Specialized cell cycles in early erythropoiesis

早期红细胞生成的特殊细胞周期

基本信息

批准号：
10214602
负责人：
Merav Socolovsky
金额：
$ 43.6万
依托单位：
UNIV OF MASSACHUSETTS MED SCH WORCESTER
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-09-15 至 2024-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10214602
关键词：
ATAC-seq Accelerated Phase Anemia Animal Model Bone Marrow Bromodeoxyuridine CFU-E Cell Cycle Cell Cycle Regulation Cell Density Cell Differentiation process Cell Proliferation Cell division Cells Chromatin Comb animal structure Coupled DNA DNA replication fork Data Development Developmental Delay Disorders Developmental Process Drosophila genus Embryonic Development Erythrocytes Erythroid Erythroid Cells Erythropoiesis Erythropoietin Receptor Event Failure Fetal Liver G1 Phase Gene Abnormality Gene Expression Genes Genetic Transcription Goals Hemoglobin Homeostasis Individual Length Life Link Mammalian Cell Measurement Mediator of activation protein Mitotic Modification Mus Mutant Strains Mice Nature Pharmaceutical Preparations Phase Process Publishing Regulation Reporter Role S Phase Science Signal Transduction Speed Stress Testing Time Validation Work Xenopus base cell growth drug testing functional outcomes genetic manipulation genome-wide in vivo inhibitor/antagonist innovation insight novel nucleotide analog prevent progenitor self-renewal single-cell RNA sequencing stem cells transcription factor transcriptome transcriptomics

项目摘要

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.

项目概要基本上在所有谱系中，干细胞的细胞周期都是静止的，并在终末期经历有丝分裂。然而，在发育期间，细胞周期被认为是分化的细胞。 “通用”，仅根据其数量进行调节，以维持体内平衡或应对压力。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果蝇或爪蟾等模型生物体的发育，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。包括小鼠红细胞轨迹的单细胞转录组分析和复制叉的研究早期红细胞生成的动力学揭示了整个哺乳动物中特化细胞周期的存在我们的主要假设是细胞的发育阶段特异性。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，以及类似开关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，在关键细胞命运转换期间精心策划，从 CFU-e 祖细胞的自我更新，到我们发现，在此转换之前，红细胞终末分化（ETD）逐渐缩短。 G1；并且，在开关处，S相突然缩短，而且，S相缩短是以下结果。一种通过整体提高复制叉速度来调节 S 期长度的新机制。建议，我们将研究这些细胞周期的机制以及功能结果在 AIM 1 中，我们将对四种红细胞调节因子进行功能分析：E2F4、KLF1、EpoR。和 Stat5。使用这些调节因子的小鼠突变体，我们将确定它们在红细胞周期中的作用。在 AIM 2 中，我们将进行单细胞 RNA-seq 分析。我们将订购细胞转录组来生成红系细胞。发育伪时间，并确定该伪时间的异常情况，包括上调失败复制基因、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关联任何异常，在 AIM 3 中，我们将确定 S 是否存在。 CFU-e/ETD切换需要缩短相位，使用多种药物和基因操作来防止或加速 S 期缩短，并检查对 CFU-e/ETD 开关的后续影响。此外，我们将研究 S 期缩短在改变 CFU-染色质可及性方面的潜在作用。 e/ETD 开关。该提案涉及可能直接调节的创新细胞周期修饰。具体来说，用细胞周期修饰药物延迟 CFU-e/ETD 转换。 CFU-e 的扩增，这是贫血治疗的转化目标。