1 17 114 5' FLANKING REGION OF VAR GENES NUCLEATE HISTONE MODIFICATION PATTERNS LINKED TO PHENOTYPIC INHERITANCE OF VIRULENCE TRAITS IN MALARIA PARASITES. IN THE HUMAN MALARIA PARASITE PLASMODIUM FALCIPARUM ANTIGENIC VARIATION FACILITATES LONG-TERM CHRONIC INFECTION OF THE HOST. THIS IS ACHIEVED BY SEQUENTIAL EXPRESSION OF A SINGLE MEMBER OF THE 60-MEMBER VAR FAMILY. HERE WE SHOW THAT THE 5' FLANKING REGION NUCLEATES EPIGENETIC EVENTS STRONGLY LINKED TO THE MAINTENANCE OF MONO-ALLELIC VAR GENE EXPRESSION PATTERN DURING PARASITE PROLIFERATION. TRI- AND DIMETHYLATION OF HISTONE H3 LYSINE 4 PEAK IN THE 5' UPSTREAM REGION OF TRANSCRIBED VAR AND DURING THE POISED STATE (NON-TRANSCRIBED PHASE OF VAR GENES DURING THE 48 H ASEXUAL LIFE CYCLE), 'BOOKMARKING' THIS MEMBER FOR RE-ACTIVATION AT THE ONSET OF THE NEXT CYCLE. HISTONE H3 LYSINE 9 TRIMETHYLATION ACTS AS AN ANTAGONIST TO LYSINE 4 METHYLATION TO ESTABLISH STABLY SILENT VAR GENE STATES ALONG THE 5' FLANKING AND CODING REGION. FURTHERMORE, WE SHOW THAT COMPETITION EXISTS BETWEEN H3K9 METHYLATION AND H3K9 ACETYLATION IN THE 5' FLANKING REGION AND THAT THESE MARKS CONTRIBUTE EPIGENETICALLY TO REPRESSING OR ACTIVATING VAR GENE EXPRESSION. OUR WORK POINTS TO A PIVOTAL ROLE OF THE HISTONE METHYL MARK WRITING AND READING MACHINERY IN THE PHENOTYPIC INHERITANCE OF VIRULENCE TRAITS IN THE MALARIA PARASITE. 2007 2 3912 20 LIFE-SPAN EXTENSION BY CALORIC RESTRICTION IS DETERMINED BY TYPE AND LEVEL OF FOOD REDUCTION AND BY REPRODUCTIVE MODE IN BRACHIONUS MANJAVACAS (ROTIFERA). WE MEASURED LIFE SPAN AND FECUNDITY OF THREE REPRODUCTIVE MODES IN A CLONE OF THE MONOGONONT ROTIFER BRACHIONUS MANJAVACAS SUBJECTED TO CHRONIC CALORIC RESTRICTION (CCR) OVER A RANGE OF FOOD CONCENTRATIONS OR TO INTERMITTENT FASTING (IF). IF INCREASED LIFE SPAN 50%-70% FOR ALL THREE MODES, WHEREAS CCR INCREASED LIFE SPAN OF ASEXUAL FEMALES DERIVED FROM SEXUALLY OR ASEXUALLY PRODUCED EGGS, BUT NOT THAT OF SEXUAL FEMALES. THE MAIN EFFECT OF CR ON BOTH ASEXUAL MODES WAS TO DELAY DEATH AT YOUNG AGES, RATHER THAN TO PREVENT DEATH AT MIDDLE AGES OR TO GREATLY EXTEND MAXIMUM LIFE SPAN; IN CONTRAST CR IN SEXUAL FEMALES GREATLY INCREASED THE LIFE SPAN OF A FEW LONG-LIVED INDIVIDUALS. LIFETIME FECUNDITY DID NOT DECREASE WITH CCR, SUGGESTING A LACK OF RESOURCE ALLOCATION TRADE-OFF BETWEEN SOMATIC MAINTENANCE AND REPRODUCTION. MULTIPLE OUTCOMES FOR A CLONAL LINEAGE INDICATE THAT DIFFERENT RESPONSES ARE ESTABLISHED THROUGH EPIGENETIC PROGRAMMING, WHEREAS DIFFERENCES IN LIFE-SPAN ALLOCATIONS SUGGEST THAT MULTIPLE GENETIC MECHANISMS MEDIATE LIFE-SPAN EXTENSION. 2013 3 5096 38 PLASMODIUM FALCIPARUM SET2 DOMAIN IS ALLOSTERICALLY REGULATED BY ITS PHD-LIKE DOMAIN TO METHYLATE AT H3K36. THE ANTIGENIC VARIATION IS AN ESSENTIAL MECHANISM EMPLOYED BY THE MALARIA PARASITE TO ESTABLISH A CHRONIC INFECTION IN HUMANS. THREE MAJOR VIRULENT PROTEINS EMP1, RIFINS, AND STEVOR HAVE BEEN IMPLICATED IN CONTRIBUTING TO THE ANTIGENIC VARIATION PROCESS AND ARE ENCODED BY MULTIGENE FAMILIES IN PLASMODIUM SPP. THE KEY VIRULENCE FACTOR PFEMP1 IS ENCODED BY VAR GENES, AND IT EXHIBITS A MUTUALLY EXCLUSIVE TRANSCRIPTIONAL SWITCHING BETWEEN VAR GENES, ENSURING AN INDIVIDUAL PARASITE ONLY TRANSCRIBES A SINGLE VAR GENE AT A TIME. EXPRESSION OF VAR GENES IS TIGHTLY REGULATED BY TWO HISTONE EPIGENETIC METHYLATION MARKS H3K36ME3 AND H3K9ME3, OF WHICH THE H3K36ME3 MARK IS HIGHLY ENRICHED ON TRANSCRIPTION START SITES (TSSS) OF SUPPRESSED VAR GENES IN P. FALCIPARUM. HOWEVER, THE MECHANISMS OF H3K36ME3 MARK PROPAGATION ON ALL THE 59 VAR GENES OF P. FALCIPARUM ARE NOT KNOWN. HERE, WE HAVE IDENTIFIED A PHD (PLANT HOMEODOMAIN-LIKE DOMAIN) LIKE DOMAIN PRESENT WITHIN THE PFSET2 PROTEIN THAT SPECIFICALLY BINDS TO THE H3K36ME2 MARK, AN INTERMEDIATE PRODUCT OF THE H3K36ME3 MARK FORMATION ON THE NUCLEOSOME. SURPRISINGLY, WE HAVE FOUND THAT PHD - H3K36ME2 INTERACTION LEADS TO STIMULATION OF SET2 DOMAIN ACTIVITY ON THE NUCLEOSOME SUBSTRATES. THE ALLOSTERIC STIMULATION OF THE PFSET2 DOMAIN BY PHD-LIKE DOMAIN PRESENT WITHIN THE SAME PROTEIN SUGGESTS A NOVEL MECHANISM OF H3K36ME3 MARK PROPAGATION ON VAR GENES OF P. FALCIPARUM. THIS STUDY PROPOSES ALLOSTERIC REGULATION OF PFSET2 PROTEIN BY H3K36ME2 MARK AS AN ESSENTIAL MECHANISM OF VAR GENES SUPPRESSION TO ENSURE SUCCESSFUL ANTIGENIC VARIATION BY THE MALARIA PARASITE. 2021 4 370 40 AN APICOMPLEXAN BROMODOMAIN PROTEIN, TGBDP1, ASSOCIATES WITH DIVERSE EPIGENETIC FACTORS TO REGULATE ESSENTIAL TRANSCRIPTIONAL PROCESSES IN TOXOPLASMA GONDII. THE PROTOZOAN PATHOGEN TOXOPLASMA GONDII RELIES ON TIGHT REGULATION OF GENE EXPRESSION TO INVADE AND ESTABLISH INFECTION IN ITS HOST. THE DIVERGENT GENE REGULATORY MECHANISMS OF TOXOPLASMA AND RELATED APICOMPLEXAN PATHOGENS RELY HEAVILY ON REGULATORS OF CHROMATIN STRUCTURE AND HISTONE MODIFICATIONS. THE IMPORTANT CONTRIBUTION OF HISTONE ACETYLATION FOR TOXOPLASMA IN BOTH ACUTE AND CHRONIC INFECTION HAS BEEN DEMONSTRATED, WHERE HISTONE ACETYLATION INCREASES AT ACTIVE GENE LOCI. HOWEVER, THE DIRECT CONSEQUENCES OF SPECIFIC HISTONE ACETYLATION MARKS AND THE CHROMATIN PATHWAY THAT INFLUENCES TRANSCRIPTIONAL REGULATION IN RESPONSE TO THE MODIFICATION ARE UNCLEAR. AS A READER OF LYSINE ACETYLATION, THE BROMODOMAIN SERVES AS A MEDIATOR BETWEEN THE ACETYLATED HISTONE AND TRANSCRIPTIONAL REGULATORS. HERE WE SHOW THAT THE BROMODOMAIN PROTEIN, TGBDP1, WHICH IS CONSERVED AMONG APICOMPLEXA AND WITHIN THE ALVEOLATA SUPERPHYLUM, IS ESSENTIAL FOR TOXOPLASMA ASEXUAL PROLIFERATION. USING CLEAVAGE UNDER TARGETS AND TAGMENTATION, WE DEMONSTRATE THAT TGBDP1 IS RECRUITED TO TRANSCRIPTIONAL START SITES OF A LARGE PROPORTION OF PARASITE GENES. TRANSCRIPTIONAL PROFILING DURING TGBDP1 KNOCKDOWN REVEALED THAT LOSS OF TGBDP1 LEADS TO MAJOR DYSREGULATION OF GENE EXPRESSION, IMPLYING MULTIPLE ROLES FOR TGBDP1 IN BOTH GENE ACTIVATION AND REPRESSION. THIS IS SUPPORTED BY INTERACTOME ANALYSIS OF TGBDP1 DEMONSTRATING THAT TGBDP1 FORMS A CORE COMPLEX WITH TWO OTHER BROMODOMAIN PROTEINS AND AN APIAP2 FACTOR. THIS CORE COMPLEX APPEARS TO INTERACT WITH OTHER EPIGENETIC FACTORS SUCH AS NUCLEOSOME REMODELING COMPLEXES. WE CONCLUDE THAT TGBDP1 INTERACTS WITH DIVERSE EPIGENETIC REGULATORS TO EXERT OPPOSING INFLUENCES ON GENE EXPRESSION IN THE TOXOPLASMA TACHYZOITE. IMPORTANCE HISTONE ACETYLATION IS CRITICAL FOR PROPER REGULATION OF GENE EXPRESSION IN THE SINGLE-CELLED EUKARYOTIC PATHOGEN TOXOPLASMA GONDII. BROMODOMAIN PROTEINS ARE "READERS" OF HISTONE ACETYLATION AND MAY LINK THE MODIFIED CHROMATIN TO TRANSCRIPTION FACTORS. HERE, WE SHOW THAT THE BROMODOMAIN PROTEIN TGBDP1 IS ESSENTIAL FOR PARASITE SURVIVAL AND THAT LOSS OF TGBDP1 RESULTS IN GLOBAL DYSREGULATION OF GENE EXPRESSION. TGBDP1 IS RECRUITED TO THE PROMOTER REGION OF A LARGE PROPORTION OF PARASITE GENES, FORMS A CORE COMPLEX WITH TWO OTHER BROMODOMAIN PROTEINS, AND INTERACTS WITH DIFFERENT TRANSCRIPTIONAL REGULATORY COMPLEXES. WE CONCLUDE THAT TGBDP1 IS A KEY FACTOR FOR SENSING SPECIFIC HISTONE MODIFICATIONS TO INFLUENCE MULTIPLE FACETS OF TRANSCRIPTIONAL REGULATION IN TOXOPLASMA GONDII. 2023 5 3827 43 INVESTIGATION OF HETEROCHROMATIN PROTEIN 1 FUNCTION IN THE MALARIA PARASITE PLASMODIUM FALCIPARUM USING A CONDITIONAL DOMAIN DELETION AND SWAPPING APPROACH. THE HUMAN MALARIA PARASITE PLASMODIUM FALCIPARUM ENCODES A SINGLE ORTHOLOG OF HETEROCHROMATIN PROTEIN 1 (PFHP1) THAT PLAYS A CRUCIAL ROLE IN THE EPIGENETIC REGULATION OF VARIOUS SURVIVAL-RELATED PROCESSES. PFHP1 IS ESSENTIAL FOR PARASITE PROLIFERATION AND THE HERITABLE SILENCING OF GENES LINKED TO ANTIGENIC VARIATION, HOST CELL INVASION, AND SEXUAL CONVERSION. HERE, WE EMPLOYED CRISPR/CAS9-MEDIATED GENOME EDITING COMBINED WITH THE DICRE/LOXP SYSTEM TO INVESTIGATE HOW THE PFHP1 CHROMODOMAIN (CD), HINGE DOMAIN, AND CHROMOSHADOW DOMAIN (CSD) CONTRIBUTE TO OVERALL PFHP1 FUNCTION. WE SHOW THAT THE 76 C-TERMINAL RESIDUES ARE RESPONSIBLE FOR TARGETING PFHP1 TO THE NUCLEUS. FURTHERMORE, WE REVEAL THAT EACH OF THE THREE FUNCTIONAL DOMAINS OF PFHP1 ARE REQUIRED FOR HETEROCHROMATIN FORMATION, GENE SILENCING, AND MITOTIC PARASITE PROLIFERATION. FINALLY, WE DISCOVERED THAT THE HINGE DOMAIN AND CSD OF HP1 ARE FUNCTIONALLY CONSERVED BETWEEN P. FALCIPARUM AND P. BERGHEI, A RELATED MALARIA PARASITE INFECTING RODENTS. IN SUMMARY, OUR STUDY PROVIDES NEW INSIGHTS INTO PFHP1 FUNCTION AND OFFERS A TOOL FOR FURTHER STUDIES ON EPIGENETIC REGULATION AND LIFE CYCLE DECISION IN MALARIA PARASITES.IMPORTANCE MALARIA IS CAUSED BY UNICELLULAR PLASMODIUM SPECIES PARASITES THAT REPEATEDLY INVADE AND REPLICATE INSIDE RED BLOOD CELLS. SOME BLOOD-STAGE PARASITES EXIT THE CELL CYCLE AND DIFFERENTIATE INTO GAMETOCYTES THAT ARE ESSENTIAL FOR MALARIA TRANSMISSION VIA THE MOSQUITO VECTOR. EPIGENETIC CONTROL MECHANISMS ALLOW THE PARASITES TO ALTER THE EXPRESSION OF SURFACE ANTIGENS AND TO BALANCE THE SWITCH BETWEEN PARASITE MULTIPLICATION AND GAMETOCYTE PRODUCTION. THESE PROCESSES ARE CRUCIAL TO ESTABLISH CHRONIC INFECTION AND OPTIMIZE PARASITE TRANSMISSION. HERE, WE PERFORMED A MUTATIONAL ANALYSIS OF HETEROCHROMATIN PROTEIN 1 (HP1) IN P. FALCIPARUM WE DEMONSTRATE THAT ALL THREE DOMAINS OF THIS PROTEIN ARE INDISPENSABLE FOR THE PROPER FUNCTION OF HP1 IN PARASITE MULTIPLICATION, HETEROCHROMATIN FORMATION, AND GENE SILENCING. MOREOVER, EXPRESSION OF CHIMERIC PROTEINS REVEALED THE FUNCTIONAL CONSERVATION OF HP1 PROTEINS BETWEEN DIFFERENT PLASMODIUM SPECIES. THESE RESULTS PROVIDE NEW INSIGHT INTO THE FUNCTION AND EVOLUTION OF HP1 AS AN ESSENTIAL EPIGENETIC REGULATOR OF PARASITE SURVIVAL. 2021 6 340 35 ALTERATIONS IN LOCAL CHROMATIN ENVIRONMENT ARE INVOLVED IN SILENCING AND ACTIVATION OF SUBTELOMERIC VAR GENES IN PLASMODIUM FALCIPARUM. PLASMODIUM FALCIPARUM ERYTHROCYTE MEMBRANE PROTEIN 1 (PFEMP1), ENCODED BY THE VAR GENE FAMILY, UNDERGOES ANTIGENIC VARIATION AND PLAYS AN IMPORTANT ROLE IN CHRONIC INFECTION AND SEVERE MALARIA. ONLY A SINGLE VAR GENE IS TRANSCRIBED PER PARASITE, AND EPIGENETIC CONTROL MECHANISMS ARE FUNDAMENTAL IN THIS STRATEGY OF MUTUALLY EXCLUSIVE TRANSCRIPTION. WE SHOW THAT SUBTELOMERIC UPSB VAR GENE PROMOTERS CARRIED ON EPISOMES ARE SILENCED BY DEFAULT, AND THAT PROMOTER ACTIVATION IS SUFFICIENT TO SILENCE ALL OTHER FAMILY MEMBERS. HOWEVER, THEY ARE ACTIVE BY DEFAULT WHEN PLACED DOWNSTREAM OF A SECOND ACTIVE VAR PROMOTER, UNDERSCORING THE SIGNIFICANCE OF LOCAL CHROMATIN ENVIRONMENT AND NUCLEAR COMPARTMENTALIZATION IN VAR PROMOTER REGULATION. NATIVE CHROMATIN COVERING THE SPE2-REPEAT ARRAY IN UPSB PROMOTERS IS RESISTANT TO NUCLEASE DIGESTION, AND INSERTION OF THESE REGULATORY ELEMENTS INTO A HETEROLOGOUS PROMOTER CAUSES LOCAL ALTERATIONS IN NUCLEOSOMAL ORGANIZATION AND PROMOTER REPRESSION. OUR FINDINGS SUGGEST A COMMON LOGIC UNDERLYING THE TRANSCRIPTIONAL CONTROL OF ALL VAR GENES, AND HAVE IMPORTANT IMPLICATIONS FOR OUR UNDERSTANDING OF THE EPIGENETIC PROCESSES INVOLVED IN THE REGULATION OF THIS MAJOR VIRULENCE GENE FAMILY. 2007 7 1218 46 CRISPR INTERFERENCE OF A CLONALLY VARIANT GC-RICH NONCODING RNA FAMILY LEADS TO GENERAL REPRESSION OF VAR GENES IN PLASMODIUM FALCIPARUM. THE HUMAN MALARIA PARASITE PLASMODIUM FALCIPARUM USES MUTUALLY EXCLUSIVE EXPRESSION OF THE PFEMP1-ENCODING VAR GENE FAMILY TO EVADE THE HOST IMMUNE SYSTEM. DESPITE PROGRESS IN THE MOLECULAR UNDERSTANDING OF THE DEFAULT SILENCING MECHANISM, THE ACTIVATION MECHANISM OF THE UNIQUELY EXPRESSED VAR MEMBER REMAINS ELUSIVE. A GC-RICH NONCODING RNA (NCRNA) GENE FAMILY HAS COEVOLVED WITH PLASMODIUM SPECIES THAT EXPRESS VAR GENES. HERE, WE SHOW THAT THIS NCRNA FAMILY IS TRANSCRIBED IN A CLONALLY VARIANT MANNER, WITH PREDOMINANT TRANSCRIPTION OF A SINGLE MEMBER OCCURRING WHEN THE NCRNA IS LOCATED ADJACENT TO AND UPSTREAM OF AN ACTIVE VAR GENE. WE DEVELOPED A SPECIFIC CRISPR INTERFERENCE (CRISPRI) STRATEGY THAT ALLOWED FOR THE TRANSCRIPTIONAL REPRESSION OF ALL GC-RICH MEMBERS. A LACK OF GC-RICH NCRNA TRANSCRIPTION LED TO THE DOWNREGULATION OF THE ENTIRE VAR GENE FAMILY IN RING-STAGE PARASITES. STRIKINGLY, IN MATURE BLOOD-STAGE PARASITES, THE GC-RICH NCRNA CRISPRI AFFECTED THE TRANSCRIPTION PATTERNS OF OTHER CLONALLY VARIANT GENE FAMILIES, INCLUDING THE DOWNREGULATION OF ALL PFMC-2TM MEMBERS. WE PROVIDE EVIDENCE FOR THE KEY ROLE OF GC-RICH NCRNA TRANSCRIPTION IN VAR GENE ACTIVATION AND DISCOVERED A MOLECULAR LINK BETWEEN THE TRANSCRIPTIONAL CONTROL OF VARIOUS CLONALLY VARIANT MULTIGENE FAMILIES INVOLVED IN PARASITE VIRULENCE. THIS WORK OPENS NEW AVENUES FOR ELUCIDATING THE MOLECULAR PROCESSES THAT CONTROL IMMUNE EVASION AND PATHOGENESIS IN P. FALCIPARUMIMPORTANCEPLASMODIUM FALCIPARUM IS THE DEADLIEST MALARIA PARASITE SPECIES, ACCOUNTING FOR THE VAST MAJORITY OF DISEASE CASES AND DEATHS. THE VIRULENCE OF THIS PARASITE IS RELIANT UPON THE MUTUALLY EXCLUSIVE EXPRESSION OF CYTOADHERENCE PROTEINS ENCODED BY THE 60-MEMBER VAR GENE FAMILY. ANTIGENIC VARIATION OF THIS MULTIGENE FAMILY SERVES AS AN IMMUNE EVASION MECHANISM, ULTIMATELY LEADING TO CHRONIC INFECTION AND PATHOGENESIS. UNDERSTANDING THE REGULATION MECHANISM OF ANTIGENIC VARIATION IS KEY TO DEVELOPING NEW THERAPEUTIC AND CONTROL STRATEGIES. OUR STUDY UNCOVERS A NOVEL LAYER IN THE EPIGENETIC REGULATION OF TRANSCRIPTION OF THIS FAMILY OF VIRULENCE GENES BY MEANS OF A MULTIGENE-TARGETING CRISPR INTERFERENCE APPROACH. 2020 8 2091 34 EPIGENETIC DYSREGULATION OF VIRULENCE GENE EXPRESSION IN SEVERE PLASMODIUM FALCIPARUM MALARIA. CHRONIC INFECTIONS WITH THE HUMAN MALARIA PARASITE PLASMODIUM FALCIPARUM DEPEND ON ANTIGENIC VARIATION. P. FALCIPARUM ERYTHROCYTE MEMBRANE PROTEIN 1 (PFEMP1), THE MAJOR ERYTHROCYTE SURFACE ANTIGEN MEDIATING PARASITE SEQUESTRATION IN THE MICROVASCULATURE, IS ENCODED IN PARASITES BY A HIGHLY DIVERSE FAMILY OF VAR GENES. ANTIGENIC SWITCHING IS MEDIATED BY CLONAL VARIATION IN VAR EXPRESSION, AND RECENT IN VITRO STUDIES HAVE DEMONSTRATED A ROLE FOR EPIGENETIC PROCESSES IN VAR REGULATION. EXPRESSION OF PARTICULAR PFEMP1 VARIANTS MAY RESULT IN PARASITE ENRICHMENT IN DIFFERENT TISSUES, A FACTOR IN THE DEVELOPMENT OF SEVERE DISEASE. HERE, WE STUDY IN VIVO HUMAN INFECTIONS AND PROVIDE EVIDENCE THAT INFECTION-INDUCED STRESS RESPONSES IN THE HOST CAN MODIFY PFEMP1 EXPRESSION VIA THE PERTURBATION OF EPIGENETIC MECHANISMS. OUR WORK SUGGESTS THAT SEVERE DISEASE MAY NOT BE THE DIRECT RESULT OF AN ADAPTIVE VIRULENCE STRATEGY TO MAXIMIZE PARASITE SURVIVAL BUT THAT IT MAY INDICATE A LOSS OF CONTROL OF THE CAREFULLY REGULATED PROCESS OF ANTIGENIC SWITCHING THAT MAINTAINS CHRONIC INFECTIONS. 2012 9 1219 38 CRISPR/CAS9 GENOME EDITING REVEALS THAT THE INTRON IS NOT ESSENTIAL FOR VAR2CSA GENE ACTIVATION OR SILENCING IN PLASMODIUM FALCIPARUM. PLASMODIUM FALCIPARUM RELIES ON MONOALLELIC EXPRESSION OF 1 OF 60 VAR VIRULENCE GENES FOR ANTIGENIC VARIATION AND HOST IMMUNE EVASION. EACH VAR GENE CONTAINS A CONSERVED INTRON WHICH HAS BEEN IMPLICATED IN PREVIOUS STUDIES IN BOTH ACTIVATION AND REPRESSION OF TRANSCRIPTION VIA SEVERAL EPIGENETIC MECHANISMS, INCLUDING INTERACTION WITH THE VAR PROMOTER, PRODUCTION OF LONG NONCODING RNAS (LNCRNAS), AND LOCALIZATION TO REPRESSIVE PERINUCLEAR SITES. HOWEVER, FUNCTIONAL STUDIES HAVE RELIED PRIMARILY ON ARTIFICIAL EXPRESSION CONSTRUCTS. USING THE RECENTLY DEVELOPED P. FALCIPARUM CLUSTERED REGULARLY INTERSPACED SHORT PALINDROMIC REPEATS (CRISPR)/CAS9 SYSTEM, WE DIRECTLY DELETED THE VAR2CSA P. FALCIPARUM 3D7_1200600 (PF3D7_1200600) ENDOGENOUS INTRON, RESULTING IN AN INTRONLESS VAR GENE IN A NATURAL, MARKER-FREE CHROMOSOMAL CONTEXT. DELETION OF THE VAR2CSA INTRON RESULTED IN AN UPREGULATION OF TRANSCRIPTION OF THE VAR2CSA GENE IN RING-STAGE PARASITES AND SUBSEQUENT EXPRESSION OF THE PFEMP1 PROTEIN IN LATE-STAGE PARASITES. INTRON DELETION DID NOT AFFECT THE NORMAL TEMPORAL REGULATION AND SUBSEQUENT TRANSCRIPTIONAL SILENCING OF THE VAR GENE IN TROPHOZOITES BUT DID RESULT IN INCREASED RATES OF VAR GENE SWITCHING IN SOME MUTANT CLONES. TRANSCRIPTIONAL REPRESSION OF THE INTRONLESS VAR2CSA GENE COULD BE ACHIEVED VIA LONG-TERM CULTURE OR PANNING WITH THE CD36 RECEPTOR, AFTER WHICH REACTIVATION WAS POSSIBLE WITH CHONDROITIN SULFATE A (CSA) PANNING. THESE DATA SUGGEST THAT THE VAR2CSA INTRON IS NOT REQUIRED FOR SILENCING OR ACTIVATION IN RING-STAGE PARASITES BUT POINT TO A SUBTLE ROLE IN REGULATION OF SWITCHING WITHIN THE VAR GENE FAMILY.IMPORTANCEPLASMODIUM FALCIPARUM IS THE MOST VIRULENT SPECIES OF MALARIA PARASITE, CAUSING HIGH RATES OF MORBIDITY AND MORTALITY IN THOSE INFECTED. CHRONIC INFECTION DEPENDS ON AN IMMUNE EVASION MECHANISM TERMED ANTIGENIC VARIATION, WHICH IN TURN RELIES ON MONOALLELIC EXPRESSION OF 1 OF ~60 VAR GENES. UNDERSTANDING ANTIGENIC VARIATION AND THE TRANSCRIPTIONAL REGULATION OF MONOALLELIC EXPRESSION IS IMPORTANT FOR DEVELOPING DRUGS AND/OR VACCINES. THE VAR GENE FAMILY ENCODES THE ANTIGENIC SURFACE PROTEINS THAT DECORATE INFECTED ERYTHROCYTES. UNTIL RECENTLY, STUDYING THE UNDERLYING GENETIC ELEMENTS THAT REGULATE MONOALLELIC EXPRESSION IN P. FALCIPARUM WAS DIFFICULT, AND MOST STUDIES RELIED ON ARTIFICIAL SYSTEMS SUCH AS EPISOMAL REPORTER GENES. OUR STUDY WAS THE FIRST TO USE CRISPR/CAS9 GENOME EDITING FOR THE FUNCTIONAL STUDY OF AN IMPORTANT, CONSERVED GENETIC ELEMENT OF VAR GENES-THE INTRON-IN AN ENDOGENOUS, EPISOME-FREE MANNER. OUR FINDINGS SHED LIGHT ON THE ROLE OF THE VAR GENE INTRON IN TRANSCRIPTIONAL REGULATION OF MONOALLELIC EXPRESSION. 2017 10 129 44 A UNIQUE VIRULENCE GENE OCCUPIES A PRINCIPAL POSITION IN IMMUNE EVASION BY THE MALARIA PARASITE PLASMODIUM FALCIPARUM. MUTUALLY EXCLUSIVE GENE EXPRESSION, WHEREBY ONLY ONE MEMBER OF A MULTI-GENE FAMILY IS SELECTED FOR ACTIVATION, IS USED BY THE MALARIA PARASITE PLASMODIUM FALCIPARUM TO ESCAPE THE HUMAN IMMUNE SYSTEM AND PERPETUATE LONG-TERM, CHRONIC INFECTIONS. A FAMILY OF GENES CALLED VAR ENCODES THE CHIEF ANTIGENIC AND VIRULENCE DETERMINANT OF P. FALCIPARUM MALARIA. VAR GENES ARE TRANSCRIBED IN A MUTUALLY EXCLUSIVE MANNER, WITH SWITCHING BETWEEN ACTIVE GENES RESULTING IN ANTIGENIC VARIATION. WHILE RECENT WORK HAS SHED CONSIDERABLE LIGHT ON THE EPIGENETIC BASIS FOR VAR GENE ACTIVATION AND SILENCING, HOW SWITCHING IS CONTROLLED REMAINS A MYSTERY. IN PARTICULAR, SWITCHING SEEMS NOT TO BE RANDOM, BUT INSTEAD APPEARS TO BE COORDINATED TO RESULT IN TIMELY ACTIVATION OF INDIVIDUAL GENES LEADING TO SEQUENTIAL WAVES OF ANTIGENICALLY DISTINCT PARASITE POPULATIONS. THE MOLECULAR BASIS FOR THIS APPARENT COORDINATION IS UNKNOWN. HERE WE SHOW THAT VAR2CSA, AN UNUSUAL AND HIGHLY CONSERVED VAR GENE, OCCUPIES A UNIQUE POSITION WITHIN THE VAR GENE SWITCHING HIERARCHY. INDUCTION OF SWITCHING THROUGH THE DESTABILIZATION OF VAR SPECIFIC CHROMATIN USING BOTH GENETIC AND CHEMICAL METHODS REPEATEDLY LED TO THE RAPID AND EXCLUSIVE ACTIVATION OF VAR2CSA. ADDITIONAL EXPERIMENTS DEMONSTRATED THAT THESE REPRESENT "TRUE" SWITCHING EVENTS AND NOT SIMPLY DE-SILENCING OF THE VAR2CSA PROMOTER, AND THAT ACTIVATION IS LIMITED TO THE UNIQUE LOCUS ON CHROMOSOME 12. COMBINED WITH TRANSLATIONAL REPRESSION OF VAR2CSA TRANSCRIPTS, FREQUENT "DEFAULT" SWITCHING TO THIS LOCUS AND DETECTION OF VAR2CSA UNTRANSLATED TRANSCRIPTS IN NON-PREGNANT INDIVIDUALS, THESE DATA SUGGEST THAT VAR2CSA COULD PLAY A CENTRAL ROLE IN COORDINATING SWITCHING, FULFILLING A PREDICTION MADE BY MATHEMATICAL MODELS DERIVED FROM POPULATION SWITCHING PATTERNS. THESE STUDIES PROVIDE THE FIRST INSIGHTS INTO THE MECHANISMS BY WHICH VAR GENE SWITCHING IS COORDINATED AS WELL AS AN EXAMPLE OF HOW A PHARMACOLOGICAL AGENT CAN DISRUPT ANTIGENIC VARIATION IN PLASMODIUM FALCIPARUM. 2015 11 2839 24 FOSB INDUCTION IN NUCLEUS ACCUMBENS BY COCAINE IS REGULATED BY E2F3A. THE TRANSCRIPTION FACTOR DELTAFOSB HAS BEEN PROPOSED AS A MOLECULAR SWITCH FOR THE TRANSITION FROM CASUAL, VOLITIONAL DRUG USE INTO A CHRONICALLY ADDICTED STATE, BUT THE UPSTREAM REGULATORY MECHANISMS GOVERNING DELTAFOSB EXPRESSION ARE INCOMPLETELY UNDERSTOOD. IN THIS STUDY, WE FIND A NOVEL REGULATORY ROLE FOR THE TRANSCRIPTION FACTOR E2F3, RECENTLY IMPLICATED IN TRANSCRIPTIONAL REGULATION BY COCAINE, IN CONTROLLING DELTAFOSB INDUCTION IN THE MOUSE NUCLEUS ACCUMBENS (NAC) FOLLOWING COCAINE ADMINISTRATION. WE FIND THAT AN E2F CONSENSUS SEQUENCE 500 BP UPSTREAM OF THE FOSB TRANSCRIPTION START SITE IS ENRICHED FOR E2F3 SPECIFICALLY OVER OTHER E2F ISOFORMS. WE FURTHER CONCLUDE THAT DELTAFOSB EXPRESSION IS REGULATED SPECIFICALLY BY E2F3A, NOT E2F3B, THAT E2F3A EXPRESSION IS SPECIFIC TO D1 RECEPTOR-EXPRESSING MEDIUM SPINY NEURONS, AND THAT E2F3A OVEREXPRESSION IN NAC RECAPITULATES THE INDUCTION OF FOSB AND DELTAFOSB MRNA EXPRESSION OBSERVED AFTER CHRONIC COCAINE EXPOSURE. E2F3A KNOCKDOWN IN NAC DOES NOT ABOLISH DELTAFOSB INDUCTION BY COCAINE, A RESULT CONSISTENT WITH PREVIOUSLY PUBLISHED DATA SHOWING THAT SINGULAR KNOCKDOWN OF UPSTREAM REGULATORS OF DELTAFOSB IS INSUFFICIENT TO BLOCK COCAINE-INDUCED EXPRESSION. FINALLY, TO ELUCIDATE POTENTIAL COMBINATORIAL EPIGENETIC MECHANISMS INVOLVED IN E2F3A'S REGULATION OF FOSB, WE EXPLORE H3K4ME3 ENRICHMENT AT THE FOSB PROMOTER AND FIND THAT IT IS NOT ENHANCED BY E2F3A OVEREXPRESSION, SUGGESTING THAT IT MAY INSTEAD BE A PRE-EXISTING PERMISSIVE MARK ALLOWING FOR E2F3A TO INTERACT WITH FOSB. TOGETHER, THESE FINDINGS SUPPORT A ROLE FOR E2F3A AS A NOVEL, UPSTREAM REGULATOR OF THE ADDICTION-MEDIATING TRANSCRIPTION FACTOR DELTAFOSB IN NAC. 2019 12 6179 41 THE HUMAN MALARIA PARASITE PLASMODIUM FALCIPARUM CAN SENSE ENVIRONMENTAL CHANGES AND RESPOND BY ANTIGENIC SWITCHING. THE PRIMARY ANTIGENIC AND VIRULENCE DETERMINANT OF THE HUMAN MALARIA PARASITE PLASMODIUM FALCIPARUM IS A VARIANT SURFACE PROTEIN CALLED PFEMP1. DIFFERENT FORMS OF PFEMP1 ARE ENCODED BY A MULTICOPY GENE FAMILY CALLED VAR, AND SWITCHING BETWEEN ACTIVE GENES ENABLES THE PARASITES TO EVADE THE ANTIBODY RESPONSE OF THEIR HUMAN HOSTS. VAR GENE SWITCHING IS KEY FOR THE MAINTENANCE OF CHRONIC INFECTIONS; HOWEVER, WHAT CONTROLS SWITCHING IS UNKNOWN, ALTHOUGH IT HAS BEEN SUGGESTED TO OCCUR AT A CONSTANT FREQUENCY WITH LITTLE OR NO ENVIRONMENTAL INFLUENCE. VAR GENE TRANSCRIPTION IS CONTROLLED EPIGENETICALLY THROUGH THE ACTIVITY OF HISTONE METHYLTRANSFERASES (HMTS). STUDIES IN MODEL SYSTEMS HAVE SHOWN THAT METABOLISM AND EPIGENETIC CONTROL OF GENE EXPRESSION ARE LINKED THROUGH THE AVAILABILITY OF INTRACELLULAR S-ADENOSYLMETHIONINE (SAM), THE PRINCIPAL METHYL DONOR IN BIOLOGICAL METHYLATION MODIFICATIONS, WHICH CAN FLUCTUATE BASED ON NUTRIENT AVAILABILITY. TO DETERMINE WHETHER ENVIRONMENTAL CONDITIONS AND CHANGES IN METABOLISM CAN INFLUENCE VAR GENE EXPRESSION, P. FALCIPARUM WAS CULTURED IN MEDIA WITH ALTERED CONCENTRATIONS OF NUTRIENTS INVOLVED IN SAM METABOLISM. WE FOUND THAT CONDITIONS THAT INFLUENCE LIPID METABOLISM INDUCE VAR GENE SWITCHING, INDICATING THAT PARASITES CAN RESPOND TO CHANGES IN THEIR ENVIRONMENT BY ALTERING VAR GENE EXPRESSION PATTERNS. GENETIC MODIFICATIONS THAT DIRECTLY MODIFIED EXPRESSION OF THE ENZYMES THAT CONTROL SAM LEVELS SIMILARLY LED TO PROFOUND CHANGES IN VAR GENE EXPRESSION, CONFIRMING THAT CHANGES IN SAM AVAILABILITY MODULATE VAR GENE SWITCHING. THESE OBSERVATIONS DIRECTLY CHALLENGE THE PARADIGM THAT ANTIGENIC VARIATION IN P. FALCIPARUM FOLLOWS AN INTRINSIC, PROGRAMED SWITCHING RATE, WHICH OPERATES INDEPENDENTLY OF ANY EXTERNAL STIMULI. 2023 13 786 30 CELL-TYPE-SPECIFIC EPIGENETIC EDITING AT THE FOSB GENE CONTROLS SUSCEPTIBILITY TO SOCIAL DEFEAT STRESS. CHRONIC SOCIAL DEFEAT STRESS REGULATES THE EXPRESSION OF FOSB IN THE NUCLEUS ACCUMBENS (NAC) TO PROMOTE THE CELL-TYPE-SPECIFIC ACCUMULATION OF DELTAFOSB IN THE TWO MEDIUM SPINY NEURON (MSN) SUBTYPES IN THIS REGION. DELTAFOSB IS SELECTIVELY INDUCED IN D1-MSNS IN THE NAC OF RESILIENT MICE, AND IN D2-MSNS OF SUSCEPTIBLE MICE. HOWEVER, LITTLE IS KNOWN ABOUT THE CONSEQUENCES OF SUCH SELECTIVE INDUCTION, PARTICULARLY IN D2-MSNS. THIS STUDY EXAMINED HOW CELL-TYPE-SPECIFIC CONTROL OF THE ENDOGENOUS FOSB GENE IN NAC REGULATES SUSCEPTIBILITY TO SOCIAL DEFEAT STRESS. HISTONE POST-TRANSLATIONAL MODIFICATIONS (HPTMS) WERE TARGETED SPECIFICALLY TO FOSB USING ENGINEERED ZINC-FINGER PROTEINS (ZFPS). FOSB-ZFPS WERE FUSED TO EITHER THE TRANSCRIPTIONAL REPRESSOR, G9A, WHICH PROMOTES HISTONE METHYLATION OR THE TRANSCRIPTIONAL ACTIVATOR, P65, WHICH PROMOTES HISTONE ACETYLATION. THESE ZFPS WERE EXPRESSED IN D1- VS D2-MSNS USING CRE-DEPENDENT VIRAL EXPRESSION IN THE NAC OF MICE TRANSGENIC FOR CRE RECOMBINASE IN THESE MSN SUBTYPES. WE FOUND THAT STRESS SUSCEPTIBILITY IS OPPOSITELY REGULATED BY THE SPECIFIC CELL TYPE AND HPTM TARGETED. WE REPORT THAT FOSB-TARGETED HISTONE ACETYLATION IN D2-MSNS OR HISTONE METHYLATION IN D1-MSNS PROMOTES A STRESS-SUSCEPTIBLE, DEPRESSIVE-LIKE PHENOTYPE, WHILE HISTONE METHYLATION IN D2-MSNS OR HISTONE ACETYLATION IN D1-MSNS INCREASES RESILIENCE TO SOCIAL STRESS AS QUANTIFIED BY SOCIAL INTERACTION BEHAVIOR AND SUCROSE PREFERENCE. THIS WORK PRESENTS THE FIRST DEMONSTRATION OF CELL- AND GENE-SPECIFIC TARGETING OF HISTONE MODIFICATIONS, WHICH MODEL NATURALLY OCCURRING TRANSCRIPTIONAL PHENOMENA THAT CONTROL SOCIAL DEFEAT STRESS BEHAVIOR. THIS EPIGENETIC-EDITING APPROACH, WHICH RECAPITULATES PHYSIOLOGICAL CHANGES IN GENE EXPRESSION, REVEALS CLEAR DIFFERENCES IN THE SOCIAL DEFEAT PHENOTYPE INDUCED BY FOSB GENE MANIPULATION IN MSN SUBTYPES. 2018 14 5970 22 TESTOSTERONE ACTS WITHIN THE MEDIAL AMYGDALA OF RATS TO REDUCE INNATE FEAR TO PREDATOR ODOR AKIN TO THE EFFECTS OF TOXOPLASMA GONDII INFECTION. RATS INFECTED WITH THE PROTOZOAN TOXOPLASMA GONDII EXHIBIT A REDUCED AVERSION TO CAT ODOR. THIS BEHAVIORAL CHANGE IS THOUGHT TO INCREASE TROPHIC TRANSMISSION OF THE PARASITE. INFECTED MALE RATS ALSO SHOW A GREATER TESTICULAR SYNTHESIS OF TESTOSTERONE AND EPIGENETIC CHANGE IN ARGININE VASOPRESSIN WITHIN THE MEDIAL AMYGDALA. HERE, WE SHOW THAT EXOGENOUS SUPPLY OF TESTOSTERONE WITHIN MEA OF UNINFECTED CASTRATES RECAPITULATES REDUCTION IN INNATE FEAR AKIN TO BEHAVIORAL CHANGE ATTRIBUTED TO THE PARASITE. WE ALSO SHOW THAT CASTRATION POST ESTABLISHMENT OF CHRONIC INFECTION PRECLUDES CHANGES IN FEAR AND MEDIAL AMYGDALA ARGININE VASOPRESSIN IN THE INFECTED MALE RATS. THESE OBSERVATIONS SUPPORT THE ROLE OF GONADAL HORMONES AND PURSUANT NEUROENDOCRINE CHANGES IN MEDIATING THE LOSS OF FEAR IN THE INFECTED RATS. THIS WORK ALSO DEMONSTRATES THAT TESTOSTERONE ACTING SPECIFICALLY WITHIN THE MEDIAL AMYGDALA SUFFICIENTLY EXPLAINS REDUCED DEFENSIVE BEHAVIORS OFTEN OBSERVED DURING THE APPETITIVE COMPONENT OF REPRODUCTIVE BEHAVIORS. 2020 15 35 23 A CHROMATIN ACTIVITY-BASED CHEMOPROTEOMIC APPROACH REVEALS A TRANSCRIPTIONAL REPRESSOME FOR GENE-SPECIFIC SILENCING. IMMUNE CELLS DEVELOP ENDOTOXIN TOLERANCE (ET) AFTER PROLONGED STIMULATION. ET INCREASES THE LEVEL OF A REPRESSION MARK H3K9ME2 IN THE TRANSCRIPTIONALLY SILENT CHROMATIN SPECIFICALLY ASSOCIATED WITH PRO-INFLAMMATORY GENES. HOWEVER, IT IS NOT CLEAR WHAT PROTEINS ARE FUNCTIONALLY INVOLVED IN THIS PROCESS. HERE WE SHOW THAT A NOVEL CHROMATIN ACTIVITY-BASED CHEMOPROTEOMIC (CHAC) APPROACH CAN DISSECT THE FUNCTIONAL CHROMATIN PROTEIN COMPLEXES THAT REGULATE ET-ASSOCIATED INFLAMMATION. USING UNC0638 THAT BINDS THE ENZYMATICALLY ACTIVE H3K9-SPECIFIC METHYLTRANSFERASE G9A/GLP, CHAC REVEALS THAT G9A IS CONSTITUTIVELY ACTIVE AT A G9A-DEPENDENT MEGA-DALTON REPRESSOME IN PRIMARY ENDOTOXIN-TOLERANT MACROPHAGES. G9A/GLP BROADLY IMPACTS THE ET-SPECIFIC REPROGRAMMING OF THE HISTONE CODE LANDSCAPE, CHROMATIN REMODELLING AND THE ACTIVITIES OF SELECT TRANSCRIPTION FACTORS. WE DISCOVER THAT THE G9A-DEPENDENT EPIGENETIC ENVIRONMENT PROMOTES THE TRANSCRIPTIONAL REPRESSION ACTIVITY OF C-MYC FOR GENE-SPECIFIC CO-REGULATION OF CHRONIC INFLAMMATION. CHAC MAY ALSO BE APPLICABLE TO DISSECT OTHER FUNCTIONAL PROTEIN COMPLEXES IN THE CONTEXT OF PHENOTYPIC CHROMATIN ARCHITECTURES. 2014 16 3368 29 HISTONE METHYLTRANSFERASE G9A DIMINISHES EXPRESSION OF CANNABINOID CB(1) RECEPTORS IN PRIMARY SENSORY NEURONS IN NEUROPATHIC PAIN. TYPE 1 CANNABINOID RECEPTORS (CB(1)RS) ARE EXPRESSED IN THE DORSAL ROOT GANGLION (DRG) AND CONTRIBUTE TO THE ANALGESIC EFFECT OF CANNABINOIDS. HOWEVER, THE EPIGENETIC MECHANISM REGULATING THE EXPRESSION OF CB(1)RS IN NEUROPATHIC PAIN IS UNKNOWN. G9A (ENCODED BY THE EHMT2 GENE), A HISTONE 3 AT LYSINE 9 METHYLTRANSFERASE, IS A KEY CHROMATIN REGULATOR RESPONSIBLE FOR GENE SILENCING. IN THIS STUDY, WE DETERMINED G9A'S ROLE IN REGULATING CB(1)R EXPRESSION IN THE DRG AND IN CB(1)R-MEDIATED ANALGESIC EFFECTS IN AN ANIMAL MODEL OF NEUROPATHIC PAIN. WE SHOW THAT NERVE INJURY PROFOUNDLY REDUCED MRNA LEVELS OF CB(1)RS BUT INCREASED THE EXPRESSION OF CB(2) RECEPTORS IN THE RAT DRG. CHIP RESULTS INDICATED INCREASED ENRICHMENT OF HISTONE 3 AT LYSINE 9 DIMETHYLATION, A G9A-CATALYZED REPRESSIVE HISTONE MARK, AT THE PROMOTER REGIONS OF THE CB(1)R GENES. G9A INHIBITION IN NERVE-INJURED RATS NOT ONLY UP-REGULATED THE CB(1)R EXPRESSION LEVEL IN THE DRG BUT ALSO POTENTIATED THE ANALGESIC EFFECT OF A CB(1)R AGONIST ON NERVE INJURY-INDUCED PAIN HYPERSENSITIVITY. FURTHERMORE, IN MICE LACKING EHMT2 IN DRG NEURONS, NERVE INJURY FAILED TO REDUCE CB(1)R EXPRESSION IN THE DRG AND TO DECREASE THE ANALGESIC EFFECT OF THE CB(1)R AGONIST. MOREOVER, NERVE INJURY DIMINISHED THE INHIBITORY EFFECT OF THE CB(1)R AGONIST ON SYNAPTIC GLUTAMATE RELEASE FROM PRIMARY AFFERENT NERVES TO SPINAL CORD DORSAL HORN NEURONS IN WT MICE BUT NOT IN MICE LACKING EHMT2 IN DRG NEURONS. OUR FINDINGS REVEAL THAT NERVE INJURY DIMINISHES THE ANALGESIC EFFECT OF CB(1)R AGONISTS THROUGH G9A-MEDIATED CB(1)R DOWN-REGULATION IN PRIMARY SENSORY NEURONS. 2020 17 3498 23 IDENTIFICATION OF NOVEL HYPOMORPHIC AND NULL MUTATIONS IN KLF1 DERIVED FROM A GENETIC SCREEN FOR MODIFIERS OF ALPHA-GLOBIN TRANSGENE VARIEGATION. POSITION-EFFECT VARIEGATION OF TRANSGENE EXPRESSION IS SENSITIVE TO THE CHROMATIN STATE. WE PREVIOUSLY REPORTED A FORWARD GENETIC SCREEN IN MICE CARRYING A VARIEGATED ALPHA-GLOBIN GFP TRANSGENE TO FIND NOVEL GENES ENCODING EPIGENETIC REGULATORS. WE NAMED THE PHENOVARIANT STRAINS "MOMMES" FOR MODIFIERS OF MURINE METASTABLE EPIALLELES. HERE WE REPORT POSITIONAL CLONING OF MUTATIONS IN TWO MOMME STRAINS WHICH RESULT IN SUPPRESSION OF VARIEGATION. BOTH STRAINS HARBOUR POINT MUTATIONS IN THE ERYTHROID TRANSCRIPTION FACTOR, KLF1. ONE (D11) GENERATES A STOP CODON IN THE ZINC FINGER DOMAIN AND A HOMOZYGOUS NULL PHENOTYPE. THE OTHER (D45) GENERATES AN AMINO ACID TRANSVERSION (H350R) WITHIN A CONSERVED LINKER BETWEEN ZINC FINGERS TWO AND THREE. HOMOZYGOUS MOMMED45 MICE HAVE CHRONIC MICROCYTIC ANAEMIA WHICH MODELS THE PHENOTYPE IN A RECENTLY DESCRIBED FAMILY. THIS IS THE FIRST GENETIC EVIDENCE THAT THE LINKERS BETWEEN THE ZINC FINGERS OF TRANSCRIPTION FACTORS HAVE A FUNCTION BEYOND THAT OF A SIMPLE SPACER. 2015 18 4919 26 PANNEXIN-1 UP-REGULATION IN THE DORSAL ROOT GANGLION CONTRIBUTES TO NEUROPATHIC PAIN DEVELOPMENT. PANNEXIN-1 (PANX1) IS A LARGE-PORE MEMBRANE CHANNEL INVOLVED IN THE RELEASE OF ATP AND OTHER SIGNALING MEDIATORS. LITTLE IS KNOWN ABOUT THE EXPRESSION AND FUNCTIONAL ROLE OF PANX1 IN THE DORSAL ROOT GANGLION (DRG) IN THE DEVELOPMENT OF CHRONIC NEUROPATHIC PAIN. IN THIS STUDY, WE DETERMINED THE EPIGENETIC MECHANISM INVOLVED IN INCREASED PANX1 EXPRESSION IN THE DRG AFTER NERVE INJURY. SPINAL NERVE LIGATION IN RATS SIGNIFICANTLY INCREASED THE MRNA AND PROTEIN LEVELS OF PANX1 IN THE DRG BUT NOT IN THE SPINAL CORD. IMMUNOCYTOCHEMICAL LABELING SHOWED THAT PANX1 WAS PRIMARILY EXPRESSED IN A SUBSET OF MEDIUM AND LARGE DRG NEURONS IN CONTROL RATS AND THAT NERVE INJURY MARKEDLY INCREASED THE NUMBER OF PANX1-IMMUNOREACTIVE DRG NEURONS. NERVE INJURY SIGNIFICANTLY INCREASED THE ENRICHMENT OF TWO ACTIVATING HISTONE MARKS (H3K4ME2 AND H3K9AC) AND DECREASED THE OCCUPANCY OF TWO REPRESSIVE HISTONE MARKS (H3K9ME2 AND H3K27ME3) AROUND THE PROMOTER REGION OF PANX1 IN THE DRG. HOWEVER, NERVE INJURY HAD NO EFFECT ON THE DNA METHYLATION LEVEL AROUND THE PANX1 PROMOTER IN THE DRG. FURTHERMORE, INTRATHECAL INJECTION OF THE PANX1 BLOCKERS OR PANX1-SPECIFIC SIRNA SIGNIFICANTLY REDUCED PAIN HYPERSENSITIVITY INDUCED BY NERVE INJURY. IN ADDITION, SIRNA KNOCKDOWN OF PANX1 EXPRESSION IN A DRG CELL LINE SIGNIFICANTLY REDUCED CASPASE-1 RELEASE INDUCED BY NEURONAL DEPOLARIZATION. OUR FINDINGS SUGGEST THAT NERVE INJURY INCREASES PANX1 EXPRESSION LEVELS IN THE DRG THROUGH ALTERED HISTONE MODIFICATIONS. PANX1 UP-REGULATION CONTRIBUTES TO THE DEVELOPMENT OF NEUROPATHIC PAIN AND STIMULATION OF INFLAMMASOME SIGNALING. 2015 19 495 22 ASSESSMENT OF THE HEALTH EFFECTS OF TRICHLOROETHYLENE. THE EPIDEMIOLOGICAL STUDIES PERFORMED THUS FAR HAVE PRESENTED ONLY LIMITED EVIDENCE FOR THE CARCINOGENICITY OF TRICHLOROETHYLENE (TRI) TO HUMANS. HOWEVER, THESE STUDIES HAD DRAWBACKS SUCH AS INSUFFICIENT SIZE OF COHORT, SHORT OBSERVATION PERIOD, AND INADEQUATE TRI EXPOSURE ASSESSMENT; THEREFORE, NO CONCRETE CONCLUSION HAS BEEN REACHED CONCERNING TRI CARCINOGENICITY TO HUMANS. DESPITE THE LIMITED EPIDEMIOLOGICAL EVIDENCE AS TO THE CARCINOGENICITY OF TRI, THE INTERNATIONAL AGENCY FOR RESEARCH ON CANCER (IARC) HAS CHANGED THE CARCINOGENICITY CLASSIFICATION OF TRI FROM GROUP 3 (NOT CLASSIFIABLE AS TO CARCINOGENICITY TO HUMANS) TO GROUP 2A (PROBABLY CARCINOGENIC TO HUMANS). IN REGARD TO THE NEW CLASSIFICATION BY THE IARC, THE COMMITTEE FOR OCCUPATIONAL EXPOSURE LIMITS OF THE JAPAN SOCIETY FOR OCCUPATIONAL HEALTH HAS MADE A PROPOSAL THAT IT IS TOO EARLY TO CLASSIFY THE CARCINOGENICITY OF TRI AS GROUP 2A AND THAT IT IS PROPER TO PROMOTE EXPOSURE CONTROL WITH THE CARCINOGENICITY BEING CLASSIFIED AS 2B FOR THE MOMENT. THERE ARE SPECIES DIFFERENCES IN TRI CARCINOGENICITY, PARTICULARLY BETWEEN RATS AND MICE. ALTHOUGH EXPERIMENTAL STUDIES HAVE FOUND NO EVIDENCE THAT TRI INDUCES LIVER CANCER IN RATS, THERE IS AMPLE EVIDENCE THAT TRI PROMOTES THE DEVELOPMENT OF LIVER CANCER IN MICE, PARTICULARLY IN B6C3F1 MICE. THE CARCINOGENICITY OF TRI IN THIS STRAIN OF MICE MAY BE BASED ON AN EPIGENETIC MECHANISM RATHER ON A GENOTOXIC MECHANISM AND THE LIVER CANCER MAY BE INDUCED ONLY AFTER TRI HAS BEEN INHALED FOR A LONG PERIOD OF TIME AT CONCENTRATIONS HIGH ENOUGH TO CAUSE CYTOTOXICITY. CONVERSELY, WITH NO REPORTS SHOWING TRI-INDUCED RENAL TUMORS IN MICE, THE POSSIBILITY HAS BEEN SUGGESTED THAT THIS CHEMICAL INDUCES SUCH TUMORS IN MALE RATS. THE SPECIES DIFFERENCES ARE MAINLY ACCOUNTED FOR BY DIFFERENCES IN THE METABOLISM OF TRI BETWEEN RATS AND MICE. FROM A GENERAL SURVEY OF THE LITERATURE, IT CAN BE CONCLUDED THAT TRI ITSELF IS NOT MUTAGENIC. HOWEVER, THE CONJUGATION OF TRI WITH GLUTATHIONE (GSH), A MINOR PATHWAY OF TRI METABOLISM, RESULTS IN MUTAGENIC METABOLITES IN THE KIDNEY OF RATS. THE ACUTE TOXICITY OF TRI IS NEUROTOXICITY BASED ON ITS ANESTHETIC ACTION. AN EXPOSURE TO EXTREMELY HIGH LEVELS OF TRI MAY CAUSE THE LIVER AND KIDNEY DISORDERS. REPEATED EXPOSURES TO HIGH LEVELS OF TRI MAY RESULT IN NEURO-, HEPATO-, AND/OR NEPHROTOXICITY. THE MAIN SYMPTOMS APPEARING AFTER CHRONIC EXPOSURE AT LOW LEVELS ARE NEUROLOGICAL CHANGES REPRESENTED BY SUBJECTIVE SYMPTOMS RELATING TO CENTRAL AND AUTONOMIC NERVOUS SYSTEMS, OR BY A LOWERED CONDUCTION VELOCITY OF THE NERVES OR A PROLONGED LATENCY OF THE NERVE RESPONSES. FOR THE PRESENT, IT IS REASONABLE TO USE THE NEUROLOGICAL FINDINGS FOR ESTABLISHING THE REFERENCE VALUES OF TRI FOR BOTH WORK AND GENERAL ENVIRONMENTS. A VALUE OF 25 PPM (135 MG/M3) IS PROPOSED AS A REFERENCE VALUE FOR WORK ENVIRONMENTS, AND 25-50 PPB (135-270 MICROGRAMS/M3) FOR THE GENERAL ENVIRONMENT (1/1,000 OF THE VALUE FOR WORK ENVIRONMENT). 1997 20 131 25 A2B ADENOSINE SIGNALING REPRESSES CIITA TRANSCRIPTION VIA AN EPIGENETIC MECHANISM IN VASCULAR SMOOTH MUSCLE CELLS. CHRONIC INFLAMMATION PLAYS A MAJOR ROLE IN THE PATHOGENESIS OF ATHEROSCLEROSIS. VASCULAR SMOOTH MUSCLE CELLS (VSMC), BY EXPRESSING AND PRESENTING MAJOR HISTOCOMPATIBILITY COMPLEX II (MHC II) MOLECULES, HELP RECRUIT T LYMPHOCYTE AND INITIATE THE INFLAMMATORY RESPONSE WITHIN THE VASCULATURE. WE HAVE PREVIOUSLY SHOWN THAT VSMCS ISOLATED FROM MICE WITH DEFICIENT ADENOSINE A2B RECEPTOR (A2B-NULL) EXHIBIT HIGHER EXPRESSION OF CLASS II TRANSACTIVATOR (CIITA), THE MASTER REGULATOR OF MHC II TRANSCRIPTION, COMPARED TO WILD TYPE LITTERMATES. HERE WE REPORT THAT ACTIVATION OF A2B ADENOSINE SIGNALING SUPPRESSES CIITA EXPRESSION IN HUMAN AORTIC SMOOTH MUSCLE CELLS. DOWN-REGULATION OF CIITA EXPRESSION WAS LARGELY ATTRIBUTABLE TO TRANSCRIPTIONAL REPRESSION OF TYPE III AND IV PROMOTERS. CHROMATIN IMMUNOPRECIPITATION (CHIP) ANALYSES REVEALED THAT A2B SIGNALING REPRESSED CIITA TRANSCRIPTION BY ATTENUATING SPECIFIC HISTONE MODIFICATIONS ON THE CIITA PROMOTERS IN A STAT1-DEPENDENT MANNER. STAT1 INTERACTED WITH PCAF/GCN5, HISTONE H3K9 ACETYLTRANSFERASES, AND WDR5, A KEY COMPONENT OF THE MAMMALIAN H3K4 METHYLTRANSFERASE COMPLEX, TO ACTIVATE CIITA TRANSCRIPTION. A2B SIGNALING PREVENTED RECRUITMENT OF PCAF/GCN5 AND WDR5 TO THE CIITA PROMOTERS IN A STAT1-DEPENDENT MANNER. IN CONCLUSION, OUR DATA SUGGEST THAT ADENOSINE A2B SIGNALING REPRESSES CIITA TRANSCRIPTION IN VSMCS BY MANIPULATING THE INTERACTION BETWEEN STAT1 AND THE EPIGENETIC MACHINERY. 2015