Supplementary Figure 5 : Mitotic conformation following depletion of Smc2 and characterization of smc2K38I allele.

From: SMC complexes differentially compact mitotic chromosomes according to genomic context

Supplementary Figure 5

(a) (Left) Hi-C data collected from M phase cells following disruption of conDensin with smc2td allele to deplete Smc2 (MDsmc2). Chromosomes XIII to XVI are shown as representative of the whole genome. (Middle) Log2 ratio of smc2 depleted M dataset over wt M dataset (MD smc2/M), respectively. (Right) P(s) of M versus MDsmc2. Data set assembled from one Hi-C data set. (b) Description and characterization of the smc2td GAL1smc2K38I allele used in Figs 5 and 6. (i) Western blot showing degradation of the degron tagged Smc2 protein and the concurrent GAL1 induced expression of Smc2K38I mutant in both nocodazole and cdc20 arrested metaphase state. Expression examined by Western blot in one experiment (ii) FACS analysis of DNA content following degradation of smc2td with/without expression of Smc2K38I. Representative profiles shown from one of two independent experiments. Expression of Smc2K38I increases the aneuploidy of cells generated following one cell division (right) as shown by increased number of cells with more than 2C and less than 1C DNA content. Profiled cells also contain V5-tagged Brn1 (as in (iii)). (iii) ChIP analysis of condensin complex enrichment as assayed by ChIP with Brn1-V5 at CEN4, in wildtype cells (wt), smc2 degron cells (smc2-td), or smc2-td combined with expression of smc2K38I shown in a boxplot format with all data points shown (sample size n = 11, 5, 3 experiments, respectively). Center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, data points are plotted as open circles. The increased penetrance of the smc2K38I phenotype with regard to aneuploidy and chromatin binding suggests that this allele approximates the null state. (c) (Left) Zoom into contact heatmaps from condensin depleted mitotically arrested cells, MD, for ChrXV (0-330 kb (CENXV is at 330 kb)). (Right) Zoom into log2 ratio of MD over M contact maps for ChrXV (0-330 kb (CENXV is at 330 kb))