The Role of RB Family Proteins in an S Phase-Dependent Erythroid Commitment Step

RB 家族蛋白在 S 相依赖性红细胞承诺步骤中的作用

• 批准号: 8446029
• 负责人: Merav Socolovsky
• 金额: $ 24.94万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-01-22 至 2014-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8446029
• 关键词: Anemia; Biology; Blood Cells; CFU-E; Cell Cycle; Cell Cycle Regulation; Cell Differentiation process; Cell division; Cell surface; Cells; Chromatin; DNA; DNA Methylation; DNA Methyltransferase; DNA Modification Methylases; Dependence; Down-Regulation; Erythrocytes; Erythroid; Erythropoiesis; Event; Family member; Fetal Liver; Figs - dietary; Gatekeeping; Gene Expression; Generations; Genes; Knockout Mice; Laboratories; Link; Locus Control Region; Mediating; Mediator of activation protein; Molecular; Mus; Phenotype; Process; Protein Family; Regulation; Retinoblastoma; Role; S Phase; S Phase Arrest; Science; Signaling Protein; Somatic Cell; TFRC gene; Testing; Transcription Repressor/Corepressor; Tumor Suppressor Proteins; Undifferentiated; Up-Regulation; Work; beta Globin; carcinogenesis; cell type; demethylation; erythroid differentiation; gene induction; gene repression; genome-wide; human GATA1 protein; leukemia; novel; progenitor; programs; promoter; self-renewal; transcription factor

DESCRIPTION (provided by applicant): The erythroid phenotype is acquired through gene induction and morphological maturation in the space of 3 to 5 'differentiation divisions'. We recently uncovered a fundamental organizational feature of fetal liver erythropoiesis, whereby a novel S phase-dependent switch controls the transition from self-renewal to differentiation divisions (Pop et al., PLoS Biology 2010). Further, this switch also triggers an unusual process of genome-wide DNA demethylation, the first known example of such a process in somatic cells (Shearstone et al., Science 2011). The S phase-dependent switch takes place at the transition from flow-cytometric "subset 0" (S0, Lin-CD71med/low) to "subset 1" (S1, Lin-CD71high) in the murine fetal liver. It comprises several erythroid commitment events, including the onset of Epo dependence, activation of the erythroid master transcriptional regulator GATA-1, and an activating chromatin reconfiguration at the beta-globin locus-control region. These events take place synchronously, during early S phase of the last generation of colony-forming-unit erythroid (CFU-e) progenitors, and are dependent on S phase progression. The S phase-dependent switch at the S0/S1 transition represents a novel, pivotal interaction between the cell cycle and differentiation programs, distinct from the well-established interaction between terminal maturation and cell-cycle exit. The mechanisms underlying the switch and the process of global DNA demethylation that it triggers are largely unknown. We identified PU.1, a transcriptional repressor of erythropoiesis, as a central regulator of this switch. We propose that PU.1 coordinates the synchronous cell cycle and differentiation events at the S0/S1 transition, through novel, antagonistic interactions between PU.1 and S phase progression. Here we propose that the tumor suppressor protein pRb, and its family members, p107 and p130, mediate the antagonistic interactions between PU.1 and S phase, and hence act as gatekeepers of the S0/S1 erythroid commitment switch. We will investigate this hypothesis with the following two aims: 1) Determine whether the S phase-dependent switch at the S0/S1 transition is dysregulated in mice deleted for one, two or three Rb family proteins or in PU.1-null mice. We will determine whether PU.1 is able to exert its dual inhibitory functions on S phase and on erythroid differentiation in mice conditionally-deleted for one, two or three of the Rb family proteins pRb, p107 or p130. Further, we will determine whether the S0/S1 transition is accelerated or becomes S phase-independent in mice deleted for PU.1 or for Rb family proteins. 2) Determine whether PU.1 and/or Rb family members interact with DNMTs and regulate global DNA methylation at the S0/S1 transition. We will ask whether global or erythroid-specific DNA demethylation takes place prematurely in S0 cells of mice deleted for PU.1 or for Rb family proteins, and whether DNMTs are directly associated with PU.1 at gene promoters. This work has the potential to uncover fundamental mechanisms of Rb and PU.1 regulation of cell cycle and differentiation, relevant to leukemia and to anemia.

描述（由申请人提供）：红细胞表型是通过基因诱导和在3至5个“分化分裂”的空间内的形态成熟而获得的。我们最近发现了胎儿肝脏红细胞生成的一个基本组织特征，即一种新颖的 S 相依赖性开关控制从自我更新到分化分裂的转变（Pop 等人，PLoS Biology 2010）。此外，这种开关还触发了一种不寻常的全基因组 DNA 去甲基化过程，这是体细胞中此类过程的第一个已知例子（Shearstone 等人，Science 2011）。 S 相依赖性转换发生在鼠胎儿肝脏中从流式细胞术“子集 0”（S0，Lin-CD71med/low）到“子集 1”（S1，Lin-CD71high）的转变时。它包括几个红细胞定型事件，包括 Epo 依赖性的开始、红细胞主转录调节因子 GATA-1 的激活以及 β-珠蛋白位点控制区域的激活染色质重构。这些事件在最后一代集落形成单位红系 (CFU-e) 祖细胞的早期 S 期同步发生，并且依赖于 S 期进展。 S0/S1 转变处的 S 相依赖性开关代表了细胞周期和分化程序之间的一种新颖的、关键的相互作用，与成熟终末和细胞周期退出之间已确立的相互作用不同。这种转换背后的机制以及它引发的全球 DNA 去甲基化过程在很大程度上尚不清楚。我们确定了 PU.1（红细胞生成的转录抑制因子）作为该开关的中央调节因子。我们提出 PU.1 通过 PU.1 和 S 期进展之间新颖的拮抗相互作用来协调 S0/S1 转变时的同步细胞周期和分化事件。在这里，我们提出肿瘤抑制蛋白 pRb 及其家族成员 p107 和 p130 介导 PU.1 和 S 期之间的拮抗相互作用，因此充当 S0/S1 红细胞承诺开关的看门人。我们将通过以下两个目标来研究这一假设：1) 确定在删除一个、两个或三个 Rb 家族蛋白的小鼠或 PU.1 缺失的小鼠中，S0/S1 转换处的 S 相依赖性开关是否失调。 老鼠。我们将确定 PU.1 是否能够在条件性删除 Rb 家族蛋白 pRb、p107 或 p130 中的一种、两种或三种的小鼠中发挥其对 S 期和红细胞分化的双重抑制功能。此外，我们将确定在缺失 PU.1 或 Rb 家族蛋白的小鼠中，S0/S1 转变是否加速或变得与 S 相无关。 2) 确定 PU.1 和/或 Rb 家族成员是否与 DNMT 相互作用并在 S0/S1 转换时调节全局 DNA 甲基化。我们将询问缺失 PU.1 或 Rb 家族蛋白的小鼠 S0 细胞中是否会过早发生全局或红细胞特异性 DNA 去甲基化，以及 DNMT 是否在基因启动子处与 PU.1 直接相关。这项工作有可能揭示 Rb 和 PU.1 调节细胞周期和分化的基本机制，与白血病和贫血相关。