Heterochromatin-mediated gene silencing provides a valuable genetic tool for the study of chromatin in Drosophila. However, although traditional genetic screens for modifiers of silencing have identified many factors that contribute to silencing, the distinction between its establishment and its maintenance remains poorly understood. To address this issue, I have performed an over-expression screen for modifiers of silencing, in which timing of expression can be easily controlled. I have focused my work on two of these factors – the Crol zinc-finger protein, and the Xnp chromatin remodeler.
Crol is special in its effect on silencing. While other modifiers of silencing alter the expression frequency of the reporter gene, early over-expression of Crol in development changes the pattern of silencing later in development. I propose that Crol over-expression switches the chromatin of the reporter gene into a heritable transcriptionally-permissive state, thus establishing 'open' chromatin.
Over-expression of the Xnp remodeler alters the frequency of silencing, and null mutants for this remodeler exhibit severe de-repression. This demonstrates that Xnp is required for proper silencing. I find that Xnp co-localizes with the replacement histone H3.3 on chromatin, and is important for its deposition into chromatin. The Hira histone chaperone also co-localizes with Xnp and H3.3 on chromatin, and is important for the deposition of the histone. While both xnp and Hira mutants are viable, flies mutant for xnp and Hira exhibit no deposition of H3.3, and die as larvae. This lethality reveals that RI nucleosome assembly is essential in Drosophila. Furthermore, I have found that both Xnp and Hira arrive to chromatin prior to the arrival of histones, suggesting that replication-independent (RI) nucleosome assembly occurs in a stepwise manner. I propose that Xnp and Hira promote RI assembly by recognizing disrupted chromatin and forming a binding platform at the site, to which soluble chaperoned histones are delivered. In the absence of RI assembly, genome-wide transcription may be compromised and DNA may be more susceptible to damage.
|Advisor:||Ahmad, Kami, Wu, Chao-Ting|
|School Location:||United States -- Massachusetts|
|Source:||DAI-B 72/04, Dissertation Abstracts International|
|Subjects:||Molecular biology, Genetics|
|Keywords:||Chromatin, Epigenetics, Histone chaperones, Nucleosome assembly|
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