Genes and signals controlling mammalian hematopoiesis.

控制哺乳动物造血的基因和信号。

• 批准号: 9348228
• 负责人: PAUL E LOVE
• 金额: $ 232.6万
• 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/9348228
• 关键词: Acute Erythroblastic Leukemia; Acute T Cell Leukemia; Adult; Antigen Receptors; Antigens; Area; Autoimmune Diseases; B-Lymphocytes; Binding; Binding Proteins; Binding Sites; Biochemical; Blood Cells; Blood Platelets; CD3 Antigens; CD3E gene; Cell Differentiation process; Cell Maintenance; Cell Maturation; Cells; ChIP-seq; Complex; DNA Binding; Development; Diagnosis; Ensure; Erythrocytes; Erythroid; Erythroid Cells; Erythroid Progenitor Cells; Erythropoiesis; Feedback; Gene Expression; Gene Mutation; Gene Proteins; Gene Targeting; Generations; Genes; Genetic Transcription; Hematopoiesis; Hematopoietic; Hematopoietic Neoplasms; Hematopoietic System; Hematopoietic stem cells; Human; ITAM; Immune System Diseases; Immunotherapy; Investigation; Knowledge; LIM Domain; LIM Domain Protein; Laboratories; Life; Lymphocyte; Lymphopoiesis; Maintenance; Malignant Childhood Neoplasm; Maps; Mature T-Lymphocyte; Membrane Proteins; Mus; Mutant Strains Mice; Myeloid Cells; 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; Surface; System; T cell regulation; T-Cell Activation; T-Cell Development; T-Cell Receptor; T-Lymphocyte; Thymocyte Development; Thymocyte Selection; Thymus Gland; Transgenes; Transgenic Organisms; Tumor Stem Cells; Tyrosine; Work; adapter protein; base; chromatin immunoprecipitation; deep sequencing; fetal; genome-wide; interest; mutant; novel; progenitor; programs; promoter; protein complex; receptor; receptor-mediated signaling; reconstitution; research study; response; screening; self-renewal; small hairpin RNA; stem; therapeutic target; thymocyte; transcription factor

Our research is focused in four main areas: 1) characterization of the role of T cell antigen receptor (TCR) signals in T cell development. 2) identification and analysis of signal 'tuning' molecules that function downstream of the TCR that augment or 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. These molecules may represent targets for immunotherapy in humans. 3) Identification and characterization of new molecules that have important roles in T cell development. 4) Studies of the genes controlling the generation, maintenance and differentiation of Hematopoietic Stem Cells (HSCs). Examining the 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 different 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 analogous 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). In current studies we are using conditional gene expression systems to analyze the importance of TCR signaling at specific stages of 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 static inhibitory co-receptor, CD5 regulation by TCR signaling provides a feedback mechanism to 'fine-tune' the overall TCR signaling response during thymocyte selection since the expression of CD5 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 threshold 'window' of positive selection. Since little is known about how CD5 regulates TCR signaling, we initiated a project to characterize CD5 function, both genetically and biochemically. We have also begun a search for additional tuning molecules using a microarray based screen. The identification of such molecules may be relevant to the diagnosis and treatment of human autoimmune diseases since these molecules function to determine the activation threshold for T cells. Identification of new genes that regulate T cell development. We identified a novel T-lineage restricted protein, designated Themis. Biochemical studies indicate that Themis functions in the TCR signaling pathway and may have an important role is helping to sustain TCR signaling. Themis-/- and conditional Themis deficient mice have been generated and their phenotype reveals an important role for this protein in late thymocyte development and selection. Themis contains a novel (CABIT) domain of unknown function. Current and projected experiments are directed at elucidating the mechanism by which Themis regulatesT cell signaling and determining the function of the CABIT domain. We also identified several other genes that are highly expressed in developing T cells (thymocytes) but have not yet been characterized. We are currently generating mice with targeted mutations of these genes to study their role in lymphopoiesis. Genes controlling Hematopoietic Stem (HSC) cell maintenance and self-renewal. 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. Several years ago 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 as prior work had suggested a function for Ldb1 in the hematopoietic system. 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 multi-subunit transcription complexes may control maintenance of lineage specific stem cells. Consistent with this, a genome-wide ChIP-seq screen identified Ldb1-complex binding sites within the promoter/gene body of a high percentage of genes known to be essential for HSC maintenance. These binding sites were frequently co-occupied by the transcription factors Tal1 (Scl) and Gata2, two proteins known to be essential for HSC maintenance. Strong co-occupancy of the same sites by Ldb1 Tal1 and Gata2 indicates that multimeric complexes that include these proteins as subunits are important for regulating the expression of maintenance-critical genes in HSCs. Deletion of Ldb1 in HSCs resulted in loss of HSCs 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 complexes in the maintenance of hematopoietic tumor stem cells particularly those that predispose to T-Cell Acute Lymphoblastic Leukemia (T-ALL). Identification of Ldb1 protein complexes as master regulators of erythropoiesis Our recent studies on Ldb1 deficient mice 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 (as opposed to Gata2 which is a subunit of Ldb1 complexes in HSCs). 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/Tal1/Gata1 complexes bind at promoters and regulatory sites within nearly all erythroid genes known to be controlled by Gata1 and Tal1. Taken together, these findings demonstrate that Gata1 and Tal1 function primarily through Ldb1 complexes to globally activate erythroid gene expression

我们的研究集中在四个主要领域：1）表征T细胞抗原受体（TCR）信号在T细胞发育中的作用。 2）鉴定和分析TCR下游功能的信号“调谐”分子增强或抑制TCR信号传导。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。这些分子可能代表人类免疫疗法的靶标。 3）在T细胞发育中具有重要作用的新分子的鉴定和表征。 4）研究控制造血干细胞（HSC）产生，维持和分化的基因。 检查T细胞抗原受体（TCR）信号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-ITAMS在功能上是类似的，但协同起来可以扩增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）。 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。 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，TCR信号通过CD5调节而不是简单地充当静态抑制性共受体，而是为“微调”胸腺细胞选择过程中总体TCR信号反应的反馈机制，因为CD5的表达取决于TCR信号的强度。 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，因为它将允许更广泛的TCR通过正选择的信号传导阈值“窗口”。 由于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。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与人类自身免疫性疾病的诊断和治疗有关，因为这些分子起作用以确定T细胞的激活阈值。 鉴定调节T细胞发育的新基因。 我们确定了一种新型的T-Linege受限蛋白，称为主题。生化研究表明，TRES在TCR信号通路中起作用，并且可能发挥重要作用是有助于维持TCR信号传导。 Themis - / - 和有条件的主体缺乏小鼠已经产生，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。 Themis包含一个未知功能的新颖（CABIT）领域。当前和投影实验旨在阐明主调节型细胞信号传导和确定CABIT域功能的机制。我们还确定了在发育中的T细胞（胸腺细胞）中高度表达的其他几个基因，但尚未表征。我们目前正在生成具有这些基因靶向突变的小鼠，以研究其在淋巴管中的作用。 控制造血茎（HSC）细胞维持和自我更新的基因。 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。几年前，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，因为先前的工作提出了造血系统中LDB1的功能。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-核核的多生成转录复合物可以控制谱系特异性干细胞的维持。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复合结合位点，这些基因的启动子/基因体中已知对HSC维持必不可少的基因所必需的基因。这些结合位点经常被转录因子TAL1（SCL）和GATA2共占据，这是两个已知对于HSC维持必不可少的蛋白质。 LDB1 TAL1和GATA2对同一位点的强共占据表明，包括这些蛋白质作为亚基的多聚体配合物对于调节HSC中维持临界基因的表达很重要。 HSC中LDB1的删除导致HSC的丢失，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”。 目前的研究集中在探索LDB1复合物在维持造血肿瘤干细胞中的潜在作用，尤其是那些易感T细胞急性淋巴细胞白血病（T-ALL）的潜在作用。 鉴定LDB1蛋白复合物是红细胞生成的主要调节剂 我们最近对LDB1缺乏小鼠的研究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，其中包括适配器蛋白LMO2和转录因子SCL（TAL1）和GATA1（与GATA2相对于HSC中LDB1复合物的亚基）。 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/TAL1/GATA1复合物在几乎所有被GATA1和TAL1控制的启动子和调节位点上结合了启动子和调节位点。综上