Specialized cell cycles in early erythropoiesis

项目摘要 在基本上所有的谱系中，干细胞的细胞周期是静止的，并且在终末经历有丝分裂退出。 分化细胞然而，在中间发育期，细胞周期被认为是 “一般性的”，仅在数量上受到调节，以维持体内平衡或对压力作出反应。的 哺乳动物细胞周期的一般观点与早期胚胎细胞的特化细胞周期形成对比。 在模式生物如果蝇或非洲爪蟾中，细胞周期控制，包括戏剧性的 细胞周期长度的变化与发育事件密切相关。我们最近发表的作品， 包括对小鼠红细胞轨迹的单细胞转录组学分析和对复制叉的研究。 早期红细胞生成的动力学已经揭示了在整个哺乳动物中存在特化的细胞周期， 红细胞发育我们的主要假设是，细胞的发育阶段特异性特化 周期是分化过程的组成部分，并调节细胞生长等增量变化， 以及类似开关的细胞命运决定。在这个提议中，我们研究了细胞周期的早期特化， 红细胞生成，围绕关键细胞命运转换的时间精心安排，从CFU-e祖细胞的自我更新， 红系终末分化（ETD）。我们发现，在这个转换之前， G1;并且在开关处，存在S相的突然缩短。此外，S相缩短是以下因素的结果： 一种新的机制，调节S相长度，通过全球增加复制叉速度。在这 建议，我们将调查的机制，以及功能的结果，这些细胞周期 专业化。在AIM 1中，我们将对E2 F4、KLF 1、EpoR四种红系调节因子进行功能分析 的Stat 5。利用这些调节因子的突变小鼠，我们将确定它们在红系细胞周期中的作用 专业化和随之而来的发展决策。在AIM 2中，我们将进行单细胞RNA-seq分析 四个调节子中每一个都有祖细胞被删除。我们会用细胞转录组生成红细胞 发育假时，并确定异常沿着这一假时，包括未能上调 复制基因，可能反映发育延迟或停滞的异常细胞密度，以及细胞周期阶段 对于每个细胞。我们将在单细胞水平上联系任何异常。在AIM 3中，我们将确定S是否 CFU-e / ETD转换需要缩短时相，使用各种药物和遗传操作， 防止或加速S期缩短，并检查对CFU-e/ETD转换的后续影响。 此外，我们还将研究S期缩短在修饰CFU-100上染色质可及性中的潜在作用。 e/ETD开关。影响：该提案涉及创新的细胞周期修饰，可能直接调节 发展过程。具体地说，用细胞周期修饰药物延迟CFU-e/ETD转换导致 CFU-e扩增，这是治疗贫血的一个转化目标。

Project Summary In essentially all lineages, the cell cycle is quiescent in stem cells, and undergoes mitotic exit in terminally differentiated cells. In the intervening developmental period, however, cell cycles have been regarded as ‘generic’, regulated only with respect to their number, so as to maintain homeostasis or respond to stress. The generic view of the mammalian cell cycle contrasts with the specialized cell cycles of early embryonic development in model organisms such as Drosophila or Xenopus, where cell cycle control, including dramatic changes in cell cycle length, are intimately linked to developmental events. Our recently published work, including a single-cell transcriptomic analysis of the mouse erythroid trajectory and a study of replication fork dynamics in early erythropoiesis has uncovered the presence of specialized cell cycles throughout mammalian erythroid development. Our principal hypothesis is that developmental-stage-specific specializations of the cell cycle are integral to the process of differentiation, and regulate both incremental changes such as cell growth, as well as switch-like cell fate decisions. In this proposal, we investigate cell cycle specialization in early erythropoiesis, orchestrated around the time of a key cell fate switch, from self-renewal of CFU-e progenitors, to erythroid terminal differentiation (ETD). We found that, preceding this switch, there is progressive shortening of G1; and that, at the switch, there is an abrupt shortening of S phase. Further, S phase shortening is the result of a novel mechanism of regulating S phase length, through a global increase in replication fork speed. In this proposal, we will investigate both the mechanisms, as well as the functional outcomes, of these cell cycle specializations. In AIM 1, we will carry out functional analysis of four erythroid regulators: E2F4, KLF1, EpoR and Stat5. Using mice mutant for each of these regulators, we will determine their roles in erythroid cell cycle specializations and consequent developmental decisions. In AIM 2, we will carry out single-cell RNA-seq analysis of progenitors deleted for each of the four regulators. We will order cell transcriptomes to generate the erythroid developmental pesudotime, and determine abnormalities along this pseudotime, including failure to upregulate replication genes, abnormal cell densities that might reflect developmental delays or arrest, and cell cycle phase for each cell. We will correlate any abnormalities at the single cell level. In AIM 3, we will determine whether S phase shortening is required for the CFU-e / ETD switch, using a variety of drugs and genetic manipulation to prevent, or accelerate, S phase shortening, and examine the consequent effect on the CFU-e/ETD switch. Further, we will examine the potential role of S phase shortening in modifying chromatin accessibility at the CFU- e/ETD switch. IMPACT: this proposal deals with innovative cell cycle modifications that might directly regulate the developmental process. Specifically, delaying the CFU-e/ETD switch with cell cycle modifying drugs results in amplification of CFU-e, a translational goal in the treatment of anemia.