The chromosomal association of Smc6 depends on cohesin.

<p>(<b>A</b>) Comparison of ChIP-on-chip and ChIP-seq. Chromosomal localization of Smc6-FLAG as determined by ChIP-on-chip (upper panel) and ChIP-seq (middle panel). The lowest panel shows a ChIP-seq map from a control experiment performed on cells lacking FLAG-tagged proteins. (<b>B</b>) Chromosomal association of Smc6-FLAG in G2/M-arrested <i>scc1-73</i> cells as determined by ChIP-seq. In both (A) and (B), cells were arrested in G2/M after a synchronous S-phase at 35°C before sample preparation. The asterisk (*) denotes a false positive peak. The Y-axis shows fold enrichment of ChIP/whole cell extract (WCE) in log scale for ChIP-on-chip and in linear scale for ChIP-seq, while the X-axis shows chromosomal positions. Blue horizontal bars in the panel describing the genomic region denote open reading frames, while red and green vertical lines denote replication origins (ARS) and centromeres (CEN), respectively. (<b>C</b>) Chromosomal association of Smc6-FLAG at selected chromosomal positions in indicated strains as determined by ChIP-qPCR. Cells were arrested in G2/M after a synchronous S-phase at 35°C, or after G1 arrest (G1), before sample preparation. Chromosome number and distance from the left telomere of each analyzed position is indicated below the corresponding group of bars. Y-axis displays the amount of DNA in ChIP fraction in relation to input. Each bar represents the mean of three independent experiments with standard deviations indicated. Statistical analysis was performed using the Student's two-tailed <i>t</i>-test. <i>P</i>-values are illustrated in the figures as follows: n. s. = not significant (<i>p</i>>0.05); * = significant (<i>p</i><0.05); ** = significant (<i>p</i><0.01); *** = significant (<i>p</i><0.001). Maps displaying Smc6 enrichment at the corresponding sites as determined by ChIP-seq are displayed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004680#pgen.1004680.s002" target="_blank">Figure S2</a>. (<b>D</b>) Chromosomal association of Smc6-HA as determined by immunofluorescence of chromosome spreads. Indicated <i>SMC6-3HA</i>-expressing cells were arrested in G2/M after a synchronous S-phase at 35°C. Samples were collected and chromosome spreads were prepared and stained to detect the HA-epitope tag on Smc6. Box whisker plots representing the signal intensity in arbitrary units (AU) from quantifications of at least 50 cells for each sample are shown. Statistical analysis was done using a two-tailed <i>t</i>-test with Welch's correction. <i>P</i>-values are as follows: Wild-type (no tag) vs. <i>SMC6-3HA</i>, <i>p</i><0.0001 (****); <i>SMC6-3HA</i> vs. <i>scc1-73 SMC6-3HA</i>, <i>p</i><0.0001 (****); <i>SMC6-3HA</i> vs. <i>top2-4 SMC6-3HA</i>, <i>p</i> = 0.0006 (***);<i>scc1-73 SMC6-3HA</i> vs. <i>top2-4 scc1-73 SMC6-3HA</i>, <i>p</i> = 0.0002 (***); <i>top2-4 SMC6-3HA</i> vs. <i>top2-4 scc1-73 SMC6-3HA</i>, <i>p</i><0.0001 (****).