Lmo2-Lyl1 and the bHLH factor network in pro-T cells

pro-T 细胞中的 Lmo2-Lyl1 和 bHLH 因子网络

• 批准号: 10299482
• 负责人: ELLEN V. ROTHENBERG
• 金额: $ 58.9万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-06-15 至 2026-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10299482
• 关键词: Acute; Acute Lymphocytic Leukemia; Affect; B-Lymphocytes; BHLH Protein; Benign; Binding; Biochemical; Cell Cycle; Cell Differentiation process; Cell Lineage; Cells; Chromatin; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Data; Development; Dose; Down-Regulation; E protein; Enzymes; Event; GATA3 gene; Gene Expression; Gene Rearrangement; Genes; Genome; Genomics; Helix-Turn-Helix Motifs; Heterodimerization; Human; Immune; Immune system; In Vitro; Inhibitor of Differentiation Proteins; Kinetics; Lymphoid Cell; Molecular; Monitor; Mutate; Normal Cell; Oncogenes; Phase; Phenotype; Population Dynamics; Process; Proliferating; Property; Proto-Oncogenes; Rag1 Mouse; Regulation; Regulator Genes; Regulatory Element; Role; Site; T cell differentiation; T-Cell Development; T-Cell Receptor; T-Cell Receptor Genes; T-Lymphocyte; TCF3 gene; Testing; Thymus Gland; Tumor stage; Work; base; dimer; gene product; genetic signature; genome-wide; in vivo; leukemia; network architecture; preservation; prevent; programs; protein complex; response; stem cells; transcription factor

PROJECT SUMMARY The progression of T cell precursors from multipotency to commitment occurs after multiple cell cycles in the thymus. These early, pre-commitment cell cycles are important for expansion of the precursors to generate enough pro-T cells to survive later selection events. However, the early stages are poorly understood, and in particular it has not been clear what controls the precise trajectory of the cells' differentiation nor the timing or irreversibility of commitment when it occurs. Genomic regulatory elements that are active after commitment tend to be characterized by motifs for basic helix-loop-helix (bHLH) E proteins, E2A or HEB, whereas those that are active before commitment have other signatures, suggesting that there is a major increase in E protein activity across this transition. In fact, the expression of E proteins is almost equally high before commitment, but our recent data show that they are occupied in substantially different roles before commitment, in complexes with other heterodimerization partners. This proposal is based on recent evidence that the alternative complex, containing Lmo2 and Lyl1 dimerized with E2A or HEB, may actually be a major controller of the commitment transition in early T cells. Our recent evidence shows that expression of Lmo2 and Lyl1 is sufficient to make committed pro-T cells reverse their differentiation in terms of gene expression. Not only does the Lmo2/Lyl1/E2A complex bind to different genomic sites than E protein dimers, but also the addition of Lmo2 and Lyl1 to committed pro-T cells is sufficient to remove E proteins from sites that they occupy after commitment and shift them to sites that are normally active only before commitment. The implication is that the kinetics of downregulation of Lmo2 and Lyl1 in normal T-cell differentiation could be vital for determining the timing of commitment and of the maturation of the pro-T cells. Lmo2 and Lyl1 have been considered as T- lineage proto-oncogenes, but our evidence suggests a potent role in normal development. This proposal is to determine the mechanism of how this works and to test its significance for the actual developmental dynamics of normal early T cells. Our preliminary work identifies the signature target genes affected by Lmo2+Lyl1 and their overlap with genes expressed in normal pro-T cells. We now propose to define: (1) the distinct molecular mechanisms that control different subsets of these signature genes, based on genome-wide mapping of the chromatin state changes caused by Lmo2+Lyl1/E protein complex binding as compared to pure E protein dimer binding; (2) whether endogenous Lmo2/Lyl1/E protein complexes indeed control differentiation kinetics and commitment of normal pro-T cells, based on acute CRISPR and monitoring in vitro and in vivo; and (3) the gene regulatory network architecture, involving factors regulated by Lmo2+Lyl1, through which Lmo2+Lyl1 exert their surprisingly broad impacts on T cell development. The results should determine whether and how a biochemical mechanism of transcription factor heterodimerization partner switching may explain a central unsolved problem in the dynamics of T cell development.

项目概要 T 细胞前体从多能性到定型的进展发生在多个细胞周期之后 胸腺。这些早期的、预承诺的细胞周期对于前体细胞的扩展非常重要，以产生 足够的前 T 细胞能够在以后的选择事件中存活下来。然而，人们对早期阶段知之甚少，并且在 特别是目前尚不清楚是什么控制着细胞分化的精确轨迹，也不清楚是什么控制着细胞分化的时间或时间。 承诺发生时不可撤销。承诺后活跃的基因组调控元件 往往以基本螺旋-环-螺旋 (bHLH) E 蛋白、E2A 或 HEB 的基序为特征，而那些 在承诺之前活跃的有其他特征，表明 E 蛋白显着增加 跨越这一转变的活动。事实上，E蛋白的表达在承诺前几乎同样高， 但我们最近的数据显示，他们在做出承诺之前所扮演的角色截然不同， 与其他异二聚化伙伴的复合物。该提议是基于最近的证据表明 另一种复合物，包含与 E2A 或 HEB 二聚化的 Lmo2 和 Lyl1，实际上可能是主要控制器 早期 T 细胞的承诺转变。我们最近的证据表明 Lmo2 和 Lyl1 的表达是 足以使定型前 T 细胞逆转其基因表达分化。不仅 Lmo2/Lyl1/E2A 复合物与 E 蛋白二聚体相比是否与不同的基因组位点结合，而且还添加了 Lmo2 和 Lyl1 作用于定型前 T 细胞足以将 E 蛋白从其占据的位点上去除 并将它们转移到通常仅在承诺之前才活跃的站点。其含义是 正常 T 细胞分化中 Lmo2 和 Lyl1 下调的动力学对于确定 前 T 细胞的定型和成熟的时间。 Lmo2 和 Lyl1 被认为是 T- 谱系原癌基因，但我们的证据表明它在正常发育中发挥着重要作用。 该提案旨在确定其工作原理并测试其对实际的意义 正常早期 T 细胞的发育动态。我们的初步工作确定了特征目标基因 受 Lmo2+Lyl1 及其与正常 pro-T 细胞中表达的基因重叠的影响。我们现在建议 定义：（1）控制这些特征基因不同子集的不同分子机制，基于 Lmo2+Lyl1/E 蛋白复合物结合引起的染色质状态变化的全基因组图谱 与纯E蛋白二聚体结合相比； (2)内源性Lmo2/Lyl1/E蛋白复合物是否确实存在 基于急性 CRISPR 和监测控制正常 pro-T 细胞的分化动力学和定向 体外和体内； (3)基因调控网络架构，涉及Lmo2+Lyl1调控的因子， Lmo2+Lyl1 通过这种方式对 T 细胞发育发挥令人惊讶的广泛影响。结果应该 确定转录因子异二聚化伙伴是否以及如何形成生化机制 转换可以解释 T 细胞发育动力学中一个未解决的核心问题。