Blanc SVSE 2 - Sciences de la vie, de la santé et des écosystèmes : Biologie cellulaire, développement

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

Deciphering the centromere «histone code«

Understanding the mechanisms that govern the fidelity of transmission of genetic material during cell division is a major issue. A lack of fidelity of this transmission leads to cell death or cancer.

What are the mechanisms involved chromosomes transmission fidelity ?

During cell division, either during embryonic development or during the replacement of dead cells of the adult organism, the chromosomes are distributed equally in the two daughter cells. Control of this distribution is carried out by molecular machines located at a particular location of the chromosome called the centromere. In humans, during cell division, chromosomes adopt a characteristic «X« shape. These chromosomes are composed of two identical parts (the arms of X, called «sister chromatids«) joined together at the junction of the «X« called «centromere.« Until cell division, sister chromatids are held jointed at the centromere. At the time of division, sister chromatids separate and are distributed into daughter cells. Control of the junction between sister chromatids and the signal for their separation, neither too early nor too late, is located at the centromere. Molecular complexes are recruited at this point to ensure good cohesion between sister chromatids, until cell division is triggered. Mechanisms that recruit these complexes are poorly understood. The chromosomal DNA is wrapped around small proteins called histones. These proteins undergo chemical modifications (acetylation, methylation, etc.) at particular positions that define a code called «histone code« or «epigenetic code« that entitle certain regions of chromosome specific properties. One of these properties is to give the centromere the ability to recruit the molecular complexes mentioned above. Our laboratory seeks to decipher the «histone code« of the centromere to understand its role in the fidelity of chromosome transmission.

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.

Project coordination


The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.



Help of the ANR 451,568 euros
Beginning and duration of the scientific project: - 36 Months

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