Replication Timing Regulation and Chromatin Structure Dynamics during the Cell Cycle and Development

Eukaryotic genomes replicate via the synchronous firing of clusters of origins that together produce multi-replicon domains, each of which replicates at a defined time during S-phase. This temporal program is termed the DNA replication-timing program. Replication Timing (RT) is a stable epigenetic property that is cell type specific and is extensively regulated during differentiation in units that range from 400-800kb called replication domains. DNA that replicates at distinct times during S-phase is also spatially separated in the nucleus. Consistent with this, the binary nuclear compartments defined by chromatin spatial proximity maps, align precisely with the replication-timing program. But the dynamics of this relationship during differentiation and cell cycle have been poorly understood. To this end, we first showed that there is a coordinated switch in nuclear compartment along with a switch in replication timing during differentiation. It was also observed that regions of the genome that switch replication timing and nuclear compartment continue to maintain their structural boundaries. Genome-wide analysis of replication domains revealed that they are indeed stable structural units corresponding to Topologically-Associating Domains (TADs) defined by Hi-C. Next we showed that the interphase chromatin structure consisting of TADs and their long-range contacts are established during early G1 coincident with the establishment of the replication-timing program. We also show that developmentally regulated regions of the genome have fundamentally different higher order structure. In G2 phase, the replication timing-program is lost while inter-phase chromatin structure acquired in early G1 was retained. This shows that interphase chromatin structure is not sufficient to dictate RT and lead us to hypothesize that the chromatin structure set-up during early G1 may act as a scaffold to seed the assembly of some factor capable of setting replication initiation thresholds. The de-coupling of chromatin structure and RT could then be due to the removal of this factor during S-phase. Consistent with this hypothesis, we discovered a protein Rif1 that enters the nucleus right after mitosis and its knockout has a profound disruptive effect on RT in both mouse and human cells. Lastly, we explored the conservation of replication timing at single cell level that revealed a highly conserved yet stochastic regulation of replication timing. Surprisingly, the intrinsic (within cell) stochasticity and the extrinsic (cell-to-cell) stochasticity were similar. This is consistent with a model of replication timing regulation where the timing is the outcome of stochastic origin firing and is not affected by the precise environment within a cell. In summary, the work descried in this thesis uncovers a model where replication-timing is regulated at the unit of chromatin structure called TADs, which are generally stable across cell-types, but the compartment that they reside in corresponds to the time of their replication. Interphase chromatin structure is established along with the establishment of RT and may act as scaffold for replication regulation factors like Rif1. Finally, replication timing and its association with chromatin structure are highly conserved and are observed even at the single chromosome level.