Mammalian genomes are spatially organized into compartments, topologically associating domains (TADs), and loops to facilitate gene regulation and other chromosomal functions. How compartments, TADs, and loops are generated is unknown. It has been proposed that cohesin forms TADs and loops by extruding chromatin loops until it encounters CTCF, but direct evidence for this hypothesis is missing. Here, we show that cohesin suppresses compartments but is required for TADs and loops, that CTCF defines their boundaries, and that the cohesin unloading factor WAPL and its PDS5 binding partners control the length of loops. In the absence of WAPL and PDS5 proteins, cohesin forms extended loops, presumably by passing CTCF sites, accumulates in axial chromosomal positions (vermicelli), and condenses chromosomes. Unexpectedly, PDS5 proteins are also required for boundary function. These results show that cohesin has an essential genome-wide function in mediating long-range chromatin interactions and support the hypothesis that cohesin creates these by loop extrusion, until it is delayed by CTCF in a manner dependent on PDS5 proteins, or until it is released from DNA by WAPL.
Mammalian genomes are spatially organized by CCCTC-binding factor (CTCF) and cohesin into chromatin loops and topologically associated domains, which have important roles in gene regulation and recombination. By binding to specific sequences, CTCF defines contact points for cohesin-mediated long-range chromosomal cis-interactions. Cohesin is also present at these sites, but has been proposed to be loaded onto DNA elsewhere and to extrude chromatin loops until it encounters CTCF bound to DNA. How cohesin is recruited to CTCF sites, according to this or other models, is unknown. Here we show that the distribution of cohesin in the mouse genome depends on transcription, CTCF and the cohesin release factor Wings apart-like (Wapl). In CTCF-depleted fibroblasts, cohesin cannot be properly recruited to CTCF sites but instead accumulates at transcription start sites of active genes, where the cohesin-loading complex is located. In the absence of both CTCF and Wapl, cohesin accumulates in up to 70 kilobase-long regions at 3'-ends of active genes, in particular if these converge on each other. Changing gene expression modulates the position of these 'cohesin islands'. These findings indicate that transcription can relocate mammalian cohesin over long distances on DNA, as previously reported for yeast cohesin, that this translocation contributes to positioning cohesin at CTCF sites, and that active genes can be freed from cohesin either by transcription-mediated translocation or by Wapl-mediated release.
Mammalian imprinted genes often cluster with long noncoding (lnc) RNAs. Three lncRNAs that induce parental-specific silencing show hallmarks indicating that their transcription is more important than their product. To test whether Airn transcription or product silences the Igf2r gene, we shortened the endogenous lncRNA to different lengths. The results excluded a role for spliced and unspliced Airn lncRNA products and for Airn nuclear size and location in silencing Igf2r. Instead, silencing only required Airn transcriptional overlap of the Igf2r promoter, which interferes with RNA polymerase II recruitment in the absence of repressive chromatin. Such a repressor function for lncRNA transcriptional overlap reveals a gene silencing mechanism that may be widespread in the mammalian genome, given the abundance of lncRNA transcripts.
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