Cis-regulatory chromatin contacts form de novo in the absence of loop extrusion

NIPBL promotes chromatin loop extrusion by the cohesin complex until it stalls at convergently oriented CTCF sites, forming structural loops. In contrast, a large fraction of loops connecting cis-regulatory elements (CREs) can be maintained in the absence of cohesin. However, whether de novo establishment of CRE loops requires loop extrusion remains unclear. To address this question, we characterized the formation of structural and CRE loops during the mitosis-to-G1-phase transition in the absence of NIPBL. Structural loop formation was impaired proportionally to loop length. Computational modeling supports these observations, suggesting that NIPBL enhances cohesin extrusion beyond NIPBL's known loading function. Importantly the majority of CRE loops, regardless of length, was established independently of loop extrusion. While globally gene activation showed little impairment, NIPBL loss delayed the formation of CRE loops involving weak enhancers. Collectively, our findings reveal that post-mitotic establishment of regulatory contacts and gene transcription can occur independently of chromatin loop extrusion. Overall design: Hi-C was performed in NIPBL-AID cells isolated in Late G1 stage. Cells were arrested in prometaphase with nocodazole. Cells were treated with auxin prior to release into G1 for 4 hours (untreated, released cells as a control). Using the G1E-ER4-derived cell lines: Lmna-mAID and Lmna-mAID/Lbr-dTag to achieve targeted degradation of Lamin A/C and/or LBR, followed by fluorescence-activated cell sorting (FACS) to isolate cell samples at various time points during post-mitotic G1 phase entry. Chromatin three-dimensional structural information of these samples was then acquired through Hi-C (High-throughput Chromosome Conformation Capture) analysis. CHIP-seq and TTseq were also performed on the treated samples to help us better understand the regulatory role of NIPBL.