Project Grant F32GM167147

HISTONE ACETYLATION AND CHROMATIN REMODELING AS EPIGENETIC DRIVERS OF MEIOTIC RECOMBINATION - PROJECT ABSTRACT MEIOTIC RECOMBINATION IS AN ESSENTIAL PROCESS FOR THE FERTILITY OF MANY ORGANISMS, INCLUDING HUMANS. ERRORS IN RECOMBINATION CAN CAUSE LOSS OF GAMETOCYTE PRODUCTION, CHROMOSOME MISSEGREGATION, STERILITY, MISCARRIAGES, AND/OR DEVELOPMENTAL DEFECTS IN CHILDREN. THE FACTORS THAT CONTROL RECOMBINATION POSITIONS IN MAMMALS REMAINS POORLY UNDERSTOOD, PARTICULARLY THE EPIGENETIC FACTORS THAT GOVERN CHROMATIN STRUCTURE AT RECOMBINATION SITES. RECOMBINATION IS INITIATED BY DNA DOUBLE STRAND BREAKS (DSBS) MADE BY THE TOPOISOMERASE-LIKE SPO11 PROTEIN. IN MOST MAMMALS, SPO11-INDUCED DSBS OCCUR IN PREFERENTIAL GENOMIC REGIONS TERMED "HOTSPOTS". HOTSPOTS HAVE DISTINCT HISTONE POST-TRANSLATIONAL MODIFICATIONS (PTMS) AND NUCLEOSOME POSITIONING EVENTS. TWO LONG- KNOWN HOTSPOT EPIGENETIC FEATURES ARE HISTONE ACETYLATION AND OPEN CHROMATIN ARCHITECTURE, BUT THE IMPACT OF THESE EPIGENETIC INPUTS ON MEIOTIC RECOMBINATION REMAINS UNKNOWN DUE TO THE LACK OF INFORMATION REGARDING THE ENZYMATIC EFFECTORS OF THESE EPIGENETIC FEATURES. THIS RESEARCH PLAN WILL ADDRESS THESE KNOWLEDGE GAPS BY CHARACTERIZING NOVEL EPIGENETIC DRIVERS OF MEIOTIC RECOMBINATION IN MICE. AIM 1 WILL FOCUS ON IDENTIFYING THE HISTONE ACETYLTRANSFERASE (HAT) RESPONSIBLE FOR DECORATING RECOMBINATION HOTSPOTS WITH ACETYLATION MARKS. A CANDIDATE-BASED APPROACH WILL BE USED TO IDENTIFY (VIA CUT-AND-RUN AND/OR CHIP-SEQ) DSB-HOTSPOT-LOCALIZED HISTONE ACETYLTRANSFERASES. PROMISING CANDIDATES FROM THIS DNA-PROTEIN INTERACTION SCREEN WILL BE SUBJECT TO KNOCKOUT OR TESTIS-SPECIFIC CONDITIONAL KNOCKOUT WITHIN MOUSE MODELS. THESE MODELS WILL BE USED TO TEST FOR HAT CANDIDATE ACETYLATION OF HOTSPOTS, THE RELEVANCE OF HOTSPOT ACETYLATION TO DSB FORMATION AND RESECTION, AND THE ROLE OF HOTSPOT ACETYLATION IN SHAPING THE CHROMATIN ARCHITECTURE AT DSB HOTSPOTS. SUCCESSFUL COMPLETION OF THIS AIM WILL DETERMINE THE WRITER(S) OF HISTONE ACETYLATION AT RECOMBINATION SITES AND WILL GENERATE A HOTSPOT HAT- DEFICIENT MODEL TO DETERMINE IF AND HOW HISTONE ACETYLATION AFFECTS KEY ASPECTS OF RECOMBINATION IN VIVO, SUCH AS CHROMATIN ORGANIZATION, DSB LOCATION, AND DSB END RESECTION. AIM 2 WILL FOCUS ON NUCLEOSOME REMODELING PROTEINS INVOLVED IN MEIOTIC RECOMBINATION. UTILIZING SIMILAR APPROACHES TO AIM 1, A CANDIDATE APPROACH WILL BE APPLIED UTILIZING CUT-AND-RUN AND/OR CHIP-SEQ TO IDENTIFY NOVEL CHROMATIN REMODELERS THAT LOCALIZE TO DSB HOTSPOTS. I WILL FOCUS ON CANDIDATES THAT MAY ACT AS POTENTIAL READERS OF HOTSPOT NUCLEOSOME POST-TRANSLATIONAL MODIFICATIONS. PROMISING CANDIDATES THAT LOCALIZE TO HOTSPOTS WILL BE SUBJECT TO KNOCKOUT OR TESTIS-SPECIFIC CONDITIONAL KNOCKOUT WITHIN MOUSE MODELS. USING THESE MODELS, I WILL APPLY GENOME WIDE CHROMATIN ACCESSIBILITY PROFILING (ATAC-SEQ/MNASE-SEQ) TO TEST CANDIDATES' ROLES IN HOTSPOT ACCESSIBILITY. I WILL ALSO PROFILE DSB FORMATION AND END-RESECTION PHENOTYPES GENOME WIDE WITH S1-SEQ. USING HAT-DEFICIENT MICE FROM AIM 1, I WILL TEST IF HISTONE ACETYLATION SUPPORTS RECRUITMENT OF CHROMATIN REMODELERS TO HOTSPOTS. TOGETHER, RESULTS FROM THESE AIMS WILL IMPROVE OUR UNDERSTANDING OF THE EPIGENETIC FACTORS ESSENTIAL FOR MEIOTIC RECOMBINATION AND OFFER A PLATFORM FOR INVESTIGATING MECHANISTIC LINKS BETWEEN NUCLEOSOME MODIFICATION AND NUCLEOSOME REMODELING.