Open Access Dissertation
Date of Award
Lori A. Pile
SIN3, the scaffold protein of a histone modifying complex is conserved from yeast to mammals. Drosophila SIN3 associates with both a histone deactylase RPD3 and a histone demethylase dKDM5/LID. Immunopurification of dKDM5/LID verifies a previously observed interaction with SIN3 and RPD3. Furthermore, deficiency of dKDM5/LID phenocopies deficiency of SIN3 in many cellular and developmental processes. Knockdown of both Sin3A and lid hinder cell proliferation in Drosophila cultured cells and developing flies. Knockdown of these genes also results in a curved wing phenotype implicating a role in wing development. Analysis of underlying gene expression changes upon decreased expression of SIN3, dKDM5/LID or both at a genome wide level determined multiple genes that are commonly regulated by SIN3 and dKDM5/LID. Common gene targets of SIN3 and dKDM5/LID are implicated in processes related to stress tolerance. Additive roles of these proteins seem important in the regulation of cell cycle associated genes. Induction of paraquat mediated oxidative stress found a higher number of genes to be regulated by both SIN3 and dKDM5/LID, with an enrichment of genes involved in cell cycle regulation. Moreover, SIN3 and dKDM5/LID were found to bind the TSS proximal regions of several regulated genes suggesting direct regulation of these targets. Determination of histone modification changes at the transcriptional start sites of target genes upon knockdown of Sin3A or lid reveal changes in histone acetylation levels at some genes with no significant changes in histone methylation levels. This suggests an important role for the histone deacetylase activity of the complex in affecting gene regulation, while, the contribution of the demethylase activity appears to be minimal. It is, however, possible that the role of demethylation is highly context specific and allows for fine tuning of gene regulation under specific conditions or during specific developmental time points. This work emphasizes the important contributions of the histone demethylase dKDM5/LID to regulation of cellular events by the SIN3 complex.
Supplementary files included are as follows:
* Supplementary Data 1_AG - Excel spreadsheet containing RNAseq differential expression analysis outputs
* Supplementary Data 2_AG - Excel spreadsheet containing Gene Ontology analysis data
Gajan, Ambikai, "Analyzing The Interactions Of Kdm5/lid And Sin3 In Drosophila Melanogaster" (2015). Wayne State University Dissertations. 1129.