Community Research and Development Information Service - CORDIS



Project ID: 281765
Funded under: FP7-IDEAS-ERC
Country: Denmark

Mid-Term Report Summary - CHROMATINREPLICATION (How to Replicate Chromatin - Maturation, Timing Control and Stress-Induced Aberrations)

Accurate epigenetic control is essential during development and to avoid disease. The challenge of propagating genetic and epigenetic information is met in S phase and entails genome-wide disruption and restoration of chromatin coupled to faithful copying of DNA. How specific chromatin structures are restored on new DNA and transmitted through mitotic cell division remains a fundamental question in biology central to understand cell fate and identity.

Chromatin restoration on new DNA involves a complex set of events including nucleosome assembly and remodelling, restoration of marks on DNA and histones, deposition of histone variants and establishment of higher order chromosomal structures. To dissect these fundamental processes, we have developed a novel technology termed nascent chromatin capture (NCC) that provides unique possibility for biochemical and proteomic analysis of chromatin replication in human cells. NCC relies on biotin-dUTP labelling of replicating DNA, affinity-purification and quantitative proteomics. We have used NCC to profile chromatin proteome dynamics during replication in human cells. Comparing nascent chromatin with mature post-replicative chromatin, we have provided association dynamics for 3995 proteins. The replication machinery and 485 chromatin factors like CAF-1, DNMT1, SUV39h1 are enriched in nascent chromatin, whereas 170 factors including histone H1, DNMT3, MBD1-3 and PRC1 show delayed association. This correlates with H4K5K12diAc removal and H3K9me1 accumulation, while H3K27me3 and H3K9me3 remain unchanged. Finally, we combined NCC enrichment with experimentally derived chromatin probabilities to predict a function in nascent chromatin for 93 uncharacterized proteins and identify new replication factors. This illustrates the power of NCC as an explorative tool for identifying function of uncharacterized proteins. Moreover, this work provides an extensive resource for the scientific community to advance our understanding of genome and epigenome maintenance.

Replication stress, including fork stalling and collapse, challenges normal chromatin replication and may trigger stochastic epigenetic aberrations affecting genome function in succeeding generations. To address how replication fork damage influence the chromatin environment, we are applying NCC to cells exposed to genotoxic agents. Our proteomic analyses have identified recruitment of a large number of DNA repair and checkpoint signalling factors. This validates the quality of the data set, which we now use to explore chromatin changes and identify new repair factors.

Modifications on histones and histone variants define chromatin structure and are considered instrumental in epigenetic regulation. However, it remains unknown how histone-based information is inherited during cell proliferation. We are currently exploiting NCC to track histone modifications during replication and across the cell cycle. This work should reveal how different histone marks are transmitted during cell division and provide a foundation to understand how epigenetic states are propagated.

Taken together, this work will improve our understanding of human development, somatic cell reprogramming and complex diseases like cancer.

Reported by

Københavns Universitet
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