforaminal epidural injections from February 2018 to June 2019 were recruited and randomized to each arm of the study - 84 patients were randomized to the 22-gauge needle arm and 78 patients to 25-gauge arm. Each transforaminal injection level was considered a separate incidence, hence total number of incidence was 249 (136 in 22-gauge arm and 113 in 25-gauge arm). The primary outcome measure was intravascular uptake during live fluoroscopy and/or blood aspiration. The secondary outcome measure was patient reported pain during the procedure on the numerical rating scale. RESULTS: Fisher exact test was used to detect differences between 2 groups in regards to intravascular uptake and paired t-tests were used to detect differences in pain scores. The incidence of intravascular uptake for a 22-gauge needle was 5.9% (95% confidence interval: 1.9 to 9.8%) and for a 25-gauge needle, 7.1% (95% confidence interval: 2.4 to 11.8%) [p = 0.701]. Average numerical rating scale scores during the initial needle entry for 22-gauge and 25-gauge needle was 3.46 (95% confidence interval: 2.94 to 3.98) and 3.13 (95% confidence interval: 2.57 to 3.69) respectively [p = 0.375]. CONCLUSIONS: The study showed no statistically significant difference in intravascular uptake or pain perception between a 22-gauge needle and 25-gauge needle during lumbosacral transforaminal epidural steroid injections. TRIAL REGISTRATION: ClinicalTrials.gov NCT04350307. Registered 4/17/2020. (Retrospectively registered).

Effects of Minimal Grouping On Implicit Prejudice, Infrahumanization, and Neural Processing Despite Orthogonal Social Categorizations.

Racial prejudice is a pervasive and pernicious form of intergroup bias. However, a mounting number of studies show that re-categorization-even into minimal groups-can overcome the typical consequences of racial and other group classifications. We tested the effects of minimal grouping on implicit prejudice and infrahumanization using a paradigm in which race was orthogonal to group membership. This allowed us to examine whether knowledge of group membership overrides obvious category differences. We found that participants infrahumanized and showed implicit bias toward the minimal out-group, despite the crosscutting presence of race, and in fact did not show any of the usual implicit racial bias. In addition, Event-Related Potentials (ERPs) showed an early race effect followed by distinct reactions on the basis of group as processing continued. This is evidence that arbitrary social classifications can engender in-group preference even in the presence of orthogonal, visually salient categorizations.

High-throughput small molecule screen identifies inhibitors of aberrant chromatin accessibility.

Mutations in chromatin-modifying proteins and transcription factors are commonly associated with a wide variety of cancers. Through gain- or loss-of-function, these mutations may result in characteristic alterations of accessible chromatin, indicative of shifts in the landscape of regulatory elements genome-wide. The identification of compounds that reverse a specific chromatin signature could lead to chemical probes or potential therapies. To explore whether chromatin accessibility could serve as a platform for small molecule screening, we adapted formaldehyde-assisted isolation of regulatory elements (FAIRE), a chemical method to enrich for nucleosome-depleted genomic regions, as a high-throughput, automated assay. After demonstrating the validity and robustness of this approach, we applied this method to screen an epigenetically targeted small molecule library by evaluating regions of aberrant nucleosome depletion mediated by EWSR1-FLI1, the chimeric transcription factor critical for the bone and soft tissue tumor Ewing sarcoma. As a class, histone deacetylase inhibitors were greatly overrepresented among active compounds. These compounds resulted in diminished accessibility at targeted sites by disrupting transcription of EWSR1-FLI1. Capitalizing on precise differences in chromatin accessibility for drug discovery efforts offers significant advantages because it does not depend on the a priori selection of a single molecular target and may detect novel biologically relevant pathways.

Molecular classification of Crohn's disease reveals two clinically relevant subtypes.

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.

Enhanced Nociception in Angelman Syndrome Model Mice.

Angelman syndrome (AS) is a severe neurodevelopmental disorder caused by mutation or deletion of the maternal UBE3A allele. The maternal UBE3A allele is expressed in nearly all neurons of the brain and spinal cord, whereas the paternal UBE3A allele is repressed by an extremely long antisense transcript (UBE3A-ATS). Little is known about expression of UBE3A in the peripheral nervous system, where loss of maternal UBE3A might contribute to AS phenotypes. Here we sought to examine maternal and paternal Ube3a expression in DRGs neurons and to evaluate whether nociceptive responses were affected in AS model mice (global deletion of maternal Ube3a allele; Ube3am-/p+). We found that most large-diameter proprioceptive and mechanosensitive DRG neurons expressed maternal Ube3a and paternal Ube3a-ATS In contrast, most small-diameter neurons expressed Ube3a biallelically and had low to undetectable levels of Ube3a-ATS Analysis of single-cell DRG transcriptomes further suggested that Ube3a is expressed monoallelically in myelinated large-diameter neurons and biallelically in unmyelinated small-diameter neurons. Behavioral responses to some noxious thermal and mechanical stimuli were enhanced in male and female AS model mice; however, nociceptive responses were not altered by the conditional deletion of maternal Ube3a in the DRG. These data suggest that the enhanced nociceptive responses in AS model mice are due to loss of maternal Ube3a in the central, but not peripheral, nervous system. Our study provides new insights into sensory processing deficits associated with AS.SIGNIFICANCE STATEMENT Angelman syndrome (AS) is a neurodevelopmental disorder caused by loss or mutation of the maternal UBE3A allele. While sensory processing deficits are frequently associated with AS, it is currently unknown whether Ube3a is expressed in peripheral sensory neurons or whether maternal deletion of Ube3a affects somatosensory responses. Here, we found that Ube3a is primarily expressed from the maternally inherited allele in myelinated large-diameter sensory neurons and biallelically expressed in unmyelinated small-diameter neurons. Nociceptive responses to select noxious thermal and mechanical stimuli were enhanced following global, but not sensory neuron-specific, deletion of maternal Ube3a in mice. These data suggest that maternal loss of Ube3a affects nociception via a central, but not peripheral mechanism, with implications for AS.

Tissue- and strain-specific effects of a genotoxic carcinogen 1,3-butadiene on chromatin and transcription.

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.

The accessible chromatin landscape of the human genome.

DNase I hypersensitive sites (DHSs) are markers of regulatory DNA and have underpinned the discovery of all classes of cis-regulatory elements including enhancers, promoters, insulators, silencers and locus control regions. Here we present the first extensive map of human DHSs identified through genome-wide profiling in 125 diverse cell and tissue types. We identify ∼2.9 million DHSs that encompass virtually all known experimentally validated cis-regulatory sequences and expose a vast trove of novel elements, most with highly cell-selective regulation. Annotating these elements using ENCODE data reveals novel relationships between chromatin accessibility, transcription, DNA methylation and regulatory factor occupancy patterns. We connect ∼580,000 distal DHSs with their target promoters, revealing systematic pairing of different classes of distal DHSs and specific promoter types. Patterning of chromatin accessibility at many regulatory regions is organized with dozens to hundreds of co-activated elements, and the transcellular DNase I sensitivity pattern at a given region can predict cell-type-specific functional behaviours. The DHS landscape shows signatures of recent functional evolutionary constraint. However, the DHS compartment in pluripotent and immortalized cells exhibits higher mutation rates than that in highly differentiated cells, exposing an unexpected link between chromatin accessibility, proliferative potential and patterns of human variation.