Low Replicative Stress Triggers Cell-Type Specific Inheritable Advanced Replication Timing

Abstract

DNA replication timing (RT), reflecting the temporal order of origin activation, is known as a robust and conserved cell-type specific process. Upon low replication stress, the slowing of replication forks induces well-documented RT delays associated to genetic instability, but it can also generate RT advances that are still uncharacterized. In order to characterize these advanced initiation events, we monitored the whole genome RT from six independent human cell lines treated with low doses of aphidicolin. We report that RT advances are cell-type-specific and involve large heterochromatin domains. Importantly, we found that some major late to early RT advances can be inherited by the unstressed next-cellular generation, which is a unique process that correlates with enhanced chromatin accessibility, as well as modified replication origin landscape and gene expression in daughter cells. Collectively, this work highlights how low replication stress may impact cellular identity by RT advances events at a subset of chromosomal domains.

The Heritability of Replication Problems

“…Cells were synchronized at the end of G1 with L-mimosin, permeabilized with detergent, and depleted of soluble proteins by extraction with highsalt buffers, allowing supercoiled DNA loops to unwind and form halos. A significant reduction in DNA loops was observed in the aphidicolin-released cells [26], suggesting that RS can modify origin selection along the genome in the next cell cycle and corroborate with the observations that the loading of some MCMs onto chromatin of daughter cells was stimulated by RS in the previous cell cycle.…”

“…One explanation relies on the nuclear lamina which participates in the chromosome architecture and dynamics within the nucleus, and controls RT of late domains called lamin-associated domains. Indeed, lamin expression was strongly affected in RKO cells compared to other cell lines [26]. A more flexible chromatin organization in RKO cells could render them more permissive to RT changes.…”

“…ORC serves as a scaffold for the subsequent association of CDC6 and CDT1, which together coordinate the loading of the MCM2-7 complex in order to form the prereplication complex (pre-RC) required for replication fork formation and activity [25]. Recently, we reported in human cell lines that aphidicolin-induced replication stress in mother cells did not modify the association with chromatin of the ORCs proteins, CDC6 and, CDT1, but can stimulate the loading of MCM2 and p-MCM2 onto the chromatin in G1/S [26].…”

“…This organization involves the formation of chromatin loops corresponding to the inter-origin DNA regions, whose size correlates with the length of the replicons, facilitating access of the initiator proteins required to activate origins. We recently reported that RS in mother cells can induce a spatial re-organization of activated origins in G1 daughter cells [26]. Indeed, the length of chromatin loops in nuclei daughter cells emanating from mock-treated and APH-treated mother cells can be monitored by the "fluorescent DNA halo" technique, allowing visualization by means of fluorescence staining of halo formation around insoluble scaffold that results from unwound DNA loops [27].…”

“…The changes in MCM loading and origin selection in daughter cells may affect the timing of activation of replication origins of a subset of domains in the subsequent S-phase. To test this hypothesis, mother cells were released from aphidicolin and the replication timing (RT) of the next generation of cells was analysed by using human whole genome microarrays [26,28]. Strikingly, in RKO cells, 5% of the genome daughter cells were replicated earlier when the mother cells were exposed to low RS.…”