GA-Binding Proteins Regulate Dosage Compensation in Drosophila Melanogaster

Kaye, Emily G.

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Year:: 2017
Contributor:: Kaye, Emily G. (creator); Freiman, Richard (Reader); Wharton, Kristi (Reader); Larschan, Erica (Advisor); Guertin, Michael (Reader); Fawzi, Nicolas (Reader); Brown University. Department of Molecular Biology, Cell Biology and Biochemistry (sponsor)
Genre:: theses
Subject:: Transcription factors; dosage compensation
Extent:: xvii, 241 p.
DOI: https://doi.org/10.26300/ek07-en15

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Abstract:: Abstract of “GA-Binding Proteins Regulate Dosage Compensation in Drosophila melanogaster” by Emily Grace Kaye, Ph.D., Brown University, May 2018 A transcription factor (TF) will bind only a subset of its potential binding sites in the genome. Which sites are bound ultimately determines the functional impact of a TF on the cell. To improve our understanding of TF targeting and transcriptional regulation, we compare the role of two essential proteins with similar consensus binding motifs: GAGA Factor (GAF), and Chromatin-Linked Adapter for MSL Proteins (CLAMP). Both target GA-rich sequences genome-wide, yet CLAMP has a specialized role in recruiting the Male-Specific Lethal (MSL) complex to the single male X-chromosome in Drosophila melanogaster for the process known as gene dosage compensation. Dosage compensation corrects the imbalance between gene expression of sex-linked genes and autosomal genes. MSL facilitates the increase in transcription of genes on the male X-chromosome by first localizing to GA-rich DNA sequence motifs clustered in Chromatin Entry Sites (CES) on the X-chromosome. CLAMP is required for MSL recruitment to CES, while GAF depletion can cause male-specific lethality but leads to loss of MSL at only a single site when measured by immunostaining. Since both TFs are essential in males and females, we first investigated differences in GA-binding site sequences, including analysis of the distribution of sequences on X versus autosomes. Next, to test their individual versus overlapping functions, we used in vitro gel-shift assays to determine that CLAMP outcompetes GAF for long GA-repeat stretches, thus contributing to the differences observed between binding patterns for the two proteins. To investigate potential interactions at shared binding sites, we performed RNA interference (RNAi) experiments followed by chromatin immunoprecipitation (ChIP) to test for relocalization of each factor after the other has been knocked-down. Finally, we identify a shared function between GAF and CLAMP as part of an insulator complex, which may explain the requirement for both proteins in MSL targeting and as well as reveals a shared non sex-specific essential function. Overall our experiments provide insight into TF targeting for dosage compensation, an example of context-specific gene regulation.
Notes:: Thesis (Ph. D.)--Brown University, 2017

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Kaye, Emily G., "GA-Binding Proteins Regulate Dosage Compensation in Drosophila Melanogaster" (2017). Molecular Biology, Cell Biology, and Biochemistry Theses and Dissertations. Brown Digital Repository. Brown University Library. https://doi.org/10.26300/ek07-en15

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Molecular Biology, Cell Biology, and Biochemistry Theses and Dissertations

Theses and Dissertations for the Molecular Biology, Cell Biology, and Biochemistry department.
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