| How the genome is shaped directly influences how it is expressed. In fact, the genome is organized at many different levels, each imparting specific regulation over transcription. Fundamental principles regulating genome organization are poorly understood. More recently, a role for the chromosome structuring complex, condensin II, in organization of the genome has been identified. However, mechanisms regulating interphase condensin II are elusive. Herein this dissertation, I present data from experiments performed in Drosophila melanogaster identifying proteins involved in the loading and removal of condensin II from interphase chromosomes. First, by using yeast 2 hybrid assays, we identify that the chromodomain protein, Mrg15, interacts with condensin II subunit, Cap-H2, through its MRG binding motif. In cultured cells and Drosophila using subcellular fractionation, we show that Mrg15 recruits Cap-H2 to chromatin. Immunofluorescence and fluorescent in-situ hybridization (FISH) demonstrate that Mrg15 recruitment allows for condensin II's chromosome compaction roles. Secondly, using an RNAi screen in cultured cells, we find that the SCFSlimb complex (Slimb) regulates nuclear morphology through condensin II. We further show through immunoprecipitation that Slimb targets Cap-H2 for ubiqutination. This results in eviction of Cap-H2 from chromatin and repressed condensin II activity as demonstrated by immunofluorescence confirmation of inhibition of chromosome dispersal. Lastly, we identified that Casein Kinase I Alpha (CK1alpha) also functions to negatively regulate Cap-H2 and condensin II. Subcellular fractionation and immunofluorescent analysis of chromatin bound Cap-H2 reveals that CK1alpha normally evicts Cap-H2 from chromatin. Also, through cytological techniques, we find that CK1alpha influence over genome organization is mediated through condensin II regulation. In summary, we elucidate condensin II regulation during interphase by identifying three previously unknown condinsin II interactors: Mrg15, Slimb, and CK1alpha. We end by describing a role for CK1alpha regulation of condensin II during the response to heat stress in Drosophila. These findings are impactful in that they further our limited knowledge of genome organization. Through a better understanding of genome organization, we can better understand mechanisms regulating transcription and with that, better understand biology. |
