Type of Document	Dissertation
Author	Gilbert, Matthew Kenneth
Author's Email Address	mgilbe6@lsu.edu
URN	etd-04132009-142305
Title	Evidence that the Boundary Element-Associated Factors BEAF-32A and BEAF-32B Affect Chromatin Structure in Drosophila melanogaster
Degree	Doctor of Philosophy (Ph.D.)
Department	Biochemistry (Biological Sciences)
Advisory Committee	Advisor Name Title Craig M. Hart Committee Chair Marcia Newcomer Committee Member Mark Batzer Committee Member Pat DiMario Committee Member Randall Hall Dean's Representative
Keywords	chromatin structure insulators
Date of Defense	2009-04-03
Availability	unrestricted
Abstract The Boundary Element-Associated Factors, BEAF-32A and BEAF-32B bind to hundreds of loci on Drosophila chromosomes. These proteins function as insulators; they can prevent promoter activation by an enhancer when placed between them and protect transgenes from chromosomal position effects. To gain insight into BEAF function we designed and expressed a transgene encoding a dominant-negative form of BEAF. This peptide, BID, consists of the BEAF self-interaction domain. We demonstrate here that this peptide interferes with BEAF’s ability to bind DNA and prevents it from functioning as an insulator. In addition, expression of BID leads to a global disruption of polytene chromosome structure. Subsequent work using a fly line with a null mutation in the BEAF gene (BEAF AB-KO) also demonstrates a perturbation to polytene chromosome structure, although it is limited to the X-chromosome. Using Micrococcal nuclease and DNase I we analyzed hypersensitive site alterations in the BEAF AB-KO line, and observed alterations that are consistent with the shifting of positioned nucleosomes. This effect appears limited to regions near promoters. Finally, using fluorescently-tagged BEAF-32A and BEAF- 32B we attempt to characterize the localization and behavior of these proteins. We find that they localize very differently on polytene chromosomes, that BEAF-32B disassociates from mitotic chromosomes while BEAF-32A remains associated, and FRAP experiments indicate different recovery dynamics. This data is consistent with a model that BEAF-dependent insulators function by affecting chromatin structure or dynamics.
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