Targeted cohesin loading characterizes the entry and exit sites of loop extrusion trajectories

Abstract

The cohesin complex (SMC1-SMC3-RAD21) shapes chromosomes by DNA loop extrusion, but individual extrusion trajectories were so far unappreciable in vivo. Here, we site-specifically induced dozens of extrusion trajectories anchored at artificial loading sites in living cells. Extruding cohesin transports loading proteins MAU2-NIPBL over megabase DNA distances to blocking CTCF sites that then loop back to the loading sequences, showing that CTCF-CTCF interactions are unnecessary for stabilized contacts between loop extrusion obstacles. When stalled, cohesin can block other extruding cohesin from either direction. Without RAD21, MAU2-NIPBL exclusively accumulate at loading sites, here genome-wide defined as enhancers. SMC1 now also selectively accumulates here, suggesting that cohesin may load modularly on chromatin. Genes inside high cohesin extrusion trajectories are collectively hindered in transcription. This work characterizes the impact, entry and exit sites of individual cohesin loop extrusion trajectories.

Cohesin organizes 3D DNA contacts surrounding active enhancers inC. elegans

“…In worms, an X-specific condensin surrounds its recruitment elements on the X (Anderson et al 2019; Rowley et al 2020; Kim et al 2022; Morao et al 2022). Targeted loading of cohesin using tetO-tetR system also showed spreading of cohesin binding at nearby CTCF sites (Han et al 2023). Thus, observation of spreading implies the presence of loading sites.…”

“…ChIP-seq signal for NIPBL does not track cohesin spreading and resembles the binding pattern of the residual SMC-1 in SMC-3 depletion (Figure 3A). While whether NIPBL is a loader and processivity factor is still debated (Ciosk et al 2000; Kagey et al 2010; Alonso-Gil et al 2023; Banigan et al 2023; Han et al 2023), the lack of spreading pattern in NIPBL track suggests that NIPBL functions as a loader rather than a processivity factor that moves with cohesin in C. elegans (discussed below after Hi-C analysis).…”

“…In vivo , recent observation of chromatin jets or plumes further supports cohesin as a two-sided loop extruding factor (Liu et al 2021a; Guo et al 2022; Galitsyna et al 2023; Isiaka et al 2023). DNA-damage induced recruitment of cohesin or artificial loading of cohesin via a tetO-tetR system generated ‘stripe’, suggesting that cohesin could also act as a one-sided loop extruding factor (Arnould et al 2021; Han et al 2023). Our population average ChIP-seq data (Figure 3) cannot distinguish whether the spreading of cohesin towards left and right occur simultaneously, which would imply two-sided loop extrusion, or independently, which would imply one-sided loop extrusion.…”

“…In zebrafish, chromatin jets correlated with enhancer activity across development, and knockdown of pioneer transcription factors eliminated chromatin jets originating at these sites (Galitsyna et al 2023). In mammalian cells, analysis of cohesin binding upon CTCF site deletion, depletion of pioneer transcription factors, and ectopic recruitment experiments suggest that cohesin is preferentially loaded at enhancers (Dowen et al 2013; Liu et al 2021b; Vos et al 2021; Han et al 2023). Our work also showed an overlap of jet origins with active enhancers in C. elegans , thus preferential loading of cohesin may be a conserved mechanism of regulating 3D DNA contacts surrounding enhancers, even in a compact genome that lacks CTCF.…”

“…In vitro, SMC complexes translocate along DNA [12,[37][38][39]. In vivo, this dynamic process manifests in ChIP-seq signal track as 'spreading', where the enrichment gradually decreases with increasing genomic distance from a loading source (33).…”