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.

项目总结 在多个细胞周期后，T细胞前体从多能性发展到承受性 胸腺。这些早期的、预先承诺的细胞周期对于前体细胞的扩张非常重要 足够的PRO-T细胞在以后的选择活动中存活。然而，人们对早期阶段知之甚少，而且在 特别是，目前还不清楚是什么控制了细胞分化的准确轨迹，也不清楚是什么控制了细胞分化的时间或时间 承诺发生时的不可逆转性。承诺后活跃的基因组调控元件 倾向于以碱性螺旋-环-螺旋(BHLH)E蛋白的基序为特征，E2a或Heb，而那些 在承诺之前活跃的人有其他特征，这表明E蛋白有很大的增加 在此过渡期间的活动。事实上，在承诺之前，E蛋白的表达几乎是同样高的， 但我们最近的数据显示，在承诺之前，他们扮演的角色有很大的不同 与其他异二聚体的络合物。这项建议是基于最近的证据，即 含有与E2a或Heb二聚化的Lmo2和Lyl1的替代复合体实际上可能是主要的控制者 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 足以使承诺的亲T细胞在基因表达方面逆转其分化。不仅 Lmo2/Lyl1/E2a复合体是否与E蛋白二聚体结合到不同的基因组位置，而且还与E蛋白二聚体的添加 LMO2和Lyl1与承诺的PRO-T细胞结合足以将E蛋白从它们占据的位置移除 并将他们转移到通常只有在承诺之前才活跃的站点。言下之意是 正常T细胞分化中Lmo2和Lyl1下调的动力学对确定 承诺的时间和前T细胞成熟的时间。Lmo2和Lyl1被认为是T- 血统原癌基因，但我们的证据表明，在正常发育中发挥着重要作用。 这项建议是为了确定这一机制是如何工作的，并测试其在实际中的意义 正常早期T细胞的发育动力学。我们的初步工作确定了标志性的目标基因 受Lmo2+Lyl1的影响以及它们与正常PRO-T细胞中表达的基因重叠。我们现在建议 定义：(1)控制这些标志性基因不同子集的不同分子机制，基于 Lmo2+Lyl1/E蛋白复合体结合AS引起染色质状态变化的全基因组图谱 与纯E蛋白二聚体结合的比较；(2)内源性Lmo2/Lyl1/E蛋白复合体是否真的存在 基于急性CRISPR和监测的控制正常PRO-T细胞的分化动力学和承诺 体外和体内；(3)基因调控网络结构，涉及Lmo2+Lyl1调控因子， 通过它，Lmo2+Lyl1对T细胞发育产生了惊人的广泛影响。结果应该是 确定转录因子异二聚化的生化机制是否以及如何 转换可能解释了T细胞发育动力学中一个中心悬而未决的问题。