New Gene Regulatory Proteins Regulating Erythroid Development

项目总结（见说明）： 使用第二代mRNA测序和生物信息学方法的组合，我们已经获得了从CFU-E阶段到去核成红细胞的每个发育阶段表达的mRNA的完整列表。我们鉴定了14个在此期间强烈上调的基因，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探索的结合蛋白：Runx 1 t1、Sertad 2、SertadS、Mxd 1、Mxd 3、Btg 2、Med 13 l、Ncoa 7、Calcocol、Asf 1b、Dedd 2、Bag 1、Hdac 11、HEXIM 1和EII 2。在目标1中，我们将确定这14个目标中的哪一个 这些蛋白质在CFU-E阶段的红系发育中起重要作用，通过系统地敲低纯化的CFU-E细胞中的每一种蛋白质并在Epo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、去核和血红蛋白和其他红细胞系重要标志物基因的蓄积来分析广泛效应。在Zon实验室的合作下，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HDAC 2、Hipk-1和-2以及在敲低时对红细胞生成具有最显著影响的新因子，我们将使用培养的敲低小鼠祖细胞的第二代mRNA测序来确定其表达受其直接和间接调节的基因。最后，在目标#3中，我们将确定这些因子对Polll与启动子区域结合、Polll延伸以及在某些情况下的表观遗传组蛋白修饰的全局作用。例如，使用其中因子已被敲低的祖细胞，我们将通过Chip-seq测量Polll和转录延伸特征的两种组蛋白修饰的全局分布。结合生物信息学分析，我们将确定这些因子中的每一个在红细胞生成期间对红细胞系重要基因转录的控制是否在Polll结合或Polll延伸的水平起作用。这些和其他研究将创建一个广泛的框架，了解表观遗传和转录调控网络活跃在终端红细胞生成。

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.