A role for condensin-mediator interaction in mitotic chromosomal organization [Hi-C]

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) Hi-C experiment in mitotic cells with wild-type Cnd1 expression from the plasmid , without exogenous Cnd1 expression , and with exogenous Cnd1-K658E expression. 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 the 60-minute time point after the release. (2) Hi-C experiment in mitotic cells with wild-type Pmc4 expression from the plasmid and without exogenous Pmc4 expression. The endogenous Pmc4 was degraded by the AID system. The mitotic cells were prepared using the cdc25-22 block-release. (3) Hi-C experiment in asynchronous culture cells of various mediator deletions. (4) Hi-C experiment in asynchronous culture cells with Cnd2 depletion. Cells were cultured in EMM liquid medium containing thiamine and auxin to induce transcriptional inhibition of cnd2 gene and post-translational degradation of endogenous Cnd2 proteins The Cnd2 WT or C703R mutant was expressed from the plasmid. (5) Hi-C experimets in motitic cells with Cnd1 WT or Cnd1 K658E expression from the endogenous locus. Mitotic cells were prepared in YEA using the cdc25-22 G2/M block-release and harvested at the 40-minute time point after the release. (6) Hi-C experiments in mitotic cells with Cnd1, Pmc4 or Cnd1/Pmc4 double depletion. Mitotic cells were prepared in YEA using the cdc25-22 G2/M block-release and harvested at the 40-minute time point after the release. (7) Hi-C experiments in mitotic cells with 5 or 10 % HD treatment for 5min before fixation. Mitotic cells were prepared in YEA using cdc25-22 G2/M block-release and harvested at the 40-minute time point after the release.