Analysis of cohesin-dependent gene regulation in fission yeast. Schizosaccharomyces pombe

In addition to its well-know function in chromosome segregation, increasing evidence implicates cohesin in the control of gene expression. It has been previously reported that inactivation of the cohesin loader Mis4 in G1-arrested cells leads to the dissociation of cohesin from chromatin. We exploited this experimental situation to ask whether this loss of cohesin would affect gene expression on a genome-wide scale. Overall design: Three independent inoculates (biological replicates) of cells carring either the thermosensitive mis4-367 or the wild-type mis4+ allele were grown at permissive temperature of 25°C. Cells were G1-arreted by titrating out the Cdc10 transcription factor by overexpression of the C-terminal fragment of its binding partner Res1, and then shifted for 2 hours at 37°C. Cells were harvested before and after the temperature shift to proceed to total RNA extraction.

除黏连蛋白（cohesin）在染色体分离中的经典功能外，越来越多的证据表明其参与基因表达的调控。此前已有研究报道，在G1期阻滞的细胞中失活黏连蛋白加载因子Mis4，会导致黏连蛋白从染色质（chromatin）上解离。本研究利用这一实验体系，探究黏连蛋白的缺失是否会在全基因组范围内影响基因表达。
实验设计：将三个独立的细胞接种物（均为生物学重复，biological replicates）分别培养，这些细胞分别携带热敏型mis4-367等位基因（allele）或野生型mis4+等位基因，培养温度为25℃的允许温度。通过过表达Cdc10的结合伴侣Res1的C端片段，滴定耗竭Cdc10转录因子（Cdc10 transcription factor），使细胞阻滞于G1期；随后将培养温度切换至37℃，处理2小时。在温度切换前后分别收集细胞，用于总RNA提取。