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How protein complexes recognize and modify chromatin: Structural studies on MSL1-MSL3-MOF Dosage Compensation Complex bound to nucleosomes

Final Report Summary - MSL1-MSL3-MOF (How protein complexes recognize and modify chromatin: Structural studies on MSL1-MSL3-MOF Dosage Compensation Complex bound to nucleosomes)

Chromosomal DNA is not naked but organised into chromatin fibres. The basic unit of the chromatin fibre is the nucleosome core particle formed by 147bp of DNA wrapped around an octamer of histone proteins. Chromatin regulates every aspect of DNA metabolism including DNA replication, transcription and repair. This regulation is mediated by multi-subunit protein complexes that slide nucleosomes, evict histones, exchange histone variants and add post-translational modifications to the histones and the DNA. These activities regulate the DNA accessibility of molecular machineries involved in DNA metabolism. The structural investigation of how protein complexes recognise and modify chromatin has been very limited so far. Indeed, even though hundreds of chromatin remodelling and modifying enzymes have been described to interact with chromatin and to modify it, only the structure of two proteins bound to the nucleosome have been solved at high resolution so far. These structures have been extremely important because they revealed molecular interactions that were not found by previous genetic and biochemical studies. Moreover, these structures explained at atomic resolution how these proteins recognise and modify the nucleosome. If we want to understand how chromatin remodelling and modifying enzymes function at the molecular level we need to solve their high-resolution structure in complex with the nucleosome. My work focuses on answering this fundamental question. To reach this goal I will use a combination of X-ray crystallography, biochemistry and electron microscopy.
I reconstituted, purified and characterised biochemically the MSL1-MSL3-MOF complex. This complex is an activator of DNA transcription and it is conserved in all eukaryotes. Moreover, miss-regulation of this complex has been associated with cancer and disease. I could isolate a minimal complex and I could map the regions of interactions between the subunit of the complex. This is very important, because the interaction between the subunits of the complex regulate its catalytic activity. I could produce a preliminary 3-D reconstruction of the complex at low resolution. I then moved to the preparation of the nucleosome substrate needed to study the activity of the complex. I set up a protocol for the assembly and the purification of chromatin-containing complexes suited for structural studies. The protocol I set up ensures high yields and high purity and homogeneity. Because X-ray crystallography of large DNA-protein complexes is very challenging and because there were no examples of nucleosome-protein complexes when I started the project I decided to set up the conditions for crystallization and structural analysis by using a domain of a well characterised protein. The MSL1-MSL3-MOF complex was not suited to set up protocols because of its complexity. I choose the BAH (Bromo Adjacent Homology Domain) domain of Sir3 (Silent Information Regulator) because it has been very well characterised in vitro and in vivo and therefore it was a suited candidate for setting up such a challenging technique. Importantly, the BAH domain I purified carries an N-terminal avetylation, a post-translational modification that is present in more than 80% of eukaryotic proteins but about which we know very little. The structure I solved showed that the N-terminal acetylation induces a conformational change in the structure of the BAH. As a consequence of this conformational change, the BAH is closer to the nucleosome compared to the not-acetylated BAH. This is very important because it suggests that the N-terminal acetylation might have a general role in favouring protein-protein interactions. The structure I solved has been published in Nature Structural and Molecular Biology and since I could follow the entire project as if I was the PI, I am last and the only corresponding author of my publication. The structure I solved is very important because it allowed me to set up conditions and protocols to investigate other chromatin-binding complexes. The next complex I will analyse will bethe MSL1-MSL3-MOF complex.
My work will have a strong impact in the chromatin and epigenetic field because many complexes that bind and modify chromatin have been described to be linked to disease and cancer. If we can solve the high resolution structure of these complexes bound to chromatin, we could design drugs to inhibit those complexes. These drugs could be used to dissect the function of these complexes and also they could be used as a starting point to design drugs to cure disease.