1 3724 144 INHIBITION OF EZH2 AMELIORATES CARTILAGE ENDPLATE DEGENERATION AND ATTENUATES THE PROGRESSION OF INTERVERTEBRAL DISC DEGENERATION VIA DEMETHYLATION OF SOX-9. BACKGROUND: CARTILAGINOUS ENDPLATE (CEP) DEGENERATION IS CONSIDERED AS ONE OF THE MAJOR CAUSES OF INTERVERTEBRAL DISC DEGENERATION (IVDD) WHICH CAUSES LOW BACK PAIN. RECENT STUDIES HAVE PROVED THAT EPIGENETIC ALTERATION IS INVOLVED IN A VARIETY OF DISEASES. THIS WORK EXPLORED THE ROLE OF HISTONE METHYLTRANSFERASE ENHANCER OF ZESTE HOMOLOGUE 2 (EZH2) IN CEP DEGENERATION, AS WELL AS ITS UNDERLYING EPIGENETIC MECHANISMS, AND CONFIRMED THE EFFECT OF EZH2 KNOCKDOWN ON DELAYING IVDD DEVELOPMENT. METHODS: WESTERN BLOTTING, IMMUNOFLUORESCENCE STAINING, AND CHIP ASSAY WERE APPLIED TO DEMONSTRATE THE MOLECULAR MECHANISM OF EZH2 IN CEP TISSUE. THE THERAPEUTIC POTENTIAL OF EZH2 WAS INVESTIGATED USING PUNCTURE-INDUCED RAT MODELS. FINDINGS: THE EZH2 EXPRESSION WAS UPREGULATED IN HUMAN AND RAT CEP TISSUE. IT WAS ALSO FOUND THAT THE OVEREXPRESSION OF EZH2 SUPPRESSED THE EXPRESSION OF COLLAGEN II, AGGRECAN AND SOX-9, AND PROMOTED THE EXPRESSION OF ADTAMTS5 AND MMP13 IN RAT ENDPLATE CHONDROCYTES (EPCS), WHICH COULD BE REVERSED BY EZH2 SILENCING. THE CORRELATION BETWEEN EZH2 AND SOX-9 WAS FURTHER EXPLORED, WHILE OVEREXPRESSION OF SOX-9 COULD REVERSE THE EFFECT OF EZH2 IN RAT EPCS. MOREOVER, INHIBITION OF EZH2 UPREGULATED THE LEVEL OF SOX-9 BY DEMETHYLATING H3K27ME3 AT SOX-9 PROMOTER SITES, REVEALING THE REGULATORY MECHANISM OF EZH2 ON SOX-9. MEANWHILE, PUNCTURE-INDUCED RAT MODELS SHOWED THAT EZH2 KNOCKDOWN EXERTED A PROTECTIVE EFFECT ON CEP AND DISC DEGENERATION. INTERPRETATION: THIS STUDY REVEALS THAT EZH2 INHIBITION IS A PROMISING STRATEGY FOR MITIGATING THE SYMPTOMS AND PROGRESSION OF IVDD. FUNDING: THIS STUDY WAS FUNDED BY THE NATURAL SCIENCE FOUNDATION OF ZHEJIANG PROVINCE (Y16H060034). AUTHORS DECLARE THAT THE FUNDERS HAD NO INVOLVEMENT IN THE STUDY DESIGN, DATA ANALYSIS AND INTERPRETATION OF THE RESULTS. 2019 2 3128 41 GIPC-REGULATED IGFBP-3 PROMOTES HSC MIGRATION IN VITRO AND PORTAL HYPERTENSION IN VIVO THROUGH A BETA1-INTEGRIN PATHWAY. BACKGROUND & AIMS: TRANSFORMING GROWTH FACTOR (TGF-BETA)-INDUCED ACTIVATION OF QUIESCENT HEPATIC STELLATE CELLS (HSCS) AND THEIR TRANSFORMATION TO MYOFIBROBLASTS IS A KEY EVENT IN LIVER FIBROSIS AND PORTAL HYPERTENSION. GIPC (ALSO REFERRED TO AS SYNECTIN) IS A DOWNSTREAM SIGNAL ACTIVATION MOLECULE OF TGF-BETA AND OTHER RECEPTORS. IN THIS STUDY, WE SOUGHT TO IDENTIFY NOVEL GENES TARGETED BY TGF-BETA AND GIPC AND ELUCIDATE IF AND HOW THEY MAY CONTRIBUTE TO LIVER FIBROSIS. METHODS: WE PERFORMED SEQUENTIAL MESSENGER RNA SEQUENCING ANALYSIS ON TGF-BETA-STIMULATED HSCS AND THEN ON TGF-BETA-STIMULATED HSCS IN THE PRESENCE AND ABSENCE OF GIPC ALSO REFERRED TO AS SYNECTIN (GIPC) KNOCKDOWN. INSULIN-LIKE GROWTH FACTOR BINDING PROTEIN-3 (IGFBP-3) TRANSPORT PROTEIN EMERGED AS A TOP ACTIVATION TARGET OF BOTH TGF-BETA AND GIPC. QUANTITATIVE POLYMERASE CHAIN REACTION, ENZYME-LINKED IMMUNOSORBENT ASSAY, TARGETED CHROMATIN IMMUNOPRECIPITATION, AND WESTERN BLOT ANALYSIS WERE DONE FOR FURTHER CONFIRMATION. RESULTS: IGFBP-3, AN INSULIN GROWTH FACTOR TRANSPORT PROTEIN, EMERGED AS A TOP ACTIVATION TARGET OF BOTH TGF-BETA AND GIPC, WHICH WAS CONFIRMED BY QUANTITATIVE POLYMERASE CHAIN REACTION, ENZYME-LINKED IMMUNOSORBENT ASSAY, AND WESTERN BLOT ANALYSIS. TARGETED CHROMATIN IMMUNOPRECIPITATION SHOWED THAT GIPC INCREASES THE HISTONE 3 LYSINE 27 (H3K27) ACETYLATION ACTIVATING MARK AND CONCURRENTLY DECREASES THE H3K27 INHIBITORY TRIMETHYLATION (H3K27M3) MARK, PROVIDING AN EPIGENETIC CORRELATE TO THE GENE REGULATION CHANGES. IN VIVO, GLOBAL KNOCKOUT OF IGFBP-3 MICE RESULTED IN ATTENUATION OF HSC ACTIVATION MARKERS AND ATTENUATION OF PORTAL PRESSURE IN RESPONSE TO CHRONIC LIVER INJURY MODELS. ANALYSIS OF SERUM LEVELS FROM CIRRHOTIC PATIENTS ALSO SHOWED AN IGFBP-3 INCREASE OF MORE THAN 2-FOLD COMPARED WITH HEALTHY CONTROLS. FINALLY, IN VITRO MECHANISM STUDIES SHOWED THAT IGFBP-3 PROMOTES HSC MIGRATION THROUGH INTEGRIN-DEPENDENT PHOSPHORYLATION OF PROTEIN KINASE B. CONCLUSIONS: TGF-BETA UP-REGULATES IGFBP-3 THROUGH GIPC, LEADING TO INCREASED HSC MIGRATION IN VITRO AND PROMOTES PORTAL HYPERTENSION IN VIVO. THESE STUDIES SUPPORT THE ROLE OF IGFBP-3 AS A POTENTIAL PATHOPHYSIOLOGIC TARGET OR BIOMARKER IN CHRONIC LIVER DISEASE. 2020 3 148 47 ABERRANT FLUID SHEAR STRESS CONTRIBUTES TO ARTICULAR CARTILAGE PATHOGENESIS VIA EPIGENETIC REGULATION OF ZBTB20 BY H3K4ME3. PURPOSE: OSTEOARTHRITIS (OA) IS A COMMON DISEASE FOR HUMAN BEINGS, CHARACTERIZED BY SEVERE INFLAMMATION, CARTILAGE DEGRADATION, AND SUBCHONDRAL BONE DESTRUCTION. HOWEVER, CURRENT THERAPIES ARE LIMITED TO RELIEVING PAIN OR JOINT REPLACEMENT AND NO EFFECTIVE TREATMENT METHODS HAVE BEEN DISCOVERED TO IMPROVE DEGENERATIVE CHANGES. CURRENTLY, A VARIETY OF EVIDENCES HAVE INDICATED THAT ABERRANT MECHANICAL STIMULI IS CLOSELY ASSOCIATED WITH ARTICULAR JOINT PATHOGENESIS, WHILE THE DETAILED UNDERLYING MECHANISM REMAINS UNELUCIDATED. IN THE PRESENT STUDY, WE DETERMINED TO INVESTIGATE THE IMPACT OF EXCESSIVE HIGH FLUID SHEAR STRESS (FSS) ON PRIMARY CHONDROCYTES AND THE UNDERLYING EPIGENETIC MECHANISMS. MATERIALS AND METHODS: PHALLOIDIN STAINING AND EDU STAINING WERE USED TO EVALUATE CELL MORPHOLOGY AND VIABILITY. THE MRNA LEVEL AND PROTEIN LEVEL OF GENES WERE DETERMINED BY QPCR, WESTERN BLOT ASSAY, AND IMMUNOFLUORESCENCE STAINING. MECHANISTIC INVESTIGATION WAS PERFORMED THROUGH RNA-SEQUENCING AND CUT&TAG SEQUENCING. IN VIVO, WE ADOPTED UNILATERAL ANTERIOR CROSSBITES (UAC) MICE MODEL TO INVESTIGATE THE EXPRESSION OF H3K4ME3 AND ZBTB20 IN ABERRANT FORCE-RELATED CARTILAGE PATHOGENESIS. RESULTS: THE RESULTS DEMONSTRATED THAT FSS GREATLY DISRUPTS CELL MORPHOLOGY AND SIGNIFICANTLY DECREASED CHONDROCYTE VIABILITY. ABERRANT FSS INDUCES REMARKABLE INFLAMMATORY MEDIATORS PRODUCTION, LEADING TO CARTILAGE DEGENERATION AND DEGRADATION. IN DEPTH MECHANISTIC STUDY SHOWED THAT FSS RESULTS IN MORE THAN 10-FOLD UPREGULATION OF H3K4ME3, AND THE MODULATORY EFFECT OF H3K4ME3 ON CARTILAGE WAS OBTAINED BY DIRECTLY TARGETING ZBTB20. FURTHERMORE, WNT SIGNALING WAS STRONGLY ACTIVATED IN HIGH FSS-INDUCED OA PATHOGENESIS, AND THE NEGATIVE IMPACT OF ZBTB20 ON CHONDROCYTES WAS ALSO ACHIEVED THROUGH ACTIVATING WNT SIGNALING PATHWAY. MOREOVER, PHARMACOLOGICAL INHIBITION OF H3K4ME3 ACTIVATION BY MM-102 OR TREATMENT WITH WNT PATHWAY INHIBITOR LF3 COULD EFFECTIVELY ALLEVIATE THE DESTRUCTIVE EFFECT OF FSS ON CHONDROCYTES. IN VIVO UAC MICE MODEL VALIDATED THE DYSREGULATION OF H3K4ME3 AND ZBTB20 IN ABERRANT FORCE-INDUCED CARTILAGE PATHOGENESIS. CONCLUSION: THROUGH THE COMBINATION OF IN VITRO FSS MODEL AND IN VIVO UAC MODEL, KMT2B-H3K4ME3-ZBTB20 AXIS WAS FIRST IDENTIFIED IN ABERRANT FSS-INDUCED CARTILAGE PATHOGENESIS, WHICH MAY PROVIDE EVIDENCES FOR EPIGENETIC-BASED THERAPY IN THE FUTURE. 2021 4 3049 30 GENOME-WIDE ANALYSIS REVEALS ZINC TRANSPORTER ZIP9 REGULATED BY DNA METHYLATION PROMOTES RADIATION-INDUCED SKIN FIBROSIS VIA THE TGF-BETA SIGNALING PATHWAY. RADIATION-INDUCED SKIN FIBROSIS IS A DETRIMENTAL AND CHRONIC DISORDER THAT OCCURS AFTER RADIATION EXPOSURE. DNA METHYLATION HAS BEEN CHARACTERIZED AS AN IMPORTANT REGULATORY MECHANISM OF MULTIPLE PATHOLOGICAL PROCESSES. IN THIS STUDY, WE COMPARED THE GENOME-WIDE DNA METHYLATION STATUS IN RADIATION-INDUCED FIBROTIC SKIN AND ADJACENT NORMAL TISSUES OF RATS BY METHYLATED DNA IMMUNOPRECIPITATION SEQUENCING. RADIATION-INDUCED FIBROTIC SKIN SHOWED DIFFERENTIALLY METHYLATED REGIONS ASSOCIATED WITH 3,650 PROTEIN-CODING GENES, 72 MICRORNAS, 5,836 LONG NONCODING RNAS AND 3 PIWI-INTERACTING RNAS. BY INTEGRATING THE MRNA AND METHYLATION PROFILES, THE ZINC TRANSPORTER SLC39A9/ZIP9 WAS INVESTIGATED IN GREATER DETAIL. THE PROTEIN LEVEL OF ZIP9 WAS INCREASED IN IRRADIATED SKIN TISSUES OF HUMANS, MONKEYS, AND RATS, ESPECIALLY IN RADIOGENIC FIBROTIC SKIN TISSUES. RADIATION INDUCED THE DEMETHYLATION OF A CPG DINUCLEOTIDE IN EXON 1 OF ZIP9 THAT RESULTED IN RECRUITMENT OF THE TRANSCRIPTIONAL FACTOR SP1 AND INCREASED ZIP9 EXPRESSION. OVEREXPRESSION OF ZIP9 RESULTED IN ACTIVATION OF THE PROFIBROTIC TRANSFORMING GROWTH FACTOR-BETA SIGNALING PATHWAY THROUGH PROTEIN KINASE B IN HUMAN FIBROBLASTS. IN ADDITION, RADIATION-INDUCED SKIN FIBROSIS WAS ASSOCIATED WITH INCREASED ZINC ACCUMULATION. THE ZINC CHELATOR N,N,N',N'-TETRAKIS(2-PYRIDYLMETHYL)-1,2-ETHYLENEDIAMINE ABROGATED ZIP9-INDUCED ACTIVATION OF THE TRANSFORMING GROWTH FACTOR-BETA SIGNALING PATHWAY AND ATTENUATED RADIATION-INDUCED SKIN FIBROSIS IN A RAT MODEL. IN SUMMARY, OUR FINDINGS ILLUSTRATE EPIGENETIC REGULATION OF ZIP9 AND ITS CRITICAL ROLE IN PROMOTING RADIATION-INDUCED SKIN FIBROSIS. 2020 5 4001 44 LOSS OF MEN1 LEADS TO RENAL FIBROSIS AND DECREASES HGF-ADAMTS5 PATHWAY ACTIVITY VIA AN EPIGENETIC MECHANISM. BACKGROUND: RENAL FIBROSIS IS A SERIOUS CONDITION THAT RESULTS IN THE DEVELOPMENT OF CHRONIC KIDNEY DISEASES. THE MEN1 GENE IS AN EPIGENETIC REGULATOR THAT ENCODES THE MENIN PROTEIN AND ITS ROLE IN KIDNEY TISSUE REMAINS UNCLEAR. METHODS: KIDNEY HISTOLOGY WAS EXAMINED ON PARAFFIN SECTIONS STAINED WITH HEMATOXYLIN-EOSIN STAINING. MASSON'S TRICHROME STAINING AND SIRIUS RED STAINING WERE USED TO ANALYZE RENAL FIBROSIS. GENE AND PROTEIN EXPRESSION WERE DETERMINED BY QUANTITATIVE REAL-TIME PCR (QPCR) AND WESTERN BLOT, RESPECTIVELY. IMMUNOHISTOCHEMISTRY STAINING IN THE KIDNEY TISSUES FROM MICE OR PATIENTS WAS USED TO EVALUATE PROTEIN LEVELS. FLOW CYTOMETRY WAS USED TO ANALYZE THE CELL CYCLE DISTRIBUTIONS AND APOPTOSIS. RNA-SEQUENCING WAS PERFORMED FOR DIFFERENTIAL EXPRESSION GENES IN THE KIDNEY TISSUES OF THE MEN1F/F AND MEN1?/? MICE. CHROMATIN IMMUNOPRECIPITATION SEQUENCING (CHIP-SEQ) WAS CARRIED OUT FOR IDENTIFICATION OF MENIN- AND H3K4ME3-ENRICHED REGIONS WITHIN THE WHOLE GENOME IN THE MOUSE KIDNEY TISSUE. CHIP-QPCR ASSAYS WERE PERFORMED FOR OCCUPANCY OF MENIN AND H3K4ME3 AT THE GENE PROMOTER REGIONS. LUCIFERASE REPORTER ASSAY WAS USED TO DETECT THE PROMOTER ACTIVITY. THE EXACERBATED UNILATERAL URETERAL OBSTRUCTION (UUO) MODELS IN THE MEN1F/F AND MEN1?/? MICE WERE USED TO ASSESS THE PHARMACOLOGICAL EFFECTS OF RH-HGF ON RENAL FIBROSIS. RESULTS: THE EXPRESSION OF MEN1 IS REDUCE IN KIDNEY TISSUES OF FIBROTIC MOUSE AND HUMAN DIABETIC PATIENTS AND TREATMENT WITH FIBROTIC FACTOR RESULTS IN THE DOWNREGULATION OF MEN1 EXPRESSION IN RENAL TUBULAR EPITHELIAL CELLS (RTECS). DISRUPTION OF MEN1 IN RTECS LEADS TO HIGH EXPRESSION OF ALPHA-SMA AND COLLAGEN 1, WHEREAS MEN1 OVEREXPRESSION RESTRAINS EPITHELIAL-TO-MESENCHYMAL TRANSITION (EMT) INDUCED BY TGF-BETA TREATMENT. CONDITIONAL KNOCKOUT OF MEN1 RESULTED IN CHRONIC RENAL FIBROSIS AND UUO-INDUCED TUBULOINTERSTITIAL FIBROSIS (TIF), WHICH IS ASSOCIATED WITH AN INCREASED INDUCTION OF EMT, G2/M ARREST AND JNK SIGNALING. MECHANISTICALLY, MENIN RECRUITS AND INCREASES H3K4ME3 AT THE PROMOTER REGIONS OF HEPATOCYTE GROWTH FACTOR (HGF) AND A DISINTEGRIN AND METALLOPROTEINASE WITH THROMBOSPONDIN MOTIFS 5 (ADAMTS5) GENES AND ENHANCES THEIR TRANSCRIPTIONAL ACTIVATION. IN THE UUO MICE MODEL, EXOGENOUS HGF RESTORED THE EXPRESSION OF ADAMTS5 AND AMELIORATED RENAL FIBROSIS INDUCED BY MEN1 DEFICIENCY. CONCLUSIONS: THESE FINDINGS DEMONSTRATE THAT MEN1 IS AN ESSENTIAL ANTIFIBROTIC FACTOR IN RENAL FIBROGENESIS AND COULD BE A POTENTIAL TARGET FOR ANTIFIBROTIC THERAPY. 2022 6 3468 44 HYPOXIA-INDUCED DNA HYPERMETHYLATION IN HUMAN PULMONARY FIBROBLASTS IS ASSOCIATED WITH THY-1 PROMOTER METHYLATION AND THE DEVELOPMENT OF A PRO-FIBROTIC PHENOTYPE. BACKGROUND: PULMONARY FIBROSIS IS A DEBILITATING AND LETHAL DISEASE WITH NO EFFECTIVE TREATMENT OPTIONS. UNDERSTANDING THE PATHOLOGICAL PROCESSES AT PLAY WILL DIRECT THE APPLICATION OF NOVEL THERAPEUTIC AVENUES. HYPOXIA HAS BEEN IMPLICATED IN THE PATHOGENESIS OF PULMONARY FIBROSIS YET THE PRECISE MECHANISM BY WHICH IT CONTRIBUTES TO DISEASE PROGRESSION REMAINS TO BE FULLY ELUCIDATED. IT HAS BEEN SHOWN THAT CHRONIC HYPOXIA CAN ALTER DNA METHYLATION PATTERNS IN TUMOUR-DERIVED CELL LINES. THIS EPIGENETIC ALTERATION CAN INDUCE CHANGES IN CELLULAR PHENOTYPE WITH PROMOTER METHYLATION BEING ASSOCIATED WITH GENE SILENCING. OF PARTICULAR RELEVANCE TO IDIOPATHIC PULMONARY FIBROSIS (IPF) IS THE OBSERVATION THAT THY-1 PROMOTER METHYLATION IS ASSOCIATED WITH A MYOFIBROBLAST PHENOTYPE WHERE LOSS OF THY-1 OCCURS ALONGSIDE INCREASED ALPHA SMOOTH MUSCLE ACTIN (ALPHA-SMA) EXPRESSION. THE INITIAL AIM OF THIS STUDY WAS TO DETERMINE WHETHER HYPOXIA REGULATES DNA METHYLATION IN NORMAL HUMAN LUNG FIBROBLASTS (CCD19LU). AS IT HAS BEEN REPORTED THAT HYPOXIA SUPPRESSES THY-1 EXPRESSION DURING LUNG DEVELOPMENT WE ALSO STUDIED THE EFFECT OF HYPOXIA ON THY-1 PROMOTER METHYLATION AND GENE EXPRESSION. METHODS: CCD19LU WERE GROWN FOR UP TO 8 DAYS IN HYPOXIA AND ASSESSED FOR GLOBAL CHANGES IN DNA METHYLATION USING FLOW CYTOMETRY. REAL-TIME PCR WAS USED TO QUANTIFY EXPRESSION OF THY-1, ALPHA-SMA, COLLAGEN I AND III. GENOMIC DNA WAS BISULPHITE TREATED AND METHYLATION SPECIFIC PCR (MSPCR) WAS USED TO EXAMINE THE METHYLATION STATUS OF THE THY-1 PROMOTER. RESULTS: SIGNIFICANT GLOBAL HYPERMETHYLATION WAS DETECTED IN HYPOXIC FIBROBLASTS RELATIVE TO NORMOXIC CONTROLS AND WAS ACCOMPANIED BY INCREASED EXPRESSION OF MYOFIBROBLAST MARKERS. THY-1 MRNA EXPRESSION WAS SUPPRESSED IN HYPOXIC CELLS, WHICH WAS RESTORED WITH THE DEMETHYLATING AGENT 5-AZA-2'-DEOXYCYTIDINE. MSPCR REVEALED THAT THY-1 BECAME METHYLATED FOLLOWING FIBROBLAST EXPOSURE TO 1% O2. CONCLUSION: THESE DATA SUGGEST THAT GLOBAL AND GENE-SPECIFIC CHANGES IN DNA METHYLATION MAY PLAY AN IMPORTANT ROLE IN FIBROBLAST FUNCTION IN HYPOXIA. 2012 7 3390 33 HOPX PLAYS A CRITICAL ROLE IN ANTIRETROVIRAL DRUGS INDUCED EPIGENETIC MODIFICATION AND CARDIAC HYPERTROPHY. PEOPLE LIVING WITH HIV (PLWH) HAVE TO TAKE AN ANTIRETROVIRAL THERAPY (ART) FOR LIFE AND SHOW NONCOMMUNICABLE ILLNESSES SUCH AS CHRONIC INFLAMMATION, IMMUNE ACTIVATION, AND MULTIORGAN DYSREGULATION. RECENT STUDIES SUGGEST THAT LONG-TERM USE OF ART INDUCES COMORBID CONDITIONS AND IS ONE OF THE LEADING CAUSES OF HEART FAILURE IN PLWH. HOWEVER, THE MOLECULAR MECHANISM OF ANTIRETROVIRAL DRUGS (ARVS) INDUCED HEART FAILURE IS UNCLEAR. TO DETERMINE THE MECHANISM OF ARVS INDUCED CARDIAC DYSFUNCTION, WE PERFORMED GLOBAL TRANSCRIPTOMIC PROFILING OF ARVS TREATED NEONATAL RAT VENTRICULAR CARDIOMYOCYTES IN CULTURE. DIFFERENTIALLY EXPRESSED GENES WERE IDENTIFIED BY RNA-SEQUENCING. OUR DATA SHOW THAT ARVS TREATMENT CAUSES UPREGULATION OF SEVERAL BIOLOGICAL FUNCTIONS ASSOCIATED WITH CARDIOTOXICITY, HYPERTROPHY, AND HEART FAILURE. GLOBAL GENE EXPRESSION DATA WERE VALIDATED IN CARDIAC TISSUE ISOLATED FROM HIV PATIENTS HAVING A HISTORY OF ART. INTERESTINGLY, WE FOUND THAT HOMEODOMAIN-ONLY PROTEIN HOMEOBOX (HOPX) EXPRESSION WAS SIGNIFICANTLY INCREASED IN CARDIOMYOCYTES TREATED WITH ARVS AND IN THE HEART TISSUE OF HIV PATIENTS. FURTHERMORE, WE FOUND THAT HOPX PLAYS A CRUCIAL ROLE IN ARVS MEDIATED CELLULAR HYPERTROPHY. MECHANISTICALLY, WE FOUND THAT HOPX PLAYS A CRITICAL ROLE IN EPIGENETIC REGULATION, THROUGH DEACETYLATION OF HISTONE, WHILE THE HDAC INHIBITOR, TRICHOSTATIN A, CAN RESTORE THE ACETYLATION LEVEL OF HISTONE 3 IN THE PRESENCE OF ARVS. 2021 8 1966 34 EPIGENETIC ALTERATION OF PRKCDBP IN COLORECTAL CANCERS AND ITS IMPLICATION IN TUMOR CELL RESISTANCE TO TNFALPHA-INDUCED APOPTOSIS. PURPOSE: PRKCDBP IS A PUTATIVE TUMOR SUPPRESSOR IN WHICH ALTERATION HAS BEEN OBSERVED IN SEVERAL HUMAN CANCERS. WE INVESTIGATED EXPRESSION AND FUNCTION OF PRKCDBP IN COLORECTAL CELLS AND TISSUES TO EXPLORE ITS CANDIDACY AS A SUPPRESSOR IN COLORECTAL TUMORIGENESIS. EXPERIMENTAL DESIGN: EXPRESSION AND METHYLATION STATUS OF PRKCDBP AND ITS EFFECT ON TUMOR GROWTH WERE EVALUATED. TRANSCRIPTIONAL REGULATION BY NF-KAPPAB SIGNALING WAS DEFINED BY LUCIFERASE REPORTER AND CHROMATIN IMMUNOPRECIPITATION ASSAYS. RESULTS: PRKCDBP EXPRESSION WAS HARDLY DETECTABLE IN 29 OF 80 (36%) PRIMARY TUMORS AND 11 OF 19 (58%) CELL LINES, AND ITS ALTERATION CORRELATED WITH TUMOR STAGE AND GRADE. PROMOTER HYPERMETHYLATION WAS COMMONLY FOUND IN CANCERS. PRKCDBP EXPRESSION INDUCED THE G(1) CELL-CYCLE ARREST AND INCREASED CELLULAR SENSITIVITY TO VARIOUS APOPTOTIC STRESSES. PRKCDBP WAS INDUCED BY TNFALPHA, AND ITS LEVEL CORRELATED WITH TUMOR CELL SENSITIVITY TO TNFALPHA-INDUCED APOPTOSIS. PRKCDBP INDUCTION BY TNFALPHA WAS DISRUPTED BY BLOCKING NF-KAPPAB SIGNALING WHILE IT WAS ENHANCED BY RELA TRANSFECTION. THE PRKCDBP PROMOTER ACTIVITY WAS INCREASED IN RESPONSE TO TNFALPHA, AND THIS RESPONSE WAS ABOLISHED BY DISRUPTION OF A KAPPAB SITE IN THE PROMOTER. PRKCDBP DELAYED THE FORMATION AND GROWTH OF XENOGRAFT TUMORS AND IMPROVED TUMOR RESPONSE TO TNFALPHA-INDUCED APOPTOSIS. CONCLUSIONS: PRKCDBP IS A PROAPOPTOTIC TUMOR SUPPRESSOR WHICH IS COMMONLY ALTERED IN COLORECTAL CANCER BY PROMOTER HYPERMETHYLATION, AND ITS GENE TRANSCRIPTION IS DIRECTLY ACTIVATED BY NF-KAPPAB IN RESPONSE TO TNFALPHA. THIS SUGGESTS THAT PRKCDBP INACTIVATION MAY CONTRIBUTE TO TUMOR PROGRESSION BY REDUCING CELLULAR SENSITIVITY TO TNFALPHA AND OTHER STRESSES, PARTICULARLY UNDER CHRONIC INFLAMMATORY MICROENVIRONMENT. 2011 9 5636 47 SERELAXIN ALLEVIATES CARDIAC FIBROSIS THROUGH INHIBITING ENDOTHELIAL-TO-MESENCHYMAL TRANSITION VIA RXFP1. RATIONALE: CARDIAC FIBROSIS IS AN INTEGRAL CONSTITUENT OF EVERY FORM OF CHRONIC HEART DISEASE, AND PERSISTENCE OF FIBROSIS REDUCES TISSUE COMPLIANCE AND ACCELERATES THE PROGRESSION TO HEART FAILURE. RELAXIN-2 IS A HUMAN HORMONE, WHICH HAS VARIOUS PHYSIOLOGICAL FUNCTIONS SUCH AS MEDIATING RENAL VASODILATION IN PREGNANCY. ITS RECOMBINANT FORM SERELAXIN HAS RECENTLY BEEN TESTED IN CLINICAL TRIALS AS A THERAPY FOR ACUTE HEART FAILURE BUT DID NOT MEET ITS PRIMARY ENDPOINTS. THE AIM OF THIS STUDY IS TO EXAMINE WHETHER SERELAXIN HAS AN ANTI-FIBROTIC EFFECT IN THE HEART AND THEREFORE COULD BE BENEFICIAL IN CHRONIC HEART FAILURE. METHODS: WE UTILIZED TWO DIFFERENT CARDIAC FIBROSIS MOUSE MODELS (ASCENDING AORTIC CONSTRICTION (AAC) AND ANGIOTENSIN II (ATII) ADMINISTRATION VIA OSMOTIC MINIPUMPS) TO ASSESS THE ANTI-FIBROTIC POTENTIAL OF SERELAXIN. HISTOLOGICAL ANALYSIS, IMMUNOFLUORESCENCE STAINING AND MOLECULAR ANALYSIS WERE PERFORMED TO ASSESS THE FIBROSIS LEVEL AND INDICATE ENDOTHELIAL CELLS WHICH ARE UNDERGOING ENDMT. IN VITRO TGFBETA1-INDUCED ENDOTHELIAL-TO-MESENCHYMAL TRANSITION (ENDMT) ASSAYS WERE PERFORMED IN HUMAN CORONARY ARTERY ENDOTHELIAL CELLS AND MOUSE CARDIAC ENDOTHELIAL CELLS (MCECS) AND WERE EXAMINED USING MOLECULAR METHODS. CHROMATIN IMMUNOPRECIPITATION-QPCR ASSAY WAS UTILIZED TO IDENTIFY THE SERELAXIN EFFECT ON CHROMATIN REMODELING IN THE RXFP1 PROMOTER REGION IN MCECS. RESULTS: OUR RESULTS DEMONSTRATE A SIGNIFICANT AND DOSE-DEPENDENT ANTI-FIBROTIC EFFECT OF SERELAXIN IN THE HEART IN BOTH MODELS. WE FURTHER SHOW THAT SERELAXIN MEDIATES THIS EFFECT, AT LEAST IN PART, THROUGH INHIBITION OF ENDMT THROUGH THE ENDOTHELIAL RELAXIN FAMILY PEPTIDE RECEPTOR 1 (RXFP1). WE FURTHER DEMONSTRATE THAT SERELAXIN ADMINISTRATION IS ABLE TO INCREASE ITS OWN RECEPTOR EXPRESSION (RXFP1) THROUGH EPIGENETIC REGULATION IN FORM OF HISTONE MODIFICATIONS BY ATTENUATING TGFBETA-PSMAD2/3 SIGNALING IN ENDOTHELIAL CELLS. CONCLUSIONS: THIS STUDY IS THE FIRST TO IDENTIFY THAT SERELAXIN INCREASES THE EXPRESSION OF ITS OWN RECEPTOR RXFP1 AND THAT THIS MEDIATES THE INHIBITION OF ENDMT AND CARDIAC FIBROSIS, SUGGESTING THAT SERELAXIN MAY HAVE A BENEFICIAL EFFECT AS ANTI-FIBROTIC THERAPY IN CHRONIC HEART FAILURE. 2020 10 918 30 CHRONIC HYPERGRAVITY INDUCES A MODIFICATION OF HISTONE H3 LYSINE 27 TRIMETHYLATION AT TCRBETA LOCUS IN MURINE THYMOCYTES. GRAVITY CHANGES ARE MAJOR STRESSORS ENCOUNTERED DURING SPACEFLIGHT THAT AFFECT THE IMMUNE SYSTEM. WE PREVIOUSLY EVIDENCED THAT HYPERGRAVITY EXPOSURE DURING GESTATION AFFECTS THE TCRBETA REPERTOIRE OF NEWBORN PUPS. TO IDENTIFY THE MECHANISMS UNDERLYING THIS OBSERVATION, WE STUDIED POST-TRANSLATIONAL HISTONE MODIFICATIONS. WE FIRST SHOWED THAT AMONG THE FOUR STUDIED POST-TRANSLATIONAL HISTONE H3 MODIFICATIONS, ONLY LYSINE 27 TRIMETHYLATION (H3K27ME3) IS DOWNREGULATED IN THE THYMUS OF MICE EXPOSED TO 2X G FOR 21 DAYS. WE THEN ASKED WHETHER THE TCRBETA LOCUS CHROMATIN STRUCTURE IS ALTERED BY HYPERGRAVITY EXPOSURE. CHIP STUDIES PERFORMED ON FOUR VBETA SEGMENTS OF THE MURINE DOUBLE-NEGATIVE SCIET27 THYMIC CELL LINE, WHICH CORRESPONDS TO THE LAST MATURATION STAGE BEFORE V(D)J RECOMBINATION, REVEALED INCREASES IN H3K27ME3 AFTER 2X G EXPOSURE. FINALLY, WE EVALUATED THE IMPLICATION FOR THE EZH2 METHYLTRANSFERASE IN THE REGULATION OF THE H3K27ME3 LEVEL AT THESE VBETA SEGMENTS BY TREATING SCIET27 CELLS WITH THE GSK126-SPECIFIC INHIBITOR. THESE EXPERIMENTS SHOWED THAT THE DOWNREGULATION OF H3K27ME3 CONTRIBUTES TO THE REGULATION OF THE VBETA GERMLINE TRANSCRIPT EXPRESSION THAT PRECEDES V(D)J RECOMBINATION. THESE DATA SHOW THAT MODIFICATIONS OF H3K27ME3 AT THE TCRBETA LOCUS LIKELY CONTRIBUTE TO AN EXPLANATION OF WHY THE TCR REPERTOIRE IS AFFECTED BY GRAVITY CHANGES AND IMPLY, FOR THE FIRST TIME, EZH2 IN THE REGULATION OF THE TCRBETA LOCUS CHROMATIN STRUCTURE. 2022 11 984 34 CHRONIC PSYCHOLOGICAL STRESS ALTERS GENE EXPRESSION IN RAT COLON EPITHELIAL CELLS PROMOTING CHROMATIN REMODELING, BARRIER DYSFUNCTION AND INFLAMMATION. CHRONIC STRESS IS COMMONLY ASSOCIATED WITH ENHANCED ABDOMINAL PAIN (VISCERAL HYPERSENSITIVITY), BUT THE CELLULAR MECHANISMS UNDERLYING HOW CHRONIC STRESS INDUCES VISCERAL HYPERSENSITIVITY ARE POORLY UNDERSTOOD. IN THIS STUDY, WE EXAMINED CHANGES IN GENE EXPRESSION IN COLON EPITHELIAL CELLS FROM A RAT MODEL USING RNA-SEQUENCING TO EXAMINE STRESS-INDUCED CHANGES TO THE TRANSCRIPTOME. FOLLOWING CHRONIC STRESS, THE MOST SIGNIFICANTLY UP-REGULATED GENES INCLUDED ATG16L1, COQ10B, DCAF13, NAT2, PTBP2, RRAS2, SPINK4 AND DOWN-REGULATED GENES INCLUDING ABAT, CITED2, CNNM2, DAB2IP, PLEKHM1, SCD2, AND TAB2. THE PRIMARY ALTERED BIOLOGICAL PROCESSES REVEALED BY NETWORK ENRICHMENT ANALYSIS WERE INFLAMMATION/IMMUNE RESPONSE, TISSUE MORPHOGENESIS AND DEVELOPMENT, AND NUCLEOSOME/CHROMATIN ASSEMBLY. THE MOST SIGNIFICANTLY DOWN-REGULATED PROCESS WAS THE DIGESTIVE SYSTEM DEVELOPMENT/FUNCTION, WHEREAS THE MOST SIGNIFICANTLY UP-REGULATED PROCESSES WERE INFLAMMATORY RESPONSE, ORGANISMAL INJURY, AND CHROMATIN REMODELING MEDIATED BY H3K9 METHYLATION. FURTHERMORE, A SUBPOPULATION OF STRESSED RATS DEMONSTRATED VERY SIGNIFICANTLY ALTERED GENE EXPRESSION AND TRANSCRIPT ISOFORMS, ENRICHED FOR THE DIFFERENTIAL EXPRESSION OF GENES INVOLVED IN THE INFLAMMATORY RESPONSE, INCLUDING UPREGULATION OF CYTOKINE AND CHEMOKINE RECEPTOR GENE EXPRESSION COUPLED WITH DOWNREGULATION OF EPITHELIAL ADHERENS AND TIGHT JUNCTION MRNAS. IN SUMMARY, THESE FINDINGS SUPPORT THAT CHRONIC STRESS IS ASSOCIATED WITH INCREASED LEVELS OF CYTOKINES AND CHEMOKINES, THEIR DOWNSTREAM SIGNALING PATHWAYS COUPLED TO DYSREGULATION OF INTESTINAL CELL DEVELOPMENT AND FUNCTION. EPIGENETIC REGULATION OF CHROMATIN REMODELING LIKELY PLAYS A PROMINENT ROLE IN THIS PROCESS. RESULTS ALSO SUGGEST THAT SUPER ENHANCERS PLAY A PRIMARY ROLE IN CHRONIC STRESS-ASSOCIATED INTESTINAL BARRIER DYSFUNCTION. 2022 12 3165 48 GRIK1-AS1 DEFICIENCY ACCELERATES ENDOMETRIOSIS PROGRESSION BY BOOSTING DNMT1-DEPENDENT SFRP1 PROMOTER METHYLATION IN ENDOMETRIAL STROMAL CELLS. BACKGROUND: ENDOMETRIOSIS, A GYNECOLOGICAL DISEASE THAT AFFECTS UP TO 10% OF WOMEN, IS A MAJOR CAUSE OF PAIN AND INFERTILITY. DEREGULATION OF THE EPIGENOME IS ACCOUNTABLE FOR THE ONSET AND PROGRESSION OF ENDOMETRIOSIS, ALTHOUGH ITS EXACT MECHANISM IS UNKNOWN. THE PURPOSE OF THE CURRENT STUDY IS TO EXAMINE THE ROLE OF THE LONG NON-CODING RNA (LNCRNA) GRIK1-AS1 IN THE EPIGENETIC REGULATION OF ENDOMETRIAL STROMAL CELL PROLIFERATION AND THE DEVELOPMENT OF ENDOMETRIOSIS. METHODS: ENDOMETRIOSIS DATASETS WERE SCREENED TO IDENTIFY GRIKI-AS1 AS DRAMATICALLY DECLINING IN ENDOMETRIOSIS. GAIN OR LOSS OF FUNCTION ENDOMETRIAL STROMAL CELL (ESC) MODELS WERE ESTABLISHED. THE ANTI-PROLIFERATION PHENOTYPE WAS INVESTIGATED USING IN VITRO AND IN VIVO EXPERIMENTS. EPIGENETIC REGULATORY NETWORK ANALYSES WERE CONDUCTED TO SUGGEST THE INTRINSIC MOLECULAR MECHANISM. RESULTS: WITH BIOINFORMATIC AND CLINICAL DATA, WE OBSERVED THAT GRIK1-AS1 AND SFRP1 WERE EXPRESSED AT LOW LEVELS IN ENDOMETRIOSIS. OVEREXPRESSED GRIK1-AS1 INHIBITED ESC PROLIFERATION, WHILE SFRP1 KNOCKDOWN RESCUED THE ANTIPROLIFERATIVE ABILITY OF GRIK1-AS1. SPECIFICALLY, METHYLATION-DEPENDENT EXPRESSION INHIBITION OF SFRP1 WAS REVEALED IN ESCS. MECHANISTICALLY, GRIK1-AS1 HAMPERS THE OCCUPANCY OF DNMT1 IN SRFP1 PROMOTER, LEADING TO HYPOMETHYLATION OF SFRP1 AND UPREGULATED SFRP1 EXPRESSION, THEREBY POTENTIALLY SUPPRESSING WNT SIGNALING AND ITS ADVERSE PROLIFERATIVE EFFECT. THERAPEUTICALLY, LENTIVIRUS-MEDIATED UPREGULATION OF GRIK1-AS1 INHIBITED ENDOMETRIOSIS DISEASE PROGRESSION IN VIVO. CONCLUSIONS: OUR STUDY IS A PROOF-OF-CONCEPT DEMONSTRATION FOR GRIKI-AS1-ASSOCIATED ENDOMETRIOSIS PATHOGENESIS AND HIGHLIGHTS A POTENTIAL INTERVENTION TARGET. 2023 13 2032 29 EPIGENETIC CHANGES IN P21 EXPRESSION IN RENAL CELLS AFTER EXPOSURE TO BROMATE. THIS STUDY TESTED THE HYPOTHESIS THAT BROMATE (KBRO3)-INDUCED RENAL CELL DEATH IS MEDIATED BY EPIGENETIC MECHANISMS. GLOBAL DNA METHYLATION, AS ASSESSED BY 5-METHYLCYTOSINE STAINING, WAS NOT CHANGED IN NORMAL RAT KIDNEY CELLS TREATED WITH ACUTE CYTOTOXIC DOSES OF KBRO3 (100 AND 200 PPM), AS COMPARED WITH CONTROLS. HOWEVER, KBRO3 TREATMENT DID INCREASE P38, P53 AND HISTONE 2AX (H2AX) PHOSPHORYLATION, AND P21 EXPRESSION. TREATMENT OF CELLS WITH INHIBITORS OF DNA METHYLTRANSFERASE (5-AZACYTIDINE OR 5-AZA) AND HISTONE DEACETYLASE (TRICHOSTATIN A OR TSA) IN ADDITION TO KBRO3 INCREASED CYTOTOXICITY, AS COMPARED WITH CELLS EXPOSED TO KBRO3 ALONE. 5-AZA AND TSA CO-TREATMENT DID NOT ALTER P38 OR P53 PHOSPHORYLATION, BUT SLIGHTLY DECREASED H2AX PHOSPHORYLATION AND SIGNIFICANTLY DECREASED P21 EXPRESSION. WE ALSO ASSESSED EPIGENETIC CHANGES IN CELLS TREATED UNDER SUB-CHRONIC CONDITIONS WITH ENVIRONMENTALLY RELEVANT CONCENTRATIONS OF KBRO3. UNDER THESE CONDITIONS (0-10PPM KBRO3 FOR UP TO 18 DAYS), WE DETECTED NO INCREASES IN CELL DEATH OR DNA DAMAGE. IN CONTRAST, SLIGHT ALTERATIONS WERE DETECTED IN THE PHOSPHORYLATION OF H2AX, P38, AND P53. SUB-CHRONIC LOW-DOSE KBRO3 TREATMENT ALSO INDUCED A BIPHASIC RESPONSE IN P21 EXPRESSION, WITH LOWER CONCENTRATIONS INCREASING EXPRESSION, BUT HIGHER CONCENTRATIONS DECREASING EXPRESSION. METHYLATION-SPECIFIC PCR DEMONSTRATED THAT SUB-CHRONIC KBRO3 TREATMENT ALTERED THE METHYLATION OF CYTOSINE BASES IN THE P21 GENE, AS COMPARED WITH CONTROLS, CORRELATING TO ALTERATIONS IN P21 PROTEIN EXPRESSION. COLLECTIVELY, THESE DATA SHOW THE NOVEL FINDING THAT KBRO3-INDUCED RENAL CELL DEATH IS ALTERED BY INHIBITORS OF EPIGENETIC MODIFYING ENZYMES AND THAT KBRO3 ITSELF INDUCES EPIGENETIC CHANGES IN THE P21 GENE. 2014 14 26 34 A 6-ALKYLSALICYLATE HISTONE ACETYLTRANSFERASE INHIBITOR INHIBITS HISTONE ACETYLATION AND PRO-INFLAMMATORY GENE EXPRESSION IN MURINE PRECISION-CUT LUNG SLICES. LYSINE ACETYLATIONS ARE POST-TRANSLATIONAL MODIFICATIONS OF CELLULAR PROTEINS, THAT ARE CRUCIAL IN THE REGULATION OF MANY CELLULAR PROCESSES. LYSINE ACETYLATIONS ON HISTONE PROTEINS ARE PART OF THE EPIGENETIC CODE REGULATING GENE EXPRESSION AND ARE INSTALLED BY HISTONE ACETYLTRANSFERASES. OBSERVATIONS THAT INFLAMMATORY LUNG DISEASES, SUCH AS ASTHMA AND CHRONIC OBSTRUCTIVE PULMONARY DISEASE, ARE CHARACTERIZED BY INCREASED HISTONE ACETYLTRANSFERASE ACTIVITY INDICATE THAT DEVELOPMENT OF SMALL MOLECULE INHIBITORS FOR THESE ENZYMES MIGHT BE A VALUABLE APPROACH TOWARDS NEW THERAPIES FOR THESE DISEASES. THE 6-ALKYLSALICYLATE MG149 IS A CANDIDATE TO EXPLORE THIS HYPOTHESIS BECAUSE IT HAS BEEN DEMONSTRATED TO INHIBIT THE MYST TYPE HISTONE ACETYLTRANSFERASES. IN THIS STUDY, WE DETERMINED THE K(I) VALUE FOR INHIBITION OF THE MYST TYPE HISTONE ACETYLTRANSFERASE KAT8 BY MG149 TO BE 39 +/- 7.7 MUM. UPON INVESTIGATING WHETHER THE INHIBITION OF HISTONE ACETYLTRANSFERASES BY MG149 CORRELATES WITH INHIBITION OF HISTONE ACETYLATION IN MURINE PRECISION-CUT LUNG SLICES, INHIBITION OF ACETYLATION WAS OBSERVED USING AN LC-MS/MS BASED ASSAY ON HISTONE H4 RES 4-17, WHICH CONTAINS THE TARGET LYSINE OF KAT8. FOLLOWING UP ON THIS, UPON TREATMENT WITH MG149, REDUCED PRO-INFLAMMATORY GENE EXPRESSION WAS OBSERVED IN LIPOPOLYSACCHARIDE AND INTERFERON GAMMA STIMULATED MURINE PRECISION-CUT LUNG SLICES. BASED ON THIS, WE PROPOSE THAT 6-ALKYLSALICYLATES SUCH AS MG149 HAVE POTENTIAL FOR DEVELOPMENT TOWARDS APPLICATIONS IN THE TREATMENT OF INFLAMMATORY LUNG DISEASES. 2017 15 3813 28 INTRAUTERINE GROWTH RESTRICTION INHIBITS EXPRESSION OF EUKARYOTIC ELONGATION FACTOR 2 KINASE, A REGULATOR OF PROTEIN TRANSLATION. NUTRIENT DEPRIVATION SUPPRESSES PROTEIN SYNTHESIS BY BLOCKING PEPTIDE ELONGATION. TRANSCRIPTIONAL UPREGULATION AND ACTIVATION OF EUKARYOTIC ELONGATION FACTOR 2 KINASE (EEF2K) BLOCKS PEPTIDE ELONGATION BY PHOSPHORYLATING EUKARYOTIC ELONGATION FACTOR 2. PREVIOUS STUDIES EXAMINING PLACENTAS FROM INTRAUTERINE GROWTH RESTRICTED (IUGR) NEWBORN INFANTS SHOW DECREASED EEF2K EXPRESSION AND ACTIVITY DESPITE CHRONIC NUTRIENT DEPRIVATION. HOWEVER, THE EFFECT OF IUGR ON HEPATIC EEF2K EXPRESSION IN THE FETUS IS UNKNOWN. WE, THEREFORE, EXAMINED THE TRANSCRIPTIONAL REGULATION OF HEPATIC EEF2K GENE EXPRESSION IN A SPRAGUE-DAWLEY RAT MODEL OF IUGR. WE FOUND DECREASED HEPATIC EEF2K MRNA AND PROTEIN LEVELS IN IUGR OFFSPRING AT BIRTH COMPARED WITH CONTROL, CONSISTENT WITH PREVIOUS PLACENTAL OBSERVATIONS. FURTHERMORE, THE CPG ISLAND WITHIN THE EEF2K PROMOTER DEMONSTRATED INCREASED METHYLATION AT A CRITICAL USF 1/2 TRANSCRIPTION FACTOR BINDING SITE. IN VITRO METHYLATION OF THIS BINDING SITE CAUSED NEAR COMPLETE LOSS OF EEF2K PROMOTER ACTIVITY, DESIGNATING THIS PROMOTER AS METHYLATION SENSITIVE. THE EEF2K PROMOTOR IN IUGR OFFSPRING ALSO LOST THE PROTECTIVE HISTONE COVALENT MODIFICATIONS ASSOCIATED WITH UNMETHYLATED CGIS. IN ADDITION, THE +1 NUCLEOSOME WAS DISPLACED 3' AND RNA POLYMERASE LOADING WAS REDUCED AT THE IUGR EEF2K PROMOTER. OUR FINDINGS PROVIDE EVIDENCE TO EXPLAIN WHY IUGR-INDUCED CHRONIC NUTRIENT DEPRIVATION DOES NOT RESULT IN THE UPREGULATION OF EEF2K GENE TRANSCRIPTION. 2016 16 272 36 AGE-DEPENDENT DECREASE IN THE INDUCTION OF REGULATORY T CELLS IS ASSOCIATED WITH DECREASED EXPRESSION OF RALDH2 IN MESENTERIC LYMPH NODE DENDRITIC CELLS. A DECLINE IN IMMUNE FUNCTION WITH AGING HAS BEEN REPORTED. REGULATORY T CELL (TREG) INDUCTION IS KNOWN TO DECREASE WITH AGE, AND ELUCIDATING THE UNDERLYING MECHANISM IS IMPORTANT FOR PREVENTING AGE-RELATED DISEASES DUE TO AGE-RELATED CHRONIC INFLAMMATION. IN THE INTESTINE, DENDRITIC CELLS (DCS) PLAY AN IMPORTANT ROLE IN INDUCING TREGS SPECIFIC TO ORAL ANTIGENS, AND THEY EFFICIENTLY INDUCE TREGS VIA PRODUCTION OF RETINOIC ACID (RA), A VITAMIN A METABOLITE, CATALYZED BY THE ENZYME RETINALDEHYDE DEHYDROGENASE 2 (RALDH2). WE HAVE PREVIOUSLY REPORTED THAT IN THE MESENTERIC LYMPH NODE (MLN), A SECONDARY LYMPHOID TISSUE IN WHICH IMMUNE RESPONSES TO ORAL ANTIGENS ARE INDUCED, FOUR DC SUBSETS EXPRESS DIFFERENT LEVELS OF CD11B, CD103, AND PD-L1, AND WE HAVE REPORTED THAT THE CD11B(-)CD103(+)PD-L1(HIGH) SUBSET EXPRESSES THE HIGHEST LEVELS OF THE RALDH2 GENE AND INDUCES TREGS IN VITRO. WE EXAMINED TREG INDUCTION IN YOUNG AND AGED MICE USING A TREG INDUCTION MODEL BY ADMINISTERING A FOOD ANTIGEN, AND WE FOUND THAT ANTIGEN-SPECIFIC TREG INDUCTION WAS DECREASED IN AGED MICE. WE FURTHER INVESTIGATED THE MLN DCS, AND A SIGNIFICANT DECREASE IN RALDH2 GENE EXPRESSION WAS OBSERVED IN MLN DCS FROM AGED MICE. AS FACTORS, WE FOUND THAT THE PROPORTION OF THE CD11B(-)CD103(+)PD-L1(HIGH) SUBSET WAS DECREASED IN AGED MICE COMPARED WITH THAT IN YOUNG MICE AND THAT RALDH ENZYME ACTIVITY WAS DECREASED IN THE CD11B(-)CD103(+)PD-L1(HIGH) AND CD11B(+)CD103(+)PD-L1(HIGH) SUBSETS. FURTHERMORE, ANALYSIS OF THE METHYLATION OF THE RALDH2 GENE PROMOTER REGION REVEALED THAT CPG MOTIFS WERE MORE METHYLATED IN THE MLN DCS OF AGED MICE, SUGGESTING THAT RALDH2 EXPRESSION WAS SUPPRESSED BY EPIGENETIC CHANGES. FINALLY, WE FOUND THAT RA TREATMENT TENDED TO INCREASE TREG INDUCTION. THESE RESULTS SUGGEST THAT THE REGULATION OF RA PRODUCTION MAY BE INVOLVED IN THE AGE-RELATED DECREASE IN ANTIGEN-SPECIFIC TREG INDUCTION. 2020 17 3718 36 INHIBITION OF BCL6B PROMOTES GASTRIC CANCER BY AMPLIFYING INFLAMMATION IN MICE. BACKGROUND: CHRONIC GASTRITIS HAS BEEN DEMONSTRATED TO BE A KEY CAUSE OF GASTRIC CANCER (GC), AND CONTROL OF GASTRIC INFLAMMATION IS REGARDED AS AN EFFECTIVE TREATMENT FOR THE CLINICAL PREVENTION OF GASTRIC CARCINOGENESIS. HOWEVER, THERE REMAINS AN UNMET NEED TO IDENTIFY THE DOMINANT REGULATORS OF GASTRIC ONCOGENESIS-ASSOCIATED INFLAMMATION IN VIVO. METHODS: THE MOUSE MODEL FOR THE STUDY OF INFLAMMATION-ASSOCIATED GC WAS INDUCED BY BENZO[A]PYRENE (BAP) INTRAGASTRIC ADMINISTRATION IN BCL6B(-/-) AND WILDTYPE MICE ON A C57BL/6 BACKGROUND. 5-AZA-2'-DEOXYCYTIDINE (5-AZA), THE DEMETHYLATION DRUG, WAS INTRAPERITONEALLY INJECTED TO RESTORE BCL6B EXPRESSION. HUMAN GC TISSUE ARRAY WAS USED TO ANALYSE PATIENT SURVIVAL BASED ON BCL6B AND CD3 PROTEIN EXPRESSION. RESULTS: BCL6B WAS GRADUALLY DOWNREGULATED BY ITS OWN PROMOTER HYPERMETHYLATION IN PARALLEL TO AN INCREASING INFLAMMATORY RESPONSE DURING THE PROGRESSION OF BAP-INDUCED GASTRIC CARCINOGENESIS IN MICE. MOREOVER, KNOCKOUT OF BCL6B DRAMATICALLY WORSENED THE SEVERITY OF GASTRIC CANCER AND AGGRAVATED THE INFLAMMATORY RESPONSE IN THE BAP-INDUCED MICE GC MODEL. RE-ACTIVATION OF BCL6B BY 5-AZA IMPEDED INFLAMMATORY AMPLIFICATION AND BAP-INDUCED GC DEVELOPMENT, PROLONGING SURVIVAL TIME IN WILDTYPE MICE, WHEREAS NO NOTABLE CURATIVE EFFECT OCCURRED IN BCL6B(-/-) MICE WITH 5-AZA TREATMENT. FINALLY, SIGNIFICANT NEGATIVE CORRELATIONS WERE DETECTED BETWEEN THE MRNA LEVELS OF BCL6B AND INFLAMMATORY CYTOKINES IN HUMAN GC TISSUES; PATIENTS HARBOURING BCL6B-NEGETIVE AND SEVERE-INFLAMMATION GC TUMOURS WERE FOUND TO EXHIBIT THE SHORTEST SURVIVAL TIME. CONCLUSIONS: EPIGENETIC INACTIVATION OF BCL6B PROMOTES GASTRIC CANCER THROUGH AMPLIFICATION OF THE GASTRIC INFLAMMATORY RESPONSE IN VIVO AND OFFERS A NEW APPROACH FOR GC TREATMENT AND REGENERATIVE MEDICINE. 2019 18 136 32 ABERRANT DNA HYPERMETHYLATION PATTERNS LEAD TO TRANSCRIPTIONAL SILENCING OF TUMOR SUPPRESSOR GENES IN UVB-EXPOSED SKIN AND UVB-INDUCED SKIN TUMORS OF MICE. OVEREXPOSURE OF THE HUMAN SKIN TO SOLAR ULTRAVIOLET (UV) RADIATION IS THE MAJOR ETIOLOGIC FACTOR FOR DEVELOPMENT OF SKIN CANCERS. HERE, WE REPORT THE RESULTS OF EPIGENETIC MODIFICATIONS IN UV-EXPOSED SKIN AND SKIN TUMORS IN A SYSTEMATIC MANNER. THE SKIN AND TUMOR SAMPLES WERE COLLECTED AFTER CHRONIC EXPOSURE OF THE SKIN OF SKH-1 HAIRLESS MICE TO UVB RADIATION USING A WELL-ESTABLISHED PHOTOCARCINOGENESIS PROTOCOL. WE FOUND A DISTINCT DNA HYPERMETHYLATION PATTERN IN THE UVB-EXPOSED EPIDERMAL SKIN AND UVB-INDUCED SKIN TUMORS THAT WAS ASSOCIATED WITH THE ELEVATED EXPRESSION AND ACTIVITY OF THE DNA METHYLTRANSFERASES (DNMT) 1, DNMT3A AND DNMT3B. TO EXPLORE THE ROLE OF HYPERMETHYLATION IN SKIN PHOTOCARCINOGENESIS, WE FOCUSED ON THE P16(INK4A) AND RASSF1A TUMOR SUPPRESSOR GENES, WHICH ARE TRANSCRIPTIONALLY SILENCED ON METHYLATION. WE ESTABLISHED THAT THE SILENCING OF THESE GENES IN UVB-EXPOSED EPIDERMIS AND UVB-INDUCED SKIN TUMORS IS ASSOCIATED WITH A NETWORK OF EPIGENETIC MODIFICATIONS, INCLUDING HYPOACETYLATION OF HISTONE H3 AND H4 AND INCREASED HISTONE DEACETYLATION, AS WELL AS RECRUITMENT OF METHYL-BINDING PROTEINS, INCLUDING MECP2 AND MBD1, TO THE METHYLATED CPGS. HIGHER LEVELS OF DNA METHYLATION AND DNMT ACTIVITY IN HUMAN SQUAMOUS CELL CARCINOMA SPECIMENS THAN IN NORMAL HUMAN SKIN SUGGEST THAT THE DATA ARE RELEVANT CLINICALLY. OUR DATA INDICATE FOR THE FIRST TIME THAT UVB-INDUCED DNA HYPERMETHYLATION, ENHANCED DNMT ACTIVITY AND HISTONE MODIFICATIONS OCCUR IN UVB-EXPOSED SKIN AND UVB-INDUCED SKIN TUMORS AND SUGGEST THAT THESE EVENTS ARE INVOLVED IN THE SILENCING OF TUMOR SUPPRESSOR GENES AND IN SKIN TUMOR DEVELOPMENT. 2011 19 6294 24 THE PROINFLAMMATORY CYTOKINE TNFALPHA INDUCES DNA DEMETHYLATION-DEPENDENT AND -INDEPENDENT ACTIVATION OF INTERLEUKIN-32 EXPRESSION. IL-32 IS A CYTOKINE INVOLVED IN PROINFLAMMATORY IMMUNE RESPONSES TO BACTERIAL AND VIRAL INFECTIONS. HOWEVER, THE ROLE OF EPIGENETIC EVENTS IN THE REGULATION OF IL-32 GENE EXPRESSION IS UNDERSTUDIED. HERE WE SHOW THAT IL-32 IS REPRESSED BY DNA METHYLATION IN HEK293 CELLS. USING CHIP SEQUENCING, LOCUS-SPECIFIC METHYLATION ANALYSIS, CRISPR/CAS9-MEDIATED GENOME EDITING, AND RT-QPCR (QUANTITATIVE RT-PCR) AND IMMUNOBLOT ASSAYS, WE FOUND THAT SHORT-TERM TREATMENT (A FEW HOURS) WITH THE PROINFLAMMATORY CYTOKINE TUMOR NECROSIS FACTOR ALPHA (TNFALPHA) ACTIVATES IL-32 IN A DNA DEMETHYLATION-INDEPENDENT MANNER. IN CONTRAST, PROLONGED TNFALPHA TREATMENT (SEVERAL DAYS) INDUCED DNA DEMETHYLATION AT THE PROMOTER AND A CPG ISLAND IN THE IL-32 GENE IN A TET (TEN-ELEVEN TRANSLOCATION) FAMILY ENZYME- AND NF-KAPPAB-DEPENDENT MANNER. NOTABLY, THE HYPOMETHYLATION STATUS OF TRANSCRIPTIONAL REGULATORY ELEMENTS IN IL-32 WAS MAINTAINED FOR A LONG TIME (SEVERAL WEEKS), CAUSING ELEVATED IL-32 EXPRESSION EVEN IN THE ABSENCE OF TNFALPHA. CONSIDERING THAT IL-32 CAN, IN TURN, INDUCE TNFALPHA EXPRESSION, WE SPECULATE THAT SUCH FEEDFORWARD EVENTS MAY CONTRIBUTE TO THE TRANSITION FROM AN ACUTE INFLAMMATORY RESPONSE TO CHRONIC INFLAMMATION. 2019 20 2395 29 EPIGENETIC REPROGRAMMING IN MIST1(-/-) MICE PREDICTS THE MOLECULAR RESPONSE TO CERULEIN-INDUCED PANCREATITIS. GENE EXPRESSION IS AFFECTED BY MODIFICATIONS TO HISTONE CORE PROTEINS WITHIN CHROMATIN. CHANGES IN THESE MODIFICATIONS, OR EPIGENETIC REPROGRAMMING, CAN DICTATE CELL FATE AND PROMOTE SUSCEPTIBILITY TO DISEASE. THE GOAL OF THIS STUDY WAS TO DETERMINE THE EXTENT OF EPIGENETIC REPROGRAMMING IN RESPONSE TO CHRONIC STRESS THAT OCCURS FOLLOWING ABLATION OF MIST1 (MIST1(-/-) ), WHICH IS REPRESSED IN PANCREATIC DISEASE. CHROMATIN IMMUNOPRECIPITATION FOR TRIMETHYLATION OF LYSINE RESIDUE 4 ON HISTONE 3 (H3K4ME3) IN PURIFIED ACINAR CELLS FROM WILD TYPE AND MIST1(-/-) MICE WAS FOLLOWED BY NEXT GENERATION SEQUENCING (CHIP-SEQ) OR CHIP-QPCR. H3K4ME3-ENRICHED GENES WERE ASSESSED FOR EXPRESSION BY QRT-PCR IN PANCREATIC TISSUE BEFORE AND AFTER INDUCTION OF CERULEIN-INDUCED PANCREATITIS. WHILE MOST OF H3K4ME3-ENRICHMENT IS RESTRICTED TO TRANSCRIPTIONAL START SITES, >25% OF ENRICHMENT SITES ARE FOUND WITHIN, DOWNSTREAM OR BETWEEN ANNOTATED GENES. LESS THAN 10% OF THESE SITES WERE ALTERED IN MIST1(-/-) ACINI, WITH MOST CHANGES IN H3K4ME3 ENRICHMENT NOT REFLECTING ALTERED GENE EXPRESSION. INGENUITY PATHWAY ANALYSIS OF GENES DIFFERENTIALLY-ENRICHED FOR H3K4ME3 REVEALED AN ASSOCIATION WITH PANCREATITIS AND PANCREATIC DUCTAL ADENOCARCINOMA IN MIST1(-/-) TISSUE. MOST OF THESE GENES WERE NOT DIFFERENTIALLY EXPRESSED BUT SEVERAL WERE READILY INDUCED BY ACUTE EXPERIMENTAL PANCREATITIS, WITH SIGNIFICANTLY INCREASED EXPRESSION IN MIST1(-/-) TISSUE RELATIVE TO WILD TYPE MICE. WE SUGGEST THAT THE CHRONIC CELL STRESS OBSERVED IN THE ABSENCE OF MIST1 RESULTS IN EPIGENETIC REPROGRAMMING OF GENES INVOLVED IN PROMOTING PANCREATITIS TO A POISED STATE, THEREBY INCREASING THE SENSITIVITY TO EVENTS THAT PROMOTE DISEASE. 2014