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（请参阅说明）：使用第二代mRNA测序和生物信息学方法的组合，我们获得了从CFU-E阶段到浓缩的红细胞的每个发育阶段表达的mRNA的完整列表。我们确定了在此期间强烈上调的14个基因，并编码转录因子，染色质修饰酶，RNA聚合酶II伸长因子或DNA 在其他发育过程中具有重要作用但在红细胞发育中起作用的结合蛋白质从未探索过：runx1t1，sertad2，sertads，sertads，mxd1，mxd3，mxd3，btg2，med13l，ncoa7，ncoa7，carcocol，carcocol，carcocol，asf1b，asf1b，deDd2，dedd2，dewd2，hdaC11，hdac11，hexim1，hexim1和eiii2。在AIM 1中，我们将确定这14个蛋白质在纯化的CFU-E细胞中系统地击倒每种细胞并在EPO存在下培养它们，从而在CFU-E阶段从CFU-E阶段发育起着重要作用。通过测量CD-71和TER-119的增殖，核凝结，摘除以及血红蛋白和其他标志物红细胞促红细胞的积累，可以测定广泛的影响。在与ZON实验室合作的情况下，我们将在斑马鱼胚胎中淘汰每一个，并评估对红细胞生成的影响。在AIM 2中，对于HDAC2，HIPK -1和-2，以及对撞倒红细胞生成的最大作用的新因素，我们将使用第二代MRNA测序对培养的MRNA测序对培养的敲低小鼠祖细胞的直接和间接调节的基因进行确定。最后，在AIM＃3中，我们将确定这些因素在对启动子区域，轮询伸长延伸的频率结合中的全球作用，在某些情况下，表观遗传组蛋白修饰为例如，使用偏移量敲低因素的祖细胞，我们将通过Chip-seq进行测量，Chip-Seq的全球polll和两种组蛋白修饰的经转录率的典型均具有转录的特征。再加上生物信息学分析，我们将确定每个因素在红细胞生成过程中对红斑中重要基因转录的控制是否在polll结合或频率伸长的水平上起作用。这些研究和其他研究将创建一个广泛的框架，以理解活跃于终末红霉病的表观遗传和转录调节网络。