1 2326 133 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 2 1826 46 EFFECTS OF HISTONE DEACETYLASE INHIBITOR ON EXTRACELLULAR MATRIX PRODUCTION IN HUMAN NASAL POLYP ORGAN CULTURES. BACKGROUND: NASAL POLYPOSIS IS ASSOCIATED WITH A CHRONIC INFLAMMATORY CONDITION OF THE SINONASAL MUCOSA AND INVOLVES MYOFIBROBLAST DIFFERENTIATION AND EXTRACELLULAR MATRIX (ECM) ACCUMULATION. EPIGENETIC MODULATION BY HISTONE DEACETYLASE (HDAC) INHIBITORS INCLUDING TRICHOSTATIN A (TSA) HAS BEEN REPORTED TO HAVE INHIBITORY EFFECTS ON MYOFIBROBLAST DIFFERENTIATION IN LUNG AND RENAL FIBROBLASTS. THE PURPOSE OF THIS STUDY WAS TO INVESTIGATE THE INHIBITORY EFFECT OF TSA ON MYOFIBROBLAST DIFFERENTIATION AND ECM PRODUCTION IN NASAL POLYP ORGAN CULTURES. METHODS: NASAL POLYP TISSUES FROM 18 PATIENTS WERE ACQUIRED DURING ENDOSCOPIC SINUS SURGERY. AFTER ORGAN CULTURE, NASAL POLYPS WERE STIMULATED WITH TGF-BETA1 AND THEN TREATED WITH TSA. ALPHA-SMOOTH MUSCLE ACTIN (ALPHA-SMA), FIBRONECTIN, AND COLLAGEN TYPE I EXPRESSION LEVELS WERE EXAMINED BY REVERSE TRANSCRIPTION-POLYMERASE CHAIN REACTION (PCR), REAL-TIME PCR, WESTERN BLOT, AND IMMUNOFLUORESCENT STAINING. HDAC2, HDAC4, AND ACETYLATED H4 EXPRESSION LEVELS WERE ASSAYED BY WESTERN BLOT. CYTOTOXICITY WAS ANALYZED BY THE TERMINAL DEOXYNUCLEOTIDYL TRANSFERASE BIOTIN-DUTP NICK END LABELING ASSAY. RESULTS: THE EXPRESSION LEVELS OF ALPHA-SMA, FIBRONECTIN, AND COLLAGEN TYPE 1 WERE INCREASED IN NASAL POLYP AFTER TRANSFORMING GROWTH FACTOR (TGF) BETA1 TREATMENT. TSA-INHIBITED TGF-BETA1 INDUCED THESE GENE AND PROTEIN EXPRESSION LEVELS. FURTHERMORE, TSA SUPPRESSED PROTEIN EXPRESSION LEVELS OF HDAC2 AND HDAC4. HOWEVER, TSA INDUCED HYPERACETYLATION OF HISTONES H4. TREATMENT WITH TGF-BETA1 WITH OR WITHOUT TSA DID NOT HAVE CYTOTOXIC EFFECT. CONCLUSION: THESE FINDINGS PROVIDE NOVEL INSIGHTS INTO THE EPIGENETIC REGULATION IN MYOFIBROBLAST DIFFERENTIATION AND ECM PRODUCTION OF NASAL POLYP. TSA COULD BE A CANDIDATE OF A THERAPEUTIC AGENT FOR REVERSING THE TGF-BETA1-INDUCED ECM SYNTHESIS THAT LEADS TO NASAL POLYP DEVELOPMENT. 2013 3 2300 30 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 4 3459 31 HYPOMETHYLATION OF NERVE GROWTH FACTOR (NGF) PROMOTES BINDING OF C/EBPALPHA AND CONTRIBUTES TO INFLAMMATORY HYPERALGESIA IN RATS. BACKGROUND: CHRONIC PAIN USUALLY ACCOMPANIED BY TISSUE DAMAGE AND INFLAMMATION. HOWEVER, THE PATHOGENESIS OF CHRONIC PAIN REMAINS UNCLEAR. METHODS: WE INVESTIGATED THE ROLE OF NERVE GROWTH FACTOR (NGF) IN CHRONIC INFLAMMATORY PAIN INDUCED BY COMPLETE FREUND'S ADJUVANT (CFA), EXPLORED THE METHYLATION STATUS OF CPG ISLANDS IN THE PROMOTER REGION OF THE NGF GENE, AND CLARIFIED THE FUNCTION AND MECHANISM OF C/EBPALPHA-NGF SIGNALING PATHWAY FROM EPIGENETIC PERSPECTIVE IN THE CHRONIC INFLAMMATORY PAIN MODEL. RESULTS: CFA INDUCED SIGNIFICANT HYPERALGESIA AND CONTINUOUS UPREGULATION OF NGF MRNA AND PROTEIN LEVELS IN THE L4-6 DORSAL ROOT GANGLIONS (DRGS) IN RATS. HYPOMETHYLATION OF CPG ISLANDS OCCURRED IN THE NGF GENE PROMOTER REGION AFTER CFA TREATMENT. AT THE SAME TIME, THE MIR-29B EXPRESSION LEVEL WAS SIGNIFICANTLY INCREASED, WHILE THE DNA METHYLTRANSFERASE 3B (DNMT3B) LEVEL REDUCED SIGNIFICANTLY. MOREOVER, CFA TREATMENT PROMOTED BINDING OF C/EBPALPHA TO THE NGF GENE PROMOTER REGION AND C/EBPALPHA SIRNA TREATMENT OBVIOUSLY DECREASED EXPRESSION OF NGF LEVELS AND ALSO ALLEVIATE INFLAMMATORY HYPERALGESIA SIGNIFICANTLY IN RATS. CONCLUSION: COLLECTIVELY, THE RESULTS INDICATED THAT CFA LEADS TO THE UPREGULATION OF MIR-29B LEVEL, WHICH REPRESSES THE EXPRESSION OF DNMT3B, ENHANCES THE DEMETHYLATION OF THE NGF GENE PROMOTER REGION, AND PROMOTES THE BINDING OF C/EBPALPHA WITH THE NGF GENE PROMOTER, THUS RESULTS IN THE UPREGULATION OF NGF GENE EXPRESSION AND MAINTENANCE OF CHRONIC INFLAMMATORY PAIN. 2020 5 6069 40 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 6 4303 42 MICRORNA-223 INHIBITS TISSUE FACTOR EXPRESSION IN VASCULAR ENDOTHELIAL CELLS. OBJECTIVE: ATHEROSCLEROSIS IS A CHRONIC INFLAMMATORY PROCESS, IN WHICH VASCULAR ENDOTHELIAL CELLS (ECS) BECOME DYSFUNCTIONAL OWING TO THE EFFECTS OF CHEMICAL SUBSTANCES, SUCH AS INFLAMMATORY FACTOR AND GROWTH FACTORS. TISSUE FACTOR (TF) EXPRESSION IS INDUCED BY THE ABOVE CHEMICAL SUBSTANCES IN ACTIVATED ECS. TF INITIATES THROMBOSIS ON DISRUPTED ATHEROSCLEROTIC PLAQUES WHICH PLAYS AN ESSENTIAL ROLE DURING THE ONSET OF ACUTE CORONARY SYNDROMES (ACS). INCREASING EVIDENCES SUGGEST THE IMPORTANT ROLE OF MICRORNAS AS EPIGENETIC REGULATORS OF ATHEROSCLEROTIC DISEASE. THE AIM OF OUR STUDY IS TO IDENTIFY IF MICRORNA-223 (MIR-223) TARGETS TF IN ECS. METHODS AND RESULTS: BIOINFORMATIC ANALYSIS SHOWED THAT TF IS A TARGET CANDIDATE OF MIR-223. WESTERN BLOTTING ANALYSIS REVEALED THAT TUMOR NECROSIS FACTOR ALPHA (TNF-ALPHA) INCREASED TF EXPRESSION IN AORTA OF C57BL/6J MICE AND CULTURED ECS (EA.HY926 CELLS AND HUVEC) AFTER 4 H TREATMENT. IN TNF-ALPHA TREATED ECS, TF MRNA WAS ALSO INCREASED MEASURED BY REAL-TIME PCR. REAL-TIME PCR RESULTS SHOWED THAT MIR-223 LEVELS WERE DOWNREGULATED IN TNF-ALPHA-TREATED AORTA OF C57BL/6J MICE AND CULTURED ECS. TRANSFECTION OF ECS WITH MIR-223 MIMIC OR MIR-223 INHIBITOR MODIFIED TF EXPRESSION BOTH IN MRNA AND PROTEIN LEVELS. LUCIFERASE ASSAYS CONFIRMED THAT MIR-223 SUPPRESSED TF EXPRESSION BY BINDING TO THE SEQUENCE OF TF 3'-UNTRANSLATED REGIONS (3'UTR). TF PROCOAGULANT ACTIVITY WAS INHIBITED BY OVEREXPRESSING MIR-223 WITH OR WITHOUT TNF-ALPHA STIMULATION. CONCLUSIONS: MIR-223-MEDIATED SUPPRESSION OF TF EXPRESSION PROVIDES A NOVEL MOLECULAR MECHANISM FOR THE REGULATION OF COAGULATION CASCADE, AND SUGGESTS A CLUE AGAINST THROMBOGENESIS DURING THE PROCESS OF ATHEROSCLEROTIC PLAQUE RUPTURE. 2014 7 2452 35 EPIGENETIC SUPPRESSION OF POTASSIUM-CHLORIDE CO-TRANSPORTER 2 EXPRESSION IN INFLAMMATORY PAIN INDUCED BY COMPLETE FREUND'S ADJUVANT (CFA). BACKGROUND: MULTIPLE MECHANISMS CONTRIBUTE TO THE STIMULUS-EVOKED PAIN HYPERSENSITIVITY THAT MAY BE EXPERIENCED AFTER PERIPHERAL INFLAMMATION. PERSISTENT PATHOLOGICAL STIMULI IN MANY PAIN CONDITIONS AFFECT THE EXPRESSION OF CERTAIN GENES THROUGH EPIGENETIC ALTERNATIONS. THE MAIN PURPOSE OF OUR STUDY WAS TO INVESTIGATE THE ROLE OF EPIGENETIC MODIFICATION ON POTASSIUM-CHLORIDE CO-TRANSPORTER 2 (KCC2) GENE EXPRESSION IN THE PERSISTENCE OF INFLAMMATORY PAIN. METHODS: PERSISTENT INFLAMMATORY PAIN WAS INDUCED THROUGH THE INJECTION OF COMPLETE FREUND'S ADJUVANT (CFA) IN THE LEFT HIND PAW OF RATS. ACETYL-HISTONE H3 AND H4 LEVEL WAS DETERMINED BY CHROMATIN IMMUNOPRECIPITATION IN THE SPINAL DORSAL HORN. PAIN BEHAVIOUR AND INHIBITORY SYNAPTIC FUNCTION OF SPINAL CORD WERE DETERMINED BEFORE AND AFTER CFA INJECTION. KCC2 EXPRESSION WAS DETERMINED BY REAL TIME RT-PCR AND WESTERN BLOT. INTRATHECAL KCC2 SIRNA (2 MUG PER 10 MUL PER RAT) OR HDAC INHIBITOR (10 MUG PER 10 MUL PER RAT) WAS INJECTED ONCE DAILY FOR 3 DAYS BEFORE CFA INJECTION. RESULTS: PERSISTENT INFLAMMATORY PAIN EPIGENETICALLY SUPPRESSED KCC2 EXPRESSION THROUGH HISTONE DEACETYLASE (HDAC)-MEDIATED HISTONE HYPOACETYLATION, RESULTING IN DECREASED INHIBITORY SIGNALLING EFFICACY. KCC2 KNOCK-DOWN CAUSED BY INTRATHECAL ADMINISTRATION OF KCC2 SIRNA IN NAIVE RATS REDUCED KCC2 EXPRESSION IN THE SPINAL CORD, LEADING TO SENSITIZED PAIN BEHAVIOURS AND IMPAIRED INHIBITORY SYNAPTIC TRANSMISSION IN THEIR SPINAL CORDS. MOREOVER, INTRATHECAL HDAC INHIBITOR INJECTION IN CFA RATS INCREASED KCC2 EXPRESSION, PARTIALLY RESTORING THE SPINAL INHIBITORY SYNAPTIC TRANSMISSION AND RELIEVING THE SENSITIZED PAIN BEHAVIOUR. CONCLUSION: THESE FINDINGS SUGGEST THAT THE TRANSCRIPTION OF SPINAL KCC2 IS REGULATED BY HISTONE ACETYLATION EPIGENETICALLY FOLLOWING CFA. SIGNIFICANCE: PERSISTENT PAIN SUPPRESSES KCC2 EXPRESSION THROUGH HDAC-MEDIATED HISTONE HYPOACETYLATION AND CONSEQUENTLY IMPAIRS THE INHIBITORY FUNCTION OF INHIBITORY INTERNEURONS. DRUGS SUCH AS HDAC INHIBITORS THAT SUPPRESS THE INFLUENCES OF PERSISTENT PAIN ON THE EXPRESSION OF KCC2 MAY SERVE AS A NOVEL ANALGESIC. 2017 8 5860 33 SULFORAPHANE PREVENTS ANGIOTENSIN II-INDUCED CARDIOMYOPATHY BY ACTIVATION OF NRF2 THROUGH EPIGENETIC MODIFICATION. NUCLEAR FACTOR ERYTHROID 2-RELATED FACTOR (NRF2) IS AN IMPORTANT REGULATOR OF CELLULAR ANTIOXIDANT DEFENCE. WE PREVIOUSLY SHOWED THAT SFN PREVENTED ANG II-INDUCED CARDIAC DAMAGE VIA ACTIVATION OF NRF2. HOWEVER, THE UNDERLYING MECHANISM OF SFN'S PERSISTENT CARDIAC PROTECTION REMAINS UNCLEAR. THIS STUDY AIMED TO EXPLORE THE POTENTIAL OF SFN IN ACTIVATING CARDIAC NRF2 THROUGH EPIGENETIC MECHANISMS. WILD-TYPE MICE WERE INJECTED SUBCUTANEOUSLY WITH ANG II, WITH OR WITHOUT SFN. ADMINISTRATION OF CHRONIC ANG II-INDUCED CARDIAC INFLAMMATORY FACTOR EXPRESSION, OXIDATIVE DAMAGE, FIBROSIS AND CARDIAC REMODELLING AND DYSFUNCTION, ALL OF WHICH WERE EFFECTIVELY IMPROVED BY SFN TREATMENT, COUPLED WITH AN UP-REGULATION OF NRF2 AND DOWNSTREAM GENES. BISULFITE GENOME SEQUENCING AND CHROMATIN IMMUNOPRECIPITATION (CHIP) WERE PERFORMED TO DETECT THE METHYLATION LEVEL OF THE FIRST 15 CPGS AND HISTONE H3 ACETYLATION (AC-H3) STATUS IN THE NRF2 PROMOTER REGION, RESPECTIVELY. THE RESULTS SHOWED THAT SFN REDUCED ANG II-INDUCED CPG HYPERMETHYLATION AND PROMOTED AC-H3 ACCUMULATION IN THE NRF2 PROMOTER REGION, ACCOMPANIED BY THE INHIBITION OF GLOBAL DNMT AND HDAC ACTIVITY, AND A DECREASED PROTEIN EXPRESSION OF KEY DNMT AND HDAC ENZYMES. TAKEN TOGETHER, SFN EXERTS ITS CARDIOPROTECTIVE EFFECT THROUGH EPIGENETIC MODIFICATION OF NRF2, WHICH MAY PARTIALLY CONTRIBUTE TO LONG-TERM ACTIVATION OF CARDIAC NRF2. 2021 9 6660 37 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 10 1620 34 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 11 3721 32 INHIBITION OF CLASS II HISTONE DEACETYLASES IN THE SPINAL CORD ATTENUATES INFLAMMATORY HYPERALGESIA. BACKGROUND: SEVERAL CLASSES OF HISTONE DEACETYLASES (HDACS) ARE EXPRESSED IN THE SPINAL CORD THAT IS A CRITICAL STRUCTURE OF THE NOCICEPTIVE PATHWAY. HDAC-REGULATED HISTONE ACETYLATION IS AN IMPORTANT COMPONENT OF CHROMATIN REMODELING LEADING TO EPIGENETIC REGULATION OF GENE TRANSCRIPTION. TO UNDERSTAND THE ROLE OF HISTONE ACETYLATION IN EPIGENETIC REGULATION OF PATHOLOGICAL PAIN, WE HAVE STUDIED THE IMPACT OF DIFFERENT CLASSES OF HDACS IN THE SPINAL CORD ON INFLAMMATORY HYPERALGESIA INDUCED BY COMPLETE FREUND'S ADJUVANT (CFA). RESULTS: WE INTRATHECALLY APPLIED INHIBITORS SPECIFIC TO DIFFERENT CLASSES OF HDACS AND EVALUATED THEIR IMPACT ON INFLAMMATORY HYPERALGESIA. PRE-INJECTED INHIBITORS TARGETING CLASS I AS WELL AS II (SAHA, TSA, LAQ824) OR IIA (VPA, 4-PB) HDACS SIGNIFICANTLY DELAYED THE THERMAL HYPERALGESIA INDUCED BY UNILATERAL CFA INJECTION IN THE HINDPAW. EXISTING HYPERALGESIA INDUCED BY CFA WAS ALSO ATTENUATED BY THE HDAC INHIBITORS (HDACIS). IN CONTRAST, THESE INHIBITORS DID NOT INTERFERE WITH THE THERMAL RESPONSE EITHER IN NAIVE ANIMALS, OR ON THE CONTRALATERAL SIDE OF INFLAMED ANIMALS. INTERESTINGLY, MS-275 THAT SPECIFICALLY INHIBITS CLASS I HDACS FAILED TO ALTER THE HYPERALGESIA ALTHOUGH IT INCREASED HISTONE 3 ACETYLATION IN THE SPINAL CORD AS SAHA DID. USING IMMUNOBLOT ANALYSIS, WE FURTHER FOUND THAT THE LEVELS OF CLASS IIA HDAC MEMBERS (HDAC4, 5, 7, 9) IN THE SPINAL DORSAL HORN WERE UPREGULATED FOLLOWING CFA INJECTION WHILE THOSE OF CLASS I HDAC MEMBERS (HDAC1, 2, 3) REMAINED STABLE OR WERE SLIGHTLY REDUCED. CONCLUSIONS: OUR DATA SUGGEST THAT ACTIVITY OF CLASS II HDACS IN THE SPINAL CORD IS CRITICAL TO THE INDUCTION AND MAINTENANCE OF INFLAMMATORY HYPERALGESIA INDUCED BY CFA, WHILE ACTIVITY OF CLASS I HDACS MAY BE UNNECESSARY. COMPARISON OF THE EFFECTS OF HDACIS SPECIFIC TO CLASS II AND IIA AS WELL AS THE EXPRESSION PATTERN OF DIFFERENT HDACS IN THE SPINAL CORD IN RESPONSE TO CFA SUGGESTS THAT THE MEMBERS OF CLASS IIA HDACS MAY BE POTENTIAL TARGETS FOR ATTENUATING PERSISTENT INFLAMMATORY PAIN. 2010 12 1320 38 DEMETHYLATION REGULATION OF BDNF GENE EXPRESSION IN DORSAL ROOT GANGLION NEURONS IS IMPLICATED IN OPIOID-INDUCED PAIN HYPERSENSITIVITY IN RATS. REPEATED ADMINISTRATION OF MORPHINE MAY RESULT IN OPIOID-INDUCED HYPERSENSITIVITY (OIH), WHICH INVOLVES ALTERED EXPRESSION OF NUMEROUS GENES, INCLUDING BRAIN-DERIVED NEUROTROPHIC FACTOR (BDNF) IN DORSAL ROOT GANGLION (DRG) NEURONS. YET, IT REMAINS UNCLEAR HOW BDNF EXPRESSION IS INCREASED IN DRG NEURONS AFTER REPEATED MORPHINE TREATMENT. DNA METHYLATION IS AN IMPORTANT MECHANISM OF EPIGENETIC CONTROL OF GENE EXPRESSION. IN THE CURRENT STUDY, WE HYPOTHESIZED THAT THE DEMETHYLATION REGULATION OF CERTAIN BDNF GENE PROMOTERS IN DRG NEURONS MAY CONTRIBUTE TO THE DEVELOPMENT OF OIH. REAL-TIME RT-PCR WAS USED TO ASSESS CHANGES IN THE MRNA TRANSCRIPTION LEVELS OF MAJOR BDNF EXONS INCLUDING EXON I, II, IV, VI, AS WELL AS TOTAL BDNF MRNA IN DRGS FROM RATS AFTER REPEATED MORPHINE ADMINISTRATION. THE LEVELS OF EXON IV AND TOTAL BDNF MRNA WERE SIGNIFICANTLY UPREGULATED BY REPEATED MORPHINE ADMINISTRATION, AS COMPARED TO THAT IN SALINE CONTROL GROUP. FURTHER, ELISA ARRAY AND IMMUNOCYTOCHEMISTRY STUDY REVEALED A ROBUST UPREGULATION OF BDNF PROTEIN EXPRESSION IN DRG NEURONS AFTER REPEATED MORPHINE EXPOSURE. CORRESPONDINGLY, THE METHYLATION LEVELS OF BDNF EXON IV PROMOTER SHOWED A SIGNIFICANT DOWNREGULATION BY MORPHINE TREATMENT. IMPORTANTLY, INTRATHECAL ADMINISTRATION OF A BDNF ANTIBODY, BUT NOT CONTROL IGG, SIGNIFICANTLY INHIBITED MECHANICAL HYPERSENSITIVITY THAT DEVELOPED IN RATS AFTER REPEATED MORPHINE TREATMENT. CONVERSELY, INTRATHECAL ADMINISTRATION OF AN INHIBITOR OF DNA METHYLATION, 5-AZA-2'-DEOXYCYTIDINE (5-AZA-DC) MARKEDLY UPREGULATED THE BDNF PROTEIN EXPRESSION IN DRG NEURONS AND ENHANCED THE MECHANICAL ALLODYNIA AFTER REPEATED MORPHINE EXPOSURE. TOGETHER, OUR FINDINGS SUGGEST THAT DEMETHYLATION REGULATION OF BDNF GENE PROMOTER MAY BE IMPLICATED IN THE DEVELOPMENT OF OIH THROUGH EPIGENETIC CONTROL OF BDNF EXPRESSION IN DRG NEURONS. 2016 13 439 45 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 14 1667 33 DOWNREGULATION OF PCAF BY MIR-181A/B PROVIDES FEEDBACK REGULATION TO TNF-ALPHA-INDUCED TRANSCRIPTION OF PROINFLAMMATORY GENES IN LIVER EPITHELIAL CELLS. ABERRANT CELLULAR RESPONSES TO PROINFLAMMATORY CYTOKINES, SUCH AS TNF-ALPHA, ARE PATHOGENIC FEATURES IN MOST CHRONIC INFLAMMATORY DISEASES. A VARIETY OF EXTRACELLULAR AND INTRACELLULAR FEEDBACK PATHWAYS HAS EVOLVED TO PREVENT AN INAPPROPRIATE CELLULAR REACTION TO THESE PROINFLAMMATORY CYTOKINES. IN THIS STUDY, WE REPORT THAT TNF-ALPHA TREATMENT OF HUMAN AND MOUSE CHOLANGIOCYTES AND HEPATOCYTES DOWNREGULATED EXPRESSION OF P300/CBP-ASSOCIATED FACTOR (PCAF), A COACTIVATOR AND AN ACETYLTRANSFERASE THAT PROMOTES HISTONE ACETYLATION AND GENE TRANSCRIPTION. OF THESE UPREGULATED MICRORNAS IN TNF-ALPHA-TREATED CELLS, MIR-181A/B (MIR-181A AND MIR-181B) SUPPRESSED TRANSLATION OF PCAF MRNA. FUNCTIONAL MANIPULATION OF MIR-181A/B CAUSED RECIPROCAL ALTERATIONS IN PCAF PROTEIN EXPRESSION IN CULTURED CHOLANGIOCYTES AND HEPATOCYTES. INHIBITION OF MIR-181A/B FUNCTION WITH ANTI-MIRS BLOCKED TNF-ALPHA-INDUCED SUPPRESSION OF PCAF EXPRESSION. PROMOTER RECRUITMENT OF PCAF WAS SHOWN TO BE ASSOCIATED WITH TNF-ALPHA-INDUCED TRANSCRIPTION OF INFLAMMATORY GENES. INTRIGUINGLY, PRETREATMENT OF CELLS WITH TNF-ALPHA INHIBITED TRANSCRIPTION OF INFLAMMATORY GENES IN RESPONSE TO SUBSEQUENT TNF-ALPHA STIMULATION. OVEREXPRESSION OF PCAF OR INHIBITION OF MIR-181A/B FUNCTION WITH ANTI-MIRS ATTENUATED THE INHIBITORY EFFECTS OF TNF-ALPHA PRETREATMENT ON EPITHELIAL INFLAMMATORY RESPONSE TO SUBSEQUENT TNF-ALPHA STIMULATION. DOWNREGULATION OF PCAF AND THE INHIBITORY EFFECTS OF TNF-ALPHA PRETREATMENT ON LIVER EPITHELIAL INFLAMMATORY RESPONSE WERE FURTHER CONFIRMED IN A MOUSE MODEL OF TNF-ALPHA I.P. INJECTION. THESE DATA SUGGEST THAT PCAF IS A TARGET FOR MIR-181A/B, AND DOWNREGULATION OF PCAF BY TNF-ALPHA PROVIDES NEGATIVE FEEDBACK REGULATION TO INFLAMMATORY REACTIONS IN LIVER EPITHELIAL CELLS, A PROCESS THAT MAY BE RELEVANT TO THE EPIGENETIC FINE-TUNING OF EPITHELIAL INFLAMMATORY PROCESSES IN GENERAL. 2012 15 5908 23 TARGETED DE-METHYLATION OF THE FOXP3-TSDR IS SUFFICIENT TO INDUCE PHYSIOLOGICAL FOXP3 EXPRESSION BUT NOT A FUNCTIONAL TREG PHENOTYPE. CD4+ REGULATORY T CELLS (TREGS) ARE KEY MEDIATORS OF IMMUNOLOGICAL TOLERANCE AND PROMISING EFFECTOR CELLS FOR IMMUNO-SUPPRESSIVE ADOPTIVE CELLULAR THERAPY TO FIGHT AUTOIMMUNITY AND CHRONIC INFLAMMATION. THEIR FUNCTIONAL STABILITY IS CRITICAL FOR THEIR CLINICAL UTILITY AND HAS BEEN CORRELATED TO THE DEMETHYLATED STATE OF THE TSDR/CNS2 ENHANCER ELEMENT IN THE TREG LINEAGE TRANSCRIPTION FACTOR FOXP3. HOWEVER, PROOF FOR A CAUSAL CONTRIBUTION OF THE TSDR DE-METHYLATION TO FOXP3 STABILITY AND TREG INDUCTION IS SO FAR LACKING. WE HERE ESTABLISHED A POWERFUL TRANSIENT-TRANSFECTION CRISPR-CAS9-BASED EPIGENETIC EDITING METHOD FOR THE SELECTIVE DE-METHYLATION OF THE TSDR WITHIN THE ENDOGENOUS CHROMATIN ENVIRONMENT OF A LIVING CELL. THE INDUCED DE-METHYLATED STATE WAS STABLE OVER WEEKS IN CLONAL T CELL PROLIFERATION CULTURES EVEN AFTER EXPRESSION OF THE EDITING COMPLEX HAD CEASED. EPIGENETIC EDITING OF THE TSDR RESULTED IN FOXP3 EXPRESSION, EVEN IN ITS PHYSIOLOGICAL ISOFORM DISTRIBUTION, PROVING A CAUSAL ROLE FOR THE DE-METHYLATED TSDR IN FOXP3 REGULATION. HOWEVER, SUCCESSFUL FOXP3 INDUCTION WAS NOT ASSOCIATED WITH A SWITCH TOWARDS A FUNCTIONAL TREG PHENOTYPE, IN CONTRAST TO WHAT HAS BEEN REPORTED FROM FOXP3 OVEREXPRESSION APPROACHES. THUS, TSDR DE-METHYLATION IS REQUIRED, BUT NOT SUFFICIENT FOR A STABLE TREG PHENOTYPE INDUCTION. THEREFORE, TARGETED DEMETHYLATION OF THE TSDR MAY BE A CRITICAL ADDITION TO PUBLISHED IN VITRO TREG INDUCTION PROTOCOLS WHICH SO FAR LACK FOXP3 STABILITY. 2020 16 5865 30 SUPPRESSION OF HDAC2 IN SPINAL CORD ALLEVIATES MECHANICAL HYPERALGESIA AND RESTORES KCC2 EXPRESSION IN A RAT MODEL OF BONE CANCER PAIN. EPIGENETIC MODULATION PARTICIPATES IN THE MECHANISM OF MULTIPLE TYPES OF PATHOLOGICAL PAIN, SO TARGETING THE INVOLVED REGULATORS MAY BE A PROMISING STRATEGY FOR PAIN TREATMENT. OUR PREVIOUS RESEARCH IDENTIFIED THE ANALGESIC EFFECT OF THE HISTONE DEACETYLASE (HDAC) INHIBITOR TRICHOSTATIN A (TSA) ON MECHANICAL HYPERALGESIA IN A RAT MODEL OF BONE CANCER PAIN (BCP) VIA RESTORATION OF MU-OPIOID RECEPTOR (MOR) EXPRESSION. HOWEVER, THE SPECIFIC TYPES OF HDACS CONTRIBUTING TO BCP HAVE NOT BEEN EXPLORED. THE PRESENT STUDY INVESTIGATED THE EXPRESSION PATTERN OF SOME COMMON HDACS AND FOUND THAT HDAC2 WAS UP-REGULATED IN A TIME-DEPENDENT MANNER IN THE LUMBAR SPINAL CORD OF BCP RATS. TSA APPLICATION SUPPRESSED HDAC2 EXPRESSION IN CULTURED PC12 CELLS AND REVERSED THE AUGMENTED HDAC2 IN BCP RATS. AN RNA-INTERFERING STRATEGY CONFIRMED THE ESSENTIAL ROLE OF HDAC2 IN THE MODULATION OF MECHANICAL HYPERALGESIA FOLLOWING TUMOR CELL INOCULATION, AND WE FURTHER EXAMINED ITS POSSIBLE DOWNSTREAM TARGETS. NOTABLY, HDAC2 KNOCK-DOWN DID NOT RESTORE MOR EXPRESSION, BUT IT ROBUSTLY REVERSED THE DOWN-REGULATION OF POTASSIUM-CHLORIDE COTRANSPORTER 2 (KCC2). THE IMPAIRED KCC2 EXPRESSION IS A VITAL MECHANISM OF MANY TYPES OF PATHOLOGICAL PAIN. THEREFORE, OUR RESULTS DEMONSTRATED THAT HDAC2 IN SPINAL CORD CONTRIBUTED TO THE MECHANICAL HYPERALGESIA IN BCP RATS, AND THIS EFFECT MAY BE ASSOCIATED WITH KCC2 MODULATION. 2018 17 826 34 CHARACTERIZATION OF K562 CELLS: UNCOVERING NOVEL CHROMOSOMES, ASSESSING TRANSFERRIN RECEPTOR EXPRESSION, AND PROBING PHARMACOLOGICAL THERAPIES. HUMAN ERYTHROLEUKEMIC K562 CELLS REPRESENT THE PROTOTYPICAL CELL CULTURE MODEL OF CHRONIC MYELOID LEUKEMIA (CML). THE CELLS ARE PSEUDO-TRIPLOID AND POSITIVE FOR THE PHILADELPHIA CHROMOSOME. THEREFORE, K562 CELLS HAVE BEEN WIDELY USED FOR INVESTIGATING THE BCR/ABL1 ONCOGENE AND THE TYROSINE KINASE INHIBITOR, IMATINIB-MESYLATE. FURTHER, K562 CELLS OVEREXPRESS TRANSFERRIN RECEPTORS (TFR) AND HAVE BEEN USED AS A MODEL FOR TARGETING CYTOTOXIC THERAPIES, VIA RECEPTOR-MEDIATED ENDOCYTOSIS. HERE, WE HAVE CHARACTERIZED K562 CELLS FOCUSING ON THE KARYOTYPE OF CELLS IN PROLONGED CULTURE, REGULATION OF EXPRESSION OF TFR IN WILDTYPE (WT) AND DOXORUBICIN-RESISTANT CELLS, AND RESPONSES TO HISTONE DEACETYLASE INHIBITION (HDACI). KARYOTYPE ANALYSIS INDICATES NOVEL CHROMOSOMES AND GENE EXPRESSION ANALYSIS SUGGESTS A SHIFT OF CULTURED K562 CELLS AWAY FROM PATIENT-DERIVED LEUKEMIC CELLS. WE CONFIRM THE HIGH EXPRESSION OF TFR ON K562 CELLS USING IMMUNOFLUORESCENCE AND CELL-SURFACE RECEPTOR BINDING RADIOASSAYS. IMPORTANTLY, HIGH TFR EXPRESSION IS OBSERVED IN PATIENT-DERIVED CELLS, AND WE HIGHLIGHT THE PERSISTENT EXPRESSION OF TFR FOLLOWING DOXORUBICIN ACQUIRED RESISTANCE. EPIGENETIC ANALYSIS INDICATES THAT PERMISSIVE HISTONE ACETYLATION AND METHYLATION AT THE PROMOTER REGION REGULATES THE TRANSCRIPTION OF TFR IN K562 CELLS. FINALLY, WE SHOW RELATIVELY HIGH EXPRESSION OF HDAC ENZYMES IN K562 CELLS AND DEMONSTRATE THE CHEMOTOXIC EFFECTS OF HDACI, USING THE FDA-APPROVED HYDROXAMIC ACID, VORINOSTAT. TOGETHER WITH A DESCRIPTION OF MORPHOLOGY, INFRARED SPECTRAL ANALYSIS, AND EXAMINATION OF METABOLIC PROPERTIES, WE PROVIDE A COMPREHENSIVE CHARACTERIZATION OF K562 CELLS. OVERALL, K562 CELL CULTURE SYSTEMS REMAIN WIDELY USED FOR THE INVESTIGATION OF NOVEL THERAPEUTICS FOR CML, WHICH IS PARTICULARLY IMPORTANT IN CASES OF IMATINIB-MESYLATE RESISTANCE. 2023 18 3082 40 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 19 2353 33 EPIGENETIC REGULATION OF OPIOID-INDUCED HYPERALGESIA, DEPENDENCE, AND TOLERANCE IN MICE. REPEATED ADMINISTRATION OF OPIOIDS SUCH AS MORPHINE INDUCES PERSISTENT BEHAVIORAL CHANGES INCLUDING OPIOID-INDUCED HYPERALGESIA (OIH), TOLERANCE, AND PHYSICAL DEPENDENCE. IN THE CURRENT WORK WE EXPLORED HOW THE BALANCE OF HISTONE ACETYLTRANSFERASE (HAT) VERSUS HISTONE DEACETYLASE (HDAC) MIGHT REGULATE THESE MORPHINE-INDUCED CHANGES. NOCICEPTIVE THRESHOLDS, ANALGESIA, AND PHYSICAL DEPENDENCE WERE ASSESSED DURING AND FOR A PERIOD OF SEVERAL WEEKS AFTER MORPHINE EXPOSURE. TO PROBE THE ROLES OF HISTONE ACETYLATION, THE HAT INHIBITOR CURCUMIN OR A SELECTIVE HDAC INHIBITOR SUBEROYLANILIDE HYDROXAMIC ACID (SAHA) WAS ADMINISTERED DAILY TO GROUPS OF ANIMALS. HISTONE ACETYLATION IN SPINAL CORD WAS ASSESSED BY WESTERN BLOT AND IMMUNOHISTOCHEMISTRY. CONCURRENT ADMINISTRATION OF CURCUMIN WITH MORPHINE FOR 4 DAYS SIGNIFICANTLY REDUCED DEVELOPMENT OF OPIOID-INDUCED MECHANICAL ALLODYNIA, THERMAL HYPERALGESIA, TOLERANCE, AND PHYSICAL DEPENDENCE. CONVERSELY, THE HDAC INHIBITOR SAHA ENHANCED THESE RESPONSES. INTERESTINGLY, SAHA TREATMENT AFTER THE TERMINATION OF OPIOID ADMINISTRATION SUSTAINED THESE BEHAVIORAL CHANGES FOR AT LEAST 4 WEEKS. HISTONE H3 ACETYLATION IN THE DORSAL HORN OF THE SPINAL CORD WAS INCREASED AFTER CHRONIC MORPHINE TREATMENT, BUT H4 ACETYLATION WAS UNCHANGED. MOREOVER, WE OBSERVED A DECREASE IN HDAC ACTIVITY IN THE SPINAL CORDS OF MORPHINE-TREATED MICE WHILE OVERALL HAT ACTIVITY WAS UNCHANGED, SUGGESTING A SHIFT TOWARD A STATE OF ENHANCED HISTONE ACETYLATION. PERSPECTIVE: THE CURRENT STUDY INDICATES THAT EPIGENETIC MECHANISMS PLAY A CRUCIAL ROLE IN OPIOID-INDUCED LONG-LASTING NEUROPLASTICITY. THESE RESULTS PROVIDE NEW SIGHT INTO UNDERSTANDING THE MECHANISMS OF OPIOID-INDUCED NEUROPLASTICITY AND SUGGEST NEW STRATEGIES TO LIMIT OPIOID ABUSE POTENTIAL AND INCREASE THE VALUE OF THESE DRUGS AS ANALGESICS. 2013 20 3832 28 INVOLVEMENT OF SPINAL SIRT1 IN DEVELOPMENT OF CHRONIC CONSTRICTION INJURY INDUCED NEUROPATHIC PAIN IN RATS. IT IS KNOWN THAT THE EPIGENETIC PROCESS OF HISTONE ACETYLATION IS INVOLVED IN THE NEUROPATHIC PAIN. THE AIM OF THIS STUDY WAS TO DETERMINE WHETHER SIRTUIN TYPE 1 (SIRT1), AN NAD(+) DEPENDENT DEACETYLASE, AFFECTED ALLODYNIA AND HYPERALGESIA IN NEUROPATHIC PAIN. THE NEUROPATHIC PAIN MODEL WAS ESTABLISHED BY LIGATURE OF THE RIGHT SCIATIC NERVE TO INDUCE CHRONIC CONSTRICTION INJURY (CCI) IN RATS. HISTONE ACETYLTRANSFERASE (HAT) ACTIVITY WAS INCREASED AND, AND HISTONE DEACETYLASE (HDAC) ACTIVITY WAS DECLINED IN TISSUE OF THE SPINAL DORSA HORN IN CCI RATES BY MEANS OF ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA). THE PERSISTENT HYPERALGESIA AND ALLODYNIA CAUSED BY CCI WERE ASSOCIATED WITH DOWNREGULATION OF SIRT1 AND UPREGULATION OF ACETYLATED-H3 (AC-H3) IN TISSUE OF THE SPINAL CORD BY WESTERN BLOT ASSAY, WHICH WAS REVERSED AFTER INTRATHECAL INJECTION OF SIRT1 AGONIST SRT1720. SRT1720 TREATMENT ACHIEVED ANALGESIC THROUGH INHIBITING THE ACETYLATION OF NUCLEAR FACTOR KAPPA B (NF-KAPPAB) AND BLOCKING THE RELEASES OF THE INFLAMMATORY FACTORS INCLUDING TUMOR NECROSIS FACTOR-ALPHA (TNF-ALPHA) AND INTERLEUKIN (IL)-6 BY MEANS OF WESTERN BLOT AND REAL-TIME QUANTITATIVE PCR (RT-PCR), RESPECTIVELY. TAKEN TOGETHER, THESE DATA SUGGEST THAT SIRT1 IN THE SPINAL CORD PLAYS AN IMPORTANT ROLE IN THE NEUROPATHIC PAIN IN THE RAT MODEL. 2018