Genome-wide analysis of RB and p130 binding in human diploid fibroblasts. Homo sapiens

The action of RB as a tumor suppressor has been difficult to define, in part, due to the redundancy of the related proteins p107 and p130. By coupling advanced RNAi technology with a genome wide analysis of gene expression and RB chromatin binding, we identified a unique and specific activity of RB in repressing DNA replication as cells exit the cell cycle into senescence, a tumor suppressive program. Overall design: Binding of RB was examined in growing, quiescent, senescent or senescent cells lackign RB. Binding of p130 was examined in quiescent or senescent cells in the presence or absence of RB. Libraries were prapered from DNA co-precipiated with RB or p130 specific antibodies or from mock (beads-only) immunoprecipiates.

视网膜母细胞瘤蛋白（Retinoblastoma protein，RB）作为肿瘤抑制因子的功能一直难以明确界定，这在一定程度上与其相关蛋白p107和p130的功能冗余有关。本研究将先进的RNA干扰（RNA interference，RNAi）技术与全基因组基因表达及RB染色质结合分析相结合，鉴定出RB在细胞退出细胞周期进入衰老（一类肿瘤抑制程序）过程中抑制DNA复制的独特且特异的活性。实验整体设计：分别在增殖态、静息态、衰老态以及RB缺失的衰老态细胞中检测RB的结合情况；同时在存在RB或缺失RB的静息态与衰老态细胞中检测p130的结合情况。测序文库均通过与RB或p130特异性抗体共沉淀的DNA，或仅使用磁珠的阴性对照（mock）免疫沉淀产物制备得到。