Cohesin-mediated loop anchors confine the locations of human replication origins

Abstract

DNA replication occurs through an intricately regulated series of molecular events and is fundamental for genome stability1,2. At present, it is unknown how the locations of replication origins are determined in the human genome. Here we dissect the role of topologically associating domains (TADs)3–6, subTADs7 and loops8 in the positioning of replication initiation zones (IZs). We stratify TADs and subTADs by the presence of corner-dots indicative of loops and the orientation of CTCF motifs. We find that high-efficiency, early replicating IZs localize to boundaries between adjacent corner-dot TADs anchored by high-density arrays of divergently and convergently oriented CTCF motifs. By contrast, low-efficiency IZs localize to weaker dotless boundaries. Following ablation of cohesin-mediated loop extrusion during G1, high-efficiency IZs become diffuse and delocalized at boundaries with complex CTCF motif orientations. Moreover, G1 knockdown of the cohesin unloading factor WAPL results in gained long-range loops and narrowed localization of IZs at the same boundaries. Finally, targeted deletion or insertion of specific boundaries causes local replication timing shifts consistent with IZ loss or gain, respectively. Our data support a model in which cohesin-mediated loop extrusion and stalling at a subset of genetically encoded TAD and subTAD boundaries is an essential determinant of the locations of replication origins in human S phase.

Three-dimensional chromatin organisation shapes origin activation and replication fork directionality

“…Together these data expand the prior observation that replication initiates at TAD boundaries (Emerson et al, 2022;Lombardi and Tarsounas, 2020;Petryk et al, 2016) and reveal a broad functional role for 3D chromatin organisation in replication dynamics with cell type specific regulation.…”

“…While HeLa carcinoma, K562 leukemia lymphoblast, and IMR90 primary fibroblasts displayed common TAD boundary initiation zones, most initiation sites were not shared across all three cell types. Together these data expand the prior observation that replication initiates at TAD boundaries (Emerson et al, 2022; Lombardi and Tarsounas, 2020; Petryk et al, 2016) and reveal a broad functional role for 3D chromatin organisation in replication dynamics with cell type specific regulation.…”

“…Possibilities include CTCF directionality or nucleosome phasing exerting influence over origin firing and RFD strength. For example, high-density arrays of CTCF motifs were recently identified at high-efficiency early S-phase initiation zones (Emerson et al, 2022). CTCF deletion, however, did not significantly alter replication timing (Sima et al, 2019), suggesting CTCF is likely not the driving factor of origin firing efficiency.…”

CTCF is a DNA-tension-dependent barrier to cohesin-mediated DNA loop extrusion

“…However, it remains unresolved which of these proposed mechanisms is used by CTCF and whether CTCF is sufficient for blocking loop extrusion by cohesin. Answering these questions is of great importance since CTCF is required for controlling enhancer-promoter interactions 1 , cell differentiation, nuclear reprogramming 7 , recombination of antigen receptor genes 5,6 , and timing of DNA replication 33 , and because CTCF mutations have been implicated in tumorigenesis 8 . CTCF boundaries are also sites at which replicated DNA molecules are connected by cohesin complexes which mediate cohesion 34 .…”

BTApep-TAT peptide inhibits ADP-ribosylation of BORIS to induce DNA damage in cancer

“…BORIS is the paralog of CTCF and binds to CTCF-like sites in the genome but is functionally different from CTCF [14]. CTCF organizes genomic DNA, colocalizes with the cohesin complex, regulates DNA replication and is essential for safeguarding the genome [58][59][60]. The abnormal DNA repair regulated by BORIS in cancer cells might disrupt the genome stability established by CTCF.…”