Meiotic homologous recombination generates genetic variability by creating new combinations of alleles, and is required for the proper segregration of homologous chromosomes in the first meiotic division. Errors in meiotic recombination contribute to aneuploidy, which is the leading cause of spontaneous pregnancy losses, congenital birth defects, and developmental disabilities. Although recombination can occur anywhere in the genome, it clusters at hotspots, which regulate its frequency and distribution. The current model of hotspot activation involves transcription factors binding at their specific DNA binding sites, and then recruiting histone modifying enzymes and chromatin remodeling enzymes which remodel chromatin around the hotspots, and thus exposing the underlying DNA to the basal recombination machinery (Rec12/Spo11) which induces DNA-double stranded breaks to initiate recombination. We tested this model at five different hotspot-activating DNA motifs (M26, CCAAT, Oligo-C, 4095, 4156) inserted independently at the same location in the ade6 locus. Each motif activates hotspot recombination, and this activation required the respective binding transcription factors (Atf1, Pcr1, Php2, Php3, Php5, and Rst2). We also analyzed the chromatin landscape at these five meiotic recombination hotspot DNA motifs at single-nucleosome resolution. The results revealed that each DNA motif triggers nucleosome displacement around its site leading to an open chromatin configuration. The respective DNA motif binding proteins are required for this chromatin remodeling. Hotspot activity of each motif also required the ATP-dependent chromatin remodeler Snf22, with lesser dependence on Gcn5, Mst2 and Hrp3. We conclude that distinct, cis-acting regulatory modules (transcription factor complexes) function redundantly to promote recombination through the same chromatin remodeling pathways. Interestingly, we also found that some hotspot-activating proteins affect recombination at hotspot DNA sequence motifs to which they do not bind. We show that this indirect regulation (cross-talk) is mediated via transcription of the hotspot-activating proteins, which are rate-limiting for hotspot activity. Lastly, we developed a DNA-afffinity capture with mass spectrometry (DAC-MS) approach to identify proteins which bind to additional, hotspot-regulating DNA sequence motifs. In summary, this dissertation work revealed that multiple, sequence-specific protein-DNA complexes activate meiotic recombination hotspots via chromatin remodeling, which allows the basal machinery of recombination access to its DNA substrates within chromatin.
|Advisor:||Wahls, Wayne P.|
|Commitee:||Baldini, Giulia, Eoff, Robert R., Glazko, Galina V., Tackett, Alan J.|
|School:||University of Arkansas for Medical Sciences|
|Department:||Biochemistry and Molecular Biology|
|School Location:||United States -- Arkansas|
|Source:||DAI-B 81/10(E), Dissertation Abstracts International|
|Subjects:||Biochemistry, Genetics, Molecular biology|
|Keywords:||Chromatin remodeling enzymes, Chromatin structure analysis with micrococcal nuclease, Fission yeast, Mass spectrometry, Meiosis, Recombination hotspots and chromatin remodeling, Protein affinity purification|
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