New Gene Regulatory Proteins Regulating Erythroid Development

项目总结(见说明)： 利用第二代信使核糖核酸测序和生物信息学相结合的方法，我们已经获得了从CFU-E阶段到去核红细胞每个发育阶段表达的完整的mRNAs列表。我们鉴定了14个基因，它们编码转录因子、染色质修饰酶、RNA聚合酶II延伸因子或DNA 在其他发育过程中具有重要作用但其在红细胞发育中的功能从未被研究过的结合蛋白：Runx1t1、Sertad2、SertadS、Mxd1、Mxd3、Btg2、Med13l、Ncoa7、Ccocolo、ASF1b、Dedd2、Bag1、Hdac11、HEXIM1和EII2。在目标1中，我们将确定以下14项中的哪一项 蛋白质在从CFU-E阶段开始的红系发育中起着重要作用，通过系统地击倒纯化的CFU-E细胞中的每一种蛋白质，并在EPO的存在下培养它们。将通过测量增殖、CD-71和Ter-119的诱导、核浓缩、去核以及血红蛋白和其他标记红系重要基因的积累来分析广泛的影响。在与Zon实验室的合作中，我们将在斑马鱼胚胎中敲除这些基因，并评估对红细胞生成的影响。在目标2中，对于HDAC2、Hipk-1和-2，以及击倒时对红细胞生成影响最大的新因子，我们将通过对培养的敲除小鼠祖细胞进行第二代mRNA测序，确定其直接和间接调控表达的基因。最后，在目标#3中，我们将确定这些因素在Polll与启动子区域结合、Polll延长，以及在某些情况下表观遗传组蛋白修饰方面的全局作用，以这些因素已被击倒的祖细胞为例，我们将通过Chip-Seq测量Polll和两种转录延长特征的组蛋白修饰的全球分布。结合生物信息学分析，我们将确定在红细胞生成过程中，这些因子中的每一个对红系重要基因转录的控制是否在Polll结合或Polll延伸的水平上起作用。这些研究和其他研究将为理解活跃在终末红细胞生成中的表观遗传和转录调控网络创造一个广泛的框架。

PROJECT SUMMARY (See instructions): Using a combination of second generation mRNA sequencing and bioinformatic approaches we have obtained a complete list of mRNAs expressed at each stage of development from the CFU-E stage to the enucleating erythroblast. We identified 14 genes that are strongly upregulated during this period and that encode transcription factors, chromatin-modifying enzymes, RNA Polymerase II elongation factors, or DNA binding proteins that have important roles in other developmental processes but whose functions in red cell development have never been explored: Runx1t1, Sertad2, SertadS, Mxd1, Mxd3, Btg2, Med13l, Ncoa7, Calcocol, Asf1b, Dedd2, Bag1, Hdac11, HEXIM1, and EII2. In Aim 1 we will determine which of these 14 proteins plays an important role in erythroid development from the CFU-E stage by systematically knocking down each in purified CFU-E cells and culturing them in the presence of Epo. Broad effects will be assayed by measuring proliferation, induction of CD-71 and Ter-119, nuclear condensation, enucleation, and accumulation of hemoglobin and other marker erythroid- important genes. In collaboration with the Zon laboratory we will knockdown each of these in zebra fish embryos and assess effects on erythropoiesis. In Aim 2, for HDAC2, Hipk-1 and -2, and the new factors that have the most dramatic effects on erythropoiesis when knocked down, we will determine the genes whose expression is directly and indirectly regulated by them, using second generation mRNA sequencing on cultured knockdown mouse progenitors. Finally, in Aim #3 we will determine the global roles of these factors on Polll binding to promoter regions, Polll elongation, and in some cases epigenetic histone modifications As example, using progenitors in which the factors have been knocked down, we will measure by Chip-seq the global distributions of Polll and two histone modifications characteristic of transcriptional elongation. Coupled with bioinformatic analysis we will determine whether control of erythroid- important gene transcription during erythropoiesis by each of these factors works at the level of Polll binding or Polll elongation. These and other studies will create an extensive framework for understanding the epigenetic and transcriptional regulatory networks active in terminal erythropoiesis.