Uncoupling global and fine-tuning replication timing determinants for mouse pericentric heterochromatin

Abstract

Mouse chromocenters are clusters of late-replicating pericentric heterochromatin containing HP1 bound to trimethylated lysine 9 of histone H3 (Me3K9H3). Using a cell-free system to initiate replication within G1-phase nuclei, we demonstrate that chromocenters acquire the property of late replication coincident with their reorganization after mitosis and the establishment of a global replication timing program. HP1 dissociated during mitosis but rebound before the establishment of late replication, and removing HP1 from chromocenters by competition with Me3K9H3 peptides did not result in early replication, demonstrating that this interaction is neither necessary nor sufficient for late replication. However, in cells lacking the Suv39h1,2 methyltransferases responsible for K9H3 trimethylation and HP1 binding at chromocenters, replication of chromocenter DNA was advanced by 10–15% of the length of S phase. Reintroduction of Suv39h1 activity restored the later replication time. We conclude that Suv39 activity is required for the fine-tuning of pericentric heterochromatin replication relative to other late-replicating domains, whereas separate factors establish a global replication timing program during early G1 phase.

Heterochromatin protein 1 (HP1) modulates replication timing of the Drosophila genome

“…Depletion of HP1 could result in the loss of the requirement for this mechanism and therefore allow slightly earlier replication of centromeric repetitive heterochromatin. This advanced replication timing of repeats is consistent with observations in mouse fibroblasts, where replication timing of centromeric repeats is advanced in cells depleted of the mouse homologs of the Drosophila histone methyltransferase SU(VAR)3-9 that methylate lysine 9 on histone H3 (Wu et al 2006). Interestingly, however, embryonic stem cells lacking the SUV39H1 and SUV39H2 H3K9 methyltransferases showed delayed replication timing of centromeric repeats (Jorgensen et al 2007), underscoring the context dependency of the interaction between chromatin and the replication timing program.…”

Genome-wide dynamics of replication timing revealed by in vitro models of mouse embryogenesis

“…Thus, we hypothesize that a key G 1 -phase event exists during epiblast development, in which a significantly altered subnuclear repositioning pattern is observed compared with G 1 in prior cell cycles. While it is tempting to speculate the roles of chromatin modifying activities in this genome reorganization process, we and others have shown that ablation of several chromatin modifying enzymes (MLL1 [also known as MLL], MBD3, EED, SUV39H1/H2, EHMT2 [also known as G9a], DNMT1/3A/3B, DICER) show only modest effects on replication timing (Wu et al 2006;Jorgensen et al 2007;Yokochi et al 2009). Moreover, the few known DNA methylation events, such as on the Pou5f1 promoter and the Xi, are downstream (Lock et al 1987;Feldman et al 2006).…”

Polycomb RING1A/RING1B-dependent histone H2A monoubiquitylation at pericentromeric regions promotes S phase progression

“…We exploited the fact that MEFs show cytologically defined Sphase stages that are easily distinguished by characteristic spatiotemporal patterns of DNA synthesis (Quivy et al, 2004;Wu et al, 2006) to determine whether altered replication in RING1A-and RING1B-deficient cells affected such patterns. We used asynchronous MEF cultures, 48 h after EtOH or 4′-OHT treatment, and pulsed them with IdU for 20 min in order to score the proportion of cells in each of the S-phase stages.…”