Genome-wide expression analysis of human hTert immortalized fibroblasts after downregulation of MCM2

Minichromosome maintenance (MCM) proteins facilitate replication by licensing origins and unwinding the DNA double strand. Interestingly, the number of MCM hexamers greatly exceeds the number of firing origins suggesting additional roles of MCMs. Here we show a hitherto unanticipated function of MCM2 in cilia formation in human cells and zebrafish that is uncoupled from replication. Zebrafish depleted of MCM2 develop ciliopathy-phenotypes including microcephaly and aberrant heart looping due to malformed cilia. In non-cycling human fibroblasts, loss of MCM2 promotes transcription of a subset of genes, which cause cilia shortening and centriole overduplication. Chromatin immunoprecipitation experiments show that MCM2 binds to transcription start sites of cilia inhibiting genes. We propose that such binding may block RNA polymerase II-mediated transcription. Depletion of a second MCM (MCM7), which functions in complex with MCM2 during its canonical functions, reveals an overlapping cilia-deficiency phenotype likely unconnected to replication, although MCM7 appears to regulate a distinct subset of genes and pathways. Our data suggests that MCM2 and 7 exert a role in ciliogenesis in post-mitotic tissues. 6 samples in total: 3 control, 3 siRNA MCM2

微染色体维持（Minichromosome maintenance, MCM）蛋白通过许可复制起始位点并解开DNA双链的方式促进DNA复制。值得注意的是，MCM六聚体的数量远多于激活的复制起始位点，这提示MCM蛋白存在额外的非经典功能。本研究首次揭示了MCM2此前未被认知的功能：其可在不依赖DNA复制的前提下，调控人类细胞与斑马鱼的纤毛形成过程。MCM2敲低的斑马鱼会出现纤毛病表型，包括因纤毛结构异常引发的小头畸形与心脏环化异常。在非增殖状态的人类成纤维细胞中，MCM2的缺失会促进一组特定基因的转录，进而导致纤毛缩短与中心粒过度复制。染色质免疫沉淀实验结果显示，MCM2可结合至纤毛抑制基因的转录起始位点，研究团队推测这种结合可能会阻断RNA聚合酶II介导的基因转录。另一种在经典功能中与MCM2形成复合物发挥作用的MCM蛋白——MCM7，其敲低也会引发类似的纤毛缺陷表型，且该表型同样可能不依赖于DNA复制；不过MCM7所调控的基因与通路子集与MCM2存在显著差异。本研究数据表明，MCM2与MCM7在有丝分裂后组织的纤毛发生过程中发挥调控作用。实验样本总计6份：3份为对照组，3份为MCM2小干扰RNA（siRNA）处理组。