Characterization of chromatin architecture and the mechanism of dosage compensation in Drosophila melanogaster
Bishop, Eric Paul
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Chromatin, the DNA/protein complex that makes up the major part of eukaryotic chromosomes, plays an important role in regulating many biological processes. Distinct patterns of histone occupancy correlate with different levels of transcriptional activity and with many functional elements. Modern high-throughput sequencing technology combined with chromatin immunoprecipitation (ChIP) has made it possible to profile histone modification patterns on a genome-wide scale for large mammalian genomes. In this thesis, I perform the first comprehensive comparison of histone modifications and nucleosome occupancy between D. melanogaster and H. sapiens. A systematic comparison among multiple organisms is challenging due to issues in integrating datasets derived from multiple platforms and technologies, but I show that proper normalization enables this comparison. My analysis reveals key differences in enrichment patterns of histone modifications, which are driven by differences in nucleosome occupancy. One biological system in which chromatin architecture plays a major role is Drosophila male dosage compensation, the process by which male X-linked genes are up-regulated to compensate for the lack of a second copy. Dosage compensation is regulated primarily by the Male-Specific Lethal (MSL) protein complex. However, the mechanisms by which MSL up-regulates X-linked genes and by which it specifically targets the X chromosome remain poorly understood. I performed analysis of Global Run-on Sequencing (GRO-seq) to determine the specific effect of MSL on the level of nascent transcripts in male Drosophila cells. My analysis indicates that MSL activity results in increased RNA polymerase density within the bodies of active X-linked genes, suggesting that regulation occurs at the level of transcriptional elongation. To further investigate the targeting mechanism of MSL, I examined expression and binding data for CLAMP (Coupling Lethal Adapter for MSL Proteins) a previously uncharacterized protein identified in a recent screen as a regulator of MSL. This analysis reveals that CLAMP exhibits a synergistic relationship with MSL and promotes X-chromosome specificity. Taken together, my results provide novel insights into the nature of the Drosophila chromatin landscape and the regulatory mechanism of dosage compensation.
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