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

摘要 多纤毛细胞（MCCs）存在于气道、室管膜和输卵管中，对呼吸、神经发生和发育至关重要。 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动。Res- 呼吸道MCC负责不断清洁呼吸道中吸入的污染物和病原体， 肺健康（粘膜纤毛清除率）。值得注意的是，气道纤毛细胞是人类SARS的主要靶点- CoV-2感染，以及作为遗传性纤毛病获得或遗传的有缺陷的MCC的清除受损， 与慢性呼吸道感染、COPD和哮喘相关。运动纤毛发生由> 600个纤毛 通过MCC转录因子的顺序和分层组织的活性来表达基因。然而，转录 多纤毛发生的调节只是部分破译，在不同的早期阶段存在重大的知识空白， 建立MCC命运和协调中心粒扩增的启动子。E2 f转录因子，进化- 从植物到哺乳动物都是保守的，是细胞周期的主要调节因子。典型的E2 f1 -6调节前 控制DNA复制和有丝分裂机制的基因的表达。相比之下，只有少数生理 已知非典型阻遏物E2 f7和E2 f8是必需的情况。我们最近发现了一个- 预期E2 f7和E2 f8在气道、室管膜和输卵管的运动性多纤毛发生中的主要新作用。我们 发现E2 f7和E2 f8仅在气道上皮的两个细胞群中表达，即循环基底细胞群和循环基底细胞群。 最早的MCCs祖细胞。值得注意的是，小鼠中E2 f7的消融导致基底体和基底膜的显著减少。 气道、室管膜和输卵管纤毛数。E2 f8的额外损失进一步加剧了表型， 表明生理上的合作。我们将测试我们的假设，即E2 f7和E2 f8作为开关， 增殖未定型的祖细胞到分化的MCC祖细胞，这从根本上需要过度分化， 严格的每细胞周期一次的中心粒复制程序。我们假设E2 f7和E2 f78 是将中心体复制与细胞分裂分离的关键。E2 f7和E2 f78通过协作实现这一点 阻断DNA复制并诱导交替的S*/G2 M * 样期，这使得中心粒生物发生成为可能。 随后主要是E2 f7调节和平衡早期MCC转录程序，从而安全地- 保护中心粒增殖。这项建议将全面和系统地确定各种机制， 分子伴侣及其在转录MCC层次结构中的位置，这些层次结构是这种新的纤毛功能的基础， E2f7/f8。目的1：对原发性乳腺癌患者的MCC中E2 f7和E2 f8进行单细胞机制和功能分析。 器官型培养目的2在MCC层次内定位E2 f7和E2 f8，并在早期细胞周期中交替细胞周期。 纤毛发生目的3鉴定E2 f7和E2 f8在多纤毛发生中的近端功能蛋白相互作用组 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见解 并为确定E2 f7/f8功能障碍在纤毛相关人类中的潜在因果作用奠定基础。 呼吸道和肺部疾病，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.