Doctor of Philosophy (PhD)
Compared to humans, the model organism Drosophila melanogaster is particularly gene dense and encodes several insulator binding proteins (IBP) to aid in its genome organization. Our focus of research is a particular IBP, Boundary Element-Associated Factor of 32kD (BEAF). BEAF primarily binds near the promoters of constitutively active housekeeping genes. It is thought to help maintain the active state of these genes by preventing the spread of repressive chromatin. Additionally, the enhancer blocking activity of BEAF is thought to prevent aberrant activation or silencing of genes. BEAF appears to be ubiquitously expressed among cells and tissues, albeit at varying levels. Despite its common expression, BEAF has an influence in certain developmental pathways, particularly in eye and ovary development. Ovaries from flies lacking BEAF are malformed and produce very few viable eggs. We isolated a mutation that rescues the ovary development phenotype and restores fertility to flies which lack functional BEAF. In this work we search for the location of this mutation we have named Tofu by the use of genomic sequencing, variant analysis, and classical genetics. From our investigations we identified mutations in a silencing element that may cause the misregulation of the ribbon gene. Evidence suggests ribbon may bind similar sequence motifs as BEAF and was identified in a Co-IP using BEAF, making it a strong candidate for Tofu. In a separate effort, we collect and analyze public genomic data relevant to BEAF, and analysis of lab generated data in order to dissect it’s role in insulation and gene activation. This includes: An analysis of BEAF binding in multiple cell and tissue types to look for differential binding and the effects on gene regulation. Analysis of BEAF-associated promoter size and motif distribution. Analysis of effects of transcription by RNA-seq and PRO-seq after BEAF knockdown. Analysis of nucleosome distribution after BEAF knockdown by MNase-seq. Analysis of chromatin conformation (Hi-C) after BEAF KD. We report that BEAF has a role in activating promoters to which it is bound, and find evidence that it insulates against activation by GAF.
McKowen, John Keller III, "Genomic Studies of BEAF-32 in Drosophila" (2021). LSU Doctoral Dissertations. 5515.
Hart, Craig M.
Available for download on Tuesday, March 15, 2022