A role for condensin-mediator interaction in mitotic chromosomal organization [ChIP-Seq]

Eukaryotic genomes are organized into layers of chromatin domains, such as topologically associating domains (TADs) and A/B compartments. TADs (i.e., cohesin-mediated chromatin domains) are restricted by CCCTC-binding factor (CTCF) and convergent genes in higher eukaryotes and fission yeast, respectively. However, molecular mechanisms underlying the formation of condensin-mediated chromatin domains remain largely unclear. Here, we investigate the role of newly identified interaction between the Cnd1 condensin and Pmc4 mediator subunits. We develop a condensin mutation, cnd1-K658E, that impairs the condensin-mediator interaction and find that this mutation diminishes condensin-mediated domains and causes defects in chromosomal segregation. The condensin-mediator interaction is involved in recruiting condensin to highly transcribed genes and mitotically activated genes, the latter of which demarcate condensin-mediated domains. Our study also predicted that the condensin-mediator interaction and its function in chromosomal segregation are potentially conserved in human cells. This study provides a novel insight into how genome-wide gene expression is connected to the mitotic chromosomal architecture via the condensin-mediator interaction. Overall design: (1) Genome-wide distributions of condensin were determined by ChIP-seq using mitotic cells with wild-type Cnd1 expression (Cnd1 WT-Pk) and with Cnd1-K658E expression (Cnd1-K658E-Pk) from the plasmids. The endogenous Cnd1 was repressed by culturing cells in EMM medium containing auxin. Mitotic cells were prepared by the cdc25-22 G2/M block-release and harvested at a 60-minute time point after the release. (2) In addition, genome-wide distributions of Cnd1-Pk in cells with wild-type and Pmc4 depletion are prepared. (3) Genome-wide distributions of the Cnd2-Flag (WT and C703R) expressed from the plasmids were determined using cells depleting the endogenous Cnd2. (4) Genome-wide distributions of Cnd1-WT-Myc and Cnd1-K658-Myc expressed from the endogenous locus. Mitotic cells were prepared in YEA using the cdc25-22 G2/M block-release and harvested at a 40-minute time point after the release. (5) Genome-wide distributions of Cnd1, Pmc4, Pol II in mitotic cells with or without 5% HD treatment for 5min. Mitotic cells were prepared in YEA using the cdc25-22 G2/M block-release and harvested at a 40-minute time point after the release.