2. Academic Validation
  3. Nascent chromatin capture proteomics determines chromatin dynamics during DNA replication and identifies unknown fork components

Nascent chromatin capture proteomics determines chromatin dynamics during DNA replication and identifies unknown fork components

  • Nat Cell Biol. 2014 Mar;16(3):281-93. doi: 10.1038/ncb2918.
Constance Alabert 1,  Jimi-Carlo Bukowski-Wills 2,  Sung-Bau Lee 1,  Georg Kustatscher 2,  Kyosuke Nakamura 1,  Flavia de Lima Alves 2,  Patrice Menard 3,  Jakob Mejlvang 3,  Juri Rappsilber 4,  Anja Groth 1
Affiliations

Affiliations

  • 1 Biotech Research and Innovation Centre (BRIC) and Centre for Epigenetics, University of Copenhagen, Ole Maaløes Vej 5, 2200 Copenhagen, Denmark.
  • 2 Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh EH9 3JR, UK.
  • 3 1] Biotech Research and Innovation Centre (BRIC) and Centre for Epigenetics, University of Copenhagen, Ole Maaløes Vej 5, 2200 Copenhagen, Denmark [2].
  • 4 1] Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh EH9 3JR, UK [2] Department of Biotechnology, Technische Universität Berlin, 13353 Berlin, Germany.
PMID: 24561620 DOI: 10.1038/ncb2918
Abstract

To maintain genome function and stability, DNA sequence and its organization into chromatin must be duplicated during cell division. Understanding how entire chromosomes are copied remains a major challenge. Here, we use nascent chromatin capture (NCC) to profile chromatin proteome dynamics during replication in human cells. NCC relies on biotin-dUTP labelling of replicating DNA, affinity purification and quantitative proteomics. Comparing nascent chromatin with mature post-replicative chromatin, we provide association dynamics for 3,995 proteins. The replication machinery and 485 chromatin factors such as CAF-1, DNMT1 and 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, whereas H3K27me3 and H3K9me3 remain unchanged. Finally, we combine NCC enrichment with experimentally derived chromatin probabilities to predict a function in nascent chromatin for 93 uncharacterized proteins, and identify FAM111A as a replication factor required for PCNA loading. Together, this provides an extensive resource to understand genome and epigenome maintenance.

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