Cohesin has been shown to occupy sites bound by CTCF and to contribute to DNA loop formation associated with gene repression or activation 24–26
. Cohesin has also been demonstrated to occupy sites independently of CTCF, but the role of Cohesin at these sites is not known 27
. We used ChIP-Seq to determine the genome-wide occupancy of the two Cohesin core complex proteins, Smc1a and Smc3, whose knockdown resulted in a loss of Oct4 (, Supplementary Fig. 3
and Supplementary Tables 4
). The results show that Cohesin occupies sites bound by CTCF, as expected, but also occupies the enhancer and core promoter sites bound by Mediator ( and Supplementary Fig. 5
). The regions co-occupied by Cohesin and Mediator were associated with RNA polymerase II whereas those co-occupied by Cohesin and CTCF were not (). These results demonstrate that there is a population of Cohesin that is associated with the enhancer and core promoter sites occupied by Mediator in many active promoters of ES cells.
The Cohesin loading factor Nipbl, which was also identified in the shRNA screen, has been implicated in transcriptional regulation and is mutated in the majority of individuals afflicted with Cornelia de Lange Syndrome (CdLS), a developmental disorder 14,28,29
. To our surprise, ChIP-Seq data revealed that Nipbl generally occupies the enhancer and core promoter regions bound by Mediator and Cohesin, but is rarely found at CTCF and Cohesin co-occupied sites ( and Supplementary Fig. 5
). The association between Nipbl and Mediator/Cohesin sites was highly significant (P-val <10−300
) whereas the association of Nipbl with CTCF/Cohesin sites was no greater than expected by chance (P-val =1). Thus, the Cohesin loading factor Nipbl is associated with Cohesin/Mediator sites but not with Cohesin/CTCF sites in ES cells. These results link Nipbl and CdLS to a form of Cohesin associated with Mediator at actively transcribed genes.
The co-occupancy of Mediator, Cohesin and Nipbl at the promoter regions of Oct4 (Pou5f1)
and other active ES cell genes () suggests that these complexes may all contribute to control of transcription. If Mediator, Cohesin and Nipbl function together to regulate the genes they occupy, then we would expect that knockdown of Nipbl or key components of the Mediator or Cohesin complexes would have similar effects on expression of these genes. Analysis of changes in mRNA levels in knockdown cells revealed that this is the case (). Of the approximately 2700 genes that are co-occupied by Mediator, Cohesin, Nipbl and Pol2 at high confidence, approximately 700 showed significant expression changes (P-val <0.01) in each of the Mediator, Cohesin and Nipbl knockdown datasets ( and Supplementary Table 3
). The three knockdowns had strikingly similar effects at this set of genes, which may explain why Mediator, Cohesin and Nipbl knockdowns cause very similar ES cell phenotypes (Supplementary Fig. 6
). These results indicate that actively transcribed genes occupied by Mediator, Cohesin and Nipbl typically depend on each of these factors for normal expression.