Degree

Doctor of Philosophy (PhD)

Department

Biological Science

Document Type

Dissertation

Abstract

BEAF (Boundary Element-Associated Factor) was originally identified as a chromatin domain insulator binding protein, suggesting that it might play a role in linking gene regulation to chromatin organization and dynamics. Genome-wide mapping found that BEAF is usually found near transcription start sites, often of housekeeping genes, suggesting that it might play a role in promoter function. This would be a nontraditional role for an insulator binding protein. To gain insight into molecular mechanisms of BEAF function, we identified proteins that interact with BEAF using yeast 2-hybrid assays. Here we focus on three identified transcription factors: Bcd, Scr and Sry-δ. Interactions were confirmed in pull-down experiments using bacterially expressed proteins, by bimolecular fluorescence complementation, and in a genetic assay based on a rough eye phenotype in transgenic flies. The interaction with Sry-δ was most robust. Distantly bound Sry-δ was able to interact with promoter-proximal BEAF to activate a reporter gene in transient transfection experiments. We used both a minimal developmental promoter (y) and housekeeping promoter (RpS12), and found that BEAF itself strongly activated the housekeeping promoter. These two new functions for BEAF implicate it in playing a direct role in gene regulation at hundreds of BEAF-associated promoters.

Polybromo is subunit of a SWI/SNF (switch/sucrose non-fermenting) chromatin remodelling complex. A yeast two hybrid screen of a Drosophila cDNA library identified the nuclear protein polybromo as interacting with BEAF-32B. It contains six bromodomains and functions as a reader to recognize acetylated lysine residues on the tails of histones. Evidence from the screen indicates bromodomain 2 to 5 or 6 is responsible for the interaction with BEAF-32B. We performed yeast two-hybrid assays, serial dilution and pull down assays to determine that bromodomain 4-5 is crucial for the interaction. Next, we used yeast two-hybrid to study the interaction between the different sections of BEAF-32B with bromodomain 4-5, and identified the long leucine zipper region from BEAF-32B interacts with bromodomain 4-5.

Considering polybromo and BEAF-32B both play a role in Drosophila oogenesis, and polybromo can help to replace H3 with the H3.3 histone variant, we hypothesize that polybromo, BEAF-32B and Sry-δ work synergistically to regulate transcription during oogenesis and embryo development. It is possible that BEAF-32B binds and recruits PBAP to open chromatin, which facilitates interaction with Sry-δ for gene activation.

Date

4-7-2020

Committee Chair

Hart, Craig M.

Available for download on Tuesday, March 15, 2022

Included in

Biochemistry Commons

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