ARCHITECTURE OF DROSOPHILA DOSAGE COMPENSATION COMPLEX

Abstract

In heterogametic organisms, male and female cells harbor structurally different sex chromosome pairs. The difference in the transcriptional output of these sex chromosomes is epigenetically balanced, a phenomenon dubbed as dosage compensation. In Drosophila, male cells up-regulate their one X chromosome roughly two times by the help of a molecular machine called MSL complex. This ribonucleoprotein enzymatic complex, specifically formed in male cells, localizes to the X-chromosome, changing its structure mostly by histone H4 Lysine 16 acetylation, thus enabling enhanced transcription. The details of how the complex finds the chromosome and how it regulates transcription are not thoroughly understood. In this thesis, with the help of X-ray crystallography, we derived point mutations on the scaffold Msl1 protein that create partial complexes to study the contribution of each subunit to X chromosome recognition, RNA integration and spreading alone the X chromosome. In the first part of this thesis, we focused on the PEHE region of Msl1 protein that binds Mof and Msl3. We generated point mutants of Msl1 that cannot bind Mof or Msl3. We showed that loss of either Mof or Msl3 prevents spreading of the MSL complex on the body of the X-linked genes whereas Msl1 promoter binding remained unaffected. We observed qualitative differences between high affinity sites (HAS), initial binding platforms of MSL complex, and noticed that promoter located HAS can bind Msl1 independent of Mof and Msl3 whereas other HAS depend on the presence of intact PEHE module for optimal binding. In the second part of the thesis, we examined the interaction of Msl1 and Msl2 and showed that Msl1 forms a homodimer through its coiled coil region and this homodimer creates a platform for Msl2 binding. Msl2 binding happens through helices surrounding the RING finger domain. We showed that Msl2 RING finger can function as a ubiquitin ligase, and Msl1 is an in vitro substrate of Msl2 ubiquitination. By point mutational analysis on Msl1 we showed that Msl1 forms a dimer independent of Msl2 in both male and female cells. Dimerization is required for Msl2 binding, roX2 RNA integration to the complex, X chromosome recognition and spreading along the body of X-linked genes. This clearly showed that functionality of MSL complex entirely depends on its dimeric configuration. We identified roX2 HAS as an elementary HAS where its recognition only happens through Msl1-Msl2 dimer interface. Furthermore we discovered that Msl1 binds to promoters in a dimer/Msl3/Mof/Msl2 independent fashion. This binding occurs also at the autosomes and in both sexes suggesting a general function of Msl1 at promoters of Drosophila. We showed that promoters are also occupied by Msl2 but not by Msl3, indicating that Msl3 can have an important role for distinguishing promoter bound complex and canonical MSL complex. In order to support the in vivo importance of amino acid residues that had been point mutated, we generated transgenic flies that express Msl1 and its mutated forms from the identical genomic location. We showed Msl1 mutants are unable to rescue the Msl1 null male lethality and also cause male specific lethality upon over-expression in wild type background confirming the importance of these mutated residues.