Access Type

Open Access Dissertation

Date of Award

January 2017

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Biological Sciences

First Advisor

LORI A. PILE

Abstract

he multisubunit SIN3 complex is a global transcriptional regulator. In Drosophila, a single Sin3A gene encodes different isoforms of SIN3, of which SIN3 187 and SIN3 220 are the major isoforms. Previous studies have demonstrated functional non-redundancy of SIN3 isoforms. The role of SIN3 isoforms in regulating distinct biological processes, however, is not well characterized. In addition, how the components of the SIN3 complex modulate the gene regulatory activity of the complex is not well understood. In this study, I identified the biological processes regulated by the SIN3 isoforms. Additionally, I explored how Caf1-55 impacts the gene regulatory activity of the SIN3 220 complex.

For the purpose of the study, I developed a highly reproducible ChIP protocol using micrococcal nuclease (MNase)-mediated chromatin preparation from Drosophila cultured cells. This protocol can be used to perform ChIP to map both histones and non-histone chromatin binding proteins locally and globally across the genome.

Next, we identified the biological processes regulated by the SIN3 isoforms. We established a Drosophila S2 cell culture model system in which cells predominantly express either SIN3 187 or SIN3 220. To identify genomic targets of SIN3 isoforms, we performed chromatin immunoprecipitation followed by deep sequencing. Our data demonstrate that upon overexpression of SIN3 187, the level of SIN3 220 decreased and the large majority of genomic sites bound by SIN3 220 were instead bound by SIN3 187. We used RNA-seq to identify genes regulated by the expression of one isoform or the other. In S2 cells, which predominantly express SIN3 220, we found that SIN3 220 directly regulates genes involved in metabolism and cell proliferation. We also determined that SIN3 187 regulates a unique set of genes and likely modulates expression of many genes also regulated by SIN3 220. Interestingly, biological pathways enriched for genes specifically regulated by SIN3 187 strongly suggest that this isoform plays an important role during the transition from the embryonic to the larval stage of development.

Finally, I investigated the function of Caf1-55 in the SIN3 220 complex. Our data demonstrate that Caf1-55 localizes to SIN3 220 gene targets and is partly required for recruiting SIN3 220 to chromatin. In addition, we show that the C-terminal domain of SIN3 220 physically interacts with Caf1-55. We found that the interaction between SIN3 and Caf1-55 is significantly reduced upon mutating the histone H4 binding pocket of Caf1-55. Surprisingly, the reduced interaction between the histone H4 binding mutant of Caf1-55 and SIN3 220 is not sufficient to cause a change in the expression of SIN3 220 regulated genes. Together, these data provide evidence of a novel role of Caf1-55 in impacting recruitment of a component of a chromatin modifying complex to genomic loci.

In summary, our research reveals important insights of how the SIN3 isoform specific complexes might function during the course of fly development.

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