Condensin II is recruited to promoters by pRb and regulates expression of divergently paired genes [ChIP-seq]. Condensin II is recruited to promoters by pRb and regulates expression of divergently paired genes [ChIP-seq]

In this study, use of a gene-targeted mouse model that is defective for pRb-condensin II interactions, Rb1L, has revealed instances where condensin II localization in interphase nuclei is dependent on pRb. Condensin II binding overlaps significantly with marks of active chromatin, and is enriched at the promoters of genes, including closely spaced divergent promoters. Interestingly, there are some bidirectional promoters where condensin II binds in an pRb-dependent manner and transcription of only one of the two genes is upregulated in Rb1L/L MEFs, suggesting the pRb-condensin II complex may be acting as a repressor or an insulator at distinct genomic locations to influence transcription. Chromatin conformations at these locations demonstrated that the pRb-condensin II complex may be responsible for maintaining more static, long-range interactions. In addition, pRb also recruits TFIIIC, another condensin II interactor, to many genomic loci. These recently discovered non-canonical transcriptional functions of the pRb-condensin II complex may represent an additional mechanism of pRb-mediated tumor suppression. Overall design: Chromatin was isolated from passage 4 MEFs cross-linked with either ethylene glycol bissuccinimidylsuccinate (EGS) and formaldehyde or formaldehypde alone, and then sonicated so most chromatin was ≤ 400 bp. DNA fragments were immunoprecipitated using different antibodies, washed, then purified. DNA was prepared for sequencing according to the NEBNext Ultra II DNA library prep kit. Each protein was immoprecpitated in both wild type and Rb1L/L to compare locatlization between genotypes.

本研究利用存在pRb-凝缩蛋白II（condensin II）互作缺陷的基因靶向小鼠模型Rb1L，揭示了间期细胞核内凝缩蛋白II的定位依赖于pRb的若干情形。凝缩蛋白II的结合区域与活性染色质标记存在显著重叠，且在基因启动子区域（包括紧密间隔的双向启动子）处富集。有趣的是，在部分双向启动子区域，凝缩蛋白II的结合呈pRb依赖性，而在Rb1L/L型小鼠胚胎成纤维细胞（Mouse Embryonic Fibroblasts，MEFs）中，仅两个基因中的一个的转录被上调，这提示pRb-凝缩蛋白II复合物可在特定基因组区域充当阻遏物或绝缘子以调控转录。这些区域的染色质构象分析显示，pRb-凝缩蛋白II复合物可能负责维持更为稳定的长距离染色质互作。此外，pRb还可将另一个凝缩蛋白II互作因子转录因子IIIC（TFIIIC）招募至众多基因组位点。此次新发现的pRb-凝缩蛋白II复合物的非经典转录功能，或代表了pRb介导的肿瘤抑制的另一重机制。实验整体设计：从第4代小鼠胚胎成纤维细胞中分离染色质，分别用乙二醇双琥珀酰亚胺琥珀酸酯（ethylene glycol bissuccinimidylsuccinate，EGS）与甲醛，或仅用甲醛进行交联，随后进行超声破碎，使绝大多数染色质片段长度≤400 bp。对DNA片段使用不同抗体进行免疫沉淀、洗涤后纯化。依照NEBNext Ultra II DNA文库制备试剂盒的操作流程，制备用于测序的DNA文库。分别在野生型与Rb1L/L型细胞中对各靶蛋白进行免疫沉淀，以比较不同基因型下的蛋白定位情况。