CHANGES OF REPLICATION TIMING INDUCED BY PML-RARA

DNA replication is a cellular process that, starting from precise genomic loci, ensures the loyal and faithful inheritance, from one parental cell to each daughter cell, of the genetic instructions contained in the double-strand DNA molecule. Due to the complexity and the crucial importance of the DNA replication, this process must be tightly regulated in both space and time. Up to now, however, the time-related features of DNA replication, together with the factors that might impact the temporal dimension of this system, are yet poorly studied and described. Given the lack of standard methods able to recognize differences in Replication Timing, we developed an innovative bioinformatic method (DART; Differential Analysis of Replication Timing) to accomplish this task. Moreover, the application of this procedure to our Repli-seq data was instrumental to investigate whether PML-RARα may fulfil its tumorigenic potential by eliciting an alteration of the normal replication timing pace in cells. As a result, we found that, after its expression, PML-RARα indeed exerts a deregulative effect on Replication Timing, inducing some regions to replicate earlier (LtoE-shifted) and some other later (EtoL-shifted), with respect to control cells. We observed a close association between these differentially replicated regions and both pre-existing, and PML-RARα-related, transcriptional status and chromatin structure. Regions presenting a EtoL-shifted replication coincide with ‘active’ chromatin foci enriched for direct down-regulated targets of PML-RARa; at the opposite, regions with a LtoE-shifted Replication Timing show moderate epigenetic ‘active’ features and are enriched for indirect up-regulated targets of PML-RARa.