Specialized cell cycles in early erythropoiesis

项目摘要 在基本上所有的谱系中，干细胞的细胞周期都是静止的，并在末期经历有丝分裂退出。 分化的细胞。然而，在中间的发育期，细胞周期被认为是 “一般的”，仅根据它们的数量进行调节，以维持内稳态或对压力做出反应。这个 哺乳动物细胞周期的一般观点与早期胚胎的特殊细胞周期形成对比 在模式生物中的发展，如果蝇或非洲爪哇，其中细胞周期控制，包括戏剧性的 细胞周期长度的变化与发育事件密切相关。我们最近出版的作品， 包括对小鼠红系轨迹的单细胞转录分析和复制分叉的研究 早期红细胞生成的动力学揭示了哺乳动物体内存在特殊的细胞周期 红系发育。我们的主要假设是，细胞的发育阶段特异性特化 周期是分化过程中不可或缺的一部分，并调节细胞生长等增量变化， 以及转换式的细胞命运决定。在这个提案中，我们研究了早期的细胞周期特化。 红细胞生成，大约在关键细胞命运转换的时间，从CFU-e祖细胞的自我更新，到 红系终末分化(ETD)。我们发现，在这种转换之前，有一个逐渐缩短的 G1；在转换处，S相突然缩短。更进一步，S的相位缩短是 一种通过全局提高复制分叉速度来调节S相长的新机制。在这 建议，我们将研究这些细胞周期的机制和功能结果 专业化认证。在AIM 1中，我们将对E2F4、KLF1、EPOR这四种红系调节因子进行功能分析 和STAT5。利用这些调节器的突变小鼠，我们将确定它们在红系细胞周期中的作用 专业化和相应的发展决策。在AIM 2中，我们将进行单细胞RNA-seq分析 四个监管机构中的每一个都删除了祖先的数量。我们将订购细胞转录本来生成红系 发育假性时间，并确定沿该假性时间的异常，包括未能上调 复制基因，可能反映发育迟缓或停滞的异常细胞密度，以及细胞周期阶段 对于每一个细胞。我们将在单个细胞水平上关联任何异常。在AIM 3中，我们将确定S是否 CFU-e/ETD转换需要缩短时相，使用各种药物和基因操作来 防止或加速S相位缩短，并检查由此对cfu-e/etd开关的影响。 此外，我们将研究S相位缩短在改变CFU染色质可及性方面的潜在作用。 E/ETD开关。影响：这项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推迟CFU-e/ETD转换的结果 在cfu-e的扩增中，这是治疗贫血的翻译目标。

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.