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

描述(申请人提供)：红系表型是通过基因诱导和形态成熟获得的，在3到5个‘分化分裂’的空间。我们最近发现了胎肝红细胞生成的一个基本组织特征，即一个新的S相依开关控制着从自我更新到分化分裂的转变(Pop等人，《公共科学图书馆·生物学》2010)。此外，这种转变还触发了一个不寻常的全基因组DNA去甲基化过程，这是体细胞中已知的第一个这种过程的例子(Searstone等人，科学，2011年)。在小鼠胎肝中，S的位相依赖转换发生在从流式细胞术的“亚组0”(S0，Lin-CD71med/low)到“亚组1”(s1，Lin-CD71高)的转变过程中。它包括几个红系承诺事件，包括EPO依赖的开始，红系主要转录调节因子GATA-1的激活，以及β-珠蛋白基因控制区的激活染色质重组。这些事件同步发生在最后一代集落形成单位红系祖细胞的早期S阶段，并依赖于S阶段的进展。S在S0/S1转换期的相位依赖开关代表了细胞周期和分化程序之间的一种新的、关键的相互作用，不同于已建立的末端成熟和细胞周期退出之间的相互作用。这种转换的潜在机制以及它引发的全球DNA去甲基化过程在很大程度上是未知的。我们发现PU.1，一种红血球生成的转录抑制因子，是这一开关的中心调节因子。我们认为PU1通过PU1和S期之间的新的、拮抗的相互作用来协调S0/S1转换期的同步细胞周期和分化事件。在本研究中，我们认为抑癌蛋白pRb及其家族成员p107和p130介导了PU1和S期之间的拮抗相互作用，从而在S0/S1红系承诺开关中起到了守门人的作用。我们将出于以下两个目的来研究这一假说：1)确定在缺失一个、两个或三个Rb家族蛋白的小鼠或在PU.1缺失的小鼠中，S0/S1转变中的S相依赖开关是否失调 老鼠。我们将确定PU.1是否能够对Rb家族蛋白pRb、p107或p130中的一个、两个或三个条件缺失的小鼠的S期和红系分化发挥双重抑制作用。此外，我们将确定在PU1或Rb家族蛋白缺失的小鼠中，S0/S1转变是加速的还是变得不依赖S的。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。我们将询问在缺失PU.1或Rb家族蛋白的小鼠的S0细胞中，是否发生全局或红系特异性DNA去甲基化，以及DNMT是否在基因启动子上与PU.1直接相关。这项工作有可能揭示Rb和PU1调节细胞周期和分化的基本机制，与白血病和贫血有关。

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.