Doctor of Philosophy (PhD)
Proper folding of eukaryotic genomes is required to allow correct interactions between different parts of chromosomes. Precise and timely interactions among different parts of a chromosome allow proper functioning inside a nucleus, including gene regulation, DNA replication and DNA repair. Eukaryotic regulatory elements that facilitate folding and interactions include enhancers, promoters and insulator elements. Insulator elements and their binding proteins play an important role in regulating correct chromatin structure and function. The Drosophila melanogaster special chromatin structure (scs’) is one such insulator. The Boundary Element Associated Factor (BEAF) binds to scs’. BEAF is a 32 kDa protein that has two isoforms, 32A and 32B. Genomic studies have indicated that BEAF binds from 1800 to 3000 sites in the Drosophila genome, usually near transcriptions start sites (TSSs) mainly of housekeeping and highly active genes. In this study, we performed a detailed analysis of scs’ to more precisely understand the role of BEAF in insulator and promoter function. We dissected the scs’ insulator to find minimal sequences required for insulator and promoter functions. We found these two functions overlap by 110 base pairs (bp) but can be separated. BEAF is necessary for both, but insulator function requires 50 bp additional downstream DNA sequences while promoter activity requires 50 bp additional upstream sequences. Attempts to identify binding proteins that might work with BEAF have so far been unsuccessful. We also took another approach to getting at BEAF function. Proteins that physically associate with BEAF were identified by co-immunoprecipitation from nuclear protein extracts followed by proteomic massspectrometry. These results suggest that in addition to classical insulator function, BEAF might play a more direct role in gene expression. Notably, chromatin remodeling proteins, histone chaperones and transcription factors were identified. This supports the idea that BEAF might vplay a role in keeping promoters active by helping to establish or maintain nucleosome depleted regions around TSSs.
Maharjan, Mukesh, "Role of the Drosophila BEAF Protein in Chromatin Domain Insulator and Promoter Function" (2019). LSU Doctoral Dissertations. 4917.
Hart, Craig M.