Histone post-translational modifications (acetylation, methylation, phosphorylation, etc.) play an important role in all chromatin-based processes. Specific combinations of histone modifications can specify various functions downstream. This chromatin “language” is known as the "histone code". Studies on the structure and function of this code have been focused on its role in regulating gene expression. However, several experimental data (including ours) show that post-translational modifications of histones play an important role in major centromeric functions such as sister chromatid cohesion or chromosome segregation. Our project aims to decypher the centromere histone code and identify the functions associated with post-translational centromeric histone modifications. Two axes, divided into three tasks are envisaged: 1 / Identification of histone modifications at the centromere (task 1). Centromeric chromatin is characterized by the presence of a centromere specific variant of histone H3, CENP-A. Previous studies have shown that centromeres are made up of blocks of nucleosomes containing CENP-A alternating with blocks of nucleosomes containing histone H3. We intend to take advantage of the physical link between the CENP-A and H3 nucleosomes to purify centromeric chromatin. We will conduct Tandem Affinity Purification (TAP) experiments using a cell line expressing a “TAP-tagged” version of CENP-A. Chromatin digestion will be controlled in order to copurify adjacent centromeric H3 nucleomes. Individual histones will then be purified by FPLC and their post-translational modifications profile will be established using FT-MS (Fourier-Transform Mass Spectrometry), a recent performant mass spectrometry technique. The respective profiles in interphase and mitotic cells will be compared. Using this approach, we will to map centromeric post-translational histone modifications and ultimately produce tools to study their functions. 2 / Function of previously described centromeric histone modifications (tasks 2 and 3). We have obtained preliminary results that suggest a role of phosphorylation of CENP-A serine 7 (CENP-SA7) in sister chromatid cohesion. CENP-A is phosphorylated by Aurora B but our preliminary results suggest multiple roles for Aurora B in sister chromatid cohesion. We will construct a “Shokat” version of Aurora B in order to precisely assess the functions of Aurora B in sister chromatid cohesion. We have developed an antibody against CENP-A acetylated on lysine 9 (CENP-AK9). We found that CENP-AK9 is actively deacetylated but we do not know the function of this acetylation / deacetylation balance. Our first experiences show that some lysine 9 substitutions of CENP-A are lethal and we want to construct cell lines expressing mutated forms of CENP-A under the control of an inducible promoter in order to understand the role of lysine 9 in centromere functions. Finally, our previous study suggested a role for centromeric histone H3 lysine 4 (H3K4) dimethylation in sister chromatid cohesion. We have identified the methyl-transferase responsible for this modification at the centromere and we want to assess its role centromere functions. We will also conduct "peptide pull down” experiments with a H3K4 dimethyl peptide and purify the retained complexes by FPLC. Retained complexes will then be analyzed by mass spectrometry to identify their components and their roles in centromere functions will be investigated.