Bcl11b in early T cell development

描述（由申请人提供）：已知T细胞谱系承诺依赖于Notch-Delta信号影响下转录因子的组合。然而，这些因素在谱系承诺的分子机制中的确切作用仍然是难以捉摸的。一个问题是，所有已确定的因子本身在干细胞维持和/或其他造血细胞类型以及T细胞中都有作用，尽管用于T细胞规格的精确组合可能是独一无二的。另一个问题是在谱系承诺过程中被排除的选择的复杂性。t细胞前体似乎保留了进入自然杀伤细胞（NK）发育和树突状细胞发育的非常独特的途径，也可能进入肥大细胞和巨噬细胞发育的途径，直到它们发育的中点，然后似乎立即失去所有这些替代潜力。与此同时，细胞似乎改变了它们的自我更新能力。很难在一步过程中解释所有这些变化，特别是在缺乏任何已知的明确的t细胞特异性调节功能的情况下。一直缺少的是一种t谱系特定的调节因子，它可以被证明控制这些事件的精确定义子集。这就是我们提出的Bcl11b所扮演的角色，Bcl11b是一种在造血系统中具有高度t谱系特异性表达模式的转录因子。Bcl11b诱导立即先于t谱系承诺，我们的条件Bcl11b敲除的初步数据表明，该因子控制谱系承诺功能的一个离散子集，涉及NK命运和干细胞或祖细胞特异性维持途径的抑制。在这个应用中，我们建议测试这个模型，并使用Bcl11b来澄清承诺过程中的监管联系。目标是：1。利用Bcl11b缺乏症来剖析t谱系承诺机制中的组成过程：(a)排除NK和干细胞调控程序，这与排除髓系、B和其他造血程序有关；(b)可能区分34个谱系T细胞的承诺机制2。确定Bcl11b与早期t细胞发育中Id/E蛋白比例之间的调控联系3。确定Bcl11b的直接作用靶点以及Bcl11b促进t谱系同一性的有序途径4。利用Bcl11b基因座的新型荧光敲入报告基因在单细胞水平上追踪t谱系的规范和承诺。

DESCRIPTION (provided by applicant): T cell lineage commitment is known to depend on a combination of transcription factors operating under the influence of Notch-Delta signaling. However, the exact roles of these factors in the molecular mechanism of lineage commitment have remained elusive. One problem is that all of the established factors themselves have roles in stem-cell maintenance and/or in other hematopoietic cell types as well as in T cells, even though the precise combination used for T-cell specification may be unique. Another problem is the complexity of the choices that are excluded during lineage commitment. T-cell precursors appear to retain access to the very distinct pathways of natural killer (NK) cell development and dendritic- cell development, possibly also to mast-cell and macrophage development, until a midpoint in their specification, and then appear to lose all these alternative potentials at once. At the same time, the cells appear to alter their self-renewal potential. It has been difficult to account for all these changes in a one- step process, especially in the absence of any known regulatory function that is clearly T-cell specific. What has been missing is a T-lineage specific regulatory factor that can be shown to control a precisely defined subset of these events. This is the role that we propose is played by Bcl11b, a recently characterized transcription factor with a highly T-lineage specific pattern of expression within the hematopoietic system. Bcl11b induction immediately precedes T-lineage commitment, and our preliminary data with a conditional Bcl11b knockout imply that this factor controls a discrete subset of lineage commitment functions, involving the repression of NK fates and stem or progenitor cell-specific maintenance pathways. In this application, we propose to test this model and to use Bcl11b to clarify regulatory linkages in the commitment process. The aims are: 1. To use Bcl11b deficiency to dissect component processes within the T-lineage commitment mechanism: (a) exclusion of NK and stem-cell regulatory programs as related to exclusion of myeloid, B, and other hematopoietic programs; and (b) commitment mechanisms that may distinguish 12 from 34 lineage T cells 2. To determine the regulatory links between Bcl11b and the Id/E protein ratio in early T-cell development 3. To determine the direct targets of Bcl11b and the ordered pathway through which Bcl11b action promotes T-lineage identity 4. To track T-lineage specification and commitment at the single cell level using new fluorescent knock- in reporters incorporated in the Bcl11b locus. PUBLIC HEALTH RELEVANCE: If stem cells are ever to be used therapeutically, it is vital to be able to understand and control commitment, the process by which stem-cell descendants give up their infinite growth potential and developmental plasticity in order to "settle down" to a defined biological role. This proposal will determine exactly how a newly recognized T-cell transcription factor, Bcl11b, may work to drive precursors from a stem-cell like state into a stable and useful T-cell identity, and the mechanisms that emerge will be applicable to controlling stem-cell based development in general.