Genes and signals controlling mammalian hematopoiesis.

控制哺乳动物造血的基因和信号。

• 批准号: 8553888
• 负责人: PAUL E LOVE
• 金额: $ 162.34万
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 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/8553888
• 关键词: Acute Erythroblastic Leukemia; Adaptor Signaling Protein; Adult; Antigens; Area; Autoimmune Diseases; Binding; Binding Proteins; Binding Sites; Biochemical; Blood Cells; Blood Platelets; CD3 Antigens; Cell Differentiation process; Cell Maintenance; Cell Maturation; Cells; ChIP-seq; Cloning; Complex; Development; Diagnosis; Distal; Embryo; Ensure; Erythrocytes; Erythroid; Erythroid Progenitor Cells; Erythropoiesis; Gene Expression; Gene Targeting; Generations; Genes; Genetic Transcription; Hematopoiesis; Hematopoietic; Hematopoietic Neoplasms; Hematopoietic System; Hematopoietic stem cells; Human; ITAM; Immune System Diseases; Individual; Investigation; LIM Domain; LIM Domain Protein; Laboratories; Life; Lymphocyte; Maintenance; Mature T-Lymphocyte; Mediating; Membrane Proteins; Mus; Mutant Strains Mice; Myeloid Cells; Organism; Pathway interactions; Phenotype; Play; Process; Protein Tyrosine Kinase; Proteins; Receptor Signaling; Recruitment Activity; Regulation; Research; Role; Signal Pathway; Signal Transduction; Signaling Molecule; Site; Staging; Stem cells; Structure; Surface; System; T-Cell Activation; T-Cell Development; T-Cell Receptor; T-Lymphocyte; Testing; Thymocyte Development; Thymocyte Selection; Thymus Gland; Transgenes; Transgenic Organisms; Tumor Stem Cells; Tyrosine; adapter protein; base; fetal; genome-wide; human CD3E protein; interest; mutant; novel; programs; promoter; protein complex; receptor; receptor-mediated signaling; reconstitution; research study; response; self-renewal; small hairpin RNA; stem cell fate specification; transcription factor

Our studies are focused in three areas. The first involves characterization of the role of T cell antigen receptor (TCR) signals, and in particular, individual TCR signal transducing subunits and signal transducing motifs in T cell development. Second, we have extended our studies to include analysis of signal transducing molecules that function downstream of the TCR or that inhibit TCR signaling. The aim of these studies is to understand how these molecules participate in TCR mediated signaling and to determine what roles they and the signaling pathways they regulate play in T cell maturation and T cell activation. Finally, the lab has initiated a new area of investigation of the genes controlling the generation and maintenance of Hematopoietic Stem Cells. Role of T cell antigen receptor (TCR) signaling in thymocyte development. Signal transduction sequences (termed Immunoreceptor Tyrosine-based Activation Motifs; ITAMs) are contained within four distinct subunits of the multimeric TCR complex (zeta, CD3-gamma, -delta, -epsilon). Di-tyrosine residues within ITAMs are phosphorylated upon TCR engagement and function to recruit signaling molecules, such as protein tyrosine kinases, to the TCR complex, thereby initiating the T cell activation cascade. To determine if TCR signal transducing subunits perform distinct or analogous functions in development, we previously generated zeta deficient and CD3-epsilon deficient mice by gene targeting, genetically reconstituted these mice with transgenes encoding wild-type or signaling-deficient (ITAM-mutant) forms of zeta and CD3-epsilon, and characterized the developmental and functional consequences of these alterations on TCR signaling. The results of these studies demonstrated that TCR-ITAMs are functionally equivalent but act in concert to amplify TCR signals. TCR signal amplification was found to be critical for thymocyte selection, the process by which potentially useful immature T cells are instructed to survive and differentiate further-(positive selection), and potentially auto-reactive cells that may cause auto-immune disease are deleted in the thymus (negative selection). Thus, the multi-subunit structure of the TCR may have evolved to enable complex organisms to develop a broad, self-restricted yet auto-tolerant T cell repertoire. In current studies we are using conditional gene expression systems to analyze the importance of TCR signaling at specific stages of development. In addition, we are using microarray and subtractive cloning to identify genes involved in T cell signaling and T cell development. Signaling molecules that function downstream of the TCR or that function to "fine-tune" the TCR signal. Our results with TCR-ITAM mutant mice suggested that other signaling molecules can compensate for the reduction in TCR signal strength. An initial FACS-based search for candidate compensatory molecules led us to CD5, a TCR associated trans-membrane protein that inhibits TCR signaling. Importantly, we found that CD5 surface expression is regulated by and parallels TCR signal intensity. Thus, rather than simply functioning as a co-receptor, CD5 acts to fine-tune TCR signals during thymocyte selection since its level of surface expression depends upon the intensity of TCR signaling. An obvious benefit of such fine-tuning of the TCR signaling response would be to enable the generation of a T cell repertoire with the maximum possible diversity since it would allow a broader range of TCRs to pass through the signaling window of positive selection. Since little was known about how CD5 regulates TCR signaling, we initiated a project to characterize CD5 function, both genetically and biochemically. The results of these experiments suggest a mechanism for CD5 mediated TCR signal inhibition that we are currently testing experimentally. We have also begun a search for additional tuning molecules using a microarray based screen. The identification of such molecules may have importance for the diagnosis and treatment of human autoimmune diseases. In another study, we identified a novel T-lineage restricted putative adaptor protein, designated Themis. Biochemical studies indicate that Themis functions at a distal point in the TCR signaling pathway and may have an important role is helping to sustain TCR signaling. Themis-/- mice have been generated and their phenotype reveals an important role for this protein in late thymocyte development and selection. Current and projected experiments are directed at elucidating the mechanism by which Themis functions in T cell signaling and development. Genes controlling Hematopoietic Stem cell specification and maintenance. The hematopoietic system is composed of a functionally diverse group of cells that originate from a common hematopoietic stem cell (HSC) capable of long-term self-renewal and multi-lineage differentiation. Self-renewal ensures that a pool of HSCs persists throughout life, whereas differentiation leads to the continuous generation of all circulating blood cells including lymphocytes, myeloid cells, erythrocytes and platelets. We initiated experiments aimed at identifying genes important for HSC generation and maintenance. Our initial studies focused on the role of LIM domain binding protein-1 (Ldb1) in hematopoiesis. The results of these experiments revealed a critical function for Ldb1 in regulating the self-renewal/differentiation cell fate decision in hematopoietic stem cells and suggest that Ldb1 nucleated transcription complexes may control maintenance of lineage specific stem cells. Consistent with this, a genome-wide ChIP-seq screen identified Ldb1 binding sites within the promoter/gene body of a high percentage of genes known to be essential for HSC maintenance. Deletion of Ldb1 also resulted in decreased expression of these genes suggesting that Ldb1 complexes function as "master regulators" of the transcriptional program regulating HSC maintenance/self-renewal. Current studies are focused on exploring the potential role of Ldb1 in the maintenance of hematopoietic tumor stem cells and the role of Ldb1 in early stages of hematopoiesis, specifically the specification of multipotent embryonic progenitor cells to the hematopoietic lineage. Identification of Ldb1 protein complexes as master regulators of erythropoiesis Our studies on Ldb1 deficient mice recently identified a critical role for this protein in both fetal and adult erythropoiesis. Ldb1 is a subunit of a multimeric protein complex in erythroid progenitor cells that includes the adapter protein Lmo2 and the transcription factors Scl (Tal1) and Gata1. Gata1 had been shown previously to have an essential function in the regulation of virtually all known erythroid genes. Our results from ChIP-seq and microarray experiments where Ldb1 expression was reduced in murine erythroleukemia cells using shRNA demonstrate that Ldb1/Gata1 complexes bind at promoters and regulatory sites in nearly all erythroid genes known to be controlled by Gata1. Taken together, these findings demonstrate that Gata1 functions primarily through Ldb1 complexes to activate erythroid gene expression.

我们的研究集中在三个领域。 首先涉及表征T细胞抗原受体（TCR）信号的作用，尤其是单个TCR信号转导亚基和信号转导基序在T细胞发育中的作用。 其次，我们扩展了研究，以包括对TCR下游或抑制TCR信号传导的信号转导分子的分析。这些研究的目的是了解这些分子如何参与TCR介导的信号传导，并确定它们在T细胞成熟和T细胞激活中调节发挥作用的信号传导途径。最后，该实验室已开始对控制造血干细胞的产生和维持的基因的新领域。 T细胞抗原受体（TCR）信号传导在胸腺细胞发育中的作用。 信号转导序列（称为免疫受体酪氨酸的活化基序； ITAMS）包含在多聚体TCR复合物的四个不同亚基中（Zeta，CD3 -Gamma，-delta，-epsilon）。 ITAM中的Di-tyrosine残基在TCR参与和功能下被磷酸化，以募集TCR复合物等信号分子，例如蛋白酪氨酸激酶，从而启动T细胞激活级联反应。 To determine if TCR signal transducing subunits perform distinct or analogous functions in development, we previously generated zeta deficient and CD3-epsilon deficient mice by gene targeting, genetically reconstituted these mice with transgenes encoding wild-type or signaling-deficient (ITAM-mutant) forms of zeta and CD3-epsilon, and characterized the developmental and functional consequences of these alterations on TCR信号传导。这些研究的结果表明，TCR-ITAM在功能上是等效的，但协同作用以扩大TCR信号。发现TCR信号扩增对于胸腺细胞选择至关重要，胸腺细胞的选择至关重要，该过程指示潜在有用的不成熟的T细胞生存和分化进一步的（阳性选择），并且可能导致可能引起自身免疫性疾病的潜在自身反应性细胞在胸膜中被删除（阴性选择）。 因此，TCR的多生产结构可能已经发展为使复杂的生物能够发展出宽阔，自限制但自动耐受性的T细胞库。在当前的研究中，我们使用条件基因表达系统来分析特定发育阶段的TCR信号传导的重要性。此外，我们正在使用微阵列和减法克隆来识别与T细胞信号传导和T细胞发育有关的基因。 TCR下游功能或该功能“微调” TCR信号的信号分子。 我们使用TCR-ITAM突变小鼠的结果表明，其他信号分子可以补偿TCR信号强度的降低。对候选补偿性分子的最初基于FACS的搜索使我们进入了CD5，CD5是一种抑制TCR信号传导的TCR相关的跨膜蛋白。重要的是，我们发现CD5表面表达受到TCR信号强度的调节。因此，CD5不简单地充当共受体，而是在胸腺细胞选择过程中对微调TCR信号起作用，因为其表面表达水平取决于TCR信号传导的强度。 TCR信号响应的这种微调的一个明显好处是，可以使具有最大可能多样性的T细胞库生成，因为它可以使更广泛的TCR通过正选择的信号传递窗口。 由于对CD5如何调节TCR信号传导知之甚少，因此我们启动了一个项目来表征CD5功能，无论是在遗传和生化上。这些实验的结果表明了CD5介导的TCR信号抑制的机制，我们目前正在实验测试。我们还开始使用基于微阵列的屏幕来搜索其他调整分子。这种分子的鉴定可能对人类自身免疫性疾病的诊断和治疗可能具有重要意义。在另一项研究中，我们确定了一种新型的T-linege受限推定适配器蛋白，该蛋白被指定为themis。生化研究表明，TRES在TCR信号通路中的远端发挥作用，并且可能具有重要作用是有助于维持TCR信号传导。已经产生了主题 - / - 小鼠，它们的表型揭示了该蛋白质在晚期胸腺细胞发育和选择中的重要作用。当前和投影实验旨在阐明Themis在T细胞信号传导和发育中起作用的机制。 控制造血干细胞规格和维持的基因。 造血系统由功能多样的细胞组成，该细胞源自能够长期自我更新和多条形分化的常见造血干细胞（HSC）。自我更新确保HSC库持续一生，而分化导致所有循环血细胞的连续产生，包括淋巴细胞，髓样细胞，红细胞和血小板。我们启动了旨在识别对HSC生成和维护重要的基因的实验。我们的初步研究集中于LIM结构域结合蛋白-1（LDB1）在造血中的作用。这些实验的结果揭示了LDB1在调节造血干细胞中自我更新/分化细胞命运决策方面具有关键功能，并表明LDB1成核转录复合物可以控制维持谱系特定干细胞的维持。与此相一致，全基因组芯片屏幕鉴定出启动子/基因体内的LDB1结合位点，这些基因的较高基因已知对于HSC维持至关重要。 LDB1的缺失也导致这些基因的表达降低，这表明LDB1复合物是调节HSC维护/自我更新的转录程序的“主调节器”。 当前的研究集中在探索LDB1在维持造血肿瘤干细胞中的潜在作用，以及LDB1在造血阶段的早期阶段的作用，特别是将多能胚胎祖细胞与血肿谱系的规定。 鉴定LDB1蛋白复合物是红细胞生成的主要调节剂 我们对LDB1缺乏小鼠的研究最近确定了该蛋白在胎儿和成人红细胞生成中的关键作用。 LDB1是红细胞祖细胞中多聚体蛋白复合物的亚基，其中包括衔接蛋白LMO2和转录因子SCL（TAL1）和GATA1。 GATA1先前已显示在几乎所有已知红细胞基因的调节中具有重要功能。我们来自CHIP-SEQ和微阵列实验的结果，其中使用SHRNA在鼠红血病细胞中降低了LDB1表达，这表明LDB1/GATA1复合物在几乎所有被GATA1控制的启动子和调节位点结合了启动子和调节位点。综上所述，这些发现表明，GATA1主要通过LDB1复合物发作以激活红系基因表达。