Mechanisms of cohesin mediated regulation of gene expression

Abstract

The cohesin complex is essential for proper sister chromatid segregation during cell division and post-replicative DNA repair. Cohesin is also important for the regulation of gene expression. However, the mechanisms by which cohesin impacts gene expression remain incompletely understood. Owing to its vital role in cell division and DNA repair, loss of cohesin can indirectly impact gene expression programme in cycling cells. Thus, in order to investigate cohesin’s role in gene regulation, a conditional knockout system was used which allowed rapid depletion of the cohesin subunit RAD21 and avoided secondary stress-induced effects on gene expression. Acute depletion of cohesin in mouse embryonic stem (ES) cells did not lead to a global collapse in pluripotency. Instead, the impact of cohesin depletion was limited to about 600 genes and was locus-specific in terms of direction of deregulation. A subset of deregulated genes was selected based on the positioning of cis-regulatory elements and relevance to the pluripotent state and the role of cohesin in mediating long-range interactions was analysed using chromosome conformation capture (3C). Interestingly, cohesin binding, DNA looping and transcriptional changes were not always correlated. At some of the loci tested, these interactions were maintained after removal of cohesin, questioning models where cohesin regulates gene expression solely by mediating long-range interactions. One of the pluripotency factors affected by cohesin depletion in ES cells was Myc. Experiments analysing the expression of Myc showed that it was post-transcriptionally upregulated, specifically in cohesin deficient ES cells growing in defined media supplemented with ERK and GSK3β inhibitors (2i media). Further investigation revealed that contrary to the previously reported downregulation of Myc upon cohesin depletion, cohesin was not essential for Myc expression in various cell types. In separate experiments, I investigated if cohesin was required for the transcriptional activation of a silent gene in response to extracellular stimuli. Results from IFNγ induction of cohesin deficient non-cycling mouse embryonic fibroblasts (MEFs) showed that cohesin was important for the activation of MHC class II genes and their master regulator Ciita. The expression of MHC class I genes and the associated regulatory factors remained unaffected by cohesin depletion. Further evidence is provided for the involvement of cohesin in regulating transcription by modulating RNA polymerase processivity and through the action of PTIP subunit of the MLL complexes. Altogether my work gives an insight into the role of cohesin in mediating long-range DNA interactions important for regulation of gene expression and explores novel mechanisms of gene activation by cohesin.