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

摘要 多纤毛细胞(MCC)存在于呼吸道、室管膜和输卵管中，对呼吸、神经发生起重要作用。 和生育能力。MCC在其顶端表面形成多达300个活动纤毛，以产生定向流体流动。资源- 海盗MCC负责不断清理呼吸道中吸入的污染物和病原体，以保持 肺健康(粘液纤毛清除)。值得注意的是，呼吸道纤毛细胞是人类SARS的主要目标- CoV-2感染和缺陷的MCC清除障碍，作为遗传性纤毛病获得或遗传，是 与慢性呼吸道感染、慢性阻塞性肺病和哮喘有关。运动纤毛发生由&gt；600纤毛指导 通过MCC转录因子的顺序和层级组织的活性来表达基因。然而，转录的 对多发性髂骨形成的调控只有部分破译，早期阶段的主要知识差距不同- 确定MCC命运和协调中心粒扩增的鉴定。E2F转录因子，进化- 从植物到哺乳动物都很保守，是细胞周期的主要调节因子。规范的E2F1-6调节EX-2 抑制控制DNA复制和有丝分裂机制的基因。相比之下，只有少数人在生理上 已知的情况是，非典型阻遏因子E2f7和E2f8是必不可少的。我们最近确认了一个没有前科的- 推测E2f7和E2f8在呼吸道、室管膜和输卵管的运动性多发性纤毛发生中具有重要的新作用。我们 发现E2f7和E2f8仅在两个呼吸道上皮细胞群中表达，即循环基底层和 最早的MCC祖先。值得注意的是，消融小鼠的E2f7导致基底体和 气管、室管膜和输卵管纤毛数量。E2f8的额外丢失进一步加剧了表型， 表明生理上的配合。我们将测试我们的假设，即E2f7和E2f8充当从 增殖未承诺的祖细胞以区分MCC祖细胞，这从根本上需要克服- 严格按照细胞周期一次的中心粒复制程序进行细胞分裂。我们假设E2f7和E2f78 是将中心体复制与细胞分裂脱钩的关键。E2f7和E2f78通过合作实现这一点 阻断DNA复制，诱导交替的S*/G2M*相，使中心粒生物发生。 其次主要是E2F7调节和平衡早期MCC转录程序，从而安全地- 守卫中心粒的增殖。这项建议将全面系统地确定机制， 分子伴侣及其在转录MCC层级中的位置，这是这一新的纤毛功能的基础 E2f7/f8。目的1对原代MCC中E2f7和E2f8的单细胞机制和功能进行分析 器官类型培养。AIM 2将E2f7和E2f8定位在MCC层级内，并在早期定位于交替细胞周期 纤毛发生。目的3鉴定E2f7和E2f8在多发性髂骨发生中的近端功能蛋白相互作用组 具有功能验证功能。这些结果将极大地扩展我们对运动性多发性髂骨发生的基本见解。 并为确定纤毛相关人类E2f7/f8功能障碍的潜在原因奠定了基础 呼吸道和肺部疾病，对于制定预防和治疗这些疾病的新的治疗策略至关重要。

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.