Cohesin-dependent compaction of mitotic chromosomes in budding yeast. Saccharomyces cerevisiae

The extreme length of chromosomal DNA requires organizing mechanisms to both promote functional genetic interactions and ensure faithful chromosome segregation when cells divide. Microscopy and genome wide contact frequency (Hi-C) analyses indicate that intra-chromosomal looping of DNA is a primary pathway of chromosomal organization during all stages of the cell cycle (Dekker, J. & Mirny, L. . Cell 164, 1110–1121 (2016). Although the enzymatic pathways required for DNA loop formation are yet to be fully characterized, the activity of the SMC family of proteins has been consistently associated with this process in interphase and mitosis. Here we use Hi-C to study the reorganization of budding yeast chromosome conformation in early mitosis and the role of SMCs in this process. Using polymer simulations, we find that the differences between interphase and mitotic Hi-C maps can be explained by the formation of intra-chromosomal (cis-) loops in mitotic chromosomes. We demonstrate that mitotic SMC cohesin activity is required for formation of cis-loops, independently of sister-chromatid cohesion. In contrast, SMC condensin is not required for loop formation in these early mitotic cells. Rather condensin activity promotes distinct higher order structures in the chromosomes at centromeres and in the rDNA proximal regions. Thus we demonstrate that cohesin-dependent cis-loops provide the primary higher order organization of budding yeast mitotic chromosomes, independently of condensin and sister chromatid cohesion. Overall design: This submission contains the following Hi-C experiments: 2 biological replicates of WT S. cerevisiae cells arrested in G1 and metaphase; 2 replicates of cohesin and condensin temperature-sensitive mutants arrested in metaphase; 1 replicate of cdc45-degron cells arrested in early mitosis and 1 replicate of temperature-sensitive cohesin mutant cdc45-degron cells arrested in early mitosis.

染色体DNA的极端长度要求细胞分裂时存在相应的组织机制，既要保障功能性遗传互作的正常进行，又要确保染色体分离的忠实性。显微镜观测与全基因组接触频率（Hi-C）分析表明，在细胞周期的所有阶段，染色体内DNA环化都是染色体组织的核心途径（Dekker, J. & Mirny, L. . Cell 164, 1110–1121 (2016)。尽管DNA环化所需的酶促通路尚未完全阐明，但SMC家族蛋白的活性在细胞间期与有丝分裂阶段始终与该过程密切相关。本研究利用Hi-C技术，探究出芽酵母在有丝分裂早期的染色体构象重塑现象，以及SMC蛋白在此过程中发挥的作用。通过聚合物模拟实验，我们发现间期与有丝分裂Hi-C图谱之间的差异，可通过有丝分裂染色体中染色体内（顺式）环的形成得到合理解释。我们证实，有丝分裂SMC黏连蛋白（cohesin）的活性是顺式环形成的必要条件，且该过程不依赖于姐妹染色单体黏连。与之相反，SMC凝缩蛋白（condensin）并非此类早期有丝分裂细胞中环形成的必需因素。凝缩蛋白的活性反而会在着丝粒区域与rDNA近端区域的染色体中，促成独特的高级结构。综上，我们证实依赖黏连蛋白的顺式环是出芽酵母有丝分裂染色体的主要高级组织结构形式，且该过程不依赖凝缩蛋白与姐妹染色单体黏连。整体实验设计：本数据集包含以下Hi-C实验：阻滞于G1期与中期的野生型酿酒酵母（S. cerevisiae）细胞的2个生物学重复；阻滞于中期的黏连蛋白与凝缩蛋白温度敏感型突变体的2个重复；阻滞于早期有丝分裂的cdc45-degron细胞的1个重复，以及阻滞于早期有丝分裂的温度敏感型黏连蛋白突变体cdc45-degron细胞的1个重复。