UBC Theses and Dissertations
Development of a cell assay to study polycomb group genes Chevalier, Jacquelyn
Silencing is a form of transcriptional repression in which specialized structures of DNA or chromatin are inherited epigenetically. In Drosophila, the Polycomb group (PcG) genes are the best model system for studying silencing. Mutations in PcG genes cause posterior transformation in embryos and adults because homeotic genes are derepressed. PcG proteins act as members of oligomeric complexes. In Drosophila, PcG proteins mediate silencing of homeotic genes through complex, modular regulator elements ranging form 1 kb to 5 kb named PcG Response Elements (PREs). In transgenic flies, PREs maintain embryonic silencing of reporter constructs containing endogenous homeotic promoters and (para)segment-specific enhancers. This assay was used to identify PREs for many homeotic genes. PcG genes are required for maintenance rather than initiation and without the PRE, the transgene exhibits correct initiation of repression, but early in embryogenesis, the transgene becomes derepressed. All known Drosophila PcG genes have mammalian homologs and it appears that PcG function is conserved in mice and flies. Unfortunately, no one has identified a mammalian PRE in any system. Making transgenic mice is expensive and time-consuming, and no group has undertaken a systematic search for mammalian PREs. There is one published report of a group identifying a response element required for maintenance in mammals (Milne et al. 2002). Their approach tested different region of the Hox c8 locus in a reporter assay system and depended on the used of immortalized mouse embryonic fibroblasts (MEF). Based on the same principles, this thesis was aimed at identifying a murine PRE using immortalized fibroblast and a system of reporter vectors. The main goal of this thesis was to establish three MEF mutant cell lines for the PcG genes: rae28 the homolog of Polyhomeotic; M33 the homolog of Polycomb, or Asxll, the homolog of Additional sex combs. The secondary goal was to use these cells to test a putative mammalian PRE. This thesis reports the successful establishment of immortalized MEF mutant cell lines for Asxll, M33 and rae28. Our data suggest that MEF cell line of different genotypes may not be useful for studying the expression of endogenous genes. However, they may be useful for short-term studies of transgenes. One of these lines, rae28-/- was tested with a putative PRE from rae28 itself. Our preliminary results suggest that a PRE exists upstream of rae28, but the overall low transfection efficiency of the MEF, and the resulting high variability in expression of the reporter, prevents definitive conclusions. The strengths and weaknesses of this system are discussed and suggestion are made for ways to improve these experiments in the future.
Item Citations and Data