Red Cell Biology

红细胞生物学

• 批准号: 7594345
• 负责人: DAVID M. BODINE
• 金额: $ 138.54万
• 依托单位: NATIONAL HUMAN GENOME RESEARCH INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7594345
• 关键词: Affect; Affinity; Alleles; Amino Acids; Anemia; Animals; Anion Exchangers (Proteins); Anions; Ankyrins; Antibodies; Apoptosis; BFU-E; Binding; Binding Sites; Biological Assay; Cell Cycle; Cell Differentiation process; Cell Maturation; Cell membrane; Cell physiology; Cells; Cellular biology; Chromatin; Chromatin Structure; Cytidine Monophosphate; Cytoplasm; Defect; Deoxyribonuclease I; Diamond-Blackfan anemia; Disease; Dominant-Negative Mutation; Ectopic Expression; Elements; Endoribonucleases; Enhancers; Erythrocyte Anion Exchange Protein 1; Erythrocytes; Erythroid; Erythroid Cells; Erythropoiesis; Evaluation; Excision; Exons; Fetal Liver; Gene Expression; Gene Mutation; Gene Targeting; Genes; Glycine; Goals; Hemoglobin; Hepatocyte; Hydration status; Immune; Inherited; Integral Membrane Protein; Ion Channel; Knock-in Mouse; Knock-out; Localized; Mediating; Membrane Proteins; Methods; Microarray Analysis; Modeling; Molecular Conformation; Mus; Mutation; Nature; Nucleotides; Numbers; Osmotic Fragility test; Pancreatic ribonuclease; Patients; Peptide Fragments; Phenotype; Physiology; Play; Polymerase Chain Reaction; Pregnancy; Production; Promoter Regions; Proteins; RPS19 gene; Regulatory Element; Relative (related person); Reverse Transcriptase Polymerase Chain Reaction; Ribonucleases; Role; Severities; Sickle Cell Anemia; Site; Sorting - Cell Movement; Staging; Staining method; Stains; Structural Protein; Structure; Structure-Activity Relationship; TFRC gene; Technology; Testing; Thinking; Transgenic Mice; Validation; Work; animal breeding; annexin A5; catalyst; cell type; day; embryonic stem cell; erythroid Kruppel-like factor; gene replacement; genetic analysis; homologous recombination; human GATA1 protein; lomustine/methotrexate/procarbazine protocol; molecular modeling; mouse model; mutant; polymerization; prevent; primitive cell; progenitor; promoter; ribosomal protein S19; sickle deoxyhemoglobin; three dimensional structure

Our overall goal is to develope mouse models to determine sdpecific aspects of red blood cell production, terminal red cell differentiation, erythroid specific gene expression and the role of the band 3 trans membrane protein in red cell physiology. Our basic approiach is to use transgenic mouse technology to create null animals by gene replacement strategies (knock out) or to introduce specific mutations into red cell genes by knock in strategies. Our work is divided into 4 specific aims. Specific Aim 1: To create a mouse model of Diamond Blackfan Anemia. We have chosen to focus on Diamond Blackfan Anemia, which is thought to affect early erythroid progenitors. Over 25% of patients with DBA have mutations in the Ribosomal Protein S19 gene (RPS19). RPS19 is a component of the large ribosomal subunit, but how mutant RPS19 mutations affects differentiating erythroid cells is unknown. Because the DBA mutations are dominant, ectopic expression of mutant RPS19 in transgenic mice produced a lethal phenotype. We are using gene targeting to place the mutant RPS19 gene downstream of the normal allele. Upon Cre mediated excision of the wild type allele, we will induce mutant RPS19 expression . We will compare wild type and mutant transgenic mice for the relative number of HSC, CMP, BFU-E and CFU-E. The terminal stages of erythroid maturation cells at each stage of erythroid maturation in mutant and WT mice will be compared using the markers CD71 and TER11936. The cell cycle status at each stage will be analyzed by PI staining and apoptosis will be analyzed with Annexin V. We anticipate that these studies will identify the stage and the nature of the defect caused by mutant RPS19 expression. Specific Aim 2; Characterization of defective erythropoiesis in EKLF deficient mice. EKLF deficient mice die at day 14.5 of gestation of a severe anemia. FACS analysis of EKLF-- fetal liver cells stained with antibodies against CD71 and Ter119 demonstrated that the terminal stages of erythroid maturation (Ter119BRIGHT) were absent. To determine which genes are regulated by EKLF in red cells, we performed microarray analysis and RT-PCR and RNase protection validation to document more than 70 genes that are misregulated in EKLF-- fetal liver cells. Among the genes down regulated in EKLF deficient fetal liver cells are those involved in the cell cycle and apoptosis pathways as well as structural proteins of the red cell membrane, and channel proteins that regulate the hydration or red blood cells. We will use FACS to sort the primitive cells and compare the number of colony forming cells, their cell cycle status and the degree of apoptosis in mutant and wild type cells. We will perform osmotic fragility assays tp compare the stability of the red cell membrane and hydration of primitive and definitive cells, using hemoglobin markers for each stage. Specific Aim 3: To generate a comprehensive profile of chromatin changes associated with the activation of specific Ankyrin promoters. Identification and Characterization of Ank-1 DNaseI Hypersensitive Sites: Regulatory elements such as promoters, barriers and enhancers often co-localize with DNsae I hypersensitive sites (HSs). We have developed a high throughput quantitative PCR assay to detect DNase I HS across a 119 kb region of the Ank-1 promoter region, which includes an neuromuscular, an erythroid and a ubiquitous promoter. We have identified 6 discrete HSs within the region, which flank the three promoters. We have shown that those surrounding the erythroid promotr are barrier elements, but do not have either enhancer or enhancer blocking activity. We will use the Chip Chromatin Conformation Capture assay (4C) to determine how these HSs interact with each other. We have already shown that the sites surrounding the erythroid promoter are in contact in erythroid cells, but separated in non-erythroid cells. We will identify the proteins associated with the HSs by Chromatin Immune Preciptiation (ChIP). We hypothesize that the deletion of the erythroid promoter may allow one of the other promoters to become active in erythroid cells. To test this we have developed a targeted deletion of this region in ES cells for evaluation. Specific Aim 4: StructureFunction analysis of band 3 protein in red cells. Band 3 (B3) is an integral membrane protein that serves as the major anion channel for red blood cells and binds ankyrin, tethering the actinspectrin network to the red cell membrane. In addition to binding ankyrin, Band 3 is the major anion exchange protein of the red cell membrane and plays a critical role in maintaining red cell hydration, which is important to prevent the concentration dependent polymerization of deoxy HbS. We have shown that the N-terminus of band 3 lies in the cytoplasm and is a high affinity binding site for deoxy hemoglobin, and that this binding serves as a catalyst for HbS polymerization. Because of these varied functions, all of which may modify the severity of SCD, we have embarked on a genetic analysis of the structure and function of band 3. Molecular models using peptide fragments suggested that a b-hairpin loop in band 3 between amino acids 175 and 185 is responsible for ankyrin binding. We have used homologous recombination in ES cells to delete the nucleotides encoding the 11 amino acid hairpin loop in exon 7 of the mouse band 3 gene and replace them with a di-glycine bridge sequence. Analysis of red cell membranes will be performed to determine whether the mutant band 3 can bind ankyrin and the consequence of disrupting the band 3ankyrin bridge. We will use a similar gene targeting approach to generate a Band 3 N-terminus deleted mouse model to test the effects of mutations in the deoxy hemoglobin binding domain of band 3. In addition to analyzing the binding of Hb to band 3, we will breed these animals to mouse models of SCD to determine whether preventing deoxy Hb binding to band 3 can modulate the severity of SCD.

我们的总体目标是建立小鼠模型，以确定红细胞产生、终末红细胞分化、红细胞特异性基因表达和红细胞生理学中带3跨膜蛋白的作用的特异性方面。我们的基本方法是利用转基因小鼠技术，通过基因置换策略（敲除）或通过敲入策略将特定突变引入红细胞基因中来创建无效动物。我们的工作分为四个具体目标。 具体目标1：建立Diamond Blackfan贫血小鼠模型。我们选择专注于钻石黑扇贫血，这被认为是影响早期红系祖细胞。超过25%的DBA患者在核糖体蛋白S19基因（RPS 19）中存在突变。RPS 19是核糖体大亚基的一个组成部分，但RPS 19突变如何影响分化的红系细胞尚不清楚。由于DBA突变是显性的，转基因小鼠中突变型RPS 19的异位表达产生了致死表型。我们正在使用基因靶向将突变型RPS 19基因置于正常等位基因的下游。在Cre介导的野生型等位基因切除后，我们将诱导突变型RPS 19表达。我们将比较野生型和突变型转基因小鼠的HSC、CMP、BFU-E和CFU-E的相对数量。将使用标志物CD 71和TER 11936比较突变型和WT小鼠中红系成熟各阶段红系成熟细胞的终末阶段。在每个阶段的细胞周期状态将通过PI染色和细胞凋亡进行分析，将与膜联蛋白V。我们预计，这些研究将确定阶段和突变型RPS 19表达所造成的缺陷的性质。 具体目的2：EKLF缺陷小鼠中缺陷性红细胞生成的表征。EKLF缺陷小鼠在妊娠第14.5天死于严重贫血。用抗CD 71和Ter 119抗体染色的EKLF-胎肝细胞的FACS分析表明，红系成熟的终末阶段（Ter 119 BRIGHT）不存在。为了确定哪些基因在红细胞中受到EKLF的调控，我们进行了微阵列分析和RT-PCR和RNA酶保护验证，以记录70多个在EKLF-胎肝细胞中被错误调控的基因。在EKLF缺陷胎肝细胞中下调的基因中，有那些参与细胞周期和凋亡途径的基因，以及红细胞膜的结构蛋白和调节水化或红细胞的通道蛋白。我们将使用流式细胞仪对原始细胞进行分选，并比较突变型和野生型细胞中集落形成细胞的数量、细胞周期状态和凋亡程度。我们将进行渗透脆性试验，以比较红细胞膜的稳定性以及原始细胞和定形细胞的水合作用，使用每个阶段的血红蛋白标记物。 具体目标3：生成与特异性锚蛋白启动子激活相关的染色质变化的综合概况。 Ank-1 DNaseI超敏位点的鉴定和表征：调控元件如启动子、屏障和增强子通常与DNaseI超敏位点（HS）共定位。我们已经开发了一种高通量的定量PCR检测DNase I HS跨越119 kb区域的Ank-1启动子区，其中包括一个神经肌肉，红细胞和普遍存在的启动子。我们已经在该区域内鉴定了6个独立的HS，它们位于三个启动子的侧翼。我们已经表明，那些围绕红细胞启动子是屏障元件，但没有增强子或增强子阻断活性。我们将使用芯片染色质构象捕获测定（4C）来确定这些HS如何彼此相互作用。我们已经表明，红系细胞启动子周围的位点在红系细胞中是接触的，但在非红系细胞中是分离的。我们将通过染色质免疫沉淀（ChIP）鉴定与HS相关的蛋白质。我们假设红细胞启动子的缺失可能会使其他启动子之一在红细胞中变得活跃。为了测试这一点，我们开发了ES细胞中该区域的靶向缺失用于评估。 具体目的4：红细胞带3蛋白的结构与功能分析。带3（B3）是一种膜整合蛋白，其作为红细胞的主要阴离子通道并结合锚蛋白，将actinspectrin网络拴系到红细胞膜。除了结合锚蛋白之外，带3是红细胞膜的主要阴离子交换蛋白，并且在维持红细胞水合中起关键作用，这对于防止脱氧HbS的浓度依赖性聚合是重要的。我们已经表明，带3的N-末端位于细胞质中，是脱氧血红蛋白的高亲和力结合位点，并且这种结合作为HbS聚合的催化剂。由于这些不同的功能，所有这些都可能改变SCD的严重程度，我们已经开始对带3的结构和功能进行遗传分析。使用肽片段的分子模型表明，氨基酸175和185之间的带3中的b-发夹环负责锚蛋白结合。我们已经在ES细胞中使用同源重组来删除编码小鼠带3基因的外显子7中的11个氨基酸发夹环的核苷酸，并用双甘氨酸桥序列替换它们。将进行红细胞膜分析，以确定突变带3是否可以结合锚蛋白以及破坏带3锚蛋白桥的后果。我们将使用类似的基因靶向方法来产生带3 N-末端缺失的小鼠模型，以测试带3的脱氧血红蛋白结合结构域中的突变的影响。除了分析Hb与带3的结合之外，我们还将这些动物培育成SCD小鼠模型，以确定阻止脱氧Hb与带3结合是否可以调节SCD的严重程度。