1 1620 116 DNA METHYLTRANSFERASE-MEDIATED TRANSCRIPTIONAL SILENCING IN MALIGNANT GLIOMA: A COMBINED WHOLE-GENOME MICROARRAY AND PROMOTER ARRAY ANALYSIS. EPIGENETIC INACTIVATION OF TUMOR SUPPRESSOR GENES IS A COMMON FEATURE IN HUMAN CANCER. PROMOTER HYPERMETHYLATION AND HISTONE DEACETYLATION ARE REVERSIBLE EPIGENETIC MECHANISMS ASSOCIATED WITH TRANSCRIPTIONAL REGULATION. DNA METHYLTRANSFERASES (DNMT1 AND DNMT3B) REGULATE AND MAINTAIN PROMOTER METHYLATION AND ARE OVEREXPRESSED IN HUMAN CANCER. WE PERFORMED WHOLE-GENOME MICROARRAY ANALYSIS TO IDENTIFY GENES WITH ALTERED EXPRESSION AFTER RNAI-INDUCED SUPPRESSION OF DNMT IN A GLIOBLASTOMA MULTIFORME (GBM) CELL LINE. WE THEN IDENTIFIED GENES WITH BOTH DECREASED EXPRESSION AND EVIDENCE OF PROMOTER CPG ISLAND HYPERMETHYLATION IN GBM TISSUE SAMPLES USING A COMBINED WHOLE-GENOME MICROARRAY TRANSCRIPTOME ANALYSIS IN CONJUNCTION WITH A PROMOTER ARRAY ANALYSIS AFTER DNA IMMUNOPRECIPITATION WITH ANTI-5-METHYLCYTIDINE. DNMT1 AND 3B KNOCKDOWN RESULTED IN THE RESTORED EXPRESSION OF 308 GENES THAT ALSO CONTAINED PROMOTER REGION HYPERMETHYLATION. OF THESE, 43 WERE ALSO FOUND TO BE DOWNREGULATED IN GBM TISSUE SAMPLES. THREE DOWNREGULATED GENES WITH HYPERMETHYLATED PROMOTERS AND RESTORED EXPRESSION IN RESPONSE TO ACUTE DNMT SUPPRESSION WERE ASSAYED FOR METHYLATION CHANGES USING BISULFITE SEQUENCE ANALYSIS OF THE PROMOTER REGION AFTER CHRONIC DNMT SUPPRESSION. RESTORATION OF GENE EXPRESSION WAS NOT ASSOCIATED WITH CHANGES IN PROMOTER REGION METHYLATION, BUT RATHER WITH CHANGES IN HISTONE METHYLATION AND CHROMATIN CONFORMATION. TWO OF THE IDENTIFIED GENES EXHIBITED GROWTH SUPPRESSIVE ACTIVITY IN IN VITRO ASSAYS. COMBINING TARGETED GENETIC MANIPULATIONS WITH COMPREHENSIVE GENOMIC AND EXPRESSION ANALYSES PROVIDES A POTENTIALLY POWERFUL NEW APPROACH FOR IDENTIFYING EPIGENETICALLY REGULATED GENES IN GBM. 2009 2 6171 30 THE HDAC INHIBITOR VALPROATE INDUCES A BIVALENT STATUS OF THE CD20 PROMOTER IN CLL PATIENTS SUGGESTING DISTINCT EPIGENETIC REGULATION OF CD20 EXPRESSION IN CLL IN VIVO. TREATMENT WITH ANTI-CD20 ANTIBODIES IS ONLY MODERATELY EFFICIENT IN CHRONIC LYMPHOCYTIC LEUKEMIA (CLL), A FEATURE WHICH HAS BEEN EXPLAINED BY THE INHERENTLY LOW CD20 EXPRESSION IN CLL. IT HAS BEEN SHOWN THAT CD20 IS EPIGENETICALLY REGULATED AND THAT HISTONE DEACETYLASE INHIBITORS (HDACIS) CAN INCREASE CD20 EXPRESSION IN VITRO IN CLL. TO ASSESS WHETHER HDACIS CAN UPREGULATE CD20 ALSO IN VIVO IN CLL, THE HDACI VALPROATE WAS GIVEN TO THREE DEL13Q/NOTCH1WT CLL PATIENTS AND CD20 LEVELS WERE ANALYSED (THE PREVAIL STUDY). VALPROATE TREATMENT RESULTED IN EXPECTED GLOBAL ACTIVATING HISTONE MODIFICATIONS SUGGESTING HDAC INHIBITORY EFFECTS. HOWEVER, ALTHOUGH VALPROATE INDUCED EXPRESSION OF CD20 MRNA AND PROTEIN IN THE DEL13Q/NOTCH1WT I83-E95 CLL CELL LINE, NO SUCH EFFECTS WERE OBSERVED IN THE PATIENTS STUDIED. IN CONTRAST TO THE CELL LINE, IN PATIENTS VALPROATE TREATMENT RESULTED IN TRANSIENT RECRUITMENT OF THE TRANSCRIPTIONAL REPRESSOR EZH2 TO THE CD20 PROMOTER, CORRELATING TO AN INCREASE OF THE REPRESSIVE HISTONE MARK H3K27ME3. THIS SUGGESTS THAT VALPROATE-MEDIATED INDUCTION OF CD20 MAY BE HAMPERED BY EZH2 MEDIATED H3K27ME3 IN VIVO IN CLL. MOREOVER, VALPROATE TREATMENT RESULTED IN INDUCTION OF EZH2 AND GLOBAL H3K27ME3 IN PATIENT CELLS, SUGGESTING TRANSCRIPTIONALLY REPRESSIVE EFFECTS OF VALPROATE IN CLL. OUR RESULTS SUGGEST NEW IN VIVO MECHANISMS OF HDACIS WHICH MAY HAVE IMPLICATIONS ON THE DESIGN OF FUTURE CLINICAL TRIALS IN B-CELL MALIGNANCIES. 2017 3 6069 37 THE DIOXIN RECEPTOR IS SILENCED BY PROMOTER HYPERMETHYLATION IN HUMAN ACUTE LYMPHOBLASTIC LEUKEMIA THROUGH INHIBITION OF SP1 BINDING. THE TRANSCRIPTION FACTOR ARYL HYDROCARBON RECEPTOR (AHR) HAS RELEVANT FUNCTIONS IN CELL PROLIFERATION. INTERESTINGLY, THE AHR CAN EITHER PROMOTE OR INHIBIT PROLIFERATION DEPENDING ON THE CELL PHENOTYPE. ALTHOUGH RECENT DATA REVEAL POTENTIAL PATHWAYS FOR AHR SIGNALING IN CELL PROLIFERATION, THE MECHANISMS THAT REGULATE ITS ACTIVITY IN TUMOR CELLS REMAIN UNKNOWN. HERE, WE HAVE ANALYZED PROMOTER HYPERMETHYLATION AS A POTENTIAL MECHANISM CONTROLLING AHR EXPRESSION IN HUMAN TUMOR CELLS. AHR PROMOTER CPG METHYLATION WAS SPORADIC IN A PANEL OF 19 TUMOR CELL LINES EXCEPT FOR THE CHRONIC MYELOID LEUKEMIA (CML) K562 AND THE ACUTE LYMPHOBLASTIC LEUKEMIA (ALL) REH. WHEN COMPARED WITH NORMAL LYMPHOCYTES, REH HAD VERY LOW CONSTITUTIVE AHR EXPRESSION THAT COULD BE ATTRIBUTED TO PROMOTER HYPERMETHYLATION SINCE TREATMENT WITH THE DNA DEMETHYLATING AGENT 5-AZA-2'-DEOXYCITIDINE (AZA) SIGNIFICANTLY INCREASED AHR MRNA AND PROTEIN. THESE RESULTS IN LEUKEMIA-DERIVED CELL LINES WERE FURTHER CONFIRMED IN PRIMARY ALL, WHERE 33% OF THE PATIENTS (7/21) HAD AHR PROMOTER HYPERMETHYLATION. CHROMATIN IMMUNOPRECIPITATION (CHIP) SHOWED THAT METHYLATION IMPAIRED BINDING OF THE TRANSCRIPTION FACTOR SP1 TO THE AHR PROMOTER, THUS PROVIDING A MECHANISM FOR AHR DOWNREGULATION IN REH CELLS. THEREFORE, PROMOTER HYPERMETHYLATION REPRESENTS A NOVEL EPIGENETIC MECHANISM DOWNREGULATING AHR ACTIVITY IN HEMATOLOGICAL MALIGNANCIES SUCH AS ALL. 2006 4 1733 37 E-CADHERIN GENE RE-EXPRESSION IN CHRONIC LYMPHOCYTIC LEUKEMIA CELLS BY HDAC INHIBITORS. BACKGROUND: THE TUMOR SUPPRESSOR GENE E-CADHERIN GENE IS FREQUENTLY SILENCED IN CHRONIC LYMPHOCYTIC LEUKEMIA (CLL) CELLS AND RESULTS IN WNT-PATHWAY ACTIVATION. WE ANALYZED THE ROLE OF HISTONE EPIGENETIC MODIFICATIONS IN E-CADHERIN GENE SILENCING. METHODS: CLL SPECIMENS WERE TREATED WITH HISTONE DEACETYLASE INHIBITOR (HDACI) MS-275 AND ANALYZED FOR E-CADHERIN EXPRESSION WITH WESTERN BLOT AND RT-PCR ANALYSIS. THE DOWNSTREAM EFFECTS OF HDACI TREATED LEUKEMIC CELLS WERE STUDIED BY ANALYZING THE EFFECT ON WNT-PATHWAY SIGNALING. HDACI INDUCED ALTERATIONS IN E-CADHERIN SPLICING WERE INVESTIGATED BY TRANSCRIPT SPECIFIC REAL TIME PCR ANALYSIS. RESULTS: TREATMENT OF CLL SPECIMENS WITH HISTONE DEACETYLASE INHIBITORS (HDACI) TREATMENT RESULTED IN AN INCREASE OF THE E-CADHERIN RNA TRANSCRIPT (5 TO 119 FOLD INCREASE, N=10) IN EIGHT OUT OF TEN CLL SPECIMENS INDICATING THAT THIS GENE IS DOWN REGULATED BY HISTONE HYPOACETYLATION IN A MAJORITY OF CLL SPECIMENS. THE E-CADHERIN RE-EXPRESSION IN CLL SPECIMENS WAS NOTED BY WESTERN BLOT ANALYSIS AS WELL. BESIDES EPIGENETIC SILENCING ANOTHER MECHANISM OF E-CADHERIN INACTIVATION IS ABERRANT EXON 11 SPLICING RESULTING IN AN ALTERNATIVELY SPLICED TRANSCRIPT THAT LACKS EXON 11 AND IS DEGRADED BY THE NON-SENSE MEDIATED DECAY (NMD) PATHWAY. OUR CHROMATIN IMMUNOPRECIPITATION EXPERIMENTS SHOW THAT HDACI INCREASED THE ACETYLATION OF HISTONES H3 AND H4 IN THE E-CADHERIN PROMOTER REGION. THIS ALSO AFFECTED THE E-CADHERIN EXON 11 SPLICING PATTERN AS HDACI TREATED CLL SPECIMENS PREFERENTIALLY EXPRESSED THE CORRECTLY SPLICED TRANSCRIPT AND NOT THE EXON 11 SKIPPED ABERRANT TRANSCRIPT. THE RE-EXPRESSED E- CADHERIN BINDS TO BETA-CATENIN WITH INHIBITION OF THE ACTIVE WNT-BETA-CATENIN PATHWAY IN THESE CELLS. THIS RESULTED IN A DOWN REGULATION OF TWO WNT TARGET GENES, LEF AND CYCLIND1 AND THE WNT PATHWAY REPORTER. CONCLUSION: THE E-CADHERIN GENE IS EPIGENETICALLY MODIFIED AND HYPOACETYLATED IN CLL LEUKEMIC CELLS. TREATMENT OF CLL SPECIMENS WITH HDACI MS-275 ACTIVATES TRANSCRIPTION FROM THIS SILENT GENE WITH EXPRESSION OF MORE CORRECTLY SPLICED E-CADHERIN TRANSCRIPTS AS COMPARED TO THE ABERRANT EXON11 SKIPPED TRANSCRIPTS THAT IN TURN INHIBITS THE WNT SIGNALING PATHWAY. THE DATA HIGHLIGHTS THE ROLE OF EPIGENETIC MODIFICATIONS IN ALTERING GENE SPLICING PATTERNS. 2013 5 3082 36 GENOME-WIDE REDISTRIBUTION OF MECP2 IN DORSAL ROOT GANGLIA AFTER PERIPHERAL NERVE INJURY. BACKGROUND: METHYL-CPG-BINDING PROTEIN 2 (MECP2), A PROTEIN WITH AFFINITY FOR METHYLATED CYTOSINES, IS CRUCIAL FOR NEURONAL DEVELOPMENT AND FUNCTION. MECP2 REGULATES GENE EXPRESSION THROUGH ACTIVATION, REPRESSION AND CHROMATIN REMODELING. MUTATIONS IN MECP2 CAUSE RETT SYNDROME, AND THESE PATIENTS DISPLAY IMPAIRED NOCICEPTION. WE OBSERVED AN INCREASE IN MECP2 EXPRESSION IN MOUSE DORSAL ROOT GANGLIA (DRG) AFTER PERIPHERAL NERVE INJURY. THE FUNCTIONAL IMPLICATION OF INCREASED MECP2 IS LARGELY UNKNOWN. TO IDENTIFY REGIONS OF THE GENOME BOUND BY MECP2 IN THE DRG AND THE CHANGES INDUCED BY NERVE INJURY, A CHROMATIN IMMUNOPRECIPITATION OF MECP2 FOLLOWED BY SEQUENCING (CHIP-SEQ) WAS PERFORMED 4 WEEKS AFTER SPARED NERVE INJURY (SNI). RESULTS: WHILE THE NUMBER OF BINDING SITES ACROSS THE GENOME REMAINED SIMILAR IN THE SNI MODEL AND SHAM CONTROL, SNI INDUCED THE REDISTRIBUTION OF MECP2 TO TRANSCRIPTIONALLY RELEVANT REGIONS. TO DETERMINE HOW DIFFERENTIAL BINDING OF MECP2 CAN AFFECT GENE EXPRESSION IN THE DRG, WE INVESTIGATED MMU-MIR-126, A MICRORNA LOCUS THAT HAD ENRICHED MECP2 BINDING IN THE SNI MODEL. ENRICHED MECP2 BINDING TO MIR-126 LOCUS AFTER NERVE INJURY REPRESSED MIR-126 EXPRESSION, AND THIS WAS NOT MEDIATED BY ALTERATIONS IN METHYLATION PATTERN AT THE MIR-126 LOCUS. DOWNREGULATION OF MIR-126 RESULTED IN THE UPREGULATION OF ITS TWO TARGET GENES DNMT1 AND VEGFA IN NEURO 2A CELLS AND IN SNI MODEL COMPARED TO CONTROL. THESE TARGET GENES WERE SIGNIFICANTLY DOWNREGULATED IN MECP2-NULL MICE COMPARED TO WILD-TYPE LITTERMATES, INDICATING A REGULATORY ROLE FOR MECP2 IN ACTIVATING DNMT1 AND VEGFA EXPRESSION. INTRATHECAL DELIVERY OF MIR-126 WAS NOT SUFFICIENT TO REVERSE NERVE INJURY-INDUCED MECHANICAL AND THERMAL HYPERSENSITIVITY, BUT DECREASED DNMT1 AND VEGFA EXPRESSION IN THE DRG. CONCLUSIONS: OUR STUDY SHOWS A REGULATORY ROLE FOR MECP2 IN THAT CHANGES IN GLOBAL REDISTRIBUTION CAN RESULT IN DIRECT AND INDIRECT MODULATION OF GENE EXPRESSION IN THE DRG. ALTERATIONS IN GENOME-WIDE BINDING OF MECP2 THEREFORE PROVIDE A MOLECULAR BASIS FOR A BETTER UNDERSTANDING OF EPIGENETIC REGULATION-INDUCED MOLECULAR CHANGES UNDERLYING NERVE INJURY. 2016 6 1906 29 ENHANCER OF ZESTE HOMOLOG 2-CATALYSED H3K27 TRIMETHYLATION PLAYS A KEY ROLE IN ACUTE-ON-CHRONIC LIVER FAILURE VIA TNF-MEDIATED PATHWAY. ACUTE-ON-CHRONIC LIVER FAILURE IS MAINLY DUE TO HOST IMMUNITY SELF-DESTRUCTION. THE HISTONE H3 LYSINE 27 (H3K27) TRIMETHYLATING ENZYME, ENHANCER OF ZESTE HOMOLOG 2 (EZH2) MEDIATES EPIGENETIC SILENCING OF GENE EXPRESSION AND REGULATES IMMUNITY, ALSO INVOLVES PATHOGENESIS OF SEVERAL LIVER DISEASES. THE CURRENT STUDY WAS TO DETERMINE THE ROLE OF METHYLTRANSFERASE EZH2 AND ITS CATALYSED H3K27 TRIMETHYLATION (H3K27ME3) IN LIVER FAILURE, AND TO FURTHER INVESTIGATE THE POTENTIAL TARGET FOR LIVER FAILURE TREATMENT. EZH2 AND ITS CATALYSED H3K27ME3 WERE DETERMINED IN PERIPHERAL BLOOD MONONUCLEAR CELLS (PBMC) FROM LIVER FAILURE PATIENTS AND KUPFFER CELLS FROM EXPERIMENTAL MICE. FURTHERMORE, GSK126 (AN INHIBITOR FOR EZH2 TRIMETHYLATION FUNCTION) WAS APPLIED IN LIVER FAILURE MICE IN VIVO, AND LIPOPOLYSACCHARIDE-STIMULATED MONONUCLEAR CELLS IN VITRO. EZH2 AND H3K27ME3 WERE SIGNIFICANTLY UPREGULATED IN HUMAN PBMC FROM LIVER FAILURE PATIENTS OR MURINE KUPFFER CELLS FROM THE LIVER FAILURE ANIMALS, RESPECTIVELY. GSK126 AMELIORATED DISEASE SEVERITY IN LIVER FAILURE MICE, WHICH MAYBE ATTRIBUTE TO DOWN-REGULATE CIRCULATING AND HEPATIC PROINFLAMMATORY CYTOKINES, ESPECIALLY TNF VIA REDUCING H3K27ME3. IN-DEPTH CHROMATIN IMMUNOPRECIPITATION ANALYSIS UNRAVELLED THAT DECREASED ENRICHMENT OF H3K27ME3 ON TNF PROMOTOR, RESULTING IN TNF ELEVATION IN KUPFFER CELLS FROM LIVER FAILURE MICE. NUCLEAR FACTOR KAPPA B (NF-KAPPAB) AND PROTEIN KINASE B (AKT) SIGNALLING PATHWAYS WERE ACTIVATED UPON LIPOPOLYSACCHARIDE STIMULATION, BUT ATTENUATED BY USING GSK126, ACCOMPANIED WITH DECREASED TNF IN VITRO. IN CONCLUSION, EZH2 AND H3K27ME3 CONTRIBUTED TO THE PATHOGENESIS OF LIVER FAILURE VIA TRIGGERING TNF AND OTHER INDISPENSABLE PROINFLAMMATORY CYTOKINES. EZH2 WAS TO MODIFY H3K27ME3 ENRICHMENT, AS WELL AS, ACTIVATION OF THE DOWNSTREAM NF-KAPPAB AND AKT SIGNALLING PATHWAYS. 2018 7 3175 33 H2AX PHOSPHORYLATION REGULATED BY P38 IS INVOLVED IN BIM EXPRESSION AND APOPTOSIS IN CHRONIC MYELOGENOUS LEUKEMIA CELLS INDUCED BY IMATINIB. INCREASING EVIDENCE SUGGESTS THAT HISTONE H2AX PLAYS A CRITICAL ROLE IN REGULATION OF TUMOR CELL APOPTOSIS AND ACTS AS A NOVEL HUMAN TUMOR SUPPRESSOR PROTEIN. HOWEVER, THE ACTION OF H2AX IN CHRONIC MYELOGENOUS LEUKEMIA (CML) CELLS IS UNKNOWN. THE DETAILED MECHANISM AND EPIGENETIC REGULATION BY H2AX REMAIN ELUSIVE IN CANCER CELLS. HERE, WE REPORT THAT H2AX WAS INVOLVED IN APOPTOSIS OF CML CELLS. OVEREXPRESSION OF H2AX INCREASED APOPTOTIC SENSITIVITY OF CML CELLS (K562) INDUCED BY IMATINIB. HOWEVER, OVEREXPRESSION OF SER139-MUTATED H2AX (BLOCKING PHOSPHORYLATION) DECREASED SENSITIVITY OF K562 CELLS TO APOPTOSIS. SIMILARLY, KNOCKDOWN OF H2AX MADE K562 CELLS RESISTANT TO APOPTOTIC INDUCTION. THESE RESULTS REVEALED THAT THE FUNCTION OF H2AX INVOLVED IN APOPTOSIS IS STRICTLY RELATED TO ITS PHOSPHORYLATION (SER139). OUR DATA FURTHER INDICATED THAT IMATINIB MAY STIMULATE MITOGEN-ACTIVATED PROTEIN KINASE (MAPK) FAMILY MEMBER P38, AND H2AX PHOSPHORYLATION FOLLOWED A SIMILAR TIME COURSE, SUGGESTING A PARALLEL RESPONSE. H2AX PHOSPHORYLATION CAN BE BLOCKED BY P38 SIRNA OR ITS INHIBITOR. THESE DATA DEMONSTRATED THAT H2AX PHOSPHORYLATION WAS REGULATED BY P38 MAPK PATHWAY IN K562 CELLS. HOWEVER, THE P38 MAPK DOWNSTREAM, MITOGEN- AND STRESS-ACTIVATED PROTEIN KINASE-1 AND -2, WHICH PHOSPHORYLATED HISTONE H3, WERE NOT REQUIRED FOR H2AX PHOSPHORYLATION DURING APOPTOSIS. FINALLY, WE PROVIDED EPIGENETIC EVIDENCE THAT H2AX PHOSPHORYLATION REGULATED APOPTOSIS-RELATED GENE BIM EXPRESSION. BLOCKING OF H2AX PHOSPHORYLATION INHIBITED BIM GENE EXPRESSION. TAKEN TOGETHER, THESE DATA DEMONSTRATED THAT H2AX PHOSPHORYLATION REGULATED BY P38 IS INVOLVED IN BIM EXPRESSION AND APOPTOSIS IN CML CELLS INDUCED BY IMATINIB. 2014 8 2300 28 EPIGENETIC REGULATION OF BDNF EXPRESSION IN THE PRIMARY SENSORY NEURONS AFTER PERIPHERAL NERVE INJURY: IMPLICATIONS IN THE DEVELOPMENT OF NEUROPATHIC PAIN. BRAIN-DERIVED NEUROTROPHIC FACTOR (BDNF) IS KNOWN TO BE UP-REGULATED IN THE DORSAL ROOT GANGLION (DRG) AFTER PERIPHERAL NERVE INJURY, AND TO CONTRIBUTE TO NEUROPATHIC PAIN. HERE, WE FOUND THAT THERMAL HYPERALGESIA AND MECHANICAL ALLODYNIA AT DAY 7 POST-INJURY WERE INHIBITED ONLY WHEN ANTI-BDNF ANTIBODY WAS INTRATHECALLY ADMINISTRATED AT DAY 2 POST-INJURY. CONSISTENT WITH BEHAVIORAL RESULTS, WESTERN BLOT ANALYSIS SHOWED THAT THE EXPRESSION LEVELS OF BDNF PROTEIN IN THE SPINAL DORSAL HORN WERE MARKEDLY INDUCED DURING EARLY STAGE POST-INJURY. MOREOVER, THE MAXIMAL INCREASE IN BDNF MRNA EXPRESSION IN THE DRG WAS OBSERVED AT DAY 1 POST-INJURY, AND SIGNIFICANTLY ELEVATED LEVELS WERE SUSTAINED FOR AT LEAST 14 DAYS. FOUR OF FIVE BDNF MRNA TRANSCRIPTS WERE UP-REGULATED AFTER NERVE INJURY, AND THE MOST INDUCIBLE TRANSCRIPT WAS EXON I. USING A CHROMATIN IMMUNOPRECIPITATION (CHIP) ASSAY, WE FOUND THAT NERVE INJURY PROMOTES HISTONE H3 AND H4 ACETYLATION, TRANSCRIPTIONALLY ACTIVE MODIFICATIONS, AT BDNF PROMOTER I AT DAY 1 POST-INJURY, AND THE LEVELS OF HISTONE ACETYLATION REMAIN ELEVATED FOR AT LEAST 7 DAYS. TAKEN TOGETHER, OUR FINDINGS SUGGEST THAT AN INITIAL INCREASE IN BDNF EXON I EXPRESSION CONTROLLED BY EPIGENETIC MECHANISMS MIGHT HAVE A CRUCIAL ROLE IN THE DEVELOPMENT OF NEUROPATHIC PAIN. 2013 9 3531 33 IMATINIB CAUSES EPIGENETIC ALTERATIONS OF PTEN GENE VIA UPREGULATION OF DNA METHYLTRANSFERASES AND POLYCOMB GROUP PROTEINS. WE HAVE RECENTLY REPORTED THE POSSIBLE IMATINIB-RESISTANT MECHANISM; LONG-TERM EXPOSURE OF LEUKEMIA CELLS TO IMATINIB DOWNREGULATED LEVELS OF PHOSPHATASE AND TENSIN HOMOLOG DELETED ON CHROMOSOME 10 (PTEN) VIA HYPERMETHYLATION OF ITS PROMOTER REGION (LEUKEMIA 2010; 24: 1631). THE PRESENT STUDY EXPLORED THE MOLECULAR MECHANISMS BY WHICH IMATINIB CAUSED METHYLATION ON THE PROMOTER REGION OF THIS TUMOR SUPPRESSOR GENE IN LEUKEMIA CELLS. REAL-TIME REVERSE TRANSCRIPTION PCR FOUND THAT LONG-TERM EXPOSURE OF CHRONIC EOSINOPHILIC LEUKEMIA EOL-1 CELLS EXPRESSING FIP1L1/PLATELET-DERIVED GROWTH FACTOR RECEPTOR-ALPHA TO IMATINIB INDUCED EXPRESSION OF DNA METHYLTRANSFERASE 3A (DNMT3A) AND HISTONE-METHYLTRANSFERASE ENHANCER OF ZESTE HOMOLOG 2 (EZH2), A FAMILY OF POLYCOMB GROUP, THEREBY INCREASING METHYLATION OF THE GENE. IMMUNOPRECIPITATION ASSAY FOUND THE INCREASED COMPLEX FORMATION OF DNMT3A AND EZH2 PROTEINS IN THESE CELLS. MOREOVER, CHROMATIN IMMUNOPRECIPITATION ASSAY SHOWED THAT AMOUNTS OF BOTH DNMT3A AND EZH2 PROTEINS BOUND AROUND THE PROMOTER REGION OF PTEN GENE WERE INCREASED IN EOL-1 CELLS AFTER EXPOSURE TO IMATINIB. FURTHERMORE, WE FOUND THAT LEVELS OF DNMT3A AND EZH2 WERE STRIKINGLY INCREASED IN LEUKEMIA CELLS ISOLATED FROM INDIVIDUALS WITH CHRONIC MYELOGENOUS LEUKEMIA (N=1) AND PHILADELPHIA CHROMOSOME-POSITIVE ACUTE LYMPHOBLASTIC LEUKEMIA (N=2), WHO RELAPSED AFTER TREATMENT WITH IMATINIB COMPARED WITH THOSE ISOLATED AT THEIR INITIAL PRESENTATION. TAKEN TOGETHER, IMATINIB COULD CAUSE DRUG-RESISTANCE VIA RECRUITMENT OF POLYCOMB GENE COMPLEX TO THE PROMOTER REGION OF THE PTEN AND DOWNREGULATION OF THIS GENE'S TRANSCRIPTS IN LEUKEMIA PATIENTS. 2011 10 439 35 ANTILEUKEMIC ACTIVITY OF VALPROIC ACID IN CHRONIC LYMPHOCYTIC LEUKEMIA B CELLS DEFINED BY MICROARRAY ANALYSIS. EPIGENETIC CODE MODIFICATIONS BY HISTONE DEACETYLASE INHIBITORS HAVE RECENTLY BEEN PROPOSED AS POTENTIAL NEW THERAPIES FOR HEMATOLOGICAL MALIGNANCIES. CHRONIC LYMPHOCYTIC LEUKEMIA (CLL) REMAINS INCURABLE DESPITE THE INTRODUCTION OF NEW TREATMENTS. CLL B CELLS ARE CHARACTERIZED BY AN APOPTOSIS DEFECT RATHER THAN EXCESSIVE PROLIFERATION, BUT PROLIFERATION CENTERS HAVE BEEN FOUND IN ORGANS SUCH AS THE BONE MARROW AND LYMPH NODES. IN THIS STUDY, WE ANALYZED GENE EXPRESSION MODIFICATIONS IN CLL B CELLS AFTER TREATMENT WITH VALPROIC ACID (VPA), A WELL-TOLERATED ANTI-EPILEPTIC DRUG WITH HDAC INHIBITORY ACTIVITY. CLL B CELLS OBTAINED FROM 14 PATIENTS WERE TREATED IN VITRO WITH A CONCENTRATION OF 1 MM VPA FOR 4 H. VPA EFFECTS ON GENE EXPRESSION WERE THEREAFTER STUDIED USING AFFYMETRIX TECHNOLOGY, AND SOME IDENTIFIED GENES WERE VALIDATED BY REAL-TIME PCR AND WESTERN BLOT. WE OBSERVED THAT VPA INDUCED APOPTOSIS BY DOWNREGULATING SEVERAL ANTI-APOPTOTIC GENES AND BY UPREGULATING PRO-APOPTOTIC GENES. FURTHERMORE, VPA SIGNIFICANTLY INCREASED CHEMOSENSITIVITY TO FLUDARABINE, FLAVOPIRIDOL, BORTEZOMIB, THALIDOMIDE AND LENALIDOMIDE. VPA INHIBITED THE PROLIFERATION OF CPG/IL2-STIMULATED CLL B CELLS AND MODULATED MANY CELL CYCLE MESSENGER RNAS. IN CONCLUSION, EXPOSURE OF CLL B CELLS TO VPA INDUCED APOPTOSIS, POTENTIATED CHEMOTHERAPEUTIC AGENT EFFECTS AND INHIBITED PROLIFERATION. THESE DATA STRONGLY SUGGEST THE USE OF VPA IN CLL TREATMENT, PARTICULARLY IN COMBINATION WITH ANTILEUKEMIA AGENTS. 2009 11 66 35 A KEY ROLE FOR EZH2 IN EPIGENETIC SILENCING OF HOX GENES IN MANTLE CELL LYMPHOMA. THE CHROMATIN MODIFIER EZH2 IS OVEREXPRESSED AND ASSOCIATED WITH INFERIOR OUTCOME IN MANTLE CELL LYMPHOMA (MCL). RECENTLY, WE DEMONSTRATED PREFERENTIAL DNA METHYLATION OF HOX GENES IN MCL COMPARED WITH CHRONIC LYMPHOCYTIC LEUKEMIA (CLL), DESPITE THESE GENES NOT BEING EXPRESSED IN EITHER ENTITY. SINCE EZH2 HAS BEEN SHOWN TO REGULATE HOX GENE EXPRESSION, TO GAIN FURTHER INSIGHT INTO ITS POSSIBLE ROLE IN DIFFERENTIAL SILENCING OF HOX GENES IN MCL VS. CLL, WE PERFORMED DETAILED EPIGENETIC CHARACTERIZATION USING REPRESENTATIVE CELL LINES AND PRIMARY SAMPLES. WE OBSERVED SIGNIFICANT OVEREXPRESSION OF EZH2 IN MCL VS. CLL. CHROMATIN IMMUNE PRECIPITATION (CHIP) ASSAYS REVEALED THAT EZH2 CATALYZED REPRESSIVE H3 LYSINE 27 TRIMETHYLATION (H3K27ME3), WHICH WAS SUFFICIENT TO SILENCE HOX GENES IN CLL, WHEREAS IN MCL H3K27ME3 IS ACCOMPANIED BY DNA METHYLATION FOR A MORE STABLE REPRESSION. MORE IMPORTANTLY, HYPERMETHYLATION OF THE HOX GENES IN MCL RESULTED FROM EZH2 OVEREXPRESSION AND SUBSEQUENT RECRUITMENT OF THE DNA METHYLATION MACHINERY ONTO HOX GENE PROMOTERS. THE IMPORTANCE OF EZH2 UPREGULATION IN THIS PROCESS WAS FURTHER UNDERSCORED BY SIRNA TRANSFECTION AND EZH2 INHIBITOR EXPERIMENTS. ALTOGETHER, THESE OBSERVATIONS IMPLICATE EZH2 IN THE LONG-TERM SILENCING OF HOX GENES IN MCL, AND ALLUDE TO ITS POTENTIAL AS A THERAPEUTIC TARGET WITH CLINICAL IMPACT. 2013 12 5319 36 PTEN IS FUNDAMENTAL FOR ELIMINATION OF LEUKEMIA STEM CELLS MEDIATED BY GSK126 TARGETING EZH2 IN CHRONIC MYELOGENOUS LEUKEMIA. PURPOSE: LEUKEMIA STEM CELLS (LSCS) ARE AN IMPORTANT SOURCE OF TYROSINE KINASE INHIBITOR RESISTANCE AND DISEASE RELAPSE IN PATIENTS WITH CHRONIC MYELOGENOUS LEUKEMIA (CML). TARGETING LSCS MAY BE AN ATTRACTIVE STRATEGY TO OVERRIDE THIS THORNY PROBLEM. GIVEN THAT EZH2 WAS OVEREXPRESSED IN PRIMARY CML CD34(+) CELLS, OUR PURPOSE IN THIS STUDY WAS TO EVALUATE THE EFFECTS OF TARGETING EZH2 ON CML LSCS AND CLARIFY ITS UNDERLYING MECHANISM.EXPERIMENTAL DESIGN: HUMAN PRIMARY CML CD34(+) CELLS AND RETROVIRALLY BCR-ABL-DRIVEN CML MOUSE MODELS WERE EMPLOYED TO EVALUATE THE EFFECTS OF SUPPRESSION OF EZH2 BY GSK126- OR EZH2-SPECIFIC SHRNA IN VITRO AND IN VIVO RECRUITMENT OF EZH2 AND H3K27ME3 ON THE PROMOTER OF TUMOR-SUPPRESSOR GENE PTEN IN CML CELLS WAS MEASURED BY CHROMATIN IMMUNOPRECIPITATION ASSAY.RESULTS: OUR RESULTS SHOWED THAT PHARMACOLOGIC INHIBITION OF EZH2 BY GSK126 NOT ONLY ELICITED APOPTOSIS AND RESTRICTED CELL GROWTH IN CML BULK LEUKEMIA CELLS, BUT ALSO DECREASED LSCS IN CML CD34(+) CELLS WHILE SPARING THOSE FROM NORMAL BONE MARROW CD34(+) CELLS. SUPPRESSION OF EZH2 BY GSK126 OR SPECIFIC SHRNA PROLONGED SURVIVAL OF CML MICE AND REDUCED THE NUMBER OF LSCS IN MICE. EZH2 KNOCKDOWN RESULTED IN ELEVATION OF PTEN AND LED TO IMPAIRED RECRUITMENT OF EZH2 AND H3K27ME3 ON THE PROMOTER OF PTEN GENE. THE EFFECT OF EZH2 KNOCKDOWN IN THE CML MICE WAS AT LEAST PARTIALLY REVERSED BY PTEN KNOCKDOWN.CONCLUSIONS: THESE FINDINGS IMPROVE THE UNDERSTANDING OF THE EPIGENETIC REGULATION OF STEMNESS IN CML LSCS AND WARRANT CLINICAL TRIAL OF GSK126 IN REFRACTORY PATIENTS WITH CML. CLIN CANCER RES; 24(1); 145-57. (C)2017 AACR. 2018 13 6660 39 UPREGULATION OF CXCR4 THROUGH PROMOTER DEMETHYLATION CONTRIBUTES TO INFLAMMATORY HYPERALGESIA IN RATS. AIM AND METHODS: CHRONIC PAIN ASSOCIATED WITH INFLAMMATION IS A COMMON CLINICAL PROBLEM, AND THE UNDERLYING MECHANISMS YET ARE INCOMPLETELY DEFINED. DNA METHYLATION HAS BEEN IMPLICATED IN THE PATHOGENESIS OF CHRONIC PAIN. HOWEVER, THE SPECIFIC GENES REGULATED BY DNA METHYLATION UNDER INFLAMMATORY PAIN CONDITION REMAIN LARGELY UNKNOWN. HERE, WE INVESTIGATED HOW CHEMOKINE RECEPTOR CXCR4 EXPRESSION IS REGULATED BY DNA METHYLATION AND HOW IT CONTRIBUTES TO INFLAMMATORY PAIN INDUCED BY COMPLETE FREUND'S ADJUVANT (CFA) IN RATS. RESULTS: INTRAPLANTAR INJECTION OF CFA COULD NOT ONLY INDUCE SIGNIFICANT HYPERALGESIA IN RATS, BUT ALSO SIGNIFICANTLY INCREASE THE EXPRESSION OF CXCR4 MRNA AND PROTEIN IN THE DORSAL ROOT GANGLION (DRG). INTRATHECAL INJECTION OF CXCR4 ANTAGONIST AMD3100 SIGNIFICANTLY RELIEVED HYPERALGESIA IN INFLAMMATORY RATS IN A TIME- AND DOSE-DEPENDENT MANNER. BISULFITE SEQUENCING AND METHYLATION-SPECIFIC PCR DEMONSTRATE THAT CFA INJECTION LED TO A SIGNIFICANT DEMETHYLATION OF CPG ISLAND AT CXCR4 GENE PROMOTER. CONSISTENTLY, THE EXPRESSION OF DNMT3B WAS SIGNIFICANTLY DOWNREGULATED AFTER CFA INJECTION. ONLINE SOFTWARE PREDICTION REVEALS THREE BINDING SITES OF P65 IN THE CPG ISLAND OF CXCR4 GENE PROMOTER, WHICH HAS CONFIRMED BY THE CHROMATIN IMMUNOPRECIPITATION ASSAY, CFA TREATMENT SIGNIFICANTLY INCREASES THE RECRUITMENT OF P65 TO CXCR4 GENE PROMOTER. INHIBITION OF NF-KB SIGNALING USING P65 INHIBITOR PYRROLIDINE DITHIOCARBAMATE SIGNIFICANTLY PREVENTED THE INCREASES OF THE CXCR4 EXPRESSION. CONCLUSION: UPREGULATION OF CXCR4 EXPRESSION DUE TO PROMOTER DEMETHYLATION FOLLOWED BY INCREASED RECRUITMENT OF P65 TO PROMOTER OF CXCR4 GENE CONTRIBUTES TO INFLAMMATORY HYPERALGESIA. THESE FINDINGS PROVIDE A THEORETICAL BASIS FOR THE TREATMENT OF CHRONIC PAIN FROM AN EPIGENETIC PERSPECTIVE. 2018 14 5459 35 RESEARCH ON THE EPIGENETIC REGULATION MECHANISM OF THE PTPN6 GENE IN ADVANCED CHRONIC MYELOID LEUKAEMIA. PTPN6, A TYROSINE PHOSPHATASE PROTEIN, PLAYS A NEGATIVE ROLE IN CELL SIGNAL TRANSDUCTION AND IS NEGATIVELY CORRELATED WITH TUMOUR FORMATION AND GROWTH. HOWEVER, EPIGENETIC REGULATION MECHANISM OF THE PTPN6 GENE IN ADVANCED CHRONIC MYELOID LEUKAEMIA (CML) REMAINS UNCLEAR. THIS STUDY INVESTIGATED BONE MARROW OR BLOOD SAMPLES FROM 44 CML PATIENTS AND 10 HEALTHY VOLUNTEERS. KCL22 AND K562 CELLS WERE CULTURED AND TREATED WITH DEMETHYLATION DRUGS AND HISTONE DEACETYLASE INHIBITORS. REAL TIME QUANTITATIVE POLYMERASE CHAIN REACTION (QPCR), METHYLATION-SPECIFIC PCR, BISULFITE SEQUENCING PCR, WESTERN BLOTTING, CO-IMMUNOPRECIPITATION AND CHROMATIN IMMUNOPRECIPITATION (CHIP) WAS PERFORMED. PTPN6 WAS DOWN-REGULATED IN CELL LINES AND PATIENTS WITH ADVANCED PHASE CML, WHEREAS DNMT1, DNMT3A, MECP2, MBD2 AND HDAC1 WERE UP-REGULATED. TREATMENT WITH 5-AZACYTIDINE, DECITABINE, SODIUM VALPROATE AND LBH589 INCREASED PTPN6 EXPRESSION, BUT DECREASED THAT OF DNMT1, DNMT3A, MECP2, MBD2 AND HDAC1. IMMUNOPRECIPITATION AND MASS SPECTROMETRY SHOWED THAT HDAC1 COMBINED DIRECTLY WITH PTPN6. CHIP-SEQ SHOWED THAT HDAC1 DID NOT COMBINE WITH THE PROMOTER REGION OF PTPN6, WHILE MAPK, AKT, STAT5, JAK2 AND MYC PROMOTER REGIONS ALL COMBINED WITH HDAC1. PTPN6 IS ASSOCIATED WITH PROGRESSION OF CML. LOW EXPRESSION LEVEL OF PTPN6 WAS ASSOCIATED WITH DNA METHYLATION AND REGULATED BY HISTONE ACETYLATION. HDAC1 PARTICIPATES IN THE REGULATION OF PTPN6. 2017 15 2326 34 EPIGENETIC REGULATION OF HOTAIR IN ADVANCED CHRONIC MYELOID LEUKEMIA. PURPOSE: CHRONIC MYELOID LEUKEMIA (CML) ACCOUNTS FOR ~10% OF LEUKEMIA CASES, AND ITS PROGRESSION INVOLVES EPIGENETIC GENE REGULATION. THIS STUDY INVESTIGATED EPIGENETIC REGULATION OF HOTAIR AND ITS TARGET MICRORNA, MIR-143, IN ADVANCED CML. PATIENTS AND METHODS: WE FIRST ISOLATED BONE MARROW MONONUCLEAR CELLS FROM 70 PATIENTS WITH DIFFERENT PHASES OF CML AND FROM HEALTHY DONORS AS NORMAL CONTROL; WE ALSO CULTURED K562 AND KCL22 CELLS, TREATED WITH DEMETHYLATION DRUG; MTT ASSAY, FLOW CYTOMETRY, QUANTITATIVE REAL-TIME POLYMERASE CHAIN REACTION (QPCR), METHYLATION-SPECIFIC POLYMERASE CHAIN REACTION (MSP), WESTERN BLOT, LUCIFERASE ASSAY, RNA PULL-DOWN ASSAY AND RNA-BINDING PROTEIN IMMUNOPRECIPITATION (RIP) ASSAY WERE PERFORMED. RESULT: AS MEASURED BY QPCR, HOTAIR EXPRESSION IN K562 CELLS, KCL22 CELLS, AND SAMPLES FROM CASES OF ADVANCED-STAGE CML INCREASED WITH LEVELS OF SEVERAL DNA METHYLTRANSFERASES AND HISTONE DEACETYLATES, INCLUDING DNMT1, DNMT3A, HDAC1, EZH2, AND LSD1, AND MIR-143 LEVELS WERE DECREASED AND HOTAIR LEVELS WERE INCREASED. TREATMENT WITH 5-AZACYTIDINE, A DNA METHYLATION INHIBITOR, DECREASED DNMT1, DNMT3A, HDAC1, EZH2, LSD1 MRNA, PROTEIN LEVELS, AND HOTAIR MRNA LEVELS BUT INCREASED MIR-143 LEVELS. HOTAIR KNOCKDOWN AND MIR-143 OVEREXPRESSION BOTH INHIBITED PROLIFERATION AND PROMOTED APOPTOSIS IN KCL22 AND K562 CELLS THROUGH THE PI3K/AKT PATHWAY. RNA PULL-DOWN, MASS SPECTROMETRY, AND RIP ASSAYS SHOWED THAT HOTAIR INTERACTED WITH EZH2 AND LSD1. A DUAL-LUCIFERASE ASSAY DEMONSTRATED THAT HOTAIR INTERACTED WITH MIR-143. CONCLUSION: OUR FINDINGS DEMONSTRATE THE KEY EPIGENETIC INTERACTIONS OF HOTAIR RELATED TO CML PROGRESSION AND SUGGEST HOTAIR AS A POTENTIAL THERAPEUTIC TARGET FOR ADVANCED CML. FURTHERMORE, OUR RESULTS SUPPORT THE USE OF DEMETHYLATION DRUGS AS A CML TREATMENT STRATEGY. 2018 16 2379 51 EPIGENETIC REGULATION OF WNT PATHWAY ANTAGONISTS IN HUMAN GLIOBLASTOMA MULTIFORME. EPIGENETIC INACTIVATION OF TUMOR SUPPRESSOR GENES IS COMMON IN HUMAN CANCER. USING A LARGE-SCALE WHOLE-GENOME APPROACH IN AN EARLIER STUDY, THE AUTHORS IDENTIFIED EPIGENETICALLY SILENCED GENES WITH POTENTIAL TUMOR SUPPRESSOR FUNCTION IN GLIOBLASTOMA (GBM). THREE GENES IDENTIFIED IN THIS ANALYSIS-DKK1, SFRP1, AND WIF1-ARE POTENT INHIBITORS OF THE WNT SIGNAL TRANSDUCTION PATHWAY. HERE, THE AUTHORS CONFIRM DECREASED EXPRESSION OF THESE GENES IN GBM TUMOR TISSUE SAMPLES RELATIVE TO NONTUMOR BRAIN TISSUE SAMPLES USING REAL-TIME PCR. THEY THEN SHOW THAT EXPRESSION OF ALL 3 GENES IS RESTORED IN T98 GBM CELLS BY TREATMENT WITH THE HISTONE DEACETYLASE INHIBITOR TRICHOSTATIN A (TSA), BUT ONLY DKK1 EXPRESSION IS RESTORED BY TREATMENT WITH THE DEMETHYLATING AGENT 5-AZACYTIDINE. BISULFITE SEQUENCING DID NOT REVEAL SIGNIFICANT METHYLATION IN THE PROMOTER REGION OF DKK1, WHEREAS HISTONE ACETYLATION AND CHROMATIN ACCESSIBILITY INCREASED SIGNIFICANTLY FOR ALL 3 GENES AFTER TSA TREATMENT. ECTOPIC EXPRESSION OF DKK1 SIGNIFICANTLY REDUCES COLONY FORMATION AND INCREASES CHEMOTHERAPY-INDUCED APOPTOSIS IN T98 CELLS. ECTOPIC EXPRESSION OF THE CANONICAL WNT PATHWAY INHIBITORS WIF1 AND SFRP1 SHOWS A RELATIVE LACK OF RESPONSE. CHRONIC WNT3A STIMULATION ONLY PARTIALLY REVERSES GROWTH SUPPRESSION AFTER DKK1 REEXPRESSION, WHEREAS A SPECIFIC INHIBITOR OF THE JNK PATHWAY SIGNIFICANTLY REVERSES THE EFFECT OF DKK1 REEXPRESSION ON COLONY FORMATION AND APOPTOSIS IN T98 CELLS. THESE RESULTS SUPPORT A POTENTIAL GROWTH-SUPPRESSIVE FUNCTION FOR EPIGENETICALLY SILENCED DKK1 IN GBM AND SUGGEST THAT DKK1 RESTORATION COULD MODULATE WNT SIGNALING THROUGH BOTH CANONICAL AND NONCANONICAL PATHWAYS. 2010 17 1211 29 CPG ISLAND METHYLATION AND EXPRESSION OF THE SECRETED FRIZZLED-RELATED PROTEIN GENE FAMILY IN CHRONIC LYMPHOCYTIC LEUKEMIA. B-CELL CHRONIC LYMPHOCYTIC LEUKEMIA (CLL) IS CHARACTERIZED BY A CLONAL ACCUMULATION OF MATURE NEOPLASTIC B CELLS INDICATING DISRUPTION OF APOPTOSIS. RESTRICTION LANDMARK GENOME SCANNING WAS DONE TO IDENTIFY NOVEL TARGET GENES SILENCED BY CPG ISLAND METHYLATION IN CLL. SECRETED FRIZZLED-RELATED PROTEIN 4 (SFRP4), A NEGATIVE REGULATOR OF THE WNT SIGNALING PATHWAY, WAS FOUND TO BE FREQUENTLY METHYLATED IN CLL SAMPLES. WNT SIGNALING HAS BEEN SHOWN TO CONTROL NORMAL APOPTOTIC BEHAVIOR AND IS REQUIRED FOR NORMAL B-CELL DEVELOPMENT WHEREAS ABERRANT ACTIVATION OF THIS PATHWAY HAS BEEN OBSERVED IN CLL. WE SHOW ABERRANT DNA METHYLATION AND SILENCING OF SFRP4, AS WELL AS OF ADDITIONAL SFRP FAMILY MEMBERS, IN PRIMARY CLL SAMPLES. INDUCTION OF THEIR EXPRESSION IN A DOSE-DEPENDENT MANNER FOLLOWING TREATMENT WITH A DEMETHYLATING AGENT, 5-AZA-2'-DEOXYCYTIDINE, WAS SHOWN. OF THE FIVE SFRP FAMILY MEMBERS STUDIED IN DETAIL, SFRP1 WAS HYPERMETHYLATED AND DOWN-REGULATED IN ALL CLL PATIENT SAMPLES STUDIED, SUGGESTING THAT THIS EPIGENETIC EVENT IS A CRITICAL STEP DURING LEUKEMOGENESIS. OUR RESULTS SUGGEST THAT SILENCING OF SFRPS BY CPG ISLAND METHYLATION IS ONE POSSIBLE MECHANISM CONTRIBUTING TO ABERRANT ACTIVATION OF WNT SIGNALING PATHWAY IN CLL. 2006 18 2825 32 FLOW-DEPENDENT EPIGENETIC REGULATION OF IGFBP5 EXPRESSION BY H3K27ME3 CONTRIBUTES TO ENDOTHELIAL ANTI-INFLAMMATORY EFFECTS. RATIONALE: ATHEROSCLEROSIS IS A CHRONIC INFLAMMATORY AND EPIGENETIC DISEASE THAT IS INFLUENCED BY DIFFERENT PATTERNS OF BLOOD FLOW. HOWEVER, THE EPIGENETIC MECHANISM WHEREBY ATHEROPROTECTIVE FLOW CONTROLS ENDOTHELIAL GENE PROGRAMMING REMAINS ELUSIVE. HERE, WE INVESTIGATED THE POSSIBILITY THAT FLOW ALTERS ENDOTHELIAL GENE EXPRESSION THROUGH EPIGENETIC MECHANISMS. METHODS: EN FACE STAINING AND WESTERN BLOT WERE USED TO DETECT PROTEIN EXPRESSION. REAL-TIME PCR WAS USED TO DETERMINE RELATIVE GENE EXPRESSION. RNA-SEQUENCING OF HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS TREATED WITH SIRNA OF ENHANCER OF ZESTE HOMOLOG 2 (EZH2) OR LAMINAR FLOW WAS USED FOR TRANSCRIPTIONAL PROFILING. RESULTS: WE FOUND THAT TRIMETHYLATION OF HISTONE 3 LYSINE 27 (H3K27ME3), A REPRESSIVE EPIGENETIC MARK THAT ORCHESTRATES GENE REPRESSION, WAS REDUCED IN LAMINAR FLOW AREAS OF MOUSE AORTA AND FLOW-TREATED HUMAN ENDOTHELIAL CELLS. THE DECREASE OF H3K27ME3 PARALLELED A REDUCTION IN THE EPIGENETIC "WRITER"-EZH2, THE CATALYTIC SUBUNIT OF THE POLYCOMB REPRESSIVE COMPLEX 2 (PRC2). MOREOVER, LAMINAR FLOW DECREASED EXPRESSION OF EZH2 VIA MECHANOSENSITIVE MIR101. GENOME-WIDE TRANSCRIPTOME PROFILING STUDIES IN ENDOTHELIAL CELLS TREATED WITH EZH2 SIRNA AND FLOW REVEALED THE UPREGULATION OF NOVEL MECHANOSENSITIVE GENE IGFBP5 (INSULIN-LIKE GROWTH FACTOR-BINDING PROTEIN 5), WHICH IS EPIGENETICALLY SILENCED BY H3K27ME3. FUNCTIONALLY, INHIBITION OF H3K27ME3 BY EZH2 SIRNA OR GSK126 (A SPECIFIC EZH2 INHIBITOR) REDUCED H3K27ME3 LEVELS AND MONOCYTE ADHESION TO ENDOTHELIAL CELLS. ADENOVIRAL OVEREXPRESSION OF IGFBP5 ALSO RECAPITULATED THE ANTI-INFLAMMATORY EFFECTS OF H3K27ME3 INHIBITION. MORE IMPORTANTLY, WE OBSERVED EZH2 UPREGULATION, AND IGFBP5 DOWNREGULATION, IN ADVANCED ATHEROSCLEROTIC PLAQUES FROM HUMAN PATIENTS. CONCLUSION: TAKEN TOGETHER, OUR FINDINGS REVEAL THAT ATHEROPROTECTIVE FLOW REDUCES H3K27ME3 AS A CHROMATIN-BASED MECHANISM TO AUGMENT THE EXPRESSION OF GENES THAT CONFER AN ANTI-INFLAMMATORY RESPONSE IN THE ENDOTHELIUM. OUR STUDY EXEMPLIFIES FLOW-DEPENDENT EPIGENETIC REGULATION OF ENDOTHELIAL GENE EXPRESSION, AND ALSO SUGGESTS THAT TARGETING THE EZH2/H3K27ME3/IGFBP5 PATHWAY MAY OFFER NOVEL THERAPEUTICS FOR INFLAMMATORY DISORDERS SUCH AS ATHEROSCLEROSIS. 2018 19 1669 35 DOWNREGULATION OF THE HISTONE METHYLTRANSFERASE SETD2 PROMOTES IMATINIB RESISTANCE IN CHRONIC MYELOID LEUKAEMIA CELLS. OBJECTIVES: EPIGENETIC MODIFIERS WERE IMPORTANT PLAYERS IN THE DEVELOPMENT OF HAEMATOLOGICAL MALIGNANCIES AND SENSITIVITY TO THERAPY. MUTATIONS OF SET DOMAIN-CONTAINING 2 (SETD2), A METHYLTRANSFERASE THAT CATALYSES THE TRIMETHYLATION OF HISTONE 3 ON LYSINE 36 (H3K36ME3), WERE FOUND IN VARIOUS MYELOID MALIGNANCIES. HOWEVER, THE DETAILED MECHANISMS THROUGH WHICH SETD2 CONFERS CHRONIC MYELOID LEUKAEMIA PROGRESSION AND RESISTANCE TO THERAPY TARGETING ON BCR-ABL REMAIN UNCLEAR. MATERIALS AND METHODS: THE LEVEL OF SETD2 IN IMATINIB-SENSITIVE AND IMATINIB-RESISTANT CHRONIC MYELOID LEUKAEMIA (CML) CELLS WAS EXAMINED BY IMMUNOBLOTTING AND QUANTITATIVE REAL-TIME PCR. WE ANALYSED CD34(+) CD38(-) LEUKAEMIC STEM CELLS BY FLOW CYTOMETRY AND COLONY FORMATION ASSAYS UPON SETD2 KNOCKDOWN OR OVEREXPRESSION. THE IMPACT OF SETD2 EXPRESSION ALTERATIONS OR SMALL-MOLECULE INHIBITOR JIB-04 TARGETING H3K36ME3 LOSS ON IMATINIB SENSITIVITY WAS ASSESSED BY IC50, CELL APOPTOSIS AND PROLIFERATION ASSAYS. FINALLY, RNA SEQUENCING AND CHIP-QUANTITATIVE PCR WERE PERFORMED TO VERIFY PUTATIVE DOWNSTREAM TARGETS. RESULTS: SETD2 WAS FOUND TO ACT AS A TUMOUR SUPPRESSOR IN CML. THE NOVEL ONCOGENIC TARGETS MYCN AND ERG WERE SHOWN TO BE THE DIRECT DOWNSTREAM TARGETS OF SETD2, WHERE THEIR OVEREXPRESSION INDUCED BY SETD2 KNOCKDOWN CAUSED IMATINIB INSENSITIVITY AND LEUKAEMIC STEM CELL ENRICHMENT IN CML CELL LINES. TREATMENT WITH JIB-04, AN INHIBITOR THAT RESTORES H3K36ME3 LEVELS THROUGH BLOCKADE OF ITS DEMETHYLATION, SUCCESSFULLY IMPROVED THE CELL IMATINIB SENSITIVITY AND ENHANCED THE CHEMOTHERAPEUTIC EFFECT. CONCLUSIONS: OUR STUDY NOT ONLY EMPHASIZES THE REGULATORY MECHANISM OF SETD2 IN CML, BUT ALSO PROVIDES PROMISING THERAPEUTIC STRATEGIES FOR OVERCOMING THE IMATINIB RESISTANCE IN PATIENTS WITH CML. 2019 20 1831 29 EFFECTS OF MATERNAL SEPARATION AND ANTIDEPRESSANT DRUG ON EPIGENETIC REGULATION OF THE BRAIN-DERIVED NEUROTROPHIC FACTOR EXON I PROMOTER IN THE ADULT RAT HIPPOCAMPUS. AIM: EARLY LIFE STRESS CAN INDUCE EPIGENETIC CHANGES THROUGH GENETIC AND ENVIRONMENTAL INTERACTIONS AND IS A RISK FACTOR FOR DEPRESSION. BRAIN-DERIVED NEUROTROPHIC FACTOR (BDNF) HAS BEEN IMPLICATED IN THE PATHOPHYSIOLOGY OF DEPRESSION AND ANTIDEPRESSANT DRUG ACTION. WE INVESTIGATED EPIGENETIC CHANGES AT THE BDNF EXON I PROMOTER IN THE HIPPOCAMPUS OF ADULT RATS SUBJECTED TO MATERNAL SEPARATION (MS) DURING EARLY LIFE AND TREATED WITH AN ANTIDEPRESSANT DRUG AS ADULTS. METHODS: RAT PUPS WERE SUBJECTED TO MS FROM POSTNATAL DAY 1 TO 21 AND RECEIVED CHRONIC ESCITALOPRAM (ESC) AS ADULTS. WE ASSESSED THE EFFECTS OF MS AND ESC ON BDNF EXON I AND DNA METHYLTRANSFERASES (DNMT) MRNA LEVELS (QUANTITATIVE REVERSE-TRANSCRIPTION POLYMERASE CHAIN REACTION), ACETYLATED HISTONE H3, AND MECP2 BINDING TO THE BDNF PROMOTER I (CHROMATIN IMMUNOPRECIPITATION FOLLOWED BY REAL-TIME POLYMERASE CHAIN REACTION), AND BDNF PROTEIN LEVELS (ENZYME-LINKED IMMUNOSORBENT ASSAY). RESULTS: THE LEVELS OF BDNF PROTEIN, EXON I MRNA, HISTONE H3 ACETYLATION, AND DNMT1 AND DNMT3A MRNA WERE ALTERED IN THE MS GROUP COMPARED WITH THE CONTROL GROUP. SIGNIFICANT DECREASES WERE OBSERVED IN THE BDNF PROTEIN, EXON I MRNA, AND HISTONE H3 ACETYLATION LEVELS AND THERE WERE SIGNIFICANT INCREASES IN DNMT1 AND DNMT3A MRNA LEVELS. THE COMPARISON BETWEEN THE MS + ESC AND MS GROUPS REVEALED SIGNIFICANT INCREASES IN BDNF PROTEIN, EXON I MRNA, AND HISTONE H3 ACETYLATION LEVELS AND SIGNIFICANT DECREASES IN MECP2 AND DNMT1 AND DNMT3A MRNA LEVELS. CONCLUSION: THESE FINDINGS INDICATE THAT MS INDUCED EPIGENETIC CHANGES AT THE BDNF EXON I PROMOTER AND THESE CHANGES WERE PREVENTED BY ANTIDEPRESSANT DRUG TREATMENT DURING ADULTHOOD. 2018