Evidence that the Boundary Element-Associated Factors BEAF-32A and BEAF-32B affect chromatin structure in Drosophila melanogaster

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.

Name thesis:degree_name

Doctor of Philosophy (PhD)

Level thesis:degree_level

Dissertation

Grantor

Biological Sciences

Year dc:date.available

2009

Author dc:creator

• Gilbert, Matthew Kenneth

• chromatin structure
• insulators

Statement dc:rights

• unrestricted
• Release the entire work immediately for access worldwide.

Identifier

etd-04132009-142305
https://repository.lsu.edu/gradschool_dissertations/1491

OAI identifier oai:identifier

oai:repository.lsu.edu:gradschool_dissertations-2490