1 2091 101 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 2 1218 47 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 3 1219 50 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 4 17 34 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 5 129 47 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 6 6179 46 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 7 3827 48 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 8 340 43 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 9 5096 43 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 10 6695 32 VARIANT GENE EXPRESSION AND ANTIGENIC VARIATION BY MALARIA PARASITES. MALARIA IS A SIGNIFICANT THREAT THROUGHOUT THE DEVELOPING WORLD. AMONG THE MOST FASCINATING ASPECTS OF THE PROTOZOAN PARASITES RESPONSIBLE FOR THIS DISEASE ARE THE METHODS THEY EMPLOY TO AVOID THE IMMUNE SYSTEM AND PERPETUATE CHRONIC INFECTIONS. KEY AMONG THESE IS ANTIGENIC VARIATION: BY SYSTEMATICALLY ALTERING ANTIGENS THAT ARE DISPLAYED TO THE HOST'S IMMUNE SYSTEM, THE PARASITE RENDERS THE ADAPTIVE IMMUNE RESPONSE INEFFECTIVE. FOR PLASMODIUM FALCIPARUM, THE SPECIES RESPONSIBLE FOR THE MOST SEVERE FORM OF HUMAN MALARIA, THIS PROCESS INVOLVES A COMPLICATED MOLECULAR MECHANISM THAT RESULTS IN CONTINUOUSLY CHANGING PATTERNS OF VARIANT-ANTIGEN-ENCODING GENE EXPRESSION. ALTHOUGH MANY FEATURES OF THIS PROCESS REMAIN OBSCURE, SIGNIFICANT PROGRESS HAS BEEN MADE IN RECENT YEARS TO DECIPHER VARIOUS MOLECULAR ASPECTS OF THE REGULATORY CASCADE THAT CAUSES CHRONIC INFECTION. 2017 11 4490 28 MONOCYTE EPIGENETICS AND INNATE IMMUNITY TO MALARIA: YET ANOTHER LEVEL OF COMPLEXITY? CHILDREN UNDER THE AGE OF 5 YEARS LIVING IN AREAS OF MODERATE TO HIGH MALARIA TRANSMISSION ARE HIGHLY SUSCEPTIBLE TO CLINICAL MALARIA WITH FEVER THAT PROMPTS TREATMENT OF BLOOD STAGE INFECTION WITH ANTI-MALARIAL DRUGS. IN CONTRAST, OLDER SCHOOL AGE CHILDREN FREQUENTLY EXPERIENCE SUBCLINICAL MALARIA, I.E. CHRONIC PLASMODIUM FALCIPARUM PARASITEMIA WITHOUT FEVER OR OTHER CLINICAL SYMPTOMS. THE ROLE OF INNATE IMMUNE CELLS IN REGULATING INFLAMMATION AT A LEVEL THAT IS SUFFICIENT TO CONTROL THE PARASITE BIOMASS, WHILE AT THE SAME TIME MAINTAINING A DISEASE-TOLERANT CLINICAL PHENOTYPE, I.E., SUBCLINICAL MALARIA, IS NOT WELL UNDERSTOOD. RECENT STUDIES SUGGEST THAT HOST EPIGENETIC MECHANISMS UNDERLIE THE INNATE IMMUNE HOMEOSTASIS ASSOCIATED WITH SUBCLINICAL MALARIA. THIS CURRENT OPINION ARTICLE PRESENTS EVIDENCE SUPPORTING THE NOTION THAT MODIFICATIONS OF THE HOST MONOCYTE/MACROPHAGE EPIGENOME REGULATE INNATE IMMUNE FUNCTIONS PERTINENT TO SUBCLINICAL MALARIA. 2022 12 1349 28 DETERMINANTS OF INNATE IMMUNITY IN VISCERAL LEISHMANIASIS AND THEIR IMPLICATION IN VACCINE DEVELOPMENT. LEISHMANIASIS IS ENDEMIC TO THE TROPICAL AND SUBTROPICAL REGIONS OF THE WORLD AND IS TRANSMITTED BY THE BITE OF AN INFECTED SAND FLY. THE MULTIFACETED INTERACTIONS BETWEEN LEISHMANIA, THE HOST INNATE IMMUNE CELLS, AND THE ADAPTIVE IMMUNITY DETERMINE THE SEVERITY OF PATHOGENESIS AND DISEASE DEVELOPMENT. LEISHMANIA PARASITES ESTABLISH A CHRONIC INFECTION BY SUBVERSION AND ATTENUATION OF THE MICROBICIDAL FUNCTIONS OF PHAGOCYTIC INNATE IMMUNE CELLS SUCH AS NEUTROPHILS, MACROPHAGES AND DENDRITIC CELLS (DCS). OTHER INNATE CELLS SUCH AS INFLAMMATORY MONOCYTES, MAST CELLS AND NK CELLS, ALSO CONTRIBUTE TO RESISTANCE AND/OR SUSCEPTIBILITY TO LEISHMANIA INFECTION. IN ADDITION TO THE CYTOKINE/CHEMOKINE SIGNALS FROM THE INNATE IMMUNE CELLS, RECENT STUDIES IDENTIFIED THE SUBTLE SHIFTS IN THE METABOLIC PATHWAYS OF THE INNATE CELLS THAT ACTIVATE DISTINCT IMMUNE SIGNAL CASCADES. THE NEXUS BETWEEN METABOLIC PATHWAYS, EPIGENETIC REPROGRAMMING AND THE IMMUNE SIGNALING CASCADES THAT DRIVE THE DIVERGENT INNATE IMMUNE RESPONSES, REMAINS TO BE FULLY UNDERSTOOD IN LEISHMANIA PATHOGENESIS. FURTHER, DEVELOPMENT OF SAFE AND EFFICACIOUS VACCINES AGAINST LEISHMANIASIS REQUIRES A BROADER UNDERSTANDING OF THE EARLY INTERACTIONS BETWEEN THE PARASITES AND INNATE IMMUNE CELLS. IN THIS REVIEW WE FOCUS ON THE CURRENT UNDERSTANDING OF THE SPECIFIC ROLE OF INNATE IMMUNE CELLS, THE METABOLOMIC AND EPIGENETIC REPROGRAMMING AND IMMUNE REGULATION THAT OCCURS DURING VISCERAL LEISHMANIASIS, AND THE STRATEGIES USED BY THE PARASITE TO EVADE AND MODULATE HOST IMMUNITY. WE HIGHLIGHT HOW SUCH PATHWAYS COULD BE EXPLOITED IN THE DEVELOPMENT OF SAFE AND EFFICACIOUS LEISHMANIA VACCINES. 2021 13 3281 25 HETEROGENEOUS TFH CELL POPULATIONS THAT DEVELOP DURING ENTERIC HELMINTH INFECTION PREDICT THE QUALITY OF TYPE 2 PROTECTIVE RESPONSE. T FOLLICULAR HELPER (TFH) CELLS ARE AN IMPORTANT COMPONENT OF GERMINAL CENTER (GC)-MEDIATED HUMORAL IMMUNITY. YET, HOW A CHRONIC TYPE 1 VERSUS PROTECTIVE TYPE 2 HELMINTH INFECTION MODULATES TFH-GC RESPONSES REMAINS POORLY UNDERSTOOD. HERE, WE EMPLOY THE HELMINTH TRICHURIS MURIS MODEL AND DEMONSTRATE THAT TFH CELL PHENOTYPES AND GC ARE DIFFERENTIALLY REGULATED IN ACUTE VERSUS CHRONIC INFECTION. THE LATTER FAILED TO INDUCE TFH-GC B CELL RESPONSES, WITH TFH CELLS EXPRESSING TAU-BET AND INTERFERON-GAMMA. IN CONTRAST, INTERLEUKIN-4-PRODUCING TFH CELLS DOMINATE RESPONSES TO AN ACUTE, RESOLVING INFECTION. HEIGHTENED EXPRESSION AND INCREASED CHROMATIN ACCESSIBILITY OF T HELPER (TH)1- AND TH2 CELL-ASSOCIATED GENES ARE OBSERVED IN CHRONIC AND ACUTE INDUCED TFH CELLS, RESPECTIVELY. BLOCKADE OF THE TH1 CELL RESPONSE BY T-CELL-INTRINSIC T-BET DELETION PROMOTED TFH CELL EXPANSION DURING CHRONIC INFECTION, POINTING TO A CORRELATION BETWEEN A ROBUST TFH CELL RESPONSE AND PROTECTIVE IMMUNITY TO PARASITES. FINALLY, BLOCKADE OF TFH-GC INTERACTIONS IMPAIRED TYPE 2 IMMUNITY, REVEALING THE CRITICAL PROTECTIVE ROLE OF GC-DEPENDENT TH2-LIKE TFH CELL RESPONSES DURING ACUTE INFECTION. COLLECTIVELY, THESE RESULTS PROVIDE NEW INSIGHTS INTO THE PROTECTIVE ROLES OF TFH-GC RESPONSES AND IDENTIFY DISTINCT TRANSCRIPTIONAL AND EPIGENETIC FEATURES OF TFH CELLS THAT EMERGE DURING RESOLVING OR CHRONIC T. MURIS INFECTION. 2023 14 6293 25 THE PRO- AND ANTI-INFLAMMATORY POTENTIAL OF IL-12: THE DUAL ROLE OF TH1 CELLS. THE DIFFERENTIATION OF T-HELPER (TH) LYMPHOCYTES INTO VARIOUS TYPES OF T-HELPER EFFECTOR AND MEMORY CELLS WITH DISTINCT FUNCTIONS DEPENDING ON THE TYPE OF CONCOMITANT SIGNALS THEY RECEIVE UPON ACTIVATION IS A CRITICAL EVENT DETERMINING THE COURSE OF AN IMMUNE REACTION. TH1 CELLS CHARACTERIZED BY THE EXPRESSION OF IFN-GAMMA AND THE RECENTLY DESCRIBED TH17 CELLS PROMOTE INFLAMMATION AND ARE CRITICALLY INVOLVED IN THE INDUCTION AND MAINTENANCE OF AUTOIMMUNITY, WHEREAS THE SECRETION OF IL-4 IS A HALLMARK OF TH2 CELLS MEDIATING PROTECTION FROM PARASITES AND ALLERGY. ORIGINAL STIMULATION IN THE PRESENCE OF IL-12 RESULTS IN THE IMPRINTING OF TH1 MEMORY CELLS FOR THE EXPRESSION OF IFN-GAMMA BY EXPRESSION OF THE TRANSCRIPTION FACTOR T-BET AND EPIGENETIC MODIFICATION OF THE IFNGAMMA GENE. IT HAS BEEN DEMONSTRATED THAT TH1 CELLS ARE POTENT INDUCERS OF INFLAMMATION. HOWEVER, IN THE CHRONIC PHASE OF SUCH INFLAMMATION, THE REGULATORY POTENTIAL OF IL-12 AND TH1 CELLS THEMSELVES MAY PLAY AN IMPORTANT ROLE IN LIMITING IMMUNOPATHOLOGY. 2007 15 5981 30 TET2 PROMOTES PATHOGEN INFECTION-INDUCED MYELOPOIESIS THROUGH MRNA OXIDATION. VARIETIES OF RNA MODIFICATION FORM THE EPITRANSCRIPTOME FOR POST-TRANSCRIPTIONAL REGULATION. 5-METHYLCYTOSINE (5-MC) IS A SPARSE RNA MODIFICATION IN MESSENGER RNA (MRNA) UNDER PHYSIOLOGICAL CONDITIONS. THE FUNCTION OF RNA 5-HYDROXYMETHYLCYTOSINE (5-HMC) OXIDIZED BY TEN-ELEVEN TRANSLOCATION (TET) PROTEINS IN DROSOPHILA HAS BEEN REVEALED MORE RECENTLY. HOWEVER, THE TURNOVER AND FUNCTION OF 5-MC IN MAMMALIAN MRNA HAVE BEEN LARGELY UNKNOWN. TET2 SUPPRESSES MYELOID MALIGNANCIES MOSTLY IN AN ENZYMATIC ACTIVITY-DEPENDENT MANNER, AND IS IMPORTANT IN RESOLVING INFLAMMATORY RESPONSE IN AN ENZYMATIC ACTIVITY-INDEPENDENT WAY. MYELOPOIESIS IS A COMMON HOST IMMUNE RESPONSE IN ACUTE AND CHRONIC INFECTIONS; HOWEVER, ITS EPIGENETIC MECHANISM NEEDS TO BE IDENTIFIED. HERE WE DEMONSTRATE THAT TET2 PROMOTES INFECTION-INDUCED MYELOPOIESIS IN AN MRNA OXIDATION-DEPENDENT MANNER THROUGH ADAR1-MEDIATED REPRESSION OF SOCS3 EXPRESSION AT THE POST-TRANSCRIPTION LEVEL. TET2 PROMOTES BOTH ABDOMINAL SEPSIS-INDUCED EMERGENCY MYELOPOIESIS AND PARASITE-INDUCED MAST CELL EXPANSION THROUGH DECREASING MRNA LEVELS OF SOCS3, A KEY NEGATIVE REGULATOR OF THE JAK-STAT PATHWAY THAT IS CRITICAL FOR CYTOKINE-INDUCED MYELOPOIESIS. TET2 REPRESSES SOCS3 EXPRESSION THROUGH ADAR1, WHICH BINDS AND DESTABILIZES SOCS3 MRNA IN A RNA EDITING-INDEPENDENT MANNER. FOR THE UNDERLYING MECHANISM OF TET2 REGULATION AT THE MRNA LEVEL, TET2 MEDIATES OXIDATION OF 5-MC IN MRNA. TET2 DEFICIENCY LEADS TO THE TRANSCRIPTOME-WIDE APPEARANCE OF METHYLATED CYTOSINES, INCLUDING ONES IN THE 3' UNTRANSLATED REGION OF SOCS3, WHICH INFLUENCES DOUBLE-STRANDED RNA FORMATION FOR ADAR1 BINDING, PROBABLY THROUGH CYTOSINE METHYLATION-SPECIFIC READERS, SUCH AS RNA HELICASES. OUR STUDY REVEALS A PREVIOUSLY UNKNOWN REGULATORY ROLE OF TET2 AT THE EPITRANSCRIPTOMIC LEVEL, PROMOTING MYELOPOIESIS DURING INFECTION IN THE MAMMALIAN SYSTEM BY DECREASING 5-MCS IN MRNAS. MOREOVER, THE INHIBITORY FUNCTION OF CYTOSINE METHYLATION ON DOUBLE-STRANDED RNA FORMATION AND ADAR1 BINDING IN MRNA REVEALS ITS NEW PHYSIOLOGICAL ROLE IN THE MAMMALIAN SYSTEM. 2018 16 5125 35 POST-TRANSLATIONAL MODIFICATIONS OF TRYPANOSOMA CRUZI CANONICAL AND VARIANT HISTONES. CHAGAS DISEASE, CAUSED BY TRYPANOSOMA CRUZI, STILL AFFECTS MILLIONS OF PEOPLE AROUND THE WORLD. NO VACCINES NOR TREATMENT FOR CHRONIC CHAGAS DISEASE ARE AVAILABLE, AND CHEMOTHERAPY FOR THE ACUTE PHASE IS HINDERED BY LIMITED EFFICACY AND SEVERE SIDE EFFECTS. THE PROCESSES BY WHICH THE PARASITE ACQUIRES INFECTIVITY AND SURVIVES IN DIFFERENT HOSTS INVOLVE TIGHT REGULATION OF GENE EXPRESSION, MAINLY POST-TRANSCRIPTIONALLY. NEVERTHELESS, CHROMATIN STRUCTURE/ORGANIZATION OF TRYPANOSOMATIDS IS SIMILAR TO OTHER EUKARYOTES, INCLUDING HISTONE VARIANTS AND POST-TRANSLATIONAL MODIFICATIONS. EMERGING EVIDENCE SUGGESTS THAT EPIGENETIC MECHANISMS ALSO PLAY AN IMPORTANT ROLE IN THE BIOLOGY/PATHOGENESIS OF THESE PARASITES, MAKING EPIGENETIC TARGETS SUITABLE CANDIDATES TO DRUG DISCOVERY. HERE, WE PRESENT THE FIRST COMPREHENSIVE MAP OF POST-TRANSLATIONAL MODIFICATIONS OF T. CRUZI CANONICAL AND VARIANT HISTONES AND SHOW THAT ITS HISTONE CODE CAN BE AS SOPHISTICATED AS THAT OF OTHER EUKARYOTES. A TOTAL OF 13 DISTINCT MODIFICATION TYPES WERE IDENTIFIED, INCLUDING RATHER NOVEL AND UNUSUAL ONES SUCH AS ALTERNATIVE LYSINE ACYLATIONS, SERINE/THREONINE ACETYLATION, AND N-TERMINAL METHYLATION. SOME HISTONE MARKS CORRELATE TO THOSE DESCRIBED FOR OTHER ORGANISMS, SUGGESTING THAT SIMILAR REGULATORY MECHANISMS MAY BE IN PLACE. OTHERS, HOWEVER, ARE UNIQUE TO T. CRUZI OR TO TRYPANOSOMATIDS AS A GROUP AND MIGHT REPRESENT GOOD CANDIDATES FOR THE DEVELOPMENT OF ANTIPARASITIC DRUGS. 2017 17 3735 22 INNATE IMMUNE MEMORY: IMPLICATIONS FOR DEVELOPMENT OF PEDIATRIC IMMUNOMODULATORY AGENTS AND ADJUVANTED VACCINES. UNIQUE FEATURES OF IMMUNITY EARLY IN LIFE INCLUDE A DISTINCT IMMUNE SYSTEM PARTICULARLY RELIANT ON INNATE IMMUNITY, WITH WEAK T HELPER (TH)1-POLARIZING IMMUNE RESPONSES, AND IMPAIRED RESPONSES TO CERTAIN VACCINES LEADING TO A HEIGHTENED SUSCEPTIBILITY TO INFECTION. TO THESE IMPORTANT ASPECTS, WE NOW ADD AN INCREASINGLY APPRECIATED CONCEPT THAT THE INNATE IMMUNE SYSTEM DISPLAYS EPIGENETIC MEMORY OF AN EARLIER INFECTION OR VACCINATION, A PHENOMENON THAT HAS BEEN NAMED "TRAINED IMMUNITY." EXPOSURE OF NEONATAL LEUKOCYTES IN VITRO OR NEONATAL ANIMALS OR HUMANS IN VIVO TO SPECIFIC INNATE IMMUNE STIMULI RESULTS IN AN ALTERED INNATE IMMUNE SET POINT. GIVEN THE PARTICULAR IMPORTANCE OF INNATE IMMUNITY EARLY IN LIFE, TRAINED IMMUNITY TO EARLY LIFE INFECTION AND/OR IMMUNIZATION MAY PLAY AN IMPORTANT ROLE IN MODULATING BOTH ACUTE AND CHRONIC DISEASES. 2014 18 6121 23 THE EPIGENETIC LANDSCAPE OF T CELL EXHAUSTION. EXHAUSTED T CELLS IN CANCER AND CHRONIC VIRAL INFECTION EXPRESS DISTINCTIVE PATTERNS OF GENES, INCLUDING SUSTAINED EXPRESSION OF PROGRAMMED CELL DEATH PROTEIN 1 (PD-1). HOWEVER, THE REGULATION OF GENE EXPRESSION IN EXHAUSTED T CELLS IS POORLY UNDERSTOOD. HERE, WE DEFINE THE ACCESSIBLE CHROMATIN LANDSCAPE IN EXHAUSTED CD8(+) T CELLS AND SHOW THAT IT IS DISTINCT FROM FUNCTIONAL MEMORY CD8(+) T CELLS. EXHAUSTED CD8(+) T CELLS IN HUMANS AND A MOUSE MODEL OF CHRONIC VIRAL INFECTION ACQUIRE A STATE-SPECIFIC EPIGENETIC LANDSCAPE ORGANIZED INTO FUNCTIONAL MODULES OF ENHANCERS. GENOME EDITING SHOWS THAT PD-1 EXPRESSION IS REGULATED IN PART BY AN EXHAUSTION-SPECIFIC ENHANCER THAT CONTAINS ESSENTIAL RAR, T-BET, AND SOX3 MOTIFS. FUNCTIONAL ENHANCER MAPS MAY OFFER TARGETS FOR GENOME EDITING THAT ALTER GENE EXPRESSION PREFERENTIALLY IN EXHAUSTED CD8(+) T CELLS. 2016 19 3662 26 INDUCTION OF STABLE HUMAN FOXP3(+) TREGS BY A PARASITE-DERIVED TGF-BETA MIMIC. IMMUNE HOMEOSTASIS IN THE INTESTINE IS TIGHTLY CONTROLLED BY FOXP3(+) REGULATORY T CELLS (TREGS), DEFECTS OF WHICH ARE LINKED TO THE DEVELOPMENT OF CHRONIC CONDITIONS, SUCH AS INFLAMMATORY BOWEL DISEASE (IBD). AS A MECHANISM OF IMMUNE EVASION, SEVERAL SPECIES OF INTESTINAL PARASITES BOOST TREG ACTIVITY. THE PARASITE HELIGMOSOMOIDES POLYGYRUS IS KNOWN TO SECRETE A MOLECULE (HP-TGM) THAT MIMICS THE ABILITY OF TGF-BETA TO INDUCE FOXP3 EXPRESSION IN CD4(+) T CELLS. THE STUDY AIMED TO INVESTIGATE WHETHER HP-TGM COULD INDUCE HUMAN FOXP3(+) TREGS AS A POTENTIAL THERAPEUTIC APPROACH FOR INFLAMMATORY DISEASES. CD4(+) T CELLS FROM HEALTHY VOLUNTEERS WERE EXPANDED IN THE PRESENCE OF HP-TGM OR TGF-BETA. TREG INDUCTION WAS MEASURED BY FLOW CYTOMETRIC DETECTION OF FOXP3 AND OTHER TREG MARKERS, SUCH AS CD25 AND CTLA-4. EPIGENETIC CHANGES WERE DETECTED USING CHIP-SEQ AND PYROSEQUENCING OF FOXP3. TREG PHENOTYPE STABILITY WAS ASSESSED FOLLOWING INFLAMMATORY CYTOKINE CHALLENGE AND TREG FUNCTION WAS EVALUATED BY CELLULAR CO-CULTURE SUPPRESSION ASSAYS AND CYTOMETRIC BEAD ARRAYS FOR SECRETED CYTOKINES. HP-TGM EFFICIENTLY INDUCED FOXP3 EXPRESSION (> 60%), IN ADDITION TO CD25 AND CTLA-4, AND CAUSED EPIGENETIC MODIFICATION OF THE FOXP3 LOCUS TO A GREATER EXTENT THAN TGF-BETA. HP-TGM-INDUCED TREGS HAD SUPERIOR SUPPRESSIVE FUNCTION COMPARED WITH TGF-BETA-INDUCED TREGS, AND RETAINED THEIR PHENOTYPE FOLLOWING EXPOSURE TO INFLAMMATORY CYTOKINES. FURTHERMORE, HP-TGM INDUCED A TREG-LIKE PHENOTYPE IN IN VIVO DIFFERENTIATED TH1 AND TH17 CELLS, INDICATING ITS POTENTIAL TO RE-PROGRAM MEMORY CELLS TO ENHANCE IMMUNE TOLERANCE. THESE DATA INDICATE HP-TGM HAS POTENTIAL TO BE USED TO GENERATE STABLE HUMAN FOXP3(+) TREGS TO TREAT IBD AND OTHER INFLAMMATORY DISEASES. 2021 20 6706 24 VIRAL GENE PRODUCTS ACTIVELY PROMOTE LATENT INFECTION BY EPIGENETIC SILENCING MECHANISMS. MANY VIRUSES UNDERGO AN ACUTE INFECTION IN THE HOST ORGANISM AND THEN ARE CLEARED BY THE ENSUING HOST IMMUNE RESPONSE, BUT OTHER VIRUSES ESTABLISH A PERSISTENT INFECTION INVOLVING A LATENT INFECTION OR A CHRONIC INFECTION. LATENT INFECTION BY THE HERPESVIRUSES OR HUMAN IMMUNODEFICIENCY VIRUS INVOLVES EPIGENETIC SILENCING OF THE DNA GENOME OR PROVIRAL GENOME, RESPECTIVELY. LATENT INFECTION WAS PREVIOUSLY THOUGHT TO BE A DEFAULT PATHWAY RESULTING FROM INFECTION OF A NONPERMISSIVE CELL, BUT RECENT STUDIES HAVE SHOWN THAT VIRAL GENE PRODUCTS CAN PROMOTE EPIGENETIC SILENCING AND LATENT INFECTION. THIS REVIEW WILL SUMMARIZE THE VIRAL GENE PRODUCTS THAT HAVE BEEN SHOWN TO PROMOTE EPIGENETIC SILENCING OF THE GENOMES AND THEIR POTENTIAL FOR THERAPEUTICS TO TARGET THESE VIRAL GENE PRODUCTS AND DISRUPT OR LOCK IN LATENT INFECTION. 2017