Hematopoietic Stem Cell Biology

造血干细胞生物学

• 批准号: 8565520
• 负责人: DAVID M. BODINE
• 金额: $ 75.5万
• 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/8565520
• 关键词: Accounting; Affect; Aftercare; Alleles; Amino Acid Substitution; Amino Acids; Anemia; Animal Model; Animals; Azacitidine; Binding Sites; Bioinformatics; Blood; Blood Cells; Blood Platelets; Cells; ChIP-seq; Codon Nucleotides; Computer software; Copy Number Polymorphism; Cytoplasm; DNA; DNA Methylation; Data; Development; Diamond-Blackfan anemia; Dominant-Negative Mutation; Enrollment; Epigenetic Process; Erythroblasts; Erythrocytes; Erythroid; Erythropoiesis; Failure; Family; Frequencies; Gene Deletion; Genes; Genetic; Genets; Genome; Goals; Hematological Disease; Hematopoiesis; Hematopoietic; Hematopoietic stem cells; Inherited; Knockout Mice; Lesion; MDM2 gene; Malignant - descriptor; Megakaryocytes; Methods; Methylation; Missense Mutation; Modeling; Molecular Diagnosis; Molecular Profiling; Mosaicism; Mus; Mutation; Myelogenous; Neoplasm Metastasis; Output; Pancytopenia; Parents; Pathway interactions; Patients; Pharmaceutical Preparations; Pharmacotherapy; Point Mutation; Population; Process; Proteins; RPS19 gene; Regulatory Element; Ribosomal Proteins; Ribosomal RNA; Role; Sampling; Sequence Analysis; Siblings; Staging; Stem cells; Syndrome; System; Systems Biology; Testing; Transgenic Mice; United States National Institutes of Health; Variant; base; cell type; comparative; craniofacial; density; exome; genome-wide; in vivo; inhibitor/antagonist; insight; interest; kindred; mouse model; novel; open source; peripheral blood; proband; progenitor; programs; research study; self-renewal; stem; stem cell biology; symposium; tool; transcription factor

All of the cells in the peripheral blood are generated from a small population of hematopoietic stem cells (HSC) through a process of proliferation and differentiation known as hematopoiesis. The hematopoietic differentiation program includes well-defined stages at which the progeny of HSC become restricted to specific fates. The goal of this project is to define specific molecular signatures associated with specific stages of hematopoiesis. Project 1. DNA methylation is an essential epigenetic mark that is required for normal development and hematopoietic differentiation. We characterized genome-wide DNA methylation in primary mouse HSC, Common Myeloid progenitors (CMP), and erythroblasts (ERY). We found that DNA methylation was most abundant in HSC, with a 40% reduction in the number of peaks present in CMP, and a 67% reduction in the number of peaks present in ERY. Over 97% of the peaks present in CMP and ERY were also present in HSC, while only 3% of peaks arise de novo during differentiation. These results demonstrate that the methylation profile of erythroblasts is present in HSC and that a specific genome-wide decrease in DNA methylation occurs during erythropoiesis. (Hogart A, Lichtenberg J, Ajay SS, Anderson S, NIH Intramural Sequencing Center, Margulies EH, Bodine DM. Genome-wide DNA methylation profiles in hematopoietic stem and progenitor cells reveal overrepresentation of ETS transcription factor binding sites. Genome Res. 22 (8) 1407-18, 2012.) The magnitude of the Methyl-Seq data required the development of a novel software package we call SigSeeker. We used SigSeeker in a horizontal analysis of methylation in monocytic cells before and after treatment with 5-aza-cytidine, a DNA methylation inhibitor. (Lichtenberg J, Hogart A, Battle S. Bodine DM. Discovery of motif-based regulatory signatures in whole genome methylation experiments. Bioinformatics Open Source Conference (BOSC), 2012, Long Beach USA.) We found that SigSeeker was equally capable of analyzing ChIP-Seq data, demonstrating the applicability of the tool to many large-scale systems biology problems. (Dworkin AM, Lee M, Lichtenberg J, Patel SJ, Gildea D, Sakthianadeswaren A, NISC Comparative Sequencing Program, Foote S, Wolfsberg TG, Bodine DM, Crawford NPS. The metastasis suppressor RRP1B interacts with TRIM28 and HP1α at the c-MYC locus to induce heterochromatinization and silencing of c-MYC expression. PNAS submitted.) Our current plans are to extend these studies to generate methylation profiles for at least two additional cell types, the Megakaryocyte/Erythroid progenitor (MEP; the progenitor that gives rise to both the erythroid and megakaryocytic platelet lineages) and fully differentiated megakaryocytes. Through these studies we anticipate that we will identify methylation changes that are specific to the erythroid and megakaryocytic (platelet) lineages. Project 2. Diamond Blackfan anemia is an inherited bone marrow failure syndrome characterized by a severe deficiency of red blood cells, despite the fact that all other hematopoietic lineages differentiate normally and are present in normal numbers. Mutations in 9 different ribosomal protein genes have been identified in multiple unrelated families, most resulting in haploinsufficiency. However, 40% of DBA-associated mutations are missense mutations that cause a single amino acid change. These can be divided into two classes. The first type of amino acid substitution (Class I) interferes with the proper folding of the ribosomal protein, resulting in endosomal degradation and functional haploinsufficiency. The second type (Class II) produces proteins that appear to be folded normally as they are stable in the cytoplasm. We hypothesized that the Class II mutations act by a dominant negative mechanism. To test this hypothesis, we generated a transgenic mouse model that expresses an RPS19 mutation at codon 62 (RPS19R62W). Conditional expression of RPS19R62W in hematopoietic cells resulted in anemia with reduced numbers of erythroid progenitors. We concluded that RPS19R62W is a dominant negative DBA mutation. (Devlin EE, Dacosta L, Mohandas N, Elliott G, Bodine DM. A transgenic mouse model demonstrates a dominant negative effect of a point mutation in the RPS19 gene associated with Diamond-Blackfan anemia. Blood. 116 (15): 2826-35, 2010.) Attempts by others to generate mouse models of DBA by knocking out the mouse Rps19 gene have been unsuccessful. Preliminary data suggests that the output of the normal mouse Rps19 allele increases to compensate for the loss of the second allele. RPS19s only function appears to be in the assembly of the small ribosomal subunit. We hypothesized that mild deficiencies in the levels of ribosomal proteins that serve additional functions might be better candidates for a mouse model of DBA. Mutations in the RPL11 gene account for 15% of known DBA mutations. In addition to its role in the assembly of the large ribosomal subunit, RPL11 also interacts with MDM2, p53 and the rRNA processing pathways, which may account for the significantly higher frequency of craniofacial and physical abnormalities seen in patients with RPL11 mutations. We are currently generating an Rpl11 conditional knockout mouse to test this hypothesis and to provide an in vivo system to study the mechanism of erythroid failure and to test potentially useful drugs. Project 3. The goal of this project is to provide a molecular diagnosis to each DBA patient. We hypothesized that copy number variations that cause the deletion of a ribosomal protein gene could cause DBA in patients with normal ribosomal protein gene sequences. We used genome-wide SNP array profiling to evaluate regions of copy variation in DBA patients. In our initial sampling, we identified deletions at known DBA-related ribosomal protein gene loci in 20% of 50 DBA patients. These data suggest that ribosomal protein gene deletions are more common than previously suspected and are a significant cause of DBA. (Farrar JE, Vlachos A, Atsidaftos E, Carlson-Donohoe H, Markello TC, Arceci RJ, Ellis SR, Lipton JM, Bodine DM. Ribosomal protein gene deletions in Diamond-Blackfan Anemia. Blood. 118 (26): 6943-51, 2011). Using a sensitive new method to detect mosaic copy number variations (Markello TC, Carlson-Donohoe H, Sincan M, Adams D, Bodine DM, Farrar JE, Vlachos A, Lipton JM, Auerbach AD, Ostrander EA, Chandrasekharappa SC, Boerkoel CF, Gahl WA. Sensitive quantification of mosaicism using high density SNP arrays and the cumulative distribution function. Mol Genet Metab. 105 (4): 665-71, 2012.), we also identified novel variable mosaic loss involving chromosomal regions containing known DBA gene regions in 5 patients from 4 different kindreds. To identify novel DBA mutations, we plan to expand our sequence analysis of those patients who have not had mutations in known DBA genes identified by sequencing and do not have copy number variations identified by SNP-CGH. We will enroll 4 unrelated family groups consisting of parents, a DBA proband and either an affected or unaffected sibling for exome capture sequencing. The inclusion of the parents and sibling will add power to the analysis of the exome sequence. After the first round of sequencing we will screen the remaining pool of undiagnosed DBA patients for any newly identified mutation(s) before enrolling a second set of 4 families who do not carry a known mutation.

外周血中的所有细胞均通过造血干细胞（HSC）的少量产生，这被称为造血的过程。造血分化计划包括定义明确的阶段，其中HSC的后代仅限于特定的命运。该项目的目的是定义与造血特定阶段相关的特定分子特征。 项目1。DNA甲基化是正常发育和造血分化所必需的必不可少的表观遗传标记。我们表征了全基因组DNA甲基化，在原代小鼠HSC，常见的髓样祖细胞（CMP）和红细胞（ERY）中。我们发现，HSC中DNA甲基化最丰富，CMP中存在的峰值减少了40％，而ERY中存在的峰值减少了67％。 HSC中也存在CMP和ERY中存在的峰的97％以上，而分化过程中只有3％的峰从头出现。这些结果表明，HSC中存在红细胞的甲基化谱，并且在红细胞生成期间，DNA甲基化的特定基因组降低。 （Hogart A，Lichtenberg J，Ajay SS，Anderson S，NIH壁内测序中心，Margulies EH，Bodine dm。造血干细胞和祖细胞中全基因组的DNA甲基化特征和祖细胞中的ETS转录因子结合位点的过度体现。 甲基序列数据的大小需要开发我们称为Sigseeker的新型软件包。我们在用5-氮杂 - 酪氨酸（一种DNA甲基化抑制剂）处理前后对单核细胞中甲基化的水平分析中使用了Sigseeker。 （Lichtenberg J，Hogart A，Battle S. Bodine DM。在整个基因组甲基化实验中发现基于图案的调节签名。BioInformatics开源会议（BOSC），2012年，美国长滩。）我们发现Sigseeker同样能够分析Chip-Seq数据，表明CHIP-SEQ数据，表明Chip-Seq数据对许多大型系统的应用程序进行了应用程序，这些都可以对许多大型系统进行应用。 （Dworkin AM，Lee M，Lichtenberg J，Patel SJ，Gildea D，Sakthianadeswaren A，NISC比较测序计划，Foote S，Wolfsberg TG，Bodine DM，CrawfordNPS。Crawfordnps。Crawfordnps。NPS。转移抑制器RRP1B与TRIM28和HP1的相互作用，并与TRIM28和HP1α相互作用。 c-myc表达。 我们目前的计划是扩展这些研究，以产生至少两种其他细胞类型的甲基化谱，分别是巨核细胞/红细胞 - 红细胞祖细胞（MEP；祖细胞，产生红细胞核酸和巨核细胞血小板谱系）和完全分化的巨核细胞。通过这些研究，我们预计我们将确定针对红细胞和巨核细胞（血小板）谱系的甲基化变化。 项目2。钻石黑芬贫血是一种遗传性的骨髓衰竭综合征，其特征是红细胞严重缺乏，尽管所有其他造血谱系都均正常区分并且存在正常数量。在多个不相关的家族中已经鉴定出了9种不同的核糖体蛋白基因的突变，大多数导致单倍症。但是，与DBA相关的突变中有40％是导致单个氨基酸变化的错义突变。这些可以分为两个类。第一种氨基酸取代（I类）会干扰核糖体蛋白的适当折叠，从而导致内体降解和功能性单倍弥倍不足。第二种类型（II类）产生的蛋白质似乎在细胞质中稳定时似乎正常折叠。我们假设II类突变通过主要的负面机制作用。为了检验该假设，我们生成了一个转基因小鼠模型，该模型在密码子62（RPS19R62W）上表达RPS19突变。 RPS19R62W在造血细胞中的有条件表达导致贫血，红斑祖细胞数量减少。我们得出的结论是，RPS19R62W是一个主要的负DBA突变。 （Devlin EE，Dacosta L，Mohandas N，Elliott G，Bodine DM。一个转基因小鼠模型在RPS19基因中表现出与Diamond-Blackfan贫血相关的点突变的显着负面影响。116（15）（15）：2826-35，2010。 他人试图通过敲除鼠标RPS19基因来生成DBA的小鼠模型的尝试没有成功。初步数据表明，正常小鼠RPS19等位基因的输出增加以补偿第二等位基因的损失。 RPS19S仅功能似乎在小核糖体亚基的组装中。我们假设在核糖体蛋白水平上具有额外功能的轻度缺陷可能是DBA小鼠模型的更好候选者。 RPL11基因中的突变占已知DBA突变的15％。除了其在大型核糖体亚基的组装中的作用外，RPL11还与MDM2，p53和RRNA加工途径相互作用，这可能是RPL11突变患者看到的颅面和身体异常的频率明显更高。目前，我们正在生成RPL11条件敲除小鼠，以检验该假设，并提供一个体内系统来研究红斑失效的机制并测试潜在有用的药物。 项目3。该项目的目的是为每个DBA患者提供分子诊断。我们假设导致核糖体蛋白基因缺失的拷贝数变化可能导致核糖体蛋白基因序列正常的患者DBA。我们使用全基因组SNP阵列分析来评估DBA患者拷贝变异区域的评估。在最初的抽样中，我们在20％的50名DBA患者中确定了已知与DBA相关的核糖体蛋白基因基因座的缺失。这些数据表明，核糖体蛋白基因缺失比以前怀疑更常见，并且是DBA的重要原因。 （Farrar JE，Vlachos A，Atsidaftos E，Carlson-Donohoe H，Markello TC，Arceci RJ，Ellis SR，Lipton JM，Bodine DM。钻石 - 黑色贫血中的核糖体蛋白基因缺失。 Using a sensitive new method to detect mosaic copy number variations (Markello TC, Carlson-Donohoe H, Sincan M, Adams D, Bodine DM, Farrar JE, Vlachos A, Lipton JM, Auerbach AD, Ostrander EA, Chandrasekharappa SC, Boerkoel CF, Gahl WA. Sensitive quantification of mosaicism using high density SNP arrays和累积分布函数。 为了鉴定新的DBA突变，我们计划扩展我们对那些未通过测序鉴定的已知DBA基因突变的患者的序列分析，并且没有SNP-CGH鉴定的拷贝数变化。我们将招募4个不相关的家庭群体，包括父母，一个DBA概率以及一个受影响或未受到影响的兄弟姐妹进行外显群捕获测序。父母和兄弟姐妹的包含将为外显子序列的分析增添力量。在第一轮测序之后，我们将筛选剩余的未诊断的DBA患者池，以筛选出任何新鉴定的突变，然后再招募第二组4个未携带已知突变的家庭。