Re-establishment of TAD boundary organization during DNA replication

A ubiquitous feature of higher order genome organization is the presence of topologically associating domains (TADs). The chromatin architectural proteins cohesin and CTCF are known critical organizers of TADs, but the mechanisms of TAD establishment and maintenance, including their accurate duplication during genome replication, are not well characterized. To address this gap, we used high-throughput imaging-based CRISPR/Cas9 knock-out screening to discover chromatin factors involved in maintenance and establishment of TADs. Among the cellular factors that affect TAD organization, we found enrichment for cell cycle proteins, especially components of the DNA replication machinery. Accordingly, we demonstrate that TADs undergo temporary unfolding during S-phase DNA replication and that interference with progression through replication impedes restoration of normal TAD structure. Mechanistically, inhibition of the RPA complex prevents cohesin and CTCF binding, delays post-replication TAD re-folding and affects TAD folding in non-cycling cells. These results provide novel insights into how TAD structures are re-established during genome duplication.