Title page for ETD etd-04132009-142305


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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