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UBC Theses and Dissertations

Functional dissection of the Drosophila melanogaster bithoraxoid Polycomb response element Argiropoulos, Bob

Abstract

During the development of Drosophila melanogaster, the Polycomb Group (PcG) proteins act through complex, modular elements termed Polycomb response elements (PREs) to maintain the silent state of the homeotic and other loci. The bithoraxoid (bxd) 5.1 PRE of the homeotic gene Ultrabithorax (Ubx) is capable of maintaining the correct Ubx expression pattern throughout embryogenesis in a PcG-dependent manner. The bxd5.l PRE is also capable of conferring pairing-sensitive repression to the mini-white gene located in the same transposon. In order to understand how PcG proteins are recruited to PREs, a gel mobility shift assay was used to identify four fragments within the bxd5.l PRE that bind protein complexes from nuclear extracts that contain the PcG protein Polyhomeotic (PH). Chapter 2 of this thesis examines the in vivo contribution of these four PH binding sites in embryonic silencing and pairing-sensitive repression. I show, using a germline transformation assay, that deletion of each PH binding site, in the context of bxd5.\, disrupts embryonic PRE activity but not pairing-sensitive repression. Double mutant analysis of sites with related binding activities indicate that sites MHS-70 and MPA-168 constitute one functional unit of PRE activity, which is disabled by either mutation. By contrast, sites MHN-90 and S1HB-90 act synergistically to promote PRE activity. Furthermore, mutation of two d(GA)3 repeat elements within MHS-70 destabilizes PH complex formation in vitro and partially abrogates PRE activity in vivo indicating that these repeat elements are essential for PRE-mediated silencing. Chapter 3 of this thesis explores the modular structure of the core maintenance element within bxd5.l, the bxd\.5 PRE. The results indicate that the bxdl.5 PRE is a complex element built up of at least three modules, UPS, PSR and DPS, that make distinct contributions to silencing by the bxd PRE. The UPS and DPS modules directly repress the Ubx promoter in a parasegment-specific and developmental stage-specific manner. The PSR and DPS modules are capable of pairing-sensitive repression. Genetic analyses reveal that each module depends on the function of a subset of PcG and trxG genes that are required specifically for embryonic or pairing-sensitive repression or for both processes. The results clearly demonstrate that embryonic and pairing-sensitive repression are separable functions of the bxdl .5 PRE. Taken together, these studies provide insight into how the bxd PRE is built and into the nature of the functional components that read and interpret the information encoded by this complex cis-regulatory element.

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