Contribution of CENP-F to FOXM1-mediated discordant centromere and kinetochore transcriptional regulation [ATAC-seq]

Proper chromosome segregation is required to ensure genomic and chromosomal stability. The centromere is a unique chromatin domain present throughout the cell cycle on each chromosome defined by the CENP-A nucleosome. Centromeres (CEN) are responsible for recruiting the kinetochore (KT) during mitosis, ultimately regulating spindle attachment and mitotic checkpoint function. Upregulation of many genes that encode CEN/KT proteins is commonly observed in cancer. Here, we show although FOXM1 occupies the promoters of many CEN/KT genes with MYBL2, occupancy is insufficient alone to drive the FOXM1 correlated transcriptional program. We show that CENP-F, a component of the outer kinetochore, functions with FOXM1 to coregulate G2/M transcription and proper chromosome segregation. Loss of CENP-F results in alteration of chromatin accessibility at G2/M genes, including CENP-A, and leads to reduced FOXM1-MBB complex formation. The FOXM1-CENP-F transcriptional coordination is a cancer-specific function. We observed that a few CEN/KT genes escape FOXM1 regulation such as CENP-C which when upregulated with CENP-A, leads to increased chromosome misegregation and cell death. Together, we show that the FOXM1 and CENP-F coordinately regulate G2/M gene expression, and this coordination is specific to a subset of genes to allow for proliferation and maintenance of chromosome stability for cancer cell survival. To evaluate the accessibility changes in normal cells, in reponse to loss of CENP-F and FOXM1, we conducted ATAC-seq in 48 hour siRNA treated hTERT-RPE1.

精准的染色体分离（chromosome segregation）是保障基因组与染色体稳定性的必要前提。着丝粒（centromere）是一类独特的染色质结构域，在整个细胞周期中存在于每条染色体上，由CENP-A核小体定义其特征。着丝粒（CEN）负责在有丝分裂过程中招募动粒（kinetochore, KT），最终调控纺锤体附着与有丝分裂检验点功能。在癌症中，众多编码CEN/KT蛋白的基因常出现表达上调现象。本研究发现，尽管FOXM1与MYBL2可共同结合于众多CEN/KT基因的启动子区域，但仅靠这种结合并不足以激活与FOXM1相关的转录程序。我们证实，外动粒组分CENP-F可与FOXM1协同调控G2/M期转录以及精准的染色体分离过程。敲低CENP-F会改变包括CENP-A在内的G2/M期基因的染色质开放状态，并减少FOXM1-MBB复合物的形成。FOXM1与CENP-F的转录协同调控是癌症特异性的功能。我们观察到少数CEN/KT基因可逃脱FOXM1的调控，例如CENP-C；当CENP-C与CENP-A同时上调时，会加剧染色体分离错误与细胞死亡。综上，本研究证实FOXM1与CENP-F可协同调控G2/M期基因的表达，且这种协同调控仅作用于特定基因子集，以保障癌细胞的增殖与染色体稳定性维持，从而促进癌细胞存活。为探究正常细胞中CENP-F与FOXM1缺失后染色质开放状态的变化，我们对经siRNA处理48小时的hTERT-RPE1细胞开展了ATAC-seq实验。