Functional role of the RSF complex in establishment of centromere-specific chromatin

先前，我发现RSF复合物与CENP-A短暂相互作用，并在CENP-A并入着丝粒中起作用。因此，我想确定1)RSF复合物如何靶向着丝粒，2)哪些是帮助RSF复合物稳定保留CENP-A染色质的协同因子，以及3)RSF复合物如何参与着丝粒组装。为了解决这些问题，我对稳定表达flag - rf -I的DT40细胞进行了抗flag染色质免疫沉淀，发现rf相关DNA沿基因组分布，但特定的尖峰与cenp -a相关DNA的峰重叠。此外，我发现RSF复合体主要与FACT复合体相互作用，FACT复合体是另一种染色质重塑复合体，我们也在着丝粒上发现了它与CENP-A相互作用。我证明在敲除FACTp80（SSRP1）的细胞中，Rsf1的数量减少。

Previously, I found that the RSF complex transiently interacts with CENP-A and acts in CENP-A incorporation into centromeres. Therefore, I wanted to determine 1) how the RSF complex targets the centromere, 2) which are the synergic factors that help the RSF complex in the stabile retention of CENP-A chromatin and 3) how the RSF complex is involved in kinetochore assembly. To address these questions, I performed anti-FLAG chromatin immunoprecipitation of DT40 cells stably expressing FLAG-Rsf-I and found that RSF-associated DNA distributes along the genome, but a specific sharp peak overlaps the peak of the CENP-A-associated DNA. In addition, I found that the RSF complex mainly interacts with the FACT complex, which is another chromatin remodeling complex that we also spotted at centromere and found that interacts with CENP-A. I demonstrated that the Rsf1 amount was reduced in cells with knockout of FACTp80(SSRP1).