1 5976 108 TET1-DEPENDENT EPIGENETIC MODIFICATION OF BDNF EXPRESSION IN DORSAL HORN NEURONS MEDIATES NEUROPATHIC PAIN IN RATS. TEN-ELEVEN TRANSLOCATION METHYLCYTOSINE DIOXYGENASE 1 (TET1) MEDIATES THE CONVERSION OF 5-METHYLCYTOSINE (5 MC) TO 5-HYDROXYMETHYLCYTOSINE (5 HMC), HENCE PROMOTING DNA DEMETHYLATION. ALTHOUGH RECENT STUDIES HAVE LINKED THE DNA DEMETHYLATION OF SPECIFIC GENES TO PAIN HYPERSENSITIVITY, THE ROLE OF SPINAL TET1-DEPENDENT DNA DEMETHYLATION IN NOCICEPTION HYPERSENSITIVITY DEVELOPMENT REMAINS ELUSIVE. HERE, WE REPORT CORRELATED WITH BEHAVIORAL ALLODYNIA, SPINAL NERVE LIGATION (SNL) UPREGULATED TET1 EXPRESSION IN DORSAL HORN NEURONS THAT HYDROXYLATE 5 MC TO 5 HMC AT CPG DINUCLEOTIDES IN THE BDNF PROMOTER TO PROMOTE SPINAL BDNF EXPRESSION AT DAY 7 AFTER OPERATION. FOCAL KNOCKDOWN OF SPINAL TET1 EXPRESSION DECREASED TET1 BINDING AND 5 HMC ENRICHMENT, FURTHER INCREASED 5 MC ENRICHMENT AT CPG SITES IN THE BDNF PROMOTER AND DECREASED SPINAL BDNF EXPRESSION ACCOMPANIED BY THE ALLEVIATION OF THE DEVELOPED ALLODYNIA. MOREOVER, AT DAY 7 AFTER OPERATION, SNL-ENHANCED TET1 EXPRESSION ALSO INHIBITED THE BINDING OF DNA METHYLTRANSFERASES (DNMTS, I.E., DNMT1, DNMT3A, AND DNMT3B) TO THE BDNF PROMOTER, A REQUIREMENT FOR TRANSCRIPTIONAL SILENCING BY CATALYSING 5-CYTOSINE (5C) TO 5 MC. TOGETHER, THESE DATA SUGGEST AT CPG SITES OF THE BDNF PROMOTER, SNL-ENHANCED TET1 EXPRESSION PROMOTES DNA DEMETHYLATION BOTH BY CONVERTING 5 MC TO 5 HMC AND INHIBITING DNMT BINDING TO REGULATE SPINAL BDNF EXPRESSION, HENCE CONTRIBUTING TO BEHAVIORAL ALLODYNIA DEVELOPMENT. 2016 2 21 24 5-HYDROXYMETHYLCYTOSINE (5HMC) AND TEN-ELEVEN TRANSLOCATION 1-3 (TET1-3) PROTEINS IN THE DORSAL ROOT GANGLIA OF MOUSE: EXPRESSION AND DYNAMIC REGULATION IN NEUROPATHIC PAIN. EPIGENETIC MECHANISMS ARE INCREASINGLY IMPLICATED IN CHRONIC PAIN PATHOLOGY. IN THIS STUDY, WE DEMONSTRATE THAT THE NOVEL EPIGENETIC MARK 5-HYDROXYMETHYLCYTOSINE (5HMC) IS PRESENT IN DORSAL ROOT GANGLIA (DRG) NEURONS AND GLIA, AND ITS LEVELS INCREASE FOLLOWING NERVE INJURY. FURTHERMORE, WE SHOW THAT THE 5HMC-GENERATING TEN-ELEVEN TRANSLOCATION 1-3 (TET1-3) PROTEINS ARE EXPRESSED IN A CELL-TYPE SPECIFIC MANNER IN THE DRG, WITH TET3 DISPLAYING DIFFERENTIAL UPREGULATION AFTER INJURY, SUGGESTING A POTENTIAL ROLE IN NEUROPATHIC PAIN. 2017 3 1813 29 EFFECTS OF CAFFEIC ACID ON EPIGENETICS IN THE BRAIN OF RATS WITH CHRONIC UNPREDICTABLE MILD STRESS. THE PRESENT STUDY HYPOTHESIZED THAT CAFFEIC ACID (3,4?DIHYDROXYCINNAMIC ACID; CAA) MAY EXERT ANTIDEPRESSANT?LIKE EFFECTS IN RATS WITH CHRONIC UNPREDICTABLE MILD STRESS VIA EPIGENETIC MECHANISMS, SUCH AS DNA METHYLATION AND HYDROXYMETHYLATION. THE CHRONIC UNPREDICTABLE MILD STRESS (CUMS) MODEL WAS USED TO ANALYZE THE EFFECTS OF CAA ON BEHAVIORAL PHENOTYPES, AND TO EVALUATE THE DISTRIBUTION OF 5?METHYLCYTOSINE (5MC) AND 5?HYDROXYMETHYLCYTOSINE (5HMC) IN THE HIPPOCAMPUS AND PREFRONTAL CORTEX USING IMMUNOHISTOCHEMISTRY AND IMMUNOFLUORESCENCE. MRNA LEVELS OF THE GENES ENCODING BRAIN?DERIVED NEUROTROPIC FACTOR (BDNF) AND CATECHOL?O?METHYLTRANSFERASE (COMT), AND KEY ENZYMES REGULATING DNA METHYLATION [DNA METHYLTRANSFERASE (DNMT)1 AND DNMT3A] AND HYDROXYMETHYLATION [TEN?ELEVEN TRANSLOCATION (TET)1?3] WERE EXAMINED USING QUANTITATIVE (Q)PCR. FURTHERMORE, ENRICHMENT OF 5MC AND 5HMC AT THE PROMOTOR REGIONS OF THE BDNF AND COMT GENES WAS QUANTIFIED USING CHROMATIN IMMUNOPRECIPITATION?QPCR. BEHAVIORAL DATA SHOWED THAT CAA EXERTED A SLIGHT ANTIDEPRESSANT?LIKE EFFECT. BDNF AND COMT GENES SHOWED DIFFERENTIAL EXPRESSION PATTERNS DUE TO CUMS. CAA INTERVENTION INDUCED DIFFERENT DNMT1/DNMT3A AND TET1/TET2 MRNA LEVELS IN THE HIPPOCAMPUS AND PREFRONTAL CORTEX, RESPECTIVELY. CAA REGULATED THE RATIO OF 5MC/5HMC AT THE PROMOTOR REGION OF THE BDNF AND COMT GENES AND THEREFORE INFLUENCED GENE EXPRESSION, WHICH MAY BE A VALUABLE THERAPEUTIC OPTION FOR MAJOR DEPRESSIVE DISORDER (MDD). IN CONCLUSION, THERE WERE EPIGENETIC CHANGES IN THE HIPPOCAMPUS AND PREFRONTAL CORTEX IN CUMS RATS, AND CAA MAY FUNCTION AS A MODULATOR OF DNA METHYLATION TO REGULATE GENE TRANSCRIPTION, THUS PROVIDING A MECHANISTIC BASIS FOR THE USE OF THIS PHYTOCHEMICAL AGENT IN THE TREATMENT OF MDD. 2020 4 3841 24 IRON SUPPLEMENTATION REVERSES THE REDUCTION OF HYDROXYMETHYLCYTOSINE IN HEPATIC DNA ASSOCIATED WITH CHRONIC ALCOHOL CONSUMPTION IN RATS. BACKGROUND: ALCOHOL IS KNOWN TO AFFECT TWO EPIGENETIC PHENOMENA, DNA METHYLATION AND DNA HYDROXYMETHYLATION, AND IRON IS A COFACTOR OF TEN-ELEVEN TRANSLOCATION (TET) ENZYMES THAT CATALYZE THE CONVERSION FROM METHYLCYTOSINE TO HYDROXYMETHYLCYTOSINE. IN THE PRESENT STUDY WE AIMED TO DETERMINE THE EFFECTS OF ALCOHOL ON DNA HYDROXYMETHYLATION AND FURTHER EFFECTS OF IRON ON ALCOHOL ASSOCIATED EPIGENETIC CHANGES. METHODS: TWENTY-FOUR MALE SPRAGUE-DAWLEY RATS WERE FED EITHER LIEBER-DECARLI ALCOHOL DIET (36% CALORIES FROM ETHANOL) OR LIEBER-DECARLI CONTROL DIET ALONG WITH OR WITHOUT IRON SUPPLEMENTATION (0.6% CARBONYL IRON) FOR 8 WEEKS. HEPATIC NON-HEME IRON CONCENTRATIONS WERE MEASURED BY COLORIMETRIC ASSAYS. PROTEIN LEVELS OF HEPATIC FERRITIN AND TRANSFERRIN RECEPTOR WERE DETERMINED BY WESTERN BLOTTING. METHYLCYTOSINE, HYDROXYMETHYLCYTOSINE AND UNMODIFIED CYTOSINE IN DNA WERE SIMULTANEOUSLY MEASURED BY LIQUID CHROMATOGRAPHY/MASS SPECTROMETRY METHOD. RESULTS: IRON SUPPLEMENTATION SIGNIFICANTLY INCREASED HEPATIC NON-HEME IRON CONTENTS (P < 0.05) BUT ALCOHOL ALONE DID NOT. HOWEVER, BOTH ALCOHOL AND IRON SIGNIFICANTLY INCREASED HEPATIC FERRITIN LEVELS AND DECREASED HEPATIC TRANSFERRIN RECEPTOR LEVELS (P < 0.05). ALCOHOL REDUCED HEPATIC DNA HYDROXYMETHYLATION (0.21% +/- 0.04% VS. 0.33% +/- 0.04%, P = 0.01) COMPARED TO CONTROL, WHILE IRON SUPPLEMENTATION TO ALCOHOL DIET DID NOT CHANGE DNA HYDROXYMETHYLATION. THERE WAS NO SIGNIFICANT DIFFERENCE IN METHYLCYTOSINE LEVELS, WHILE UNMODIFIED CYTOSINE LEVELS WERE SIGNIFICANTLY INCREASED IN ALCOHOL-FED GROUPS COMPARED TO CONTROL (95.61% +/- 0.08% VS. 95.26% +/- 0.12%, P = 0.03), SUGGESTING THAT ALCOHOL FURTHER INCREASES THE CONVERSION FROM HYDROXYMETHYLCYTOSINE TO UNMODIFIED CYTOSINE. CONCLUSIONS: CHRONIC ALCOHOL CONSUMPTION ALTERS GLOBAL DNA HYDROXYMETHYLATION IN THE LIVER BUT IRON SUPPLEMENTATION REVERSES THE EPIGENETIC EFFECT OF ALCOHOL. 2016 5 3433 40 HYDROXYMETHYLATION OF MICRORNA-365-3P REGULATES NOCICEPTIVE BEHAVIORS VIA KCNH2. DNA 5-HYDROXYLMETHYLCYTOSINE (5HMC) CATALYZED BY TEN-ELEVEN TRANSLOCATION METHYLCYTOSINE DIOXYGENASE (TET) OCCURS ABUNDANTLY IN NEURONS OF MAMMALS. HOWEVER, THE IN VIVO CAUSAL LINK BETWEEN TET DYSREGULATION AND NOCICEPTIVE MODULATION HAS NOT BEEN ESTABLISHED. HERE, WE FOUND THAT SPINAL TET1 AND TET3 WERE SIGNIFICANTLY INCREASED IN THE MODEL OF FORMALIN-INDUCED ACUTE INFLAMMATORY PAIN, WHICH WAS ACCOMPANIED WITH THE AUGMENT OF GENOME-WIDE 5HMC CONTENT IN SPINAL CORD. KNOCKDOWN OF SPINAL TET1 OR TET3 ALLEVIATED THE FORMALIN-INDUCED NOCICEPTIVE BEHAVIOR AND OVEREXPRESSION OF SPINAL TET1 OR TET3 IN NAIVE MICE PRODUCED PAIN-LIKE BEHAVIOR AS EVIDENCED BY DECREASED THERMAL PAIN THRESHOLD. FURTHERMORE, WE FOUND THAT TET1 OR TET3 REGULATED THE NOCICEPTIVE BEHAVIOR BY TARGETING MICRORNA-365-3P (MIR-365-3P). FORMALIN INCREASED 5HMC IN THE MIR-365-3P PROMOTER, WHICH WAS INHIBITED BY KNOCKDOWN OF TET1 OR TET3 AND MIMICKED BY OVEREXPRESSION OF TET1 OR TET3 IN NAIVE MICE. NOCICEPTIVE BEHAVIOR INDUCED BY FORMALIN OR OVEREXPRESSION OF SPINAL TET1 OR TET3 COULD BE PREVENTED BY DOWNREGULATION OF MIR-365-3P, AND MIMICKED BY OVEREXPRESSION OF SPINAL MIR-365-3P. FINALLY, WE DEMONSTRATED THAT A POTASSIUM CHANNEL, VOLTAGE-GATED EAG-RELATED SUBFAMILY H MEMBER 2 (KCNH2), VALIDATED AS A TARGET OF MIR-365-3P, PLAYED A CRITICAL ROLE IN NOCICEPTIVE MODULATION BY SPINAL TET OR MIR-365-3P. TOGETHER, WE CONCLUDED THAT TET-MEDIATED HYDROXYMETHYLATION OF MIR-365-3P REGULATES NOCICEPTIVE BEHAVIOR VIA KCNH2. SIGNIFICANCE STATEMENT: MOUNTING EVIDENCE INDICATES THAT EPIGENETIC MODIFICATIONS IN THE NOCICEPTIVE PATHWAY CONTRIBUTE TO PAIN PROCESSES AND ANALGESIA RESPONSE. HERE, WE FOUND THAT THE INCREASE OF 5HMC CONTENT MEDIATED BY TET1 OR TET3 IN MIR-365-3P PROMOTER IN THE SPINAL CORD IS INVOLVED IN NOCICEPTIVE MODULATION THROUGH TARGETING A POTASSIUM CHANNEL, KCNH2. OUR STUDY REVEALS A NEW EPIGENETIC MECHANISM UNDERLYING NOCICEPTIVE INFORMATION PROCESSING, WHICH MAY BE A NOVEL TARGET FOR DEVELOPMENT OF ANTINOCICEPTIVE DRUGS. 2016 6 4212 33 METHAMPHETAMINE DOWNREGULATES STRIATAL GLUTAMATE RECEPTORS VIA DIVERSE EPIGENETIC MECHANISMS. BACKGROUND: CHRONIC METHAMPHETAMINE (METH) EXPOSURE CAUSES NEUROADAPTATIONS AT GLUTAMATERGIC SYNAPSES. METHODS: TO IDENTIFY THE METH-INDUCED EPIGENETIC UNDERPINNINGS OF THESE NEUROADAPTATIONS, WE INJECTED INCREASING METH DOSES TO RATS FOR 2 WEEKS AND MEASURED STRIATAL GLUTAMATE RECEPTOR EXPRESSION. WE THEN QUANTIFIED THE EFFECTS OF METH EXPOSURE ON HISTONE ACETYLATION. WE ALSO MEASURED METH-INDUCED CHANGES IN DNA METHYLATION AND DNA HYDROXYMETHYLATION. RESULTS: CHRONIC METH DECREASED TRANSCRIPT AND PROTEIN EXPRESSION OF GLUA1 AND GLUA2 ALPHA-AMINO-3-HYDROXY-5-METHYL-4-ISOXAZOLE PROPIONIC ACID RECEPTOR (AMPAR) AND GLUN1 N-METHYL-D-ASPARTATE RECEPTOR SUBUNITS. THESE CHANGES WERE ASSOCIATED WITH ALTERED ELECTROPHYSIOLOGICAL GLUTAMATERGIC RESPONSES IN STRIATAL NEURONS. CHROMATIN IMMUNOPRECIPITATION-POLYMERASE CHAIN REACTION REVEALED THAT METH DECREASED ENRICHMENT OF ACETYLATED HISTONE H4 ON GLUA1, GLUA2, AND GLUN1 PROMOTERS. METHAMPHETAMINE EXPOSURE ALSO INCREASED REPRESSOR ELEMENT-1 SILENCING TRANSCRIPTION FACTOR (REST) COREPRESSOR 1, METHYLATED CPG BINDING PROTEIN 2, AND HISTONE DEACETYLASE 2 ENRICHMENT, BUT NOT OF SIRTUIN 1 OR SIRTUIN 2, ONTO GLUA1 AND GLUA2 GENE SEQUENCES. MOREOVER, METH CAUSED INTERACTIONS OF REST COREPRESSOR 1 AND METHYLATED CPG BINDING PROTEIN 2 WITH HISTONE DEACETYLASE 2 AND OF REST WITH HISTONE DEACETYLASE 1. SURPRISINGLY, METHYLATED DNA IMMUNOPRECIPITATION AND HYDROXYMETHYLATED DNA IMMUNOPRECIPITATION-POLYMERASE CHAIN REACTION REVEALED METH-INDUCED DECREASED ENRICHMENT OF 5-METHYLCYTOSINE AND 5-HYDROXYMETHYLCYTOSINE AT GLUA1 AND GLUA2 PROMOTER SEQUENCES. IMPORTANTLY, THE HISTONE DEACETYLASE INHIBITOR, VALPROIC ACID, BLOCKED METH-INDUCED DECREASED EXPRESSION OF AMPAR AND N-METHYL-D-ASPARTATE RECEPTOR SUBUNITS. FINALLY, VALPROIC ACID ALSO ATTENUATED METH-INDUCED DECREASE H4K16AC RECRUITMENT ON AMPAR GENE SEQUENCES. CONCLUSIONS: THESE OBSERVATIONS SUGGEST THAT HISTONE H4 HYPOACETYLATION MAY BE THE MAIN DETERMINANT OF METH-INDUCED DECREASED STRIATAL GLUTAMATE RECEPTOR EXPRESSION. 2014 7 1492 36 DNA HYDROXYMETHYLATION BY TEN-ELEVEN TRANSLOCATION METHYLCYTOSINE DIOXYGENASE 1 AND 3 REGULATES NOCICEPTIVE SENSITIZATION IN A CHRONIC INFLAMMATORY PAIN MODEL. BACKGROUND: TEN-ELEVEN TRANSLOCATION METHYLCYTOSINE DIOXYGENASE CONVERTS 5-METHYLCYTOSINE IN DNA TO 5-HYDROXYMETHYLCYTOSINE, WHICH PLAYS AN IMPORTANT ROLE IN GENE TRANSCRIPTION. ALTHOUGH 5-HYDROXYMETHYLCYTOSINE IS ENRICHED IN MAMMALIAN NEURONS, ITS REGULATORY FUNCTION IN NOCICEPTIVE INFORMATION PROCESSING IS UNKNOWN. METHODS: THE GLOBAL LEVELS OF 5-HYDROXYMETHYLCYTOSINE AND TEN-ELEVEN TRANSLOCATION METHYLCYTOSINE DIOXYGENASE WERE MEASURED IN SPINAL CORDS IN MICE TREATED WITH COMPLETE FREUND'S ADJUVANT. IMMUNOBLOTTING, IMMUNOHISTOCHEMISTRY, AND BEHAVIORAL TESTS WERE USED TO EXPLORE THE DOWNSTREAM TEN-ELEVEN TRANSLOCATION METHYLCYTOSINE DIOXYGENASE-DEPENDENT SIGNALING PATHWAY. RESULTS: COMPLETE FREUND'S ADJUVANT-INDUCED NOCICEPTION INCREASED THE MEAN LEVELS (+/- SD) OF SPINAL 5-HYDROXYMETHYLCYTOSINE (178 +/- 34 VS. 100 +/- 21; P = 0.0019), TEN-ELEVEN TRANSLOCATION METHYLCYTOSINE DIOXYGENASE-1 (0.52 +/- 0.11 VS. 0.36 +/- 0.064; P = 0.0088), AND TEN-ELEVEN TRANSLOCATION METHYLCYTOSINE DIOXYGENASE-3 (0.61 +/- 0.13 VS. 0.39 +/- 0.08; P = 0.0083) COMPARED WITH LEVELS IN CONTROL MICE (N = 6/GROUP). THE KNOCKDOWN OF TEN-ELEVEN TRANSLOCATION METHYLCYTOSINE DIOXYGENASE-1 OR TEN-ELEVEN TRANSLOCATION METHYLCYTOSINE DIOXYGENASE-3 ALLEVIATED THERMAL HYPERALGESIA AND MECHANICAL ALLODYNIA, WHEREAS OVEREXPRESSION CYTOSINETHEM IN NAIVE MICE (N = 6/GROUP). DOWN-REGULATION OF SPINAL TEN-ELEVEN TRANSLOCATION METHYLCYTOSINE DIOXYGENASE-1 AND TEN-ELEVEN TRANSLOCATION METHYLCYTOSINE DIOXYGENASE-3 ALSO REVERSED THE INCREASES IN FOS EXPRESSION (123 +/- 26 VS. 294 +/- 6; P = 0.0031; AND 140 +/- 21 VS. 294 +/- 60; P = 0.0043, RESPECTIVELY; N = 6/GROUP), 5-HYDROXYMETHYLCYTOSINE LEVELS IN THE STAT3 PROMOTER (75 +/- 16.1 VS. 156 +/- 28.9; P = 0.0043; AND 91 +/- 19.1 VS. 156 +/- 28.9; P = 0.0066, RESPECTIVELY; N = 5/GROUP), AND CONSEQUENT STAT3 EXPRESSION (93 +/- 19.6 VS. 137 +/- 27.5; P = 0.035; AND 72 +/- 15.2 VS. 137 +/- 27.5; P = 0.0028, RESPECTIVELY; N = 5/GROUP) IN COMPLETE FREUND'S ADJUVANT-TREATED MICE. CONCLUSIONS: THIS STUDY REVEALS A NOVEL EPIGENETIC MECHANISM FOR TEN-ELEVEN TRANSLOCATION METHYLCYTOSINE DIOXYGENASE-1 AND TEN-ELEVEN TRANSLOCATION METHYLCYTOSINE DIOXYGENASE-3 IN THE MODULATION OF SPINAL NOCICEPTIVE INFORMATION VIA TARGETING OF STAT3. 2017 8 434 37 ANTIDEPRESSANT-LIKE EFFECT OF SODIUM BUTYRATE IS ASSOCIATED WITH AN INCREASE IN TET1 AND IN 5-HYDROXYMETHYLATION LEVELS IN THE BDNF GENE. BACKGROUND: EPIGENETIC DRUGS LIKE SODIUM BUTYRATE (NAB) SHOW ANTIDEPRESSANT-LIKE EFFECTS IN PRECLINICAL STUDIES, BUT THE EXACT MOLECULAR MECHANISMS OF THE ANTIDEPRESSANT EFFECTS REMAIN UNKNOWN. WHILE RESEARCH USING NAB HAS MAINLY FOCUSED ON ITS ROLE AS A HISTONE DEACETYLASE INHIBITOR (HDACI), THERE IS ALSO EVIDENCE THAT NAB AFFECTS DNA METHYLATION. METHODS: THE PURPOSE OF THIS STUDY WAS TO EXAMINE NAB'S PUTATIVE ANTIDEPRESSANT-LIKE EFFICACY IN RELATION TO DNA METHYLATION CHANGES IN THE PREFRONTAL CORTEX OF AN ESTABLISHED GENETIC RAT MODEL OF DEPRESSION (THE FLINDERS SENSITIVE LINE [FSL]) AND ITS CONTROLS (THE FLINDERS RESISTANT LINE). RESULTS: THE FSL RATS HAD LOWER LEVELS OF TEN-ELEVEN TRANSLOCATION METHYLCYTOSINE DIOXYGENASE 1 (TET1), WHICH CATALYZES THE CONVERSION OF DNA METHYLATION TO HYDROXYMETHYLATION. AS INDICATED BY THE BEHAVIORAL DESPAIR TEST, CHRONIC ADMINISTRATION OF NAB HAD ANTIDEPRESSANT-LIKE EFFECTS IN THE FSL AND WAS ACCOMPANIED BY INCREASED LEVELS OF TET1. THE TET1 UPREGULATION WAS ALSO ASSOCIATED WITH AN INCREASE OF HYDROXYMETHYLATION AND A DECREASE OF METHYLATION IN BRAIN-DERIVED NEUROTROPHIC FACTOR (BDNF), A GENE ASSOCIATED WITH NEUROGENESIS AND SYNAPTIC PLASTICITY. THESE EPIGENETIC CHANGES WERE ASSOCIATED WITH A CORRESPONDING BDNF OVEREXPRESSION. CONCLUSIONS: OUR DATA SUPPORT THE ANTIDEPRESSANT EFFICACY OF HDACIS AND SUGGEST THAT THEIR EPIGENETIC EFFECTS MAY ALSO INCLUDE DNA METHYLATION CHANGES THAT ARE MEDIATED BY DEMETHYLATION-FACILITATING ENZYMES LIKE TET1. 2014 9 286 20 AGING AND ALCOHOL INTERACT TO ALTER HEPATIC DNA HYDROXYMETHYLATION. BACKGROUND: AGING AND CHRONIC ALCOHOL CONSUMPTION ARE BOTH MODIFIERS OF DNA METHYLATION, BUT IT IS NOT YET KNOWN WHETHER CHRONIC ALCOHOL CONSUMPTION ALSO ALTERS DNA HYDROXYMETHYLATION, A NEWLY DISCOVERED EPIGENETIC MARK PRODUCED BY OXIDATION OF METHYLCYTOSINE. FURTHERMORE, IT HAS NOT BEEN TESTED WHETHER AGING AND ALCOHOL INTERACT TO MODIFY THIS EPIGENETIC PHENOMENON, THEREBY HAVING AN INDEPENDENT EFFECT ON GENE EXPRESSION. METHODS: OLD (18 MONTHS) AND YOUNG (4 MONTHS) MALE C57BL/6 MICE WERE PAIR-FED EITHER A LIEBER-DECARLI LIQUID DIET WITH ALCOHOL (18% OF ENERGY) OR AN ISOCALORIC LIEBER-DECARLI CONTROL DIET FOR 5 WEEKS. GLOBAL DNA HYDROXYMETHYLATION AND DNA METHYLATION WERE ANALYZED FROM HEPATIC DNA USING A NEW LIQUID CHROMATOGRAPHY-TANDEM MASS SPECTROMETRY METHOD. HEPATIC MRNA EXPRESSION OF THE TET ENZYMES WERE MEASURED VIA QUANTITATIVE REAL-TIME POLYMERASE CHAIN REACTION. RESULTS: IN YOUNG MICE, MILD CHRONIC ALCOHOL EXPOSURE SIGNIFICANTLY REDUCED GLOBAL DNA HYDROXYMETHYLATION COMPARED WITH CONTROL MICE (0.22 +/- 0.01 VS. 0.29 +/- 0.06%, P = 0.004). ALCOHOL DID NOT SIGNIFICANTLY ALTER HYDROXYMETHYLCYTOSINE LEVELS IN OLD MICE. OLD MICE FED THE CONTROL DIET SHOWED DECREASED GLOBAL DNA HYDROXYMETHYLATION COMPARED WITH YOUNG MICE FED THE CONTROL DIET (0.24 +/- 0.02 VS. 0.29 +/- 0.06%, P = 0.04). THIS MODEL SUGGESTS AN INTERACTION BETWEEN AGING AND ALCOHOL IN DETERMINING DNA HYDROXYMETHYLATION (PINTERACTION = 0.009). EXPRESSION OF TET2 AND TET3 WAS DECREASED IN THE OLD MICE RELATIVE TO THE YOUNG (P < 0.005). CONCLUSIONS: THE OBSERVATION THAT ALCOHOL ALTERS DNA HYDROXYMETHYLATION INDICATES A NEW EPIGENETIC EFFECT OF ALCOHOL. THIS IS THE FIRST STUDY DEMONSTRATING THE INTERACTIVE EFFECTS OF CHRONIC ALCOHOL CONSUMPTION AND AGING ON DNA HYDROXYMETHYLATION. 2014 10 1875 29 EMERGING ROLE OF ONE-CARBON METABOLISM AND DNA METHYLATION ENRICHMENT ON DELTA-CONTAINING GABAA RECEPTOR EXPRESSION IN THE CEREBELLUM OF SUBJECTS WITH ALCOHOL USE DISORDERS (AUD). BACKGROUND: CEREBELLUM IS AN AREA OF THE BRAIN PARTICULARLY SENSITIVE TO THE EFFECTS OF ACUTE AND CHRONIC ALCOHOL CONSUMPTION. ALCOHOL EXPOSURE DECREASES CEREBELLAR PURKINJE CELL OUTPUT BY INCREASING GABA RELEASE FROM GOLGI CELLS ONTO EXTRASYNAPTIC ALPHA6/DELTA-CONTAINING GABAA RECEPTORS LOCATED ON GLUTAMATERGIC GRANULE CELLS. HERE, WE STUDIED WHETHER CHRONIC ALCOHOL CONSUMPTION INDUCES CHANGES IN GABAA RECEPTOR SUBUNIT EXPRESSION AND WHETHER THESE CHANGES ARE ASSOCIATED WITH ALTERATIONS IN EPIGENETIC MECHANISMS VIA DNA METHYLATION. METHODS: WE USED A COHORT OF POSTMORTEM CEREBELLUM FROM CONTROL AND CHRONIC ALCOHOLICS, HERE DEFINED AS ALCOHOL USE DISORDERS SUBJECTS (N=25/GROUP). S-ADENOSYL-METHIONINE/S-ADENOSYL-HOMOCYSTEINE WERE MEASURED BY HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY. MRNA LEVELS OF VARIOUS GENES WERE ASSESSED BY REVERSE TRANSCRIPTASE-QUANTITATIVE POLYMERASE CHAIN REACTION. PROMOTER METHYLATION ENRICHMENT WAS ASSESSED USING METHYLATED DNA IMMUNOPRECIPITATION AND HYDROXY-METHYLATED DNA IMMUNOPRECIPITATION ASSAYS. RESULTS: MRNAS ENCODING KEY ENZYMES OF 1-CARBON METABOLISM THAT DETERMINE THE S-ADENOSYL-METHIONINE/S-ADENOSYL-HOMOCYSTEINE RATIO WERE INCREASED, INDICATING HIGHER "METHYLATION INDEX" IN ALCOHOL USE DISORDER SUBJECTS. WE FOUND THAT INCREASED METHYLATION OF THE PROMOTER OF THE DELTA SUBUNIT GABAA RECEPTOR WAS ASSOCIATED WITH REDUCED MRNA AND PROTEIN LEVELS IN THE CEREBELLUM OF ALCOHOL USE DISORDER SUBJECTS. NO CHANGES WERE OBSERVED IN ALPHA1- OR ALPHA6-CONTAINING GABAA RECEPTOR SUBUNITS. THE EXPRESSION OF DNA-METHYLTRANSFERASES (1, 3A, AND 3B) WAS UNALTERED, WHEREAS THE MRNA LEVEL OF TET1, WHICH PARTICIPATES IN THE DNA DEMETHYLATION PATHWAY, WAS DECREASED. HENCE, INCREASED METHYLATION OF THE DELTA SUBUNIT GABAA RECEPTOR PROMOTER MAY RESULT FROM ALCOHOL-INDUCED REDUCTION OF DNA DEMETHYLATION. CONCLUSION: TOGETHER, THESE RESULTS SUPPORT THE HYPOTHESIS THAT ABERRANT DNA METHYLATION PATHWAYS MAY BE INVOLVED IN CEREBELLAR PATHOPHYSIOLOGY OF ALCOHOLISM. FURTHERMORE, THIS WORK PROVIDES NOVEL EVIDENCE FOR A CENTRAL ROLE OF DNA METHYLATION MECHANISMS IN THE ALCOHOL-INDUCED NEUROADAPTIVE CHANGES OF HUMAN CEREBELLAR GABAA RECEPTOR FUNCTION. 2017 11 2750 26 EXPRESSION LEVELS OF THE TYROSINE HYDROXYLASE GENE AND HISTONE MODIFICATIONS AROUND ITS PROMOTER IN THE LOCUS COERULEUS AND VENTRAL TEGMENTAL AREA OF RATS DURING FORCED ABSTINENCE FROM MORPHINE. BACKGROUND: EPIGENETIC MECHANISMS SUCH AS HISTONE MODIFICATIONS MAY BE INVOLVED IN THE STRUCTURAL AND BEHAVIORAL CHANGES ASSOCIATED WITH ADDICTION. WE STUDIED WHETHER MORPHINE-INDUCED CHANGES IN MRNA LEVELS OF THE CATECHOLAMINE BIOSYNTHESIS ENZYME, TYROSINE HYDROXYLASE (TH), ARE ASSOCIATED WITH HISTONE MODIFICATIONS AROUND THE PROMOTER OF THIS GENE IN THE LOCUS COERULEUS (LC) AND VENTRAL TEGMENTAL AREA (VTA) OF RATS. METHODS: DEPENDENCE WAS INDUCED IN RATS BY INTRAPERITONEAL INJECTIONS OF MORPHINE FOR 11 DAYS. THE ANIMALS WERE KILLED 2 H (CHRONIC MORPHINE), 24 H AND 7 DAYS (SPONTANEOUS WITHDRAWAL) AFTER THE LAST INJECTION OF MORPHINE. RESULTS: ANALYSIS OF OUR REAL-TIME QUANTITATIVE REVERSE TRANSCRIPTION PCR RESULTS BY 1-WAY ANOVA SHOWED SIGNIFICANT UPREGULATION (5.13 +/- 0.39 FOLDS) OF LC LEVELS OF THE TH TRANSCRIPT 24 H AFTER THE LAST INJECTION OF MORPHINE TO RATS, WHEN COMPARED WITH 2 H AND 7 DAYS TIME POINTS. CHRONIC MORPHINE AND MORPHINE ABSTINENCE FAILED TO CAUSE ANY SIGNIFICANT CHANGES IN THE LEVELS OF TH MRNA IN THE VTA AFTER CESSATION OF MORPHINE. CONSISTENTLY, CHROMATIN IMMUNOPRECIPITATION REAL-TIME QUANTITATIVE PCR ASSAYS REVEALED THAT 24 H AFTER THE LAST INJECTION OF MORPHINE, LEVELS OF H3 ACETYLATION WERE SIGNIFICANTLY INCREASED (4.12 +/- 0.38 FOLDS) AT THE PROMOTER OF THE TH GENE IN THE LC BUT NOT IN THE VTA. OUR DATA ALSO SHOWED THAT HISTONE H3 TRIMETHYLATION FAILED TO CHANGE AROUND THE TH GENE PROMOTER EITHER IN THE VTA OR IN THE LC AFTER MORPHINE ABSTINENCE. CONCLUSIONS: RESULTS OF THE PRESENT STUDY, FOR THE FIRST TIME, DEMONSTRATE THE INVOLVEMENT OF HISTONE H3 ACETYLATION IN THE REGULATION OF TH GENE EXPRESSION IN THE LC OF RATS DURING FORCED ABSTINENCE FROM MORPHINE. 2018 12 3132 29 GLOBAL DISTRIBUTION OF DNA HYDROXYMETHYLATION AND DNA METHYLATION IN CHRONIC LYMPHOCYTIC LEUKEMIA. BACKGROUND: CHRONIC LYMPHOCYTIC LEUKEMIA (CLL) HAS BEEN A GOOD MODEL SYSTEM TO UNDERSTAND THE FUNCTIONAL ROLE OF 5-METHYLCYTOSINE (5-MC) IN CANCER PROGRESSION. MORE RECENTLY, AN OXIDIZED FORM OF 5-MC, 5-HYDROXYMETHYLCYTOSINE (5-HMC) HAS GAINED LOT OF ATTENTION AS A REGULATORY EPIGENETIC MODIFICATION WITH PROGNOSTIC AND DIAGNOSTIC IMPLICATIONS FOR SEVERAL CANCERS. HOWEVER, THERE IS NO GLOBAL STUDY EXPLORING THE ROLE OF 5-HYDROXYMETHYLCYTOSINE (5-HMC) LEVELS IN CLL. HEREIN, USING MASS SPECTROMETRY AND HMEDIP-SEQUENCING, WE ANALYSED THE DYNAMICS OF 5-HMC DURING B CELL MATURATION AND CLL PATHOGENESIS. RESULTS: WE SHOW THAT NAIVE B-CELLS HAD HIGHER LEVELS OF 5-HMC AND 5-MC COMPARED TO NON-CLASS SWITCHED AND CLASS-SWITCHED MEMORY B-CELLS. WE FOUND A SIGNIFICANT DECREASE IN GLOBAL 5-MC LEVELS IN CLL PATIENTS (N = 15) COMPARED TO NAIVE AND MEMORY B CELLS, WITH NO CHANGES DETECTED BETWEEN THE CLL PROGNOSTIC GROUPS. ON THE OTHER HAND, GLOBAL 5-HMC LEVELS OF CLL PATIENTS WERE SIMILAR TO MEMORY B CELLS AND REDUCED COMPARED TO NAIVE B CELLS. INTERESTINGLY, 5-HMC LEVELS WERE INCREASED AT REGULATORY REGIONS SUCH AS GENE-BODY, CPG ISLAND SHORES AND SHELVES AND 5-HMC DISTRIBUTION OVER THE GENE-BODY POSITIVELY CORRELATED WITH DEGREE OF TRANSCRIPTIONAL ACTIVITY. IMPORTANTLY, CLL SAMPLES SHOWED ABERRANT 5-HMC AND 5-MC PATTERN OVER GENE-BODY COMPARED TO WELL-DEFINED PATTERNS IN NORMAL B-CELLS. INTEGRATED ANALYSIS OF 5-HMC AND RNA-SEQUENCING FROM CLL DATASETS IDENTIFIED THREE NOVEL ONCOGENIC DRIVERS THAT COULD HAVE POTENTIAL ROLES IN CLL DEVELOPMENT AND PROGRESSION. CONCLUSIONS: THUS, OUR STUDY SUGGESTS THAT THE GLOBAL LOSS OF 5-HMC, ACCOMPANIED BY ITS SIGNIFICANT INCREASE AT THE GENE REGULATORY REGIONS, CONSTITUTE A NOVEL HALLMARK OF CLL PATHOGENESIS. OUR COMBINED ANALYSIS OF 5-MC AND 5-HMC SEQUENCING PROVIDED INSIGHTS INTO THE POTENTIAL ROLE OF 5-HMC IN MODULATING GENE EXPRESSION CHANGES DURING CLL PATHOGENESIS. 2019 13 6419 26 THE TET2-UPF1 COMPLEX MODULATES MRNA STABILITY UNDER STRESS CONDITIONS. INTRODUCTION: ENVIRONMENTAL STRESS PROMOTES EPIGENETIC ALTERATIONS THAT IMPACT GENE EXPRESSION AND SUBSEQUENTLY PARTICIPATE IN THE PATHOLOGICAL PROCESSES OF THE DISORDER. AMONG EPIGENETIC REGULATIONS, TEN-ELEVEN TRANSLOCATION (TET) ENZYMES OXIDIZE 5-METHYLCYTOSINE (5MC) TO 5-HYDROXYMETHYLCYTOSINE (5HMC) IN DNA AND RNA AND FUNCTION AS CRITICAL PLAYERS IN THE PATHOGENESIS OF DISEASES. OUR PREVIOUS RESULTS SHOWED THAT CHRONIC STRESS INCREASES THE EXPRESSION OF CYTOPLASMIC TET2 IN THE HIPPOCAMPUS OF MICE EXPOSED TO CHRONIC MILD STRESS (CMS). WHETHER THE CYTOPLASMIC TET2 ALTERS RNA 5HMC MODIFICATION IN CHRONIC STRESS-RELATED PROCESSES REMAINS LARGELY UNKNOWN. METHODS: TO EXPLORE THE ROLE OF CYTOPLASMIC TET2 UNDER CMS CONDITIONS, WE ESTABLISHED CMS MICE MODEL AND DETECTED THE EXPRESSION OF RNA 5HMC BY DOT BLOT. WE VERIFIED THE INTERACTION OF TET2 AND ITS INTERACTING PROTEIN BY CO-IMMUNOPRECIPITATION COMBINED WITH MASS SPECTROMETRY AND SCREENED DOWNSTREAM TARGET GENES BY CLUSTER ANALYSIS OF TET2 AND UPSTREAM FRAMESHIFT 1 (UPF1) INTERACTING RNA. THE EXPRESSION OF PROTEIN WAS DETECTED BY WESTERN BLOT AND THE EXPRESSION OF THE SCREENED TARGET GENES WAS DETECTED BY QRT-PCR. RESULTS: IN THIS STUDY, WE FOUND THAT INCREASED CYTOPLASMIC TET2 EXPRESSION UNDER CMS CONDITIONS LEADS TO INCREASE IN TOTAL RNA 5HMC MODIFICATION. TET2 INTERACTED WITH THE KEY NON-SENSE-MEDIATED MRNA DECAY (NMD) FACTOR UPF1, REGULATED THE STABILITY OF STRESS-RELATED GENES SUCH AS UNC5B MRNA, AND MIGHT THEREBY AFFECT NEURODEVELOPMENT. DISCUSSION: IN SUMMARY, THIS STUDY REVEALED THAT TET2-MEDIATED RNA 5HMC MODIFICATION IS INVOLVED IN STRESS-RELATED MRNA STABILITY REGULATION AND MAY SERVE AS A POTENTIAL THERAPEUTIC TARGET FOR CHRONIC STRESS-RELATED DISEASES SUCH AS DEPRESSION. 2023 14 4236 25 METHYLATION OF THE TYROSINE HYDROXYLASE GENE IS DYSREGULATED BY COCAINE DEPENDENCE IN THE HUMAN STRIATUM. COCAINE DEPENDENCE IS A CHRONIC, RELAPSING DISORDER CAUSED BY LASTING CHANGES IN THE BRAIN. ANIMAL STUDIES HAVE IDENTIFIED COCAINE-RELATED ALTERATIONS IN STRIATAL DNA METHYLATION; HOWEVER, IT IS UNCLEAR HOW METHYLATION IS RELATED TO COCAINE DEPENDENCE IN HUMANS. WE GENERATED METHYLOMIC PROFILES OF THE NUCLEUS ACCUMBENS USING HUMAN POSTMORTEM BRAINS FROM A COHORT OF INDIVIDUALS WITH COCAINE DEPENDENCE AND HEALTHY CONTROLS (N = 25 PER GROUP). WE FOUND HYPERMETHYLATION IN A CLUSTER OF CPGS WITHIN THE GENE BODY OF TYROSINE HYDROXYLASE (TH), CONTAINING A PUTATIVE BINDING SITE FOR THE EARLY GROWTH RESPONSE 1 (EGR1) TRANSCRIPTION FACTOR, WHICH IS HYPERMETHYLATED IN THE CAUDATE NUCLEUS OF COCAINE-DEPENDENT INDIVIDUALS. WE REPLICATED THIS FINDING AND FOUND IT TO BE SPECIFIC TO STRIATAL NEURONAL NUCLEI. FURTHERMORE, THIS LOCUS DEMONSTRATES ENHANCER ACTIVITY WHICH IS ATTENUATED BY METHYLATION AND ENHANCED BY EGR1 OVEREXPRESSION. THESE RESULTS SUGGEST THAT COCAINE DEPENDENCE ALTERS THE EPIGENETIC REGULATION OF DOPAMINERGIC SIGNALING GENES. 2021 15 5975 28 TET1 IS AN IMPORTANT TRANSCRIPTIONAL ACTIVATOR OF TNFALPHA EXPRESSION IN MACROPHAGES. ACTIVATION OF MACROPHAGES AND OVEREXPRESSION OF TNFALPHA IS ASSOCIATED WITH THE PATHOGENESIS OF CHRONIC INFLAMMATORY DISEASES. HOWEVER, THE MECHANISMS LEADING TO TNFALPHA OVEREXPRESSION ARE STILL UNKNOWN. 5-METHYLOCYTOSINE (5-MC) IS AN EPIGENETIC MODIFICATION THAT IS ASSOCIATED WITH SILENCED GENES. RECENT STUDIES SHOWED THAT IT IS CONVERTED TO 5-HYDROXYLMETHYLOCYTOSINE (5-HMC) AND REACTIVATES GENE EXPRESSION THROUGH THE ACTION OF THE FAMILY OF TEN-ELEVEN-TRANSLOCATION (TET1-3) ENZYMES. IN THIS STUDY, WE SHOW THAT 5-HMC LEVELS ARE INCREASED GLOBALLY AND SPECIFICALLY IN THE TNFALPHA PROMOTER DURING THE DIFFERENTIATION OF MONOCYTES TO MACROPHAGES. IN ADDITION, THE LEVELS OF 5-HMC ARE INCREASED UPON LPS STIMULATION OF MACROPHAGES. FURTHERMORE, CRIPSR STABLE KNOCKOUT OF TET1 DECREASES THE EXPRESSION OF TNFALPHA AND OTHER PRO-INFLAMMATORY CYTOKINES. IN CONCLUSION, WE SHOWED THAT TET1 CONTRIBUTES TO THE ACTIVATION OF MACROPHAGES POSSIBLY THROUGH REGULATION OF 5-HYDROXYMETHYLATION IN THE PROMOTER OF PRO-INFLAMMATORY CYTOKINE GENES. THE TET1 ENZYME COULD BE A PROMISING THERAPEUTIC TARGET TO INHIBIT THE PERSISTENT INFLAMMATION CAUSED BY MACROPHAGES IN CHRONIC INFLAMMATORY DISEASES. 2019 16 5617 36 SARCOSINE SUPPRESSES EPILEPTOGENESIS IN RATS WITH EFFECTS ON HIPPOCAMPAL DNA METHYLATION. EPILEPTOGENESIS IS A COMMON CONSEQUENCE OF BRAIN INSULTS, HOWEVER, THE PREVENTION OR DELAY OF THE EPILEPTOGENIC PROCESS REMAINS AN IMPORTANT UNMET MEDICAL CHALLENGE. OVEREXPRESSION OF GLYCINE TRANSPORTER 1 (GLYT1) IS PROPOSED AS A PATHOLOGICAL HALLMARK IN THE HIPPOCAMPUS OF PATIENTS WITH TEMPORAL LOBE EPILEPSY (TLE), AND WE PREVIOUSLY DEMONSTRATED IN RODENT EPILEPSY MODELS THAT AUGMENTATION OF GLYCINE SUPPRESSED CHRONIC SEIZURES AND ALTERED ACUTE SEIZURE THRESHOLDS. IN THE PRESENT STUDY WE EVALUATED THE EFFECT OF THE GLYT1 INHIBITOR, SARCOSINE (AKA N-METHYLGLYCINE), ON EPILEPTOGENESIS AND ALSO INVESTIGATED POSSIBLE MECHANISMS. WE DEVELOPED A MODIFIED RAPID KINDLING MODEL OF EPILEPTOGENESIS IN RATS COMBINED WITH SEIZURE SCORE MONITORING TO EVALUATE THE ANTIEPILEPTOGENIC EFFECT OF SARCOSINE. WE USED IMMUNOHISTOCHEMISTRY AND WESTERN BLOT ANALYSIS FOR THE EVALUATION OF GLYT1 EXPRESSION AND EPIGENETIC CHANGES OF 5-METHYLCYTOSINE (5MC) AND 5-HYDROXYMETHYLCYTOSINE (5HMC) IN THE EPILEPTOGENIC HIPPOCAMPI OF RATS, AND FURTHER EVALUATED EXPRESSION CHANGES IN ENZYMES INVOLVED IN THE REGULATION OF DNA METHYLATION, TEN-ELEVEN TRANSLOCATION METHYLCYTOSINE DIOXYGENASE 1 (TET1), DNA-METHYLTRANSFERASE 1 (DNMT1), AND DNMT3A. OUR RESULTS DEMONSTRATED: (I) EXPERIMENTAL EVIDENCE THAT SARCOSINE (3 G/KG, I.P. DAILY) SUPPRESSED KINDLING EPILEPTOGENESIS IN RATS; (II) THE SARCOSINE-INDUCED ANTIEPILEPTOGENIC EFFECT WAS ACCOMPANIED BY A SUPPRESSED HIPPOCAMPAL GLYT1 EXPRESSION AS WELL AS A REDUCTION OF HIPPOCAMPAL 5MC LEVELS AND A CORRESPONDING INCREASE IN 5HMC; AND (III) SARCOSINE TREATMENT CAUSED DIFFERENTIAL EXPRESSION CHANGES OF TET1 AND DNMTS. TOGETHER, THESE FINDINGS SUGGEST THAT SARCOSINE HAS UNPRECEDENTED DISEASE-MODIFYING PROPERTIES IN A KINDLING MODEL OF EPILEPTOGENESIS IN RATS, WHICH WAS ASSOCIATED WITH ALTERED HIPPOCAMPAL DNA METHYLATION. THUS, MANIPULATION OF THE GLYCINE SYSTEM IS A POTENTIAL THERAPEUTIC APPROACH TO ATTENUATE THE DEVELOPMENT OF EPILEPSY. 2020 17 277 31 AGE-RELATED EPIGENOME-WIDE DNA METHYLATION AND HYDROXYMETHYLATION IN LONGITUDINAL MOUSE BLOOD. DNA METHYLATION AT CYTOSINE-PHOSPHATE-GUANINE (CPG) DINUCLEOTIDES CHANGES AS A FUNCTION OF AGE IN HUMANS AND ANIMAL MODELS, A PROCESS THAT MAY CONTRIBUTE TO CHRONIC DISEASE DEVELOPMENT. RECENT STUDIES HAVE INVESTIGATED THE ROLE OF AN OXIDIZED FORM OF DNA METHYLATION - 5-HYDROXYMETHYLCYTOSINE (5HMC) - IN THE EPIGENOME, BUT ITS CONTRIBUTION TO AGE-RELATED DNA METHYLATION REMAINS UNCLEAR. WE TESTED THE HYPOTHESIS THAT 5HMC CHANGES WITH AGE, BUT IN A DIRECTION OPPOSITE TO 5-METHYLCYTOSINE (5MC), POTENTIALLY PLAYING A DISTINCT ROLE IN AGING. TO CHARACTERIZE EPIGENETIC AGING, GENOME-WIDE 5MC AND 5HMC WERE MEASURED IN LONGITUDINAL BLOOD SAMPLES (2, 4, AND 10 MONTHS OF AGE) FROM ISOGENIC MICE USING TWO SEQUENCING METHODS - ENHANCED REDUCED REPRESENTATION BISULFITE SEQUENCING AND HYDROXYMETHYLATED DNA IMMUNOPRECIPITATION SEQUENCING. EXAMINING THE EPIGENOME BY AGE, WE IDENTIFIED 28,196 UNIQUE DIFFERENTIALLY METHYLATED CPGS (DMCS) AND 8,613 DIFFERENTIALLY HYDROXYMETHYLATED REGIONS (DHMRS). MOUSE BLOOD SHOWED A GENERAL PATTERN OF EPIGENOME-WIDE HYPERMETHYLATION AND HYPO-HYDROXYMETHYLATION WITH AGE. COMPARING AGE-RELATED DMCS AND DHMRS, 1,854 ANNOTATED GENES SHOWED BOTH DIFFERENTIAL 5MC AND 5HMC, INCLUDING ONE GENE - NFIC - AT FIVE CPGS IN THE SAME 250 BP CHROMOSOMAL REGION. AT THIS REGION, 5MC AND 5HMC LEVELS BOTH DECREASED WITH AGE. REFLECTING THESE AGE-RELATED EPIGENETIC CHANGES, NFIC RNA EXPRESSION IN BLOOD DECREASED WITH AGE, SUGGESTING THAT AGE-RELATED REGULATION OF THIS GENE MAY BE DRIVEN BY 5HMC, NOT CANONICAL DNA METHYLATION. COMBINED, OUR GENOME-WIDE RESULTS SHOW AGE-RELATED DIFFERENTIAL 5MC AND 5HMC, AS WELL AS SOME EVIDENCE THAT CHANGES IN 5HMC MAY DRIVE AGE-RELATED DNA METHYLATION AND GENE EXPRESSION. 2018 18 3729 36 INHIBITION OF TET1 PREVENTS THE DEVELOPMENT OF OSTEOARTHRITIS AND REVEALS THE 5HMC LANDSCAPE THAT ORCHESTRATES PATHOGENESIS. OSTEOARTHRITIS (OA) IS A DEGENERATIVE DISEASE OF THE JOINT, WHICH RESULTS IN PAIN, LOSS OF MOBILITY, AND, EVENTUALLY, JOINT REPLACEMENT. CURRENTLY, NO DISEASE-MODIFYING DRUGS EXIST, PARTLY BECAUSE OF THE MULTIPLE LEVELS AT WHICH CARTILAGE HOMEOSTASIS IS DISRUPTED. RECENT STUDIES HAVE HIGHLIGHTED THE IMPORTANCE OF EPIGENETIC DYSREGULATION IN OA, SPARKING INTEREST IN THE EPIGENETIC MODULATION FOR THIS DISEASE. IN OUR PREVIOUS WORK, WE CHARACTERIZED A FIVEFOLD INCREASE IN CYTOSINE HYDROXYMETHYLATION (5HMC), AN OXIDIZED DERIVATIVE OF CYTOSINE METHYLATION (5MC) ASSOCIATED WITH GENE ACTIVATION, ACCUMULATING AT OA-ASSOCIATED GENES. TO TEST THE ROLE OF 5HMC IN OA, HERE, WE USED A MOUSE MODEL OF SURGICALLY INDUCED OA AND FOUND THAT OA ONSET WAS ACCOMPANIED BY A GAIN OF ~40,000 DIFFERENTIALLY HYDROXYMETHYLATED SITES BEFORE THE NOTABLE HISTOLOGICAL APPEARANCE OF DISEASE. WE DEMONSTRATED THAT TEN-ELEVEN-TRANSLOCATION ENZYME 1 (TET1) MEDIATES THE 5HMC DEPOSITION BECAUSE 98% OF SITES ENRICHED FOR 5HMC IN OA WERE LOST IN TET1(-/-) MICE. LOSS OF TET1-MEDIATED 5HMC PROTECTED THE TET1(-/-) MICE FROM OA DEVELOPMENT, INCLUDING DEGENERATION OF THE CARTILAGE SURFACE AND OSTEOPHYTE FORMATION, BY DIRECTLY PREVENTING THE ACTIVATION OF MULTIPLE OA PATHWAYS. LOSS OF TET1 IN HUMAN OA CHONDROCYTES REDUCED THE EXPRESSION OF THE MATRIX METALLOPROTEINASES MMP3 AND MMP13 AND MULTIPLE INFLAMMATORY CYTOKINES. INTRA-ARTICULAR INJECTIONS OF A DIOXYGENASES INHIBITOR, 2-HYDROXYGLUTARATE, ON MICE AFTER SURGICAL INDUCTION OF OA STALLED DISEASE PROGRESSION. TREATMENT OF HUMAN OA CHONDROCYTES WITH THE SAME INHIBITOR ALSO PHENOCOPIED TET1 LOSS. COLLECTIVELY, THESE DATA DEMONSTRATE THAT TET1-MEDIATED 5HMC DEPOSITION REGULATES MULTIPLE OA PATHWAYS AND CAN BE MODULATED FOR THERAPEUTIC INTERVENTION. 2020 19 5974 32 TET1 IN NUCLEUS ACCUMBENS OPPOSES DEPRESSION- AND ANXIETY-LIKE BEHAVIORS. DEPRESSION IS A LEADING CAUSE OF DISEASE BURDEN, YET CURRENT THERAPIES FULLY TREAT <50% OF AFFECTED INDIVIDUALS. INCREASING EVIDENCE IMPLICATES EPIGENETIC MECHANISMS IN DEPRESSION AND ANTIDEPRESSANT ACTION. HERE WE EXAMINED A POSSIBLE ROLE FOR THE DNA DIOXYGENASE, TEN-ELEVEN TRANSLOCATION PROTEIN 1 (TET1), IN DEPRESSION-RELATED BEHAVIORAL ABNORMALITIES. WE APPLIED CHRONIC SOCIAL DEFEAT STRESS, AN ETHOLOGICALLY VALIDATED MOUSE MODEL OF DEPRESSION-LIKE BEHAVIORS, AND EXAMINED TET1 EXPRESSION CHANGES IN NUCLEUS ACCUMBENS (NAC), A KEY BRAIN REWARD REGION. WE SHOW DECREASED TET1 EXPRESSION IN NAC IN STRESS-SUSCEPTIBLE MICE ONLY. SURPRISINGLY, SELECTIVE KNOCKOUT OF TET1 IN NAC NEURONS OF ADULT MICE PRODUCED ANTIDEPRESSANT-LIKE EFFECTS IN SEVERAL BEHAVIORAL ASSAYS. TO IDENTIFY TET1 TARGETS THAT MEDIATE THESE ACTIONS, WE PERFORMED RNASEQ ON NAC AFTER CONDITIONAL DELETION OF TET1 AND FOUND THAT IMMUNE-RELATED GENES ARE THE MOST HIGHLY DYSREGULATED. MOREOVER, MANY OF THESE GENES ARE ALSO UPREGULATED IN THE NAC OF RESILIENT MICE AFTER CHRONIC SOCIAL DEFEAT STRESS. THESE FINDINGS REVEAL A NOVEL ROLE FOR TET1, AN ENZYME IMPORTANT FOR DNA HYDROXYMETHYLATION, IN THE BRAIN'S REWARD CIRCUITRY IN MODULATING STRESS RESPONSES IN MICE. WE ALSO IDENTIFY A SUBSET OF GENES THAT ARE REGULATED BY TET1 IN THIS CIRCUITRY. THESE FINDINGS PROVIDE NEW INSIGHT INTO THE PATHOPHYSIOLOGY OF DEPRESSION, WHICH CAN AID IN FUTURE ANTIDEPRESSANT DRUG DISCOVERY EFFORTS. 2017 20 579 37 BEHAVIORAL AND MOLECULAR NEUROEPIGENETIC ALTERATIONS IN PRENATALLY STRESSED MICE: RELEVANCE FOR THE STUDY OF CHROMATIN REMODELING PROPERTIES OF ANTIPSYCHOTIC DRUGS. WE HAVE RECENTLY REPORTED THAT MICE BORN FROM DAMS STRESSED DURING PREGNANCY (PRS MICE), IN ADULTHOOD, HAVE BEHAVIORAL DEFICITS REMINISCENT OF BEHAVIORS OBSERVED IN SCHIZOPHRENIA (SZ) AND BIPOLAR (BP) DISORDER PATIENTS. FURTHERMORE, WE HAVE SHOWN THAT THE FRONTAL CORTEX (FC) AND HIPPOCAMPUS OF ADULT PRS MICE, LIKE THAT OF POSTMORTEM CHRONIC SZ PATIENTS, ARE CHARACTERIZED BY INCREASES IN DNA-METHYLTRANSFERASE 1 (DNMT1), TEN-ELEVEN METHYLCYTOSINE DIOXYGENASE 1 (TET1) AND EXHIBIT AN ENRICHMENT OF 5-METHYLCYTOSINE (5MC) AND 5-HYDROXYMETHYLCYTOSINE (5HMC) AT NEOCORTICAL GABAERGIC AND GLUTAMATERGIC GENE PROMOTERS. HERE, WE SHOW THAT THE BEHAVIORAL DEFICITS AND THE INCREASED 5MC AND 5HMC AT GLUTAMIC ACID DECARBOXYLASE 67 (GAD1), REELIN (RELN) AND BRAIN-DERIVED NEUROTROPHIC FACTOR (BDNF) PROMOTERS AND THE REDUCED EXPRESSION OF THE MESSENGER RNAS (MRNAS) AND PROTEINS CORRESPONDING TO THESE GENES IN FC OF ADULT PRS MICE IS REVERSED BY TREATMENT WITH CLOZAPINE (5 MG KG(-1) TWICE A DAY FOR 5 DAYS) BUT NOT BY HALOPERIDOL (1 MG KG(-1) TWICE A DAY FOR 5 DAYS). INTERESTINGLY, CLOZAPINE HAD NO EFFECT ON EITHER THE BEHAVIOR, PROMOTER METHYLATION OR THE EXPRESSION OF THESE MRNAS AND PROTEINS WHEN ADMINISTERED TO OFFSPRING OF NONSTRESSED PREGNANT MICE. CLOZAPINE, BUT NOT HALOPERIDOL, REDUCED THE ELEVATED LEVELS OF DNMT1 AND TET1, AS WELL AS THE ELEVATED LEVELS OF DNMT1 BINDING TO GAD1, RELN AND BDNF PROMOTERS IN PRS MICE SUGGESTING THAT CLOZAPINE, UNLIKE HALOPERIDOL, MAY LIMIT DNA METHYLATION BY INTERFERING WITH DNA METHYLATION DYNAMICS. WE CONCLUDE THAT THE PRS MOUSE MODEL MAY BE USEFUL PRECLINICALLY IN SCREENING FOR THE POTENTIAL EFFICACY OF ANTIPSYCHOTIC DRUGS ACTING ON ALTERED EPIGENETIC MECHANISMS. FURTHERMORE, PRS MICE MAY BE INVALUABLE FOR UNDERSTANDING THE ETIOPATHOGENESIS OF SZ AND BP DISORDER AND FOR PREDICTING TREATMENT RESPONSES AT EARLY STAGES OF THE ILLNESS ALLOWING FOR EARLY DETECTION AND REMEDIAL INTERVENTION. 2016