A novel role for E2fs: E2f7 and E2f8 control motile ciliogenesis

摘要 多纤毛细胞 (MCC) 存在于气道、室管膜和输卵管中，对于呼吸、神经发生至关重要 和生育能力。 MCC 在其顶端表面成核多达 300 个活动纤毛，以产生定向流体流。雷斯- 呼吸 MCC 负责不断清除呼吸道中的吸入污染物和病原体，以维持 肺部健康（粘液纤毛清除）。值得注意的是，气道纤毛细胞是人类 SARS 的主要目标 CoV-2 感染以及有缺陷的 MCC 清除功能受损（作为遗传性纤毛病获得或遗传）是 与慢性气道感染、慢性阻塞性肺病和哮喘有关。运动纤毛发生由 > 600 个纤毛引导 基因通过MCC转录因子的连续和分层组织的活性。然而，转录 多纤毛发生的调控仅被部分破译，在不同的早期阶段存在重大知识差距 建立 MCC 命运并协调中心粒放大的作用。 E2f转录因子，进化- 从植物到哺乳动物都高度保守，是细胞周期的主要调节因子。规范的E2f1-6调节前 控制 DNA 复制和有丝分裂机制的基因的抑制。相比之下，只有少数生理学 已知非典型阻遏蛋白 E2f7 和 E2f8 必不可少的情况。我们最近发现了一个未执行的 预计E2f7和E2f8在气道、室管膜和输卵管的活动性多纤毛发生中发挥重要的新作用。我们 发现 E2f7 和 E2f8 仅在气道上皮的两个细胞群（循环基底细胞和气道上皮细胞）中表达。 最早的 MCC 祖细胞。值得注意的是，消除小鼠中的 E2f7 会导致基底体和 气道、室管膜和输卵管中的纤毛数量。 E2f8 的额外损失进一步加剧了表型， 表明生理上的配合。我们将检验我们的假设，即 E2f7 和 E2f8 充当从 增殖未定型祖细胞以分化 MCC 祖细胞，这从根本上需要克服 严格的分裂细胞每个细胞周期一次中心粒复制程序。我们假设 E2f7 和 E2f78 对于将中心体复制与细胞分裂分离至关重要。 E2f7 和 E2f78 通过合作实现这一目标 阻断 DNA 复制并诱导交替的 S*/G2M* 样阶段，从而实现中心粒的生物发生。 接下来主要是 E2f7 调节和平衡早期 MCC 转录程序，从而安全- 保护中心粒增殖。该提案将全面系统地确定机制， 分子伴侣及其在转录 MCC 层次结构中的位置，这是这一新的纤毛功能的基础 E2f7/f8。目标 1 对原代 MCC 中的 E2f7 和 E2f8 进行单细胞机制和功能分析 器官型培养物。目标 2 在 MCC 层次结构中映射 E2f7 和 E2f8 以及早期的交替细胞周期 纤毛发生。目标 3 鉴定多纤毛发生中 E2f7 和 E2f8 的近端功能蛋白相互作用组 并进行功能验证。这些结果将极大地扩展我们对运动性多纤毛发生的基本认识 并为确定功能失调的 E2f7/f8 在纤毛相关人类中的潜在因果作用奠定基础 气道和肺部疾病，对于开发预防和治疗这些疾病的新治疗策略至关重要。

ABSTRACT Multiciliated cells (MCCs) occur in airways, ependyma and oviduct and are crucial for respiration, neurogenesis and fertility. MCCs nucleate up to 300 motile cilia at their apical surface to generate directional fluid flow. Res- piratory MCCs are responsible for constantly cleaning airways from inhaled pollutants and pathogens to maintain pulmonary health (mucociliary clearance). Notably, airway ciliated cells are the primary targets for human SARS- CoV-2 infections, and impaired clearance by defective MCCs, acquired or inherited as genetic ciliopathies, is associated with chronic airway infections, COPD and asthma. Motile ciliogenesis is directed by > 600 ciliary genes via sequential and hierarchically organized activity of MCC transcription factors. However, transcriptional regulation of multiciliogenesis is only partly deciphered, with major knowledge gaps in the early phases of differ- entiation which establish MCC fate and orchestrate centriolar amplification. E2f transcription factors, evolution- arily conserved from plants to mammals, are major regulators of cell cycle. The canonical E2f1-6 regulate ex- pression of genes controlling DNA replication and the mitotic machinery. In contrast, only a few physiological scenarios are known where the atypical repressors E2f7 and E2f8 are essential. We recently identified an unex- pected major new role of E2f7 and E2f8 in motile multiciliogenesis of airways, ependyma and oviducts. We find E2f7 and E2f8 exclusively expressed in two cell populations of airway epithelium, the cycling basal and the earliest MCCs progenitors. Notably, ablation of E2f7 in mice causes a profound reduction in basal bodies and cilia numbers in airways, ependyma and oviduct. Additional loss of E2f8 further exacerbates the phenotype, indicating physiological cooperation. We will test our hypothesis that E2f7 and E2f8 act as the switch from proliferating uncommitted progenitors to differentiating MCC progenitors which fundamentally requires overcom- ing the strict once-per-cell-cycle centriole duplication program of dividing cells. We postulate that E2f7 and E2f78 are critical for decoupling centrosome duplication from cell division. E2f7 and E2f78 achieve this by cooperatively blocking DNA replication and inducing the alternate S*/G2M*-like phases which enables centriolar biogenesis. This is followed by mainly E2f7 regulating and balancing the early MCC transcriptional program, thereby safe- guarding centriolar multiplication. This proposal will comprehensively and systematically identify mechanisms, molecular partners and their place in the transcriptional MCC hierarchy that underlie this new ciliary function of E2f7/f8. Aim 1 performs single cell mechanistic and functional analyses of E2f7 and E2f8 in MCCs in primary organotypic cultures. Aim 2 maps E2f7 and E2f8 within the MCC hierarchy and the alternate cell cycle in early ciliogenesis. Aim 3 identifies the proximal functional protein interactome of E2f7 and E2f8 in multiciliogenesis with functional validation. The results will greatly expand our fundamental insights into motile multiciliogenesis and lay the groundwork for identifying potentially causal roles of dysfunctional E2f7/f8 in cilia-related human airway and lung diseases, crucial for developing novel therapeutic strategies for their prevention and treatment.