Control of the Erythroid Terminal Differentiation Decision

描述(申请人提供)：本项目的总体目标是阐明控制早期红系祖细胞进行终末分化的决定的因素和机制。许多证据表明，差异化决策的一个关键方面 是与细胞增殖计划相协调的一项要求。然而，我们目前对细胞增殖和分化程序之间的联系的了解非常有限。我们实验室和其他实验室的工作已经确定ETS家族转录因子PU.1是早期红系祖细胞末端分化决定的关键调节因子。然而，控制PU1在这些早期红系祖细胞中表达和活性的因素 而将分化决定与细胞周期联系起来的机制尚不清楚。最近，我们发现了两种类型的因子，它们有很强的潜力将PU1和末端分化决定与细胞周期联系起来。其中一个因素是CDK6，我们发现它像PU.1一样抑制红系分化。我们还发现：1)CDK6是红系祖细胞中活跃的G1期D-细胞周期蛋白激酶；2)PU1控制CDK6基因的转录；3)CDK6使PU1磷酸化。其他转录因子是E2F2和E2F4，它们在G1-S期细胞周期进程中起主要作用。我们发现E2F2和E2F4在红系祖细胞中占据了PU1基因的启动子和上游调控元件(URE)。它们还在这些细胞中的许多其他PU.1靶基因上与PU.1结合得非常近。在目标1和目标2中，我们提出了一系列实验，以确定CDK6和E2F2和E2F4如何影响PU.1的表达和活性，以及它们如何在红系终末分化决策中与PU.1合作。我们还建议研究CDK6和E2F2在应激性红细胞生成和红系前体细胞体外自我更新中的作用。我们和其他实验室的工作表明，PU.1用于调控红系分化决定的主要机制之一是抑制红系特异基因的表达。最近，我们发现PU.1与染色质重塑ATPase Snf2和维持DNA甲基转移酶DNMT1形成复合体，我们假设这两种酶帮助PU.1抑制红系基因的表达。在目标3中，我们提出了一些研究，以阐明Snf2h和DNMT1在PU1介导的红系特异性基因抑制和红系终末分化抑制中的作用。这些研究的成功完成将使人们对造血细胞的增殖和分化程序之间的联系机制有了新的认识，并对造血系统如何调节红细胞的产生，包括在个体发育和应激条件下的情况有了更深入的了解。拟议的工作还将提供重要的信息，使人们能够开发新的方法来管理在慢性和急性贫血中出现的红细胞产生缺陷。

DESCRIPTION (provided by applicant): The overall goal of this project is to elucidate the factors and mechanisms that control the decision of early erythroid progenitors to undertake terminal differentiation. Much evidence suggests that a crucial aspect of differentiation decisions is a requirement for coordination with the cell proliferation program. However, our current knowledge about the connections between the cell proliferation and differentiation programs is very limited. Work from our lab and others has identified the Ets - family transcription factor PU.1 as a key regulator of the terminal differentiation decision in early erythroid progenitors. And yet, the factors that control PU.1 expression and activity in these early erythroid progenitors and the mechanisms that link the differentiation decision to the cell cycle are not known. Recently, we identified two types of factors that have a very strong potential for connecting PU.1 and the terminal differentiation decision to the cell cycle. One factor is CDK6, which we discovered inhibits erythroid differentiation, like PU.1. We also showed that: 1) CDK6 is the active G1 phase D-cyclin kinase in erythroid progenitors; 2) PU.1 controls transcription of the CDK6 gene; 3) CDK6 phosphorylates PU.1. The other factors are E2F2 and E2F4, transcriptions factors that play major roles in G1 to S phase cell cycle progression. We found that E2F2 and E2F4 occupy the promoter and upstream regulatory element (URE) of the PU.1 gene in erythroid progenitors. They also bind very close to PU.1 at many other PU.1 target genes in these cells. In Aims 1 and 2, we propose a series of experiments to determine how CDK6 and E2F2 and E2F4 influence PU.1 expression and activity and how they collaborate with PU.1 in the erythroid terminal differentiation decision. We also propose to investigate roles for CDK6 and E2F2 in stress erythropoiesis and in ex vivo self-renewal of erythroid precursors. Work from our lab and others showed that one of the principal mechanisms used by PU.1 to regulate the erythroid differentiation decision is repression of erythroid-specific gene expression Recently, we discovered that PU.1 forms a complex with the chromatin remodeling ATPase SNF2H and the maintenance DNA methyltransferase DNMT1; two enzymes that we hypothesize help PU.1 to repress erythroid gene expression. In Aim 3, we propose studies to elucidate the role of SNF2H and DNMT1 in PU.1-mediated repression of erythroid-specific genes and inhibition of erythroid terminal differentiation. The successful completion of the proposed studies will lead to new insights into the mechanisms connecting the proliferation and differentiation programs in hematopoietic cells and a much deeper understanding of how the hematopoietic system regulates red blood cell production, including during ontogeny and in stress conditions. The proposed work will also provide important information enabling the development of new approaches to managing defects in red blood cell production that occur in chronic and acute anemia.