1 3616 98 IN VITRO MODELING OF CD8 T CELL EXHAUSTION ENABLES CRISPR SCREENING TO REVEAL A ROLE FOR BHLHE40. IDENTIFYING NOVEL MOLECULAR MECHANISMS OF EXHAUSTED CD8 T CELLS (T (EX) ) IS A KEY GOAL OF IMPROVING IMMUNOTHERAPY OF CANCER AND OTHER DISEASES. HOWEVER, HIGH-THROUGHPUT INTERROGATION OF IN VIVO T (EX) CAN BE COSTLY AND INEFFICIENT. IN VITRO MODELS OF T (EX) ARE EASILY CUSTOMIZABLE AND QUICKLY GENERATE HIGH CELLULAR YIELD, OFFERING AN OPPORTUNITY TO PERFORM CRISPR SCREENING AND OTHER HIGH-THROUGHPUT ASSAYS. WE ESTABLISHED AN IN VITRO MODEL OF CHRONIC STIMULATION AND BENCHMARKED KEY PHENOTYPIC, FUNCTIONAL, TRANSCRIPTIONAL, AND EPIGENETIC FEATURES AGAINST BONA FIDE IN VIVO T (EX) . WE LEVERAGED THIS MODEL OF IN VITRO CHRONIC STIMULATION IN COMBINATION WITH POOLED CRISPR SCREENING TO UNCOVER TRANSCRIPTIONAL REGULATORS OF T CELL EXHAUSTION. THIS APPROACH IDENTIFIED SEVERAL TRANSCRIPTION FACTORS, INCLUDING BHLHE40. IN VITRO AND IN VIVO VALIDATION DEFINED A ROLE FOR BHLHE40 IN REGULATING A KEY DIFFERENTIATION CHECKPOINT BETWEEN PROGENITOR AND INTERMEDIATE SUBSETS OF T (EX) . BY DEVELOPING AND BENCHMARKING AN IN VITRO MODEL OF T (EX) , WE DEMONSTRATE THE UTILITY OF MECHANISTICALLY ANNOTATED IN VITRO MODELS OF T (EX) , IN COMBINATION WITH HIGH-THROUGHPUT APPROACHES, AS A DISCOVERY PIPELINE TO UNCOVER NOVEL T (EX) BIOLOGY. 2023 2 775 32 CELL TYPE-SPECIFIC WHOLE-GENOME LANDSCAPE OF DELTAFOSB BINDING IN THE NUCLEUS ACCUMBENS AFTER CHRONIC COCAINE EXPOSURE. BACKGROUND: THE ABILITY OF NEURONS TO RESPOND TO EXTERNAL STIMULI INVOLVES ADAPTATIONS OF GENE EXPRESSION. INDUCTION OF THE TRANSCRIPTION FACTOR DELTAFOSB IN THE NUCLEUS ACCUMBENS, A KEY BRAIN REWARD REGION, IS IMPORTANT FOR THE DEVELOPMENT OF DRUG ADDICTION. HOWEVER, A COMPREHENSIVE MAP OF DELTAFOSB'S GENE TARGETS HAS NOT YET BEEN GENERATED. METHODS: WE USED CUT&RUN (CLEAVAGE UNDER TARGETS AND RELEASE USING NUCLEASE) TO MAP THE GENOME-WIDE CHANGES IN DELTAFOSB BINDING IN THE 2 MAIN TYPES OF NUCLEUS ACCUMBENS NEURONS-D1 OR D2 MEDIUM SPINY NEURONS-AFTER CHRONIC COCAINE EXPOSURE. TO ANNOTATE GENOMIC REGIONS OF DELTAFOSB BINDING SITES, WE ALSO EXAMINED THE DISTRIBUTIONS OF SEVERAL HISTONE MODIFICATIONS. RESULTING DATASETS WERE LEVERAGED FOR MULTIPLE BIOINFORMATIC ANALYSES. RESULTS: THE MAJORITY OF DELTAFOSB PEAKS OCCUR OUTSIDE PROMOTER REGIONS, INCLUDING INTERGENIC REGIONS, AND ARE SURROUNDED BY EPIGENETIC MARKS INDICATIVE OF ACTIVE ENHANCERS. BRG1, THE CORE SUBUNIT OF THE SWI/SNF CHROMATIN REMODELING COMPLEX, OVERLAPS WITH DELTAFOSB PEAKS, A FINDING CONSISTENT WITH EARLIER STUDIES OF DELTAFOSB'S INTERACTING PROTEINS. CHRONIC COCAINE USE INDUCES BROAD CHANGES IN DELTAFOSB BINDING IN BOTH D1 AND D2 NUCLEUS ACCUMBENS MEDIUM SPINY NEURONS OF MALE AND FEMALE MICE. IN ADDITION, IN SILICO ANALYSES PREDICT THAT DELTAFOSB COOPERATIVELY REGULATES GENE EXPRESSION WITH HOMEOBOX AND T-BOX TRANSCRIPTION FACTORS. CONCLUSIONS: THESE NOVEL FINDINGS UNCOVER KEY ELEMENTS OF DELTAFOSB'S MOLECULAR MECHANISMS IN TRANSCRIPTIONAL REGULATION AT BASELINE AND IN RESPONSE TO CHRONIC COCAINE EXPOSURE. FURTHER CHARACTERIZATION OF DELTAFOSB'S COLLABORATIVE TRANSCRIPTIONAL AND CHROMATIN PARTNERS SPECIFICALLY IN D1 AND D2 MEDIUM SPINY NEURONS WILL REVEAL A BROADER PICTURE OF THE FUNCTION OF DELTAFOSB AND THE MOLECULAR BASIS OF DRUG ADDICTION. 2023 3 5702 24 SINGLE-CELL GENOMICS FOR INVESTIGATING PATHOGENESIS OF INFLAMMATORY DISEASES. RECENT TECHNICAL ADVANCES HAVE ENABLED UNBIASED TRANSCRIPTOMIC AND EPIGENETIC ANALYSIS OF EACH CELL, KNOWN AS "SINGLE-CELL ANALYSIS". SINGLE-CELL ANALYSIS HAS A VARIETY OF TECHNICAL APPROACHES TO INVESTIGATE THE STATE OF EACH CELL, INCLUDING MRNA LEVELS (TRANSCRIPTOME), THE IMMUNE REPERTOIRE (IMMUNE REPERTOIRE ANALYSIS), CELL SURFACE PROTEINS (SURFACE PROTEOME ANALYSIS), CHROMATIN ACCESSIBILITY (EPIGENOME), AND ACCORDANCE WITH GENOME VARIANTS (EQTLS; EXPRESSION QUANTITATIVE TRAIT LOCI). AS AN EFFECTIVE TOOL FOR INVESTIGATING ROBUST IMMUNE RESPONSES IN CORONAVIRUS DISEASE 2019 (COVID-19), MANY RESEARCHERS PERFORMED SINGLE-CELL ANALYSIS TO CAPTURE THE DIVERSE, UNBIASED IMMUNE CELL ACTIVATION AND DIFFERENTIATION. DESPITE CHALLENGES ELUCIDATING THE COMPLICATED IMMUNE MICROENVIRONMENTS OF CHRONIC INFLAMMATORY DISEASES USING EXISTING EXPERIMENTAL METHODS, IT IS NOW POSSIBLE TO CAPTURE THE SIMULTANEOUS IMMUNE FEATURES OF DIFFERENT CELL TYPES ACROSS INFLAMED TISSUES USING VARIOUS SINGLE-CELL TOOLS. IN THIS REVIEW, WE INTRODUCE PATIENT-BASED AND EXPERIMENTAL MOUSE MODEL RESEARCH UTILIZING SINGLE-CELL ANALYSES IN THE FIELD OF CHRONIC INFLAMMATORY DISEASES, AS WELL AS MULTI-ORGAN ATLAS TARGETING IMMUNE CELLS. 2023 4 3831 30 INVOLVEMENT OF INFLAMMATORY CYTOKINES AND EPIGENETIC MODIFICATION OF THE MTTFA COMPLEX IN T-HELPER CELLS OF PATIENTS' SUFFERING FROM NON-SMALL CELL LUNG CANCER AND CHRONIC OBSTRUCTIVE PULMONARY DISEASE. DYSREGULATED INFLAMMATORY RESPONSE PLAYS A CRUCIAL ROLE IN THE PATHOGENESIS OF CHRONIC OBSTRUCTIVE PULMONARY DISEASE (COPD) AND NON-SMALL CELL LUNG CANCER (NSCLC). HENCE, THE PURPOSE OF THIS RESEARCH IS TO UNCOVER THE LINK BETWEEN ALTERATIONS IN INFLAMMATORY CYTOKINE LEVELS AND DISEASE PROGRESSION IN CD4(+)T CELLS OF PATIENTS SUFFERING FROM COPD AND LUNG CANCER. WE ALSO INVESTIGATED THE EPIGENETIC REGULATION OF MTTFA TO DELINEATE THE ROLE OF OXIDATIVE STRESS-MEDIATED INFLAMMATION IN LUNG CANCER AND COPD. THE RT(2) PROFILER PCR ARRAY WAS USED TO EXAMINE THE DIFFERENTIAL EXPRESSION PATTERN OF INFLAMMATORY GENES IN CD4(+) T HELPER (TH) CELLS FROM COPD, NSCLC, AND CONTROL SUBJECTS. CANDIDATE INFLAMMATORY GENE LOCI WERE SELECTED AND THE ENRICHMENT OF TRANSCRIPTIONAL FACTOR AND HISTONE MODIFIERS WAS ANALYSED USING CHIP-QPCR. IN COMPARISON TO CONTROL SUBJECTS, A SET OF GENES (E.G., BMP2, CCL2, IL5, VEGFA, ETC.) ARE OVER-EXPRESSED WHEREAS ANOTHER SET OF GENES (E.G., AIMP1, IFNG, LTA, LTB, TNF, ETC.) ARE UNDER-EXPRESSED IN BOTH COPD AND NSCLC PATIENTS. THE INCREASED PERCENT ENRICHMENT OF INFLAMMATION-ASSOCIATED TRANSCRIPTION FACTORS INCLUDING NF-KB, CREB, HIF1, AND MYC AT THE LOCI OF INFLAMMATORY GENES WAS REVEALED BY OUR CHROMATIN IMMUNOPRECIPITATION (CHIP) DATA. H3K4ME3, H3K9ME3, H3K14AC, HDAC1, 2, 3, 6 ALL SHOWED DYSREGULATED ENRICHMENT AT THE VEGFA GENE LOCUS. ONE OF THE EPIGENETIC MODIFICATIONS, HISTONE METHYLATION, WAS FOUND TO BE ABNORMAL IN THE MTTFA COMPLEX IN COPD AND NSCLC PATIENTS IN COMPARISON TO CONTROLS. ALTHOUGH THERE IS MOUNTING EVIDENCE OF SEVERAL LINKS BETWEEN THESE DISORDERS, THERAPEUTIC OPTIONS REMAIN INADEQUATE. OUR FINDINGS CONTRIBUTE TO THE BODY OF KNOWLEDGE ABOUT THERAPEUTIC TECHNIQUES THAT USE INFLAMMATORY CYTOKINES AS A PROGNOSTIC MARKER AND HIGHLIGHT THE NEED FOR EPIGENETIC THERAPY FOR THESE DEBILITATING LUNG DISEASES. 2022 5 6687 19 VALIDATION OF THE EPIGENETIC READER BROMODOMAIN-CONTAINING PROTEIN 4 (BRD4) AS A THERAPEUTIC TARGET FOR TREATMENT OF AIRWAY REMODELING. STRUCTURAL REMODELING IS CENTRAL TO THE INITIATION AND PROGRESSION OF MANY CHRONIC LUNG DISEASES, REPRESENTING AN IMPORTANT UNMET NEED. WE EXAMINE THE EVIDENCE SUPPORTING BROMODOMAIN-CONTAINING PROTEIN 4 (BRD4) AS A VALIDATED BIOLOGICAL TARGET FOR TREATMENT OF AIRWAY REMODELING. IN EPITHELIAL CELLS AND FIBROBLASTS, BRD4 SERVES AS A SCAFFOLD FOR CHROMATIN REMODELING COMPLEXES IN ACTIVE SUPER-ENHANCERS. IN RESPONSE TO INFLAMMATORY STIMULI, BRD4 IS REPOSITIONED TO INNATE AND MESENCHYMAL GENES ACTIVATING THEIR PRODUCTION. PROOF-OF-CONCEPT STUDIES SHOW PROMISING BENEFIT OF SELECTIVE BRD4 INHIBITORS IN DISRUPTING EPITHELIAL MESENCHYMAL TRANSITION AND MYOFIBROBLAST TRANSITION IN DIVERSE MODELS OF LUNG INJURY. RECENT IDENTIFICATION OF BIOMARKERS OF BRD4 PROVIDES A BASIS FOR FURTHER DRUG DEVELOPMENT FOR APPLICATION IN VIRAL-INDUCED AIRWAY INFLAMMATION, COPD AND INTERSTITIAL LUNG DISEASES. 2020 6 2278 33 EPIGENETIC REGULATION BY SUV4-20H1 IN CARDIOPULMONARY PROGENITOR CELLS IS REQUIRED TO PREVENT PULMONARY HYPERTENSION AND CHRONIC OBSTRUCTIVE PULMONARY DISEASE. BACKGROUND: THE PATHOGENESIS OF LIFE-THREATENING CARDIOPULMONARY DISEASES SUCH AS PULMONARY HYPERTENSION (PH) AND CHRONIC OBSTRUCTIVE PULMONARY DISEASE (COPD) ORIGINATES FROM A COMPLEX INTERPLAY OF ENVIRONMENTAL FACTORS AND GENETIC PREDISPOSITIONS THAT IS NOT FULLY UNDERSTOOD. LIKEWISE, LITTLE IS KNOWN ABOUT DEVELOPMENTAL ABNORMALITIES OR EPIGENETIC DYSREGULATIONS THAT MIGHT PREDISPOSE FOR PH OR COPD IN ADULT INDIVIDUALS. METHODS: TO IDENTIFY PATHOLOGY-ASSOCIATED EPIGENETIC ALTERATION IN DISEASED LUNG TISSUES, WE SCREENED A COHORT OF HUMAN PATIENTS WITH PH AND COPD FOR CHANGES OF HISTONE MODIFICATIONS BY IMMUNOFLUORESCENCE STAINING. TO ANALYZE THE FUNCTION OF H4K20ME2/3 IN LUNG PATHOGENESIS, WE DEVELOPED A SERIES OF SUV4-20H1 KNOCKOUT MOUSE LINES TARGETING CARDIOPULMONARY PROGENITOR CELLS AND DIFFERENT HEART AND LUNG CELL TYPES, FOLLOWED BY HEMODYNAMIC STUDIES AND MORPHOMETRIC ASSESSMENT OF TISSUE SAMPLES. MOLECULAR, CELLULAR, AND BIOCHEMICAL TECHNIQUES WERE APPLIED TO ANALYZE THE FUNCTION OF SUV4-20H1-DEPENDENT EPIGENETIC PROCESSES IN CARDIOPULMONARY PROGENITOR CELLS AND THEIR DERIVATIVES. RESULTS: WE DISCOVERED A STRONG REDUCTION OF THE HISTONE MODIFICATIONS OF H4K20ME2/3 IN HUMAN PATIENTS WITH COPD BUT NOT PATIENTS WITH PH THAT DEPEND ON THE ACTIVITY OF THE H4K20 DI-METHYLTRANSFERASE SUV4-20H1. LOSS OF SUV4-20H1 IN CARDIOPULMONARY PROGENITOR CELLS CAUSED A COPD-LIKE/PH PHENOTYPE IN MICE INCLUDING THE FORMATION OF PERIVASCULAR TERTIARY LYMPHOID TISSUE AND GOBLET CELL HYPERPLASIA, HYPERPROLIFERATION OF SMOOTH MUSCLE CELLS/MYOFIBROBLASTS, IMPAIRED ALVEOLARIZATION AND MATURATION DEFECTS OF THE MICROVASCULATURE LEADING TO MASSIVE RIGHT VENTRICULAR DILATATION AND PREMATURE DEATH. MECHANISTICALLY, SUV4-20H1 BINDS DIRECTLY TO THE 5'-UPSTREAM REGULATORY ELEMENT OF THE SUPEROXIDE DISMUTASE 3 (SOD3) GENE TO REPRESS ITS EXPRESSION. INCREASED LEVELS OF THE EXTRACELLULAR SOD3 ENZYME IN SUV4-20H1 MUTANTS INCREASES HYDROGEN PEROXIDE CONCENTRATIONS, CAUSING VASCULAR DEFECTS AND IMPAIRING ALVEOLARIZATION. CONCLUSIONS: OUR FINDINGS REVEAL A PIVOTAL ROLE OF THE HISTONE MODIFIER SUV4-20H1 IN CARDIOPULMONARY CODEVELOPMENT AND UNCOVER THE DEVELOPMENTAL ORIGINS OF CARDIOPULMONARY DISEASES. WE ASSUME THAT THE STUDY WILL FACILITATE THE UNDERSTANDING OF PATHOGENIC EVENTS CAUSING PH AND COPD AND AID THE DEVELOPMENT OF EPIGENETIC DRUGS FOR THE TREATMENT OF CARDIOPULMONARY DISEASES. 2021 7 4111 32 MECHANISMS CONTRIBUTING TO PERSISTENTLY ACTIVATED CELL PHENOTYPES IN PULMONARY HYPERTENSION. CHRONIC PULMONARY HYPERTENSION (PH) IS CHARACTERIZED BY THE ACCUMULATION OF PERSISTENTLY ACTIVATED CELL TYPES IN THE PULMONARY VESSEL EXHIBITING ABERRANT EXPRESSION OF GENES INVOLVED IN APOPTOSIS RESISTANCE, PROLIFERATION, INFLAMMATION AND EXTRACELLULAR MATRIX (ECM) REMODELLING. CURRENT THERAPIES FOR PH, FOCUSING ON VASODILATATION, DO NOT NORMALIZE THESE ACTIVATED PHENOTYPES. FURTHERMORE, CURRENT APPROACHES TO DEFINE ADDITIONAL THERAPEUTIC TARGETS HAVE FOCUSED ON DETERMINING THE INITIATING SIGNALS AND THEIR DOWNSTREAM EFFECTORS THAT ARE IMPORTANT IN PH ONSET AND DEVELOPMENT. ALTHOUGH THESE APPROACHES HAVE PRODUCED A LARGE NUMBER OF COMPELLING PH TREATMENT TARGETS, MANY PROMISING HUMAN DRUGS HAVE FAILED IN PH CLINICAL TRIALS. HEREIN, WE PROPOSE THAT ONE CONTRIBUTING FACTOR TO THESE FAILURES IS THAT PROCESSES IMPORTANT IN PH DEVELOPMENT MAY NOT BE GOOD TREATMENT TARGETS IN THE ESTABLISHED PHASE OF CHRONIC PH. WE HYPOTHESIZE THAT THIS IS DUE TO ALTERATIONS OF CHROMATIN STRUCTURE IN PH CELLS, RESULTING IN FUNCTIONAL DIFFERENCES BETWEEN THE SAME FACTOR OR PATHWAY IN NORMAL OR EARLY PH CELLS VERSUS CELLS IN CHRONIC PH. WE PROPOSE THAT THE HIGH EXPRESSION OF GENES INVOLVED IN THE PERSISTENTLY ACTIVATED PHENOTYPE OF PH VASCULAR CELLS IS PERPETUATED BY AN OPEN CHROMATIN STRUCTURE AND MULTIPLE TRANSCRIPTION FACTORS (TFS) VIA THE RECRUITMENT OF HIGH LEVELS OF EPIGENETIC REGULATORS INCLUDING THE HISTONE ACETYLASES P300/CBP, HISTONE ACETYLATION READERS INCLUDING BRDS, THE MEDIATOR COMPLEX AND THE POSITIVE TRANSCRIPTION ELONGATION FACTOR (ABSTRACT FIGURE). THUS, DETERMINING HOW GENE EXPRESSION IS CONTROLLED BY EXAMINING CHROMATIN STRUCTURE, TFS AND EPIGENETIC REGULATORS ASSOCIATED WITH ABERRANTLY EXPRESSED GENES IN PULMONARY VASCULAR CELLS IN CHRONIC PH, MAY UNCOVER NEW PH THERAPEUTIC TARGETS. 2019 8 4116 27 MECHANISMS OF AIRWAY EPITHELIAL INJURY AND ABNORMAL REPAIR IN ASTHMA AND COPD. THE AIRWAY EPITHELIUM COMPRISES OF DIFFERENT CELL TYPES AND ACTS AS A PHYSICAL BARRIER PREVENTING PATHOGENS, INCLUDING INHALED PARTICLES AND MICROBES, FROM ENTERING THE LUNGS. GOBLET CELLS AND SUBMUCOSAL GLANDS PRODUCE MUCUS THAT TRAPS PATHOGENS, WHICH ARE EXPELLED FROM THE RESPIRATORY TRACT BY CILIATED CELLS. BASAL CELLS ACT AS PROGENITOR CELLS, DIFFERENTIATING INTO DIFFERENT EPITHELIAL CELL TYPES, TO MAINTAIN HOMEOSTASIS FOLLOWING INJURY. ADHERENS AND TIGHT JUNCTIONS BETWEEN CELLS MAINTAIN THE EPITHELIAL BARRIER FUNCTION AND REGULATE THE MOVEMENT OF MOLECULES ACROSS IT. IN THIS REVIEW WE DISCUSS HOW ABNORMAL EPITHELIAL STRUCTURE AND FUNCTION, CAUSED BY CHRONIC INJURY AND ABNORMAL REPAIR, DRIVES AIRWAY DISEASE AND SPECIFICALLY ASTHMA AND CHRONIC OBSTRUCTIVE PULMONARY DISEASE (COPD). IN BOTH DISEASES, INHALED ALLERGENS, POLLUTANTS AND MICROBES DISRUPT JUNCTIONAL COMPLEXES AND PROMOTE CELL DEATH, IMPAIRING THE BARRIER FUNCTION AND LEADING TO INCREASED PENETRATION OF PATHOGENS AND A CONSTANT AIRWAY IMMUNE RESPONSE. IN ASTHMA, THE INFLAMMATORY RESPONSE PRECIPITATES THE EPITHELIAL INJURY AND DRIVES ABNORMAL BASAL CELL DIFFERENTIATION. THIS LEADS TO REDUCED CILIATED CELLS, GOBLET CELL HYPERPLASIA AND INCREASED EPITHELIAL MESENCHYMAL TRANSITION, WHICH CONTRIBUTE TO IMPAIRED MUCOCILIARY CLEARANCE AND AIRWAY REMODELLING. IN COPD, CHRONIC OXIDATIVE STRESS AND INFLAMMATION TRIGGER PREMATURE EPITHELIAL CELL SENESCENCE, WHICH CONTRIBUTES TO LOSS OF EPITHELIAL INTEGRITY AND AIRWAY INFLAMMATION AND REMODELLING. INCREASED NUMBERS OF BASAL CELLS SHOWING DEREGULATED DIFFERENTIATION, CONTRIBUTES TO CILIARY DYSFUNCTION AND MUCOUS HYPERPRODUCTION IN COPD AIRWAYS. DEFECTIVE ANTIOXIDANT, ANTIVIRAL AND DAMAGE REPAIR MECHANISMS, POSSIBLY DUE TO GENETIC OR EPIGENETIC FACTORS, MAY CONFER SUSCEPTIBILITY TO AIRWAY EPITHELIAL DYSFUNCTION IN THESE DISEASES. THE CURRENT EVIDENCE SUGGESTS THAT A CONSTANT CYCLE OF INJURY AND ABNORMAL REPAIR OF THE EPITHELIUM DRIVES CHRONIC AIRWAY INFLAMMATION AND REMODELLING IN ASTHMA AND COPD. MECHANISTIC UNDERSTANDING OF INJURY SUSCEPTIBILITY AND DAMAGE RESPONSE MAY LEAD TO IMPROVED THERAPIES FOR THESE DISEASES. 2023 9 3918 23 LINKING ABERRANT CHROMATIN FEATURES IN CHRONIC LYMPHOCYTIC LEUKEMIA TO TRANSCRIPTION FACTOR NETWORKS. IN CHRONIC LYMPHOCYTIC LEUKEMIA (CLL), A DIVERSE SET OF GENETIC MUTATIONS IS EMBEDDED IN A DEREGULATED EPIGENETIC LANDSCAPE THAT DRIVES CANCEROGENESIS. TO ELUCIDATE THE ROLE OF ABERRANT CHROMATIN FEATURES, WE MAPPED DNA METHYLATION, SEVEN HISTONE MODIFICATIONS, NUCLEOSOME POSITIONS, CHROMATIN ACCESSIBILITY, BINDING OF EBF1 AND CTCF, AS WELL AS THE TRANSCRIPTOME OF B CELLS FROM CLL PATIENTS AND HEALTHY DONORS. A GLOBALLY INCREASED HISTONE DEACETYLASE ACTIVITY WAS DETECTED AND HALF OF THE GENOME COMPRISED TRANSCRIPTIONALLY DOWNREGULATED PARTIALLY DNA METHYLATED DOMAINS DEMARCATED BY CTCF CLL SAMPLES DISPLAYED A H3K4ME3 REDISTRIBUTION AND NUCLEOSOME GAIN AT PROMOTERS AS WELL AS CHANGES OF ENHANCER ACTIVITY AND ENHANCER LINKAGE TO TARGET GENES. A DNA BINDING MOTIF ANALYSIS IDENTIFIED TRANSCRIPTION FACTORS THAT GAINED OR LOST BINDING IN CLL AT SITES WITH ABERRANT CHROMATIN FEATURES. THESE FINDINGS WERE INTEGRATED INTO A GENE REGULATORY ENHANCER CONTAINING NETWORK ENRICHED FOR B-CELL RECEPTOR SIGNALING PATHWAY COMPONENTS. OUR STUDY PREDICTS NOVEL MOLECULAR LINKS TO TARGETS OF CLL THERAPIES AND PROVIDES A VALUABLE RESOURCE FOR FURTHER STUDIES ON THE EPIGENETIC CONTRIBUTION TO THE DISEASE. 2019 10 1678 29 DRUG REPURPOSING AT THE INTERFACE OF MELANOMA IMMUNOTHERAPY AND AUTOIMMUNE DISEASE. CANCER CELLS HAVE A REMARKABLE ABILITY TO EVADE RECOGNITION AND DESTRUCTION BY THE IMMUNE SYSTEM. AT THE SAME TIME, CANCER HAS BEEN ASSOCIATED WITH CHRONIC INFLAMMATION, WHILE CERTAIN AUTOIMMUNE DISEASES PREDISPOSE TO THE DEVELOPMENT OF NEOPLASIA. ALTHOUGH CANCER IMMUNOTHERAPY HAS REVOLUTIONIZED ANTITUMOR TREATMENT, IMMUNE-RELATED TOXICITIES AND ADVERSE EVENTS DETRACT FROM THE CLINICAL UTILITY OF EVEN THE MOST ADVANCED DRUGS, ESPECIALLY IN PATIENTS WITH BOTH, METASTATIC CANCER AND PRE-EXISTING AUTOIMMUNE DISEASES. HERE, THE COMBINATION OF MULTI-OMICS, DATA-DRIVEN COMPUTATIONAL APPROACHES WITH THE APPLICATION OF NETWORK CONCEPTS ENABLES IN-DEPTH ANALYSES OF THE DYNAMIC LINKS BETWEEN CANCER, AUTOIMMUNE DISEASES, AND DRUGS. IN THIS REVIEW, WE FOCUS ON MOLECULAR AND EPIGENETIC METASTASIS-RELATED PROCESSES WITHIN CANCER CELLS AND THE IMMUNE MICROENVIRONMENT. WITH MELANOMA AS A MODEL, WE UNCOVER VULNERABILITIES FOR DRUG DEVELOPMENT TO CONTROL CANCER PROGRESSION AND IMMUNE RESPONSES. THEREBY, DRUG REPURPOSING ALLOWS TAKING ADVANTAGE OF EXISTING SAFETY PROFILES AND ESTABLISHED PHARMACOKINETIC PROPERTIES OF APPROVED AGENTS. THESE PROCEDURES PROMISE FASTER ACCESS AND OPTIMAL MANAGEMENT FOR CANCER TREATMENT. TOGETHER, THESE APPROACHES PROVIDE NEW DISEASE-BASED AND DATA-DRIVEN OPPORTUNITIES FOR THE PREDICTION AND APPLICATION OF TARGETED AND CLINICALLY USED DRUGS AT THE INTERFACE OF IMMUNE-MEDIATED DISEASES AND CANCER TOWARDS NEXT-GENERATION IMMUNOTHERAPIES. 2022 11 3617 87 IN VITRO MODELING OF CD8(+) T CELL EXHAUSTION ENABLES CRISPR SCREENING TO REVEAL A ROLE FOR BHLHE40. IDENTIFYING MOLECULAR MECHANISMS OF EXHAUSTED CD8 T CELLS (T(EX)) IS A KEY GOAL OF IMPROVING IMMUNOTHERAPY OF CANCER AND OTHER DISEASES. HOWEVER, HIGH-THROUGHPUT INTERROGATION OF IN VIVO T(EX) CAN BE COSTLY AND INEFFICIENT. IN VITRO MODELS OF T(EX) ARE EASILY CUSTOMIZABLE AND QUICKLY GENERATE HIGH CELLULAR YIELD, ENABLING CRISPR SCREENING AND OTHER HIGH-THROUGHPUT ASSAYS. WE ESTABLISHED AN IN VITRO MODEL OF CHRONIC STIMULATION AND BENCHMARKED KEY PHENOTYPIC, FUNCTIONAL, TRANSCRIPTIONAL, AND EPIGENETIC FEATURES AGAINST BONA FIDE IN VIVO T(EX). WE LEVERAGED THIS MODEL OF IN VITRO CHRONIC STIMULATION IN COMBINATION WITH CRISPR SCREENING TO IDENTIFY TRANSCRIPTIONAL REGULATORS OF T CELL EXHAUSTION. THIS APPROACH IDENTIFIED SEVERAL TRANSCRIPTION FACTORS, INCLUDING BHLHE40. IN VITRO AND IN VIVO VALIDATION DEFINED A ROLE FOR BHLHE40 IN REGULATING A KEY DIFFERENTIATION CHECKPOINT BETWEEN PROGENITOR AND INTERMEDIATE T(EX) SUBSETS. BY DEVELOPING AND BENCHMARKING AN IN VITRO MODEL OF T(EX), THEN APPLYING HIGH-THROUGHPUT CRISPR SCREENING, WE DEMONSTRATE THE UTILITY OF MECHANISTICALLY ANNOTATED IN VITRO MODELS OF T(EX). 2023 12 610 24 BEYOND THE GENOME: EPIGENETIC MECHANISMS IN LUNG REMODELING. THE LUNG DEVELOPS FROM A VERY SIMPLE OUTPOUCHING OF THE FOREGUT INTO A HIGHLY COMPLEX, FINELY STRUCTURED ORGAN WITH MULTIPLE SPECIALIZED CELL TYPES THAT ARE REQUIRED FOR ITS NORMAL PHYSIOLOGICAL FUNCTION. DURING BOTH THE DEVELOPMENT OF THE LUNG AND ITS REMODELING IN THE CONTEXT OF DISEASE OR RESPONSE TO INJURY, GENE EXPRESSION MUST BE ACTIVATED AND SILENCED IN A COORDINATED MANNER TO ACHIEVE THE TREMENDOUS PHENOTYPIC HETEROGENEITY OF CELL TYPES REQUIRED FOR HOMEOSTASIS AND PATHOGENESIS. EPIGENETIC MECHANISMS, CONSISTING OF DNA BASE MODIFICATIONS SUCH AS METHYLATION, ALTERATION OF HISTONES RESULTING IN CHROMATIN MODIFICATION, AND THE ACTION OF NONCODING RNA, CONTROL THE REGULATION OF INFORMATION "BEYOND THE GENOME" REQUIRED FOR BOTH LUNG MODELING AND REMODELING. EPIGENETIC REGULATION IS SUBJECT TO MODIFICATION BY ENVIRONMENTAL STIMULI, SUCH AS OXIDATIVE STRESS, INFECTION, AND AGING, AND IS THUS CRITICALLY IMPORTANT IN CHRONIC REMODELING DISORDERS SUCH AS IDIOPATHIC PULMONARY FIBROSIS (IPF), CHRONIC OBSTRUCTIVE PULMONARY DISEASE (COPD), BRONCHOPULMONARY DYSPLASIA (BPD), AND PULMONARY HYPERTENSION (PH). TECHNOLOGICAL ADVANCES HAVE MADE IT POSSIBLE TO EVALUATE GENOME-WIDE EPIGENETIC CHANGES (EPIGENOMICS) IN DISEASES OF LUNG REMODELING, CLARIFYING EXISTING PATHOPHYSIOLOGICAL PARADIGMS AND UNCOVERING NOVEL MECHANISMS OF DISEASE. MANY OF THESE REPRESENT NEW THERAPEUTIC TARGETS. ADVANCES IN EPIGENOMIC TECHNOLOGY WILL ACCELERATE OUR UNDERSTANDING OF LUNG DEVELOPMENT AND REMODELING, AND LEAD TO NOVEL TREATMENTS FOR CHRONIC LUNG DISEASES. 2014 13 2455 27 EPIGENETIC TARGETS FOR NOVEL THERAPIES OF LUNG DISEASES. IN SPITE OF SUBSTANTIAL ADVANCES IN DEFINING THE IMMUNOBIOLOGY AND FUNCTION OF STRUCTURAL CELLS IN LUNG DISEASES THERE IS STILL INSUFFICIENT KNOWLEDGE TO DEVELOP FUNDAMENTALLY NEW CLASSES OF DRUGS TO TREAT MANY LUNG DISEASES. FOR EXAMPLE, THERE IS A COMPELLING NEED FOR NEW THERAPEUTIC APPROACHES TO ADDRESS SEVERE PERSISTENT ASTHMA THAT IS INSENSITIVE TO INHALED CORTICOSTEROIDS. ALTHOUGH THE PREVALENCE OF STEROID-RESISTANT ASTHMA IS 5-10%, SEVERE ASTHMATICS REQUIRE A DISPROPORTIONATE LEVEL OF HEALTH CARE SPENDING AND CONSTITUTE A MAJORITY OF FATAL ASTHMA EPISODES. NONE OF THE ESTABLISHED DRUG THERAPIES INCLUDING LONG-ACTING BETA AGONISTS OR INHALED CORTICOSTEROIDS REVERSE ESTABLISHED AIRWAY REMODELING. OBSTRUCTIVE AIRWAYS REMODELING IN PATIENTS WITH CHRONIC OBSTRUCTIVE PULMONARY DISEASE (COPD), RESTRICTIVE REMODELING IN IDIOPATHIC PULMONARY FIBROSIS (IPF) AND OCCLUSIVE VASCULAR REMODELING IN PULMONARY HYPERTENSION ARE SIMILARLY UNRESPONSIVE TO CURRENT DRUG THERAPY. THEREFORE, DRUGS ARE NEEDED TO ACHIEVE LONG-ACTING SUPPRESSION AND REVERSAL OF PATHOLOGICAL AIRWAY AND VASCULAR REMODELING. NOVEL DRUG CLASSES ARE EMERGING FROM ADVANCES IN EPIGENETICS. NOVEL MECHANISMS ARE EMERGING BY WHICH CELLS ADAPT TO ENVIRONMENTAL CUES, WHICH INCLUDE CHANGES IN DNA METHYLATION, HISTONE MODIFICATIONS AND REGULATION OF TRANSCRIPTION AND TRANSLATION BY NONCODING RNAS. IN THIS REVIEW WE WILL SUMMARIZE CURRENT EPIGENETIC APPROACHES BEING APPLIED TO PRECLINICAL DRUG DEVELOPMENT ADDRESSING IMPORTANT THERAPEUTIC CHALLENGES IN LUNG DISEASES. THESE CHALLENGES ARE BEING ADDRESSED BY ADVANCES IN LUNG DELIVERY OF OLIGONUCLEOTIDES AND SMALL MOLECULES THAT MODIFY THE HISTONE CODE, DNA METHYLATION PATTERNS AND MIRNA FUNCTION. 2015 14 3064 27 GENOME-WIDE DNA METHYLATION ENCODES CARDIAC TRANSCRIPTIONAL REPROGRAMMING IN HUMAN ISCHEMIC HEART FAILURE. ISCHEMIC CARDIOMYOPATHY (ICM) IS THE CLINICAL ENDPOINT OF CORONARY HEART DISEASE AND A LEADING CAUSE OF HEART FAILURE. DESPITE GROWING DEMANDS TO DEVELOP PERSONALIZED APPROACHES TO TREAT ICM, PROGRESS IS LIMITED BY INADEQUATE KNOWLEDGE OF ITS PATHOGENESIS. SINCE EPIGENETICS HAS BEEN IMPLICATED IN THE DEVELOPMENT OF OTHER CHRONIC DISEASES, THE CURRENT STUDY WAS DESIGNED TO DETERMINE WHETHER TRANSCRIPTIONAL AND/OR EPIGENETIC CHANGES ARE SUFFICIENT TO DISTINGUISH ICM FROM OTHER ETIOLOGIES OF HEART FAILURE. SPECIFICALLY, WE HYPOTHESIZE THAT GENOME-WIDE DNA METHYLATION ENCODES TRANSCRIPTIONAL REPROGRAMMING IN ICM. RNA-SEQUENCING ANALYSIS WAS PERFORMED ON HUMAN ISCHEMIC LEFT VENTRICULAR TISSUE OBTAINED FROM PATIENTS WITH END-STAGE HEART FAILURE, WHICH ENRICHED KNOWN TARGETS OF THE POLYCOMB METHYLTRANSFERASE EZH2 COMPARED TO NON-ISCHEMIC HEARTS. COMBINED RNA SEQUENCING AND GENOME-WIDE DNA METHYLATION ANALYSIS REVEALED A ROBUST GENE EXPRESSION PATTERN CONSISTENT WITH SUPPRESSION OF OXIDATIVE METABOLISM, INDUCED ANAEROBIC GLYCOLYSIS, AND ALTERED CELLULAR REMODELING. LASTLY, KLF15 WAS IDENTIFIED AS A PUTATIVE UPSTREAM REGULATOR OF METABOLIC GENE EXPRESSION THAT WAS ITSELF REGULATED BY EZH2 IN A SET DOMAIN-DEPENDENT MANNER. OUR OBSERVATIONS THEREFORE DEFINE A NOVEL ROLE OF DNA METHYLATION IN THE METABOLIC REPROGRAMMING OF ICM. FURTHERMORE, WE IDENTIFY EZH2 AS AN EPIGENETIC REGULATOR OF KLF15 ALONG WITH DNA HYPERMETHYLATION, AND WE PROPOSE A NOVEL MECHANISM THROUGH WHICH CORONARY HEART DISEASE REPROGRAMS THE EXPRESSION OF BOTH INTERMEDIATE ENZYMES AND UPSTREAM REGULATORS OF CARDIAC METABOLISM SUCH AS KLF15. 2019 15 5898 28 T(H)2 CELL DEVELOPMENT AND FUNCTION. T HELPER 2 (T(H)2) CELLS ORCHESTRATE PROTECTIVE TYPE 2 IMMUNE RESPONSES, SUCH AS THOSE THAT TARGET HELMINTHS AND FACILITATE TISSUE REPAIR, BUT ALSO CONTRIBUTE TO CHRONIC INFLAMMATORY DISEASES, SUCH AS ASTHMA AND ALLERGY. HERE, WE REVIEW RECENT INSIGHTS INTO HOW DIVERSE MOLECULAR SIGNALS FROM CELLULAR SOURCES, INCLUDING DENDRITIC CELLS, INNATE LYMPHOID CELLS AND THE EPITHELIUM, ARE INTEGRATED BY T CELLS TO GUIDE THE TRANSCRIPTIONAL AND EPIGENETIC CHANGES NECESSARY FOR T(H)2 CELL DIFFERENTIATION. OUR IMPROVED UNDERSTANDING OF THESE PATHWAYS HAS OPENED NEW AVENUES FOR THERAPEUTICALLY TARGETING T(H)2 CELLS IN ASTHMA AND ALLERGY. THE ADVENT OF COMPREHENSIVE SINGLE-CELL TRANSCRIPTOMICS ALONG WITH IMPROVEMENTS IN SINGLE-CELL PROTEOMICS AND THE GENERATION OF NOVEL IN VIVO CELL FATE MAPPING TECHNIQUES PROMISE TO EXPAND OUR UNDERSTANDING OF T CELL DIVERSITY AND OFFER NEW INSIGHT INTO DISEASE-RELATED HETEROGENEITY AND PLASTICITY OF T(H) CELL RESPONSES. 2018 16 5798 33 STEM CELLS AND LUNG REGENERATION. THE ABILITY TO REPLACE DEFECTIVE CELLS IN AN AIRWAY WITH CELLS THAT CAN ENGRAFT, INTEGRATE, AND RESTORE A FUNCTIONAL EPITHELIUM COULD POTENTIALLY CURE A NUMBER OF LUNG DISEASES. PROGRESS TOWARD THE DEVELOPMENT OF STRATEGIES TO REGENERATE THE ADULT LUNG BY EITHER IN VIVO OR EX VIVO TARGETING OF ENDOGENOUS STEM CELLS OR PLURIPOTENT STEM CELL DERIVATIVES IS LIMITED BY OUR FUNDAMENTAL LACK OF UNDERSTANDING OF THE MECHANISMS CONTROLLING HUMAN LUNG DEVELOPMENT, THE PRECISE IDENTITY AND FUNCTION OF HUMAN LUNG STEM AND PROGENITOR CELL TYPES, AND THE GENETIC AND EPIGENETIC CONTROL OF HUMAN LUNG FATE. IN THIS REVIEW, WE INTEND TO DISCUSS THE KNOWN STEM/PROGENITOR CELL POPULATIONS, THEIR RELATIVE DIFFERENCES BETWEEN RODENTS AND HUMANS, THEIR ROLES IN CHRONIC LUNG DISEASE, AND THEIR THERAPEUTIC PROSPECTS. ADDITIONALLY, WE HIGHLIGHT THE RECENT BREAKTHROUGHS THAT HAVE INCREASED OUR UNDERSTANDING OF THESE CELL TYPES. THESE ADVANCEMENTS INCLUDE NOVEL LINEAGE-TRACED ANIMAL MODELS AND SINGLE-CELL RNA SEQUENCING OF HUMAN AIRWAY CELLS, WHICH HAVE PROVIDED CRITICAL INFORMATION ON THE STEM CELL SUBTYPES, TRANSITION STATES, IDENTIFYING CELL MARKERS, AND INTRICATE PATHWAYS THAT COMMIT A STEM CELL TO DIFFERENTIATE OR TO MAINTAIN PLASTICITY. AS OUR CAPACITY TO MODEL THE HUMAN LUNG EVOLVES, SO WILL OUR UNDERSTANDING OF LUNG REGENERATION AND OUR ABILITY TO TARGET ENDOGENOUS STEM CELLS AS A THERAPEUTIC APPROACH FOR LUNG DISEASE. 2020 17 4254 38 METHYLOME-BASED CELL-OF-ORIGIN MODELING (METHYL-COOM) IDENTIFIES ABERRANT EXPRESSION OF IMMUNE REGULATORY MOLECULES IN CLL. BACKGROUND: IN CANCER, NORMAL EPIGENETIC PATTERNS ARE DISTURBED AND CONTRIBUTE TO GENE EXPRESSION CHANGES, DISEASE ONSET, AND PROGRESSION. THE CANCER EPIGENOME IS COMPOSED OF THE EPIGENETIC PATTERNS PRESENT IN THE TUMOR-INITIATING CELL AT THE TIME OF TRANSFORMATION, AND THE TUMOR-SPECIFIC EPIGENETIC ALTERATIONS THAT ARE ACQUIRED DURING TUMOR INITIATION AND PROGRESSION. THE PRECISE DISSECTION OF THESE TWO COMPONENTS OF THE TUMOR EPIGENOME WILL FACILITATE A BETTER UNDERSTANDING OF THE BIOLOGICAL MECHANISMS UNDERLYING MALIGNANT TRANSFORMATION. CHRONIC LYMPHOCYTIC LEUKEMIA (CLL) ORIGINATES FROM DIFFERENTIATING B CELLS, WHICH UNDERGO EXTENSIVE EPIGENETIC PROGRAMMING. THIS POSES THE CHALLENGE TO PRECISELY DETERMINE THE EPIGENOMIC GROUND STATE OF THE CELL-OF-ORIGIN IN ORDER TO IDENTIFY CLL-SPECIFIC EPIGENETIC ABERRATIONS. METHODS: WE DEVELOPED A LINEAR REGRESSION MODEL, METHYLOME-BASED CELL-OF-ORIGIN MODELING (METHYL-COOM), TO MAP THE CELL-OF-ORIGIN FOR INDIVIDUAL CLL PATIENTS BASED ON THE CONTINUUM OF EPIGENOMIC CHANGES DURING NORMAL B CELL DIFFERENTIATION. RESULTS: METHYL-COOM ACCURATELY MAPS THE CELL-OF-ORIGIN OF CLL AND IDENTIFIES CLL-SPECIFIC ABERRANT DNA METHYLATION EVENTS THAT ARE NOT CONFOUNDED BY PHYSIOLOGIC EPIGENETIC B CELL PROGRAMMING. FURTHERMORE, METHYL-COOM UNMASKS ABNORMAL ACTION OF TRANSCRIPTION FACTORS, ALTERED SUPER-ENHANCER ACTIVITIES, AND ABERRANT TRANSCRIPT EXPRESSION IN CLL. AMONG THE ABERRANTLY REGULATED TRANSCRIPTS WERE MANY GENES THAT HAVE PREVIOUSLY BEEN IMPLICATED IN T CELL BIOLOGY. FLOW CYTOMETRY ANALYSIS OF THESE MARKERS CONFIRMED THEIR ABERRANT EXPRESSION ON MALIGNANT B CELLS AT THE PROTEIN LEVEL. CONCLUSIONS: METHYL-COOM ANALYSIS OF CLL IDENTIFIED DISEASE-SPECIFIC ABERRANT GENE REGULATION. THE ABERRANTLY EXPRESSED GENES IDENTIFIED IN THIS STUDY MIGHT PLAY A ROLE IN IMMUNE-EVASION IN CLL AND MIGHT SERVE AS NOVEL TARGETS FOR IMMUNOTHERAPY APPROACHES. IN SUMMARY, WE PROPOSE A NOVEL FRAMEWORK FOR IN SILICO MODELING OF REFERENCE DNA METHYLOMES AND FOR THE IDENTIFICATION OF CANCER-SPECIFIC EPIGENETIC CHANGES, A CONCEPT THAT CAN BE BROADLY APPLIED TO OTHER HUMAN MALIGNANCIES. 2020 18 3 26 "EPIGENOME-WIDE METHYLATION PROFILE OF CHRONIC KIDNEY DISEASE-DERIVED ARTERIAL DNA UNCOVERS NOVEL PATHWAYS IN DISEASE-ASSOCIATED CARDIOVASCULAR PATHOLOGY.". CHRONIC KIDNEY DISEASE (CKD) RELATED CARDIOVASCULAR DISEASE (CVD) IS CHARACTERIZED BY VASCULAR REMODELLING WITH WELL-ESTABLISHED STRUCTURAL AND FUNCTIONAL CHANGES IN THE VASCULAR WALL SUCH AS ARTERIAL STIFFNESS, MATRIX DEPOSITION, AND CALCIFICATION. THESE PHENOTYPIC CHANGES RESEMBLE PATHOLOGY SEEN IN AGEING, AND ARE LIKELY TO BE MEDIATED BY SUSTAINED ALTERATIONS IN GENE EXPRESSION, WHICH MAY BE CAUSED BY EPIGENETIC CHANGES SUCH AS TISSUE-SPECIFIC DNA METHYLATION. WE AIMED TO INVESTIGATE TISSUE SPECIFIC CHANGES IN DNA METHYLATION THAT OCCUR IN CKD-RELATED CVD. GENOME-WIDE DNA METHYLATION CHANGES WERE EXAMINED IN BISULPHITE CONVERTED GENOMIC DNA ISOLATED FROM THE VASCULAR MEDIA OF CKD AND HEALTHY ARTERIES. METHYLATION-SPECIFIC PCR WAS USED TO VALIDATE THE ARRAY DATA, AND THE ASSOCIATION BETWEEN DNA METHYLATION AND GENE AND PROTEIN EXPRESSION WAS EXAMINED. THE DNA METHYLATION AGE WAS COMPARED TO THE CHRONOLOGICAL AGE IN BOTH CASES AND CONTROLS. THREE HUNDRED AND NINETEEN DIFFERENTIALLY METHYLATED REGIONS (DMR) WERE IDENTIFIED SPREAD ACROSS THE GENOME. PATHWAY ANALYSIS REVEALED THAT DMRS ASSOCIATED WITH GENES WERE INVOLVED IN EMBRYONIC AND VASCULAR DEVELOPMENT, AND SIGNALLING PATHWAYS SUCH AS TGFBETA AND FGF. EXPRESSION OF TOP DIFFERENTIALLY METHYLATED GENE HOXA5 SHOWED A SIGNIFICANT NEGATIVE CORRELATION WITH DNA METHYLATION. INTERESTINGLY, DNA METHYLATION AGE AND CHRONOLOGICAL AGE WERE HIGHLY CORRELATED, BUT THERE WAS NO EVIDENCE OF ACCELERATED AGE-RELATED DNA METHYLATION IN THE ARTERIES OF CKD PATIENTS. IN CONCLUSION, WE DEMONSTRATED THAT DIFFERENTIAL DNA METHYLATION IN THE ARTERIAL TISSUE OF CKD PATIENTS REPRESENTS A POTENTIAL MEDIATOR OF ARTERIAL PATHOLOGY AND MAY BE USED TO UNCOVER NOVEL PATHWAYS IN THE GENESIS OF CKD-ASSOCIATED COMPLICATIONS. 2021 19 4856 23 OPTIMIZING RETROVIRAL GENE EXPRESSION FOR EFFECTIVE THERAPIES. WITH THEIR ABILITY TO INTEGRATE THEIR GENETIC MATERIAL INTO THE TARGET CELL GENOME, RETROVIRAL VECTORS (RV) OF BOTH THE GAMMA-RETROVIRAL (GAMMA-RV) AND LENTIVIRAL VECTOR (LV) CLASSES CURRENTLY REMAIN THE MOST EFFICIENT AND THUS THE SYSTEM OF CHOICE FOR ACHIEVING TRANSGENE RETENTION AND THEREFORE POTENTIALLY LONG-TERM EXPRESSION AND THERAPEUTIC BENEFIT. HOWEVER, GAMMA-RV AND LV INTEGRATION COMES AT A COST IN THAT TRANSCRIPTION UNITS WILL BE PRESENT WITHIN A NATIVE CHROMATIN ENVIRONMENT AND THUS BE SUBJECT TO EPIGENETIC EFFECTS (DNA METHYLATION, HISTONE MODIFICATIONS) THAT CAN NEGATIVELY IMPACT ON THEIR FUNCTION. INDEED, HIGHLY VARIABLE EXPRESSION AND SILENCING OF GAMMA-RV AND LV TRANSGENES ESPECIALLY RESULTING FROM PROMOTER DNA METHYLATION IS WELL DOCUMENTED AND WAS THE CAUSE OF THE FAILURE OF GENE THERAPY IN A CLINICAL TRIAL FOR X-LINKED CHRONIC GRANULOMATOUS DISEASE. THIS REVIEW WILL CRITICALLY EXPLORE THE USE OF DIFFERENT CLASSES OF GENETIC CONTROL ELEMENTS THAT CAN IN PRINCIPLE REDUCE VECTOR INSERTION SITE POSITION EFFECTS AND EPIGENETIC-MEDIATED SILENCING. THESE TRANSCRIPTIONAL REGULATORY ELEMENTS BROADLY DIVIDE THEMSELVES INTO EITHER THOSE WITH A CHROMATIN BOUNDARY OR BORDER FUNCTION (SCAFFOLD/MATRIX ATTACHMENT REGIONS, INSULATORS) OR THOSE WITH A DOMINANT CHROMATIN REMODELING AND TRANSCRIPTIONAL ACTIVATING CAPABILITY (LOCUS CONTROL REGIONS,, UBIQUITOUS CHROMATIN OPENING ELEMENTS). ALL THESE TYPES OF ELEMENTS HAVE THEIR STRENGTHS AND WEAKNESSES WITHIN THE CONSTRAINTS OF A GAMMA-RV AND LV BACKBONE, SHOWING VARYING DEGREES OF EFFICACY IN IMPROVING REPRODUCIBILITY AND STABILITY OF TRANSGENE FUNCTION. COMBINATIONS OF BOUNDARY AND CHROMATIN REMODELING; TRANSCRIPTIONAL ACTIVATING ELEMENTS, WHICH DO NOT IMPEDE VECTOR PRODUCTION; TRANSDUCTION EFFICIENCY; AND STABILITY ARE MOST LIKELY TO MEET THE REQUIREMENTS WITHIN A GENE THERAPY CONTEXT ESPECIALLY WHEN TARGETING A STEM CELL POPULATION. 2013 20 3027 28 GENETICS OF CHRONIC OBSTRUCTIVE PULMONARY DISEASE. CHRONIC OBSTRUCTIVE PULMONARY DISEASE (COPD) IS A COMPLEX DISEASE WITH MULTIFACTORIAL BACKGROUND, BASED ON THE INTERACTION OF ENVIRONMENTAL AND GENETIC FACTORS. ENVIRONMENTAL FACTORS ARE CLEARLY RELATED TO THE DEVELOPMENT OF THE DISEASE. HOWEVER, FAMILY AND TWIN STUDIES SUGGESTED GENETICS FACTORS TO BE ONE OF THE IMPORTANT DETERMINANTS FOR THE DEVELOPMENT OF COPD. DIFFERENT APPROACHES HAVE BEEN USED TO IDENTIFY GENES OF INTEREST. GENOMEWIDE LINKAGE ANALYSIS FOUND AREAS OF INTEREST ON DIFFERENT CHROMOSOMES, WITH SOME GENES LOCATED IN THIS REGIONS BEING IDENTIFIED AND REPLICATED AS SUSCEPTIBILITY GENES. NUMEROUS OF CANDIDATE GENES THAT COULD BE LINKED TO DISEASE PATHOGENESIS HAVE BEEN IMPLICATED IN COPD GENETICS. HOWEVER, THE CANDIDATE GENE APPROACH IS OFTEN LIMITED BY INCONSISTENT RESULTS IN OTHER STUDY POPULATIONS. RECENTLY, A COMBINATION OF DIFFERENT METHODS IS USED GIVING MORE EVIDENCE FOR SOME CANDIDATE GENES, INCLUDING TGFBETA-1, SURFACTANT, SERPINE2 AND MICROSOMAL EPOXIDE HYDROLASE. IN THE FUTURE ONGOING EXACT PHENOTYPE DEFINITION, COMBINATION OF SEVERAL APPROACHES, GENOME-WIDE ASSOCIATION STUDIES AND ANIMAL MODEL GENETICS WILL LEAD TO NEW INSIGHTS INTO THE GENETICS OF COPD, WITH EPIGENETIC FACTORS NEEDS TO BE FURTHER INVESTIGATED AND CONSIDERED IN CONCERT WITH GENETIC FINDINGS. 2007