Developmental regulation of cohesin positioning on mammalian chromosome arms

Abstract

Cohesin has a well-­established role in sister chromatid cohesion and postreplicative DNA repair. In addition, previous work in our laboratory suggested a positive correlation between cohesin binding and gene expression (Parelho et al. 2008). We decided to establish the ChIP-­seq technique to address the relationship between cohesin binding and gene expression at the genome-­wide level. However, in the meantime, several other groups reported genome-­wide binding maps of cohesin in embryonic stem (ES) cells, demonstrating cell-­type-­specific cohesin binding and its correlation with gene expression (Schmidt et al. 2010; Kagey et al. 2010; Nitzsche et al. 2011). Kagey and colleagues argue that cohesin is required for the expression of pluripotency-­associated genes, based on cohesin downregulation for an extended time period. We believe this method generates indirect effects such as cell stress, death and enrichment for slowly cycling differentiating cells, biasing the results towards differentiated cells. We have generated ES cells homozygous for conditional Rad21 alleles and have found that, unlike the Kagey approach, rapid 24-­hour cohesin depletion does not induce cell stress responses. We detect a stronger correlation between cohesin-­bound genes and gene expression changes, suggesting our approach is more accurate in understanding the role of cohesin in gene expression. We have expanded our analysis of cohesin binding by generating ES cells expressing epitope-­tagged BORIS, a paralogue of CTCF. We have mapped BORIS binding sites in ES cells and data suggest that BORIS, unlike CTCF, does not recruit cohesin. To study the specific involvement of cohesin in gene expression, two developmentally regulated models, T cell receptor α (Tcrα) rearrangement and X chromosome inactivation (XCI), have been used. Cohesin loss in non-­cycling developing thymocytes leads to impaired Tcrα rearrangement. Finally, we present evidence that cohesin contributes to creating chromatin boundaries that segregate facultative heterochromatin from active chromatin on the inactive X chromosome in differentiating female ES cells.