Identification and Characterization of Novel Regulators of Heterochromatin Structure and Function

The term `chromatin' refers to the packaging of DNA with proteins and RNA, which is the native state of the genome in the nucleus that is critical for proper regulation of genome functions. Heterochromatin is a chromatin state that is condensed, less accessible and generally repressed for transcription compared to euchromatin. Heterochromatin has been observed in all multicellular and unicellular eukaryotes studied to date, is enriched for repetitive sequences and comprises approximately 30% of the Drosophila and Homo sapiens genomes. Heterochromatin is defined molecularly by enrichment for histone H3 containing di- and tri-methylation of lysine 9 (H3K9me2/3) and its binding partner Heterochromatin Protein 1a (HP1a)./nHP1a interacts with a wide range of proteins and directs a diverse set of heterochromatin functions, including repeat and genome stability, telomere protection, gene silencing, proper chromosome segregation and the heterochromatic DNA damage response (hDDR). In order to better understand how heterochromatin functions (especially the hDDR) are mediated, we purified HP1a before and after induced-DNA damage, and mass spectrometry analysis identified 118 putative novel HP1a-interacting proteins (HPips) and 10 putative HP1a post-translational modifications (PTMs). We determined that two of these novel binding proteins (CG8108 and CG7357) colocalize with HP1a in tissue culture cells and implicated roles for Kdm4A and Dre4 in the hDDR. Additionally, we show that a HP1a PTM (K4) and a HPip (HP5) regulate the volume of the heterochromatin domain. Finally, we identified SUMO as a HP1a PTM and show that the E3 SUMO ligase Su(var)2-10 is necessary for genomic stability and regulating the hDDR./nIn addition to identifying novel components of heterochromatin we identified novel functional regulators of HP1a by performing a genome-wide RNAi screen which assayed HP1a levels by immunofluorescence. We identified 374 candidate regulators of heterochromatin including 18 genes whose function in regulating HP1a had only previously been inferred by indirect measures. We characterized one of these genes MBD-like as regulating H3K9me2 and HP1a localization to heterochromatin./nOne exciting finding was the identification of CG8108, a novel HPip that negatively regulates HP1a levels in the nucleus. To better understand the function of this unusual protein we investigated the molecular and organismal affects of misregulating CG8108. We found that overexpression of CG8108 alters the nuclear distribution of the HP1a domain, and depletion of CG8108 redistributes HP1a occupancy at heterochromatic DNA, resulting in suppression of heterochromatin-mediated silencing. Consistent with these observations, CG8108 negatively regulates HP1a solubility on chromatin, which may indicate increased HP1a compaction or molecular crowding. Finally, CG8108 is required for the hDDR and organismal viability./nThese experiments provide a foundation for building a more complete model of heterochromatin structure and function through more detailed analysis of novel HPips and HP1a regulating proteins. Additionally, we suggest that a deeper understanding of non-histone PTMs and the heterochromatin `microenvironment' must be included to generate a complete model of heterochromatin structure and function.

Advisor: Karpen, Gary H