Purification and biochemical characterisation of novel MOF-containing NSL complexes

Abstract

Every cell of a eukaryotic organism contains the whole genome in a membrane-bound nucleus where the DNA is compacted in highly organized chromatin fibers. The association of DNA with histone proteins allows for efficient packaging of the genetic material. Chromatin is a dynamic polymer that is packed and unwrapped in a highly regulated manner to match the multiple tasks that it encounters during the lifetime of a cell. Regulation of DNA accessibility is achieved by the concerted action of chromatin-associated proteins, such as chromatin remodeling enzymes, variant histone proteins and chromatin modifying enzymes. The latter class of enzymes brings about post-translational modifications of histones. Modifications, such as acetylation, can lead to changes in chromatin structure either directly or by recruiting other effector proteins. A process that is linked to histone acetylation is dosage compensation in the fruit-fly Drosophila melanogaster which is studied as a model system for the regulation of chromatin. The multi-protein complex involved, harbours an enzyme, MOF (males absent on the first), with histone acetyltransferase activity directed towards histone H4 lysine 16. Other proteins that have previously been known to associate with it are the MSL (male specific lethal) proteins. Recent biochemical purification of MOF containing complexes revealed that, in addition to the MSL proteins, a number of novel proteins co-purified with MOF in Drosophila and mammals (Mendjan et al., 2006). During my PhD I was therefore interested to study whether these novel proteins, which we named NSL proteins (non-specific lethal), exist in a complex similar to the MSL proteins in Drosophila and if so, what might be their function. Interestingly, we found that one of the NSL proteins, NSL1 has a very similar domain architecture to MSL-1. A major part of this thesis has therefore been to study the NSL1 protein in detail. Using co-immunoprecipitation as well as in vitro interaction assays, I was able to show that NSL1 indeed interacts directly with MOF. By applying an affinity purification strategy tagging the NSL1 protein, my PhD work has demonstrated that NSL1 co-purifies with other NSL proteins in a complex that is distinct from the MSL complex. I have also been able to show by immunofluoresence microscopy that two components of this complex, NSL1 and MCRS2, co-localise on hundreds of sites on all polytene chromosomes, suggesting that very likely these proteins not only interact biochemically but may also function together in vivo. This part of my work has therefore provided novel insights into the existence of the NSL complex in Drosophila and has showed that MOF associates with two distinct sets of proteins, namely the MSLs and the NSLs. In the second part of my PhD work I studied the overall contribution of MSL and NSL complexes in modulating histone acetylation using quantitative mass spectrometric analysis of endogenous histones from Drosophila cells that were RNAi-depleted of MOF, MSL-1 and NSL1. This work revealed that MOF contributes to a majority of histone H4 K16 acetylation in Drosophila cell lines and that MSL-1 plays an important role in modulating the activity of MOF in vivo. Surprisingly, we did not observe any major changes in histone acetylation levels upon NSL1 depletion in vivo. Interestingly, NSL1 depleted cells displayed cell proliferation and segregation defects. The in vivo function of NSL1 remains elusive. Future work will therefore be required to elucidate the mechanism of action of these novel proteins in Drosophila. The foundations for it were layed by this work.