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EpiMechanism Report Summary

Project ID: 615638
Funded under: FP7-IDEAS-ERC
Country: Portugal

Mid-Term Report Summary - EPIMECHANISM (Mechanisms of Chromatin-based Epigenetic Inheritance)

This ERC-funded project aims to further our understanding of chromatin-based epigenetic mechanisms. The project proposed to dissect 3 major aspects of epigenetic mechanisms, 1) The stability of a chromatin mark 2), the cell cycle relegated propagation of such a mark and 3), the determination of the size of the chromatin mark. We focus on the centromere which is defined by a unique chromatin domain, identified by the presence of the histone H3 variant CENP-A. This histone H3 variant is a stable member of chromatin and is assembled in a cell cycle-specific manner in order to epigenetically maintain centromere identity. In the first stage of the project we performed a genetic screen to identify novel proteins that are responsible for the stable transmission of CENP-A chromatin in human cells. One factor, CENP-C is a member of the centromere and is bound directly to CENP-A nucleosomes and we contributed to the finding that this protein is critical to stabilize CENP-A in cells (Falk et al., 2015, Science). This process is essential to maintain centromere function. We extended our effort to determine chromatin stability for general chromatin by developing a tool that allows us to measure the rate of histone inheritance across the genome. This method that we call time-ChIP, was applied to determine histone dynamics in mouse ES cells and during differentiation. This work (published in Deaton et al., 2016, eLife) identified regions of fast and slow turnover and how they change during development. Importantly, it identified previously unrecognized regions that are likely novel enhancers involved in cellular differentiation.
Our work of cell cycle control of the centromeres has now revealed the mechanisms that ensure that the centromeric histone CENP-A is assembled into chromatin only after successful passage through mitosis. During the initial phase of the project we identified two key proteins involved in CENP-A assembly that we found to be inhibited through phosphorylation specifically in S, G2 and mitotic phases of the cell cycle by the major cell cycle kinases Cdk1 and 2. This provides a mechanism for how centromere assembly is tightly linked to the cell cycle (Published in Stankovic et al., 2017, Molecular Cell).
Finally in the first term of this project we achieved insight into the molecular size of centromeric chromatin and the copy number of CENP-A chromatin. We found that while CENP-A is highly enriched at centromeres, it is still a minority with only 1 in 25 nucleosome endowed with a CENP-A histone, totaling about 100 nucleosomes on a mitotic human centromere (published in Bodor et al., 2014). Thus far, our work defined the size of human centromeres, how its levels are maintained along the cell cycle and we have preliminary insights into how CENP-A nucleosomes are stabilized in chromatin to ensure epigenetic propagation of centromere identity.

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