Interplay of DNA replication, repair and chromatin: structure versus function

Hierarchical levels of chromatin organization allow different genomic functions to be spatio-temporally regulated within mammalian nuclei. Both DNA replication and DNA repair are global genomic processes. Their chromatin units have remarkable structural similarities and microscopically appear as clusters of nanofocal structures, each in the size range of chromatin loops. The present work aimed at relating genomic functions with the underlying structural organization by the two chromatin architectural proteins CTCF and cohesin, which cooperate to shape the genome into chromatin loops and domains. Here, CTCF was shown to be critical for cellular survival after ionizing irradiation in a CTCF-dose dependent way. The results obtained in different cell lines upon CTCF-depletion were integrated into a biophysical model. The decreased clonogenic potential showed to derive from the increased probability of double strand breaks to cluster in larger chromatin domains lacking CTCF at their borders. Moreover, CTCF proved to be enriched at the sites and at the time of DNA replication. CTCF intensity within replication foci was shown to decrease over a chase time after replication labeling, suggesting the occurrence of CTCF accumulation during ongoing DNA replication. The depletion of CTCF correlated with an impairment in cell cycle progression. CTCF-depleted cells stalled in G1 in a CTCF-dose dependent way, indicating that the chromatin structure provided by CTCF might be needed to properly enter S-phase. Additionally, CTCF resulted to be particularly enriched at the replicating inactive X and Y chromosomes. The depletion of CTCF led to the loss of synchrony in the DNA replication of the Y chromosome. Additionally, Y chromosome architecture showed changes of volume and shape upon CTCF reduction. In the second part of this work, the cohesin subunit RAD21 was shown essential to determine the structure of chromatin loops. RAD21-depleted cells exhibited an increase in the size and shape heterogeneity of chromatin loops. The cohesin component SA1 was investigated for a role in DNA damage signaling. SA1 KO cells showed an impairment of the γH2AX foci at all tested X-ray doses. The repair functional units decreased in number, volume and intensity in the absence of SA1. In conclusion, the results presented here led to propose that the functions of DNA replication and repair are determined by the chromatin architecture, with the structure dictating the function. Future work should further investigate the mechanisms behind the regulation of global genomic functions by these two chromatin architectural proteins and define the precise interplay between cohesin and CTCF within such regulation