CIS-REGULATORY ANALYSIS OF PU.1 BY IN VITRO ES CELL DIFFERENTIATION

通过体外 ES 细胞分化对 PU.1 进行 CIS 调控分析

• 批准号: 7229890
• 负责人: ELLEN V. ROTHENBERG
• 金额: $ 18.54万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-02-01 至 2009-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7229890
• 关键词: Biological Models; Cell Differentiation process; Cells; Complex; DNA-Protein Interaction; Data; Development; Effectiveness; Equilibrium; Gene Expression Regulation; Gene Transfer Techniques; Genes; Hematopoiesis; Hematopoietic; In Vitro; Lymphoid; Maps; Monitor; Mus; Myelogenous; Pathway interactions; Pattern; Plasmids; Regulator Genes; Regulatory Element; Reporter; Role; System; Systems Analysis; T-Lymphocyte; Testing; Undifferentiated; base; cell type; embryonic stem cell; interest; leukemia; novel strategies; research study; transcription factor

DESCRIPTION (provided by applicant): Developmental choices in mammalian hematopoietic cells depend on the balance among the expression levels of tightly regulated transcription factors. Extremely accurate regulation of these genes is not only critical for normal hematopoietic development but also to avoid leukemia. In terms of mechanism, the question is what DNA-protein interactions determine the level of expression of each transcription factor itself. PU.l in particular has a complex pattern of expression in different hematopoietic lineages that is crucial for normal differentiation. Qualitative as well as quantitative differences in PU.l expression pattern distinguish myeloid and T-lymphocyte developmental pathways. This could indicate that PU.l depends on a variety of different regulatory sequences to activate or repress it in different cellular contexts, as hinted by our preliminary data. However, in mammalian hematopoiesis there has not been any adequate system to map the functional roles of any potential regulatory sequence of interest across multiple hematopoietic cell types. This proposal is to develop a new model system for these analyses that should enable the same clonal integration of a regulatory sequence-reporter construct to be monitored in diverse hematopoietic lineages on a relatively high-throughput basis as compared to transgenesis. We will exploit a new in vitro differentiation system that enables embryonic stem cells (ES cells) to develop directly into both lymphoid and erythromyeloid cells. BAG and plasmid regulatory-sequence reporter constructs will be transfected into undifferentiated ES cells and then tested for activity as those cells differentiate in distinct developmental pathways. We plan to demonstrate the effectiveness of this system by using it to map the combined positive and negative regulatory elements of the PU.l (Sfpil) gene that govern its divergent patterns of expression in T-lymphoid precursors as contrasted with myeloid precursors. Our specific aims are: (1) to optimize the ES cell system for analysis of reporter expression in the myeloid and lymphoid lineages; (2) to use this system to define the cis-regulatory elements that control the pattern of PU.l expression in T-lineage development; and (3) to verify these conclusions by analysis of reporter expression in mice derived from the ES cell transfectants. Summary: What regulates the regulators? Here we propose to develop a new approach to this important question, using a very recent advance in cultures of mouse embryonic stem cells to overcome past technical barriers. This should permit faster and cheaper experiments to reveal how powerful regulatory genes are themselves controlled.

描述（由申请人提供）：哺乳动物造血细胞的发育选择取决于严格调控的转录因子表达水平之间的平衡。对这些基因的极其精确的调控不仅对正常的造血发育至关重要，而且对避免白血病也至关重要。在机制方面，问题是什么DNA-蛋白质相互作用决定每个转录因子本身的表达水平。PU. 1特别地在不同造血谱系中具有复杂的表达模式，这对于正常分化至关重要。PU. 1表达模式的定性和定量差异区分了髓样和T淋巴细胞发育途径。这可能表明PU. 1依赖于各种不同的调控序列来在不同的细胞环境中激活或抑制它，正如我们的初步数据所暗示的那样。然而，在哺乳动物造血中，还没有任何适当的系统来映射跨多种造血细胞类型的任何潜在的感兴趣的调控序列的功能作用。这个建议是开发一个新的模型系统，这些分析，应使相同的克隆整合的调控序列报告构建体进行监测，在不同的造血谱系在一个相对高通量的基础上相比，转基因。我们将开发一种新的体外分化系统，使胚胎干细胞（ES细胞）直接发育成淋巴细胞和红骨髓细胞。将BAG和质粒调节序列报告基因构建体转染到未分化的ES细胞中，然后测试这些细胞在不同发育途径中分化时的活性。我们计划证明该系统的有效性，通过使用它来映射的PU. 1（Sfestival）基因，管理其不同的模式，在T-淋巴前体的表达与髓系前体的组合的正和负调控元件。我们的具体目标是：（1）优化ES细胞系统以分析髓系和淋巴系中的报告基因表达;（2）使用该系统来定义控制T系发育中PU. 1表达模式的顺式调节元件;和（3）通过分析源自ES细胞转染子的小鼠中的报告基因表达来验证这些结论。摘要：监管机构的监管内容是什么？在这里，我们建议开发一种新的方法来解决这个重要的问题，利用小鼠胚胎干细胞培养的最新进展来克服过去的技术障碍。这将允许更快和更便宜的实验来揭示调控基因本身是如何被控制的。