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

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.

Two distinct modes for propagation of histone PTMs across the cell cycle

“…In NCC, biotin-dUTP labeling of newly replicated DNA and cross-linking of associated proteins allow the isolation of nascent chromatin (Supplemental Fig. S1A,B; Alabert et al 2014). We combined NCC with pulsed SILAC (stable isotope labeling with amino acids in cell culture) to differentiate newly synthesized histones (heavy) and old recycled histones (light) ( Fig.…”

“…For an outline of the experimental design, see Supplemental Figure S7C. may require recruitment of enzyme cofactors (Alabert et al 2014;Kalb et al 2014). Such a tight regulation of PTM establishment could limit unwarranted silencing, which is jeopardized in cancers carrying EZH2-activating mutations (McCabe et al 2012).…”

“…Cells were synchronized by thymidine in light medium and released into S phase in heavy medium for 3 h. Newly synthesized DNA was pulselabeled for 15 min with b-dUTP, and NCC was performed as described (Alabert et al 2014). In brief, cells were labeled with b-dUTP for 5 min in hypotonic buffer (50 mM KCl, 10 mM HEPES) and fixed 15 min later in 2% formaldehyde.…”

The effects of SUUR protein suggest its role in repressive chromatin renewal during replication inDrosophila

“…In mammals, some modifications are cast immediately after the replication fork by chromatin factors including chromatin remodelers and histone chaperones; other modifications are introduced later during chromatin maturation, when the replication fork has already passed. 8 In higher eukaryotes, genomic regions replicate asynchronously -transcriptionally active regions usually replicate earlier in the S-phase of cell cycle than do repressed heterochromatic regions. 9 Primarily, this replication schedule is defined by the pattern of active replication initiation sites (origins).…”

“…For example, an extensive study in human cells revealed that in contrast to other histone modifications, H3K9me3 and H3K27me3 are inherited via the incorporation of the histones from the template chromatin (parental histones) to the nascent chromatin. 8 The mechanism that distinguishes H3K9me3 and H3K27me3 from the rest of the histone modifications during replication remains unknown.…”