Extended Data Fig. 3: Haploid genetic screen in ∆HDAC8 cells.
From: The cohesin acetylation cycle controls chromatin loop length through a PDS5A brake mechanism
(a) Plot depicting the screen results for wild type cells and ∆HDAC8 cells. Several cohesin regulators are highlighted. (b) Gene-trap integration patterns for PDS5A in anti-sense (blue) or sense (red) orientation in wild type and ∆HDAC8 cells. ∆HDAC8 cells harbour an increase in sense insertions along the entire gene. (c) Gene-trap integration patterns for SCC2NIPBL in anti-sense (blue) or sense (red) orientation in wild type and ∆HDAC8 cells. The sense insertions in ∆HDAC8 cells appear to be tolerated until exon 10, while exons 11 - 47 appear to remain essential. This pattern much resembles the pattern found in ∆WAPL cells3. (d) Pulldown experiment on the core cohesin subunit SMC1 in cells lacking ESCO1 (∆1) or HDAC8 (∆8). We find that cohesin in ∆HDAC8 cells is enriched for binding to PDS5A, PDS5B, and WAPL. Cohesin’s binding to these factors appears to be less evidently affected in ∆ESCO1 cells. We note that in wild type cells only a small fraction of cohesin complexes is acetylated. These low acetylation levels could explain why it is relatively difficult to assess differences in binding of the mentioned proteins in ∆ESCO1 cells. This experiment was performed 3 times with similar results. (e) Pulldown experiment on the cohesin regulator SCC2NIPBL in cells lacking ESCO1 (∆1) or HDAC8 (∆8). The upper three rows belong to one experiment and the lower two rows to another experiment. Both a short exposure (se) and long exposure (le) are shown for the core cohesin subunit SMC1. We find that the amount of cohesin acetylation does not affect SCC2NIPBL binding to cohesin. We also find that SCC2NIPBL pulls along acetylated cohesin complexes, suggesting that cohesin acetylation and cohesin’s binding to SCC2NIPBL are not mutually exclusive. This experiment was performed 3 times with similar results.
