Red Cell Biology

红细胞生物学

• 批准号: 10267104
• 负责人: DAVID M. BODINE
• 金额: $ 101.32万
• 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/10267104
• 关键词: ATAC-seq; Adrenal Cortex Hormones; Adult; Age; Anemia; Animal Model; Animals; Anions; Basophilic Erythroblast; Binding; Biogenesis; Bone Marrow; CD34 gene; CFU-E; Cell Line; Cell Nucleus; Cell physiology; Cells; Cellular Metabolic Process; Cellular biology; Characteristics; Chromatin; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Collaborations; Complex; Congenital Abnormality; Coupled; Cytoplasmic Tail; DNA Methylation; Data; Defect; Deoxygenated Sickle Hemoglobin; Dependence; Diamond; Diamond-Blackfan anemia; Diet; Disease; Disease remission; Environment; Enzymes; Epigenetic Process; Erythroblasts; Erythrocyte Anion Exchange Protein 1; Erythrocyte Membrane; Erythrocytes; Erythroid; Erythroid Cells; Erythroid Progenitor Cells; Erythropoiesis; Failure; Family; Fanconi' s Anemia; Frequencies; Functional disorder; GATA1 gene; GYPA gene; Gene Mutation; Gene Proteins; Genes; Genetic; Genotype; Globin; Glycolysis; Glycolysis Pathway; Goals; Hematopoietic; Heme; Hemoglobin; Hypoxia; Impairment; In Vitro; Inherited; Introns; Iron; Knock-out; Knockout Mice; Lead; Lesion; Live Birth; Lung; Lysosomes; Malaria; Malignant Neoplasms; Membrane Transport Proteins; Messenger RNA; Metabolism; Methods; Mitochondria; Molecular Diagnosis; Mus; Mutation; North America; Oxygen; Paper; Parasites; Pathogenesis; Pathway interactions; Patients; Penetrance; Pentoses; Phenotype; Physiology; Polymers; Population; Predisposition; Preparation; Production; Proteins; RNA Splicing; Recycling; Registries; Reporter; Reproducibility; Reticulocytes; Ribosomal Proteins; Ribosomal RNA; Ribosomes; Role; Series; Shapes; Sickle Cell Anemia; Solid Neoplasm; Spontaneous Remission; Steroids; Syndrome; System; Tissues; Toxic effect; Transferrin; Transfusion; Transgenic Mice; Translations; Variant; bone marrow failure syndrome; causal variant; cohort; crystallinity; deoxyhemoglobin; epigenetic profiling; erythroid differentiation; exome; exome sequencing; gene discovery; genome sequencing; heme a; hemozoin; human male; improved; iron deficiency; iron metabolism; macrophage; mitochondrial membrane; mouse model; multiorgan damage; novel; polymerization; predictive modeling; programs; protein expression; recruit; response; restoration; senescence; sensor; sickling; small molecule; stem cells; targeted sequencing; whole genome

There are four related projects in this program: Project 1: Genome sequencing to identify DBA mutations. Project 2: In vitro culture and epigenetic profiling of DBA patient cells. Project 3: Novel reporter cells for screen on compounds that increase ribosomal protein expression Project 4. Mouse models of red blood cell functions. DBA is an inherited bone marrow failure syndrome in which ribosome defects specifically block erythropoiesis while cells of the other hematopoietic lineages are normal in both number and function. In addition to anemia, DBA shares several clinical features with other bone marrow failure syndromes, e.g. Fanconi anemia and Schwachman Diamond syndrome, including: 1) a high frequency of birth defects, 2) a predisposition to cancer (both solid tumors and AML) and 3) variable penetrance and clinical course, ranging from asymptomatic to severe. DBA is inherited as an autosomal dominant with incomplete penetrance and 60% of DBA mutations arise de novo. Despite the phenotypic variation observed among patients with DBA, only a few confirmed genotype/phenotype correlations have been established. Project 1. Mutations in at least 24 different ribosomal protein (RP) genes have been identified in 85% of DBA patients. These mutations lead to aberrant rRNA processing, impaired production of either the small or large ribosomal subunit and reduced numbers of mature 80S ribosomes. Targeted sequencing of the 80 ribosomal protein genes in >75 DBA patients without a molecular diagnosis failed to identify causative mutations in 35% of DBA patients. Exome sequencing of DBA patients showed that 80-85% have mutations in one of the 80 RP genes. Identifying and characterizing the remaining 15-20% of DBA mutations is the goal of Project 1. We are using whole genome sequencing to screen DBA patients for small deletions in and around ribosomal protein genes. In our first 6 patients we have identified 4 deletions which are too small to be detected by other methods, which we believe cause DBA in these patients. These mutations, which are in introns, interfere with the normal splicing of the RP mRNA, validating them as causative mutations We will be submitting a paper describing these results shortly. (Vlachos et al, in preparation). Project 2. An unresolved question regarding the pathogenesis of DBA is the precise stage at which erythroid differentiation is blocked. Our single cell studies show that the failure of erythropoiesis in DBA may involve a more primitive Pre-MEP or HSC population. We are currently using our in vitro differentiation system to characterize the epigenetic profiles of control and BA patients to determine the stage at which the first signs of RP haploinsufficiency are detected. DBA patients are treated with red cell transfusions, usually followed by a trial of corticosteroid therapy around the age of three years. Durable steroid-induced erythropoiesis is seen in approximately 30-40% of DBA patients. In addition, 15% of DBA patients undergo spontaneous remission and begin relatively normal erythropoiesis for periods of several months to many years. We hypothesize that the variable response to steroid treatment and spontaneous remission is due to changes in the epigenetic profile in DBA progenitor cells. We have preliminary data from over 35 patients demonstrating specific and reproducible differences in DNA methylation and chromatin accessibility (ATAC-Seq) that correlate with transfusion dependence, steroid responsiveness and remission. We are recruiting more steroid-responsive DBA patients and DBA patients in remission to identify specific epigenetic changes associated with the restoration of erythropoiesis. Project 3. We are using CRISPR to introduce RP mutations into the erythroid cell line Bel-A. We will use CRISPR to introduce a marker into the glycophorin A locus, a key marker of erythroid differentiation. The marker will fluoresce when the gene is activated, allowing small molecule screens to be performed in collaboration with NCATS. Project 4. We are investigating two novel hypotheses about erythrocyte production and function and red cell metabolism. Mammalian red cell deformability and metabolism changes between low oxygen (tissues) and high oxygen (lung) environments. In hypoxic conditions red cells show increased deformability, increased ATP release and pentose pathway activity, while in normoxic conditions red cells revert to normal deformability and activate the glycolysis pathway to produce ATP. A candidate for the oxygen sensor is band 3, the protein encoded by the Slc4a1 gene. Band 3 is the most abundant protein in the mammalian erythrocyte membrane and functions as an anion exchange channel. In vitro, the N-terminus of the cytoplasmic domain of band 3 reversibly binds glycolytic enzymes in normoxic conditions and deoxyhemoglobin in hypoxic conditions and we have shown that this leads to specific changes in ATP levels, and either glycolytic or pentose pathway intermediates depending on whether the mutation is a gain or loss of Hb binding. In sickle cell disease (SCD), polymerization of deoxyhemoglobin S is the fundamental lesion underlying the multi-organ damage that characterizes the disease. DeoxyHbS forms long insoluble polymers that distort the red cell into the characteristic sickle shape. A paradox in the study of SCD is that the polymerization of HbS within red blood cells occurs at 10-fold higher concentrations of oxygen than the polymerization of HbS in vitro. We have observed that our Band3 mutations alter the rate of HbS polymerization in the Townes mouse model. We are evaluating whether the altered physiology of these cells is a modifier of sickling. The red blood cells of an average adult human male store collectively about 2.5 grams of iron, representing about 75 percent of the total body iron. The continuous production of new red blood cells requires iron, some supplied by the diet and the remainder by macrophages which engulf senescent red blood cells and recycle iron to maturing red cells. Iron metabolism models predict that the iron required for heme synthesis (and therefore hemoglobin assembly) is imported into erythroid cells as transferrin and subsequently incorporated into heme by a series of enzymes associated with the mitochondrial membrane. To keep the levels of heme and globin chains balanced (excess heme is toxic), immature red cells express a heme exporter, FlvcR, which is expressed at its highest levels at the CFU-E stage before declining during terminal erythroid maturation (basophilic erythroblast to reticulocyte). However, the translation of globin mRNA peaks during the most terminal stages of erythroid maturation (orthochromatic erythroblast and reticulocyte) between which the nucleus is extruded and most of the heme producing mitochondria degrade. How then do reticulocytes generate enough heme to complete hemoglobin assembly? We have knocked out a novel heme transporter, HRG1 to answer this question and found that HRG1 deficient mice sequester heme in the lysosomes of splenic and bone marrow macrophages. The sequestered heme is in the form of hemozoin a crystalline form of heme that is non-toxic and heretofore only observed in malaria parasites. We are currently examining the role of HRG1 in terminally differentiating erythroid cells in conditional HRG1 knockout mice we have generated. We hypothesize that the mild anemia in HRG1 deficient mice is due to heme toxicity in the maturing erythroblast.

该计划中有四个相关项目： 项目1：鉴定DBA突变的基因组测序。 项目2：DBA患者细胞的体外培养和表观遗传分析。 项目3：在增加核糖体蛋白表达的化合物上筛选的新闻报道细胞 项目4。红细胞功能的小鼠模型。 DBA是一种遗传性的骨髓衰竭综合征，其中核糖体缺陷特异性地阻断了红细胞生成，而其他造血谱系的细胞在数量和功能方面都是正常的。除贫血外，DBA还与其他骨髓衰竭综合症具有多种临床特征，例如Fanconi贫血和Schwachman Diamond综合征，包括：1）高频率出生缺陷，2）癌症的易感性（实体瘤和AML）和3）可变的渗透和临床过程，从无症状到严重。 DBA被遗传为常染色体显性剂，并具有不完全的外渗性，而DBA突变的60％是从头出现的。尽管在DBA患者中观察到表型差异，但仅建立了少数确认的基因型/表型相关性。 项目1。在85％的DBA患者中已经鉴定出至少24种不同的核糖体蛋白（RP）基因的突变。这些突变导致异常的rRNA加工，小或大的核糖体亚基的产生受损以及成熟的80S核糖体数量减少。在没有分子诊断的> 75例DBA患者中，对80个核糖体蛋白基因的靶向测序未能鉴定35％的DBA患者的病因突变。 DBA患者的外显子组测序表明80-85％的80 RP基因中有突变。项目1的目标是确定和表征其余15-20％的DBA突变。 我们正在使用整个基因组测序来筛选DBA患者，以在核糖体蛋白基因和周围进行小缺失。在我们的前6名患者中，我们发现了4个缺失，这些缺失太小而无法通过其他方法检测到，我们认为这些方法会导致这些患者的DBA。这些突变是内含子中的，干扰了RP mRNA的正常剪接，将它们验证为病因突变，我们将在不久的将来提交一篇论文。 （Vlachos等人，在准备中）。 项目2。关于DBA发病机理的一个尚未解决的问题是将红斑分化阻塞的确切阶段。我们的单细胞研究表明，DBA中红细胞生成的失败可能涉及更原始的MEP或HSC种群。我们目前正在使用我们的体外分化系统来表征对照组和BA患者的表观遗传谱，以确定检测到RP单倍不足迹象的阶段。 DBA患者接受红细胞输血治疗，通常在三岁左右进行皮质类固醇治疗的试验。大约30-40％的DBA患者可以看到耐用类固醇引起的促红细胞生成。此外，有15％的DBA患者经历了自发缓解，并开始相对正常的红细胞生成几个月至多年。我们假设对类固醇治疗和自发缓解的可变反应是由于DBA祖细胞中表观遗传谱的变化所致。我们有来自35位患者的初步数据，这些数据表明DNA甲基化和染色质可及性（ATAC-SEQ）的特定和可重复的差异与输血依赖，类固醇反应性和缓解相关。 我们正在招募更多类固醇反应性的DBA患者和DBA患者，以识别与恢复红细胞生成的特定表观遗传变化。 项目3。我们使用CRISPR将RP突变引入红细胞系BEL-A。我们将使用CRISPR将标记物引入糖蛋白A座基因座，这是红斑分化的关键标记。当激活基因时，标记将荧光，从而可以与NCAT合作进行小分子筛选。 项目4。我们正在研究两个有关红细胞生产和功能以及红细胞代谢的新型假设。低氧（组织）和高氧（肺）环境之间的哺乳动物红细胞的可变形性和代谢变化。在低氧条件下，红细胞显示出增加的变形性，ATP释放增加和五氧化杆菌途径活性，而在常态的条件下，红细胞恢复为正常的变形性，并激活产生ATP的糖酵解途径。氧气传感器的候选者是带3，该蛋白质由SLC4A1基因编码。条带3是哺乳动物红细胞膜中最丰富的蛋白质，作为阴离子交换通道。在体外，条带3的细胞质结构域的N末端可逆地结合低氧条件下的差异条件下的糖酵解酶和脱氧血红蛋白，并且我们已经表明，这会导致ATP水平的特定变化，以及糖酵解途径或垂体途径途径依赖于METIDE的损失是造成的。在镰状细胞疾病（SCD）中，脱氧血红蛋白S的聚合是表征该疾病的多器官损害的基本病变。 Deoxyhbs形成长长的不溶性聚合物，将红细胞扭曲成特征性的镰刀形状。 SCD研究中的悖论是，红细胞内HBS的聚合的发生在氧气浓度高10倍以上，而不是体外HBS的聚合。我们已经观察到我们的频带3突变改变了小鼠小鼠模型中HBS聚合的速率。我们正在评估这些细胞的生理改变是否是可恶的修饰。 普通成年男性商店的红细胞统称约2.5克铁，约占人体总铁的75％。连续产生新的红细胞需要铁，其中一些由饮食提供，其余由巨噬细胞吞噬衰老的红细胞并将铁回收到成熟的红细胞。铁代谢模型预测，将血红素合成所需的铁（以及因此血红蛋白组装）导入到红细胞细胞中，作为转铁蛋白，随后通过与线粒体膜相关的一系列酶掺入血红素。为了保持血红素和球蛋白链的水平平衡（过量血红素是有毒的），未成熟的红细胞表达血红素出口剂FLVCR，在CFU-E阶段以其最高水平表示，然后在终末红细胞化成熟期间下降（亚嗜碱性粒细胞嗜（嗜碱性粒细胞至网状细胞））。然而，在核中最终端阶段（正色的红细胞和网状细胞）中，球蛋白mRNA峰的翻译在其之间被挤出，大多数血红素产生线粒体降级。那么网状细胞如何产生足够的血红素以完成血红蛋白组件？我们已经淘汰了一种新型的血红素转运蛋白HRG1来回答这个问题，并发现HRG1缺乏小鼠在脾和骨髓巨噬细胞的溶酶体中隔离血红素。隔离的血红素是血红蛋白的形式，一种晶体形式的血红素形式，无毒，迄今仅在疟疾寄生虫中观察到。 我们目前正在研究我们已经生成的条件HRG1基因敲除小鼠中HRG1在终末分化的红细胞细胞中的作用。我们假设HRG1缺乏小鼠中的轻度贫血是由于成熟的红细胞毒性引起的。