New Gene Regulatory Proteins Regulating Erythroid Development

调节红细胞发育的新基因调节蛋白

基本信息

批准号：
8205182
负责人：
Harvey F Lodish
金额：
$ 50.03万
依托单位：
BOSTON CHILDREN'S HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-08-15 至 2016-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8205182
关键词：
Affect Anemia Aplastic Anemia Binding Bioinformatics Biological Assay CFU-E Cell Culture Techniques Cell Cycle Cell Proliferation Cell Size Cell division Cells Characteristics Chromatin Collaborations Coupled DNA-Binding Proteins Databases Development Developmental Biology Developmental Process Diamond-Blackfan anemia Disease Down-Regulation Elongation Factor Embryo Enzymes Epigenetic Process Erythroblasts Erythrocytes Erythroid Erythroid Cells Erythropoiesis Erythropoietin Exportins Fetal Liver Fibronectins Gene Expression Generations Genes Genetic Transcription Glycophorin Goals HDAC2 gene Hemoglobin Histones Human Instruction Knockout Mice Knowledge Laboratories Measures Messenger RNA Molecular Profiling Mus Nuclear Nuclear Export Phosphotransferases Physical condensation Play Promoter Regions Proteins RNA Polymerase II Role Sideroblastic Anemia Site Staging Surface TFRC gene Thalassemia Transcriptional Regulation Transferrin Receptor Withdrawal Work Zebrafish base erythroid differentiation genetic regulatory protein histone modification in vivo knock-down leukemia mRNA Expression progenitor promoter research study transcription factor

项目摘要

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.

项目摘要（参见说明）：通过结合第二代 mRNA 测序和生物信息学方法，我们获得了从 CFU-E 阶段到去核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过的结合蛋白：Runx1t1、Sertad2、SertadS、Mxd1、Mxd3、Btg2、Med13l、Ncoa7、Calcocol、Asf1b、Dedd2、Bag1、Hdac11、HEXIM1、和EII2。在目标 1 中，我们将确定这 14 个中的哪一个通过系统地敲除纯化的 CFU-E 细胞中的每种蛋白并在 Epo 存在的情况下培养它们，Epo 蛋白在 CFU-E 阶段的红细胞发育中发挥着重要作用。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细胞重要基因的积累来分析广泛的影响。我们将与 Zon 实验室合作，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，以及当被敲低时对红细胞生成影响最显着的新因子，我们将使用第二代mRNA测序确定其表达直接和间接受它们调节的基因对培养的敲低小鼠祖细胞的影响。最后，在目标 3 中，我们将确定这些因子对 Poll 与启动子区域结合、Poll 延伸以及在某些情况下表观遗传组蛋白修饰的全局作用。例如，使用因子已被击倒的祖细胞，我们将通过以下方法进行测量Chip-seq 检测 Poll 的全局分布和转录延伸特征的两个组蛋白修饰。结合生物信息学分析，我们将确定这些因素在红细胞生成过程中对红细胞重要基因转录的控制是否在 Poll 结合或 Poll 延伸水平上起作用。这些和其他研究将为理解终末红细胞生成中活跃的表观遗传和转录调控网络创建一个广泛的框架。