Epigenetic control of centromere functions : The centromere "histone code". – EpiCentr

The main difficulty of this project lies in the purification of centromeric DNA and associated histones. In order to obtain pure fractions of centromeres, we chose to use an antibody that specifically recognizes a histone incorporated only at the centromere of chromosomes. This antibody is coupled with magnetic beads and incubated with chromosomes fragmented with an enzyme that digests DNA. One of the challenges is to control the DNA fragmentation in order not to obtain fragments too small (and lose important histones) or too large (and contaminate the sample with histones that do not belong to the centromere) .
Histones are individually isolated and purified by protein biochemistry techniques and analyzed by mass spectrometry. This technique allows to determine the exact mass of fragments of histones and to determine whether these histone carry chemical modification(s). Eventually, after identification of histone modifications at the centromere, we will search their (s) role (s) in the functions of the centromere and, particularly, in the fidelity of chromosome transmission during cell division.

Our project is at the technological development stage. We have obtained pure fractions of centromeres, consistent with the analysis by mass spectrometry and we are currently adapting this technique for larger sample volumes (10 liters of cultured cells).

This project is a basic research project mainly aimed at understanding the molecular mechanisms that govern cell division. Medical perspectives in the medium or long-term concern mainly the fight against cancer. Indeed, the main characteristic of a cancer cell is its rapid rate of division. This feature represents a «launch window« for potential treatments. Knowledge of the mechanisms controlling chromosome transmission can indeed identify new potential therapeutic targets. By disrupting these mechanisms, it should be possible to force tumor cells to spread their genetic material incorrectly in the daughter cells, resulting in the death of most of these cells.

Our studies, in the course of this project, have shown that a protein known for its role in DNA replication also had a role in cell division. We recently published this work:
Rouzeau, S., Cordelieres, F.P., Buhagiar-Labarchede, G., Hurbain, I., Onclercq-Delic, R., Gemble, S., Magnaghi-Jaulin, L., Jaulin, C., and Amor-Gueret, (2012) M. Bloom's syndrome and PICH helicases cooperate with topoisomerase IIalpha in centromere disjunction before anaphase. PLoS One 7(4):e33905.

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.