Quantitative proteomics of human chromatin
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The work presented in this thesis aims at unravelling human chromatin composition by quantitative proteomics to outline the functional and structural changes occurring during the life of human cells. Chromatin is the structure formed by proteins and RNAs interacting with the genetic material. At present, chromatin is not well defined. It is not easy to investigate either the composition of its constituent proteins or how this arrangement changes. We set out to analyse the chromatin composition changes occurring during the cell cycle. Our procedure couples a SILAC mass spectrometry-based approach with a newly developed biochemical chromatin purification method, which involves fixation of proteins to DNA. By testing two different fixation times (5 and 10 minutes) and three phases of the cell cycle (G1/S, G2, M), we quantified ~3000 proteins providing a broad picture of the global changes on chromatin protein composition. Surprisingly, chromatin seems to be occupied by many unexpected proteins (40%) that appear to be increased during mitosis. We hypothesized that these unexpected proteins come into contact with DNA during mitosis when the nuclear envelope breaks down and the highly negatively charged DNA can be found in proximity to extra nuclear proteins. We used Pulse-SILAC technique that allows to distinguish newly synthesized proteins to test this possibility. By comparing in a single cell cycle and during G0 arrest the incorporation of new proteins into chromatin with their synthesis in the cytoplasm and in the whole cell, we could not find a different behaviour for the unexpected proteins as result of mitosis. Despite the efforts in tracking down the origin of these unexpected proteins, it is still uncertain whether their presence on chromatin is the result of a biological process or, in part, a drawback of the purification methods adopted. However, proving their genuine presence on chromatin will be important to elucidate how chromatin functions.