Dissecting Distinct and Redundant Roles of ThPOK and LRF, Key Regulators of Hematopoiesis

 描述(由申请人提供)：一个基本的悬而未决的问题是，掌握世系承诺的调控者控制不同命运决定的能力主要是来自独特的功能能力，还是来自表达这些决定的细胞环境。ThPOK和LRF是POK转录因子家族的相关成员，在不同的造血过程中起着重要的作用。因此，ThPOK控制CD4T细胞的分泌，而LRF控制B细胞的分泌。这些因子在造血的其他阶段也起着关键作用，包括维持造血干细胞(见初步结果)。尽管它们具有多重关键作用，但这些因子的具体功能的机制/S仍有待充分阐明，甚至包括它们不同的作用主要是由于独特的功能能力还是表达模式的差异这一根本问题。在这里，我们建议根据两个特定的目标来阐明这些问题：目的1.ThPOK和LRF在造血过程中是否介导了固有的不同功能？ThPOK和LRF的非冗余角色可能反映了固有的功能差异，这是由它们不同的氨基酸序列或不同的表达模式决定的。为了解决这个问题，我们创造了新的敲打小鼠，其中ThPOK编码的外显子被LRF(LRF敲打小鼠，或Lki小鼠)的外显子精确替换。由于所有DNA顺式调控元件以及5‘和3’未翻译(UT)的信使核糖核酸(UT)信使核糖核酸(UT)区域都来自ThPOK基因座，因此LKI小鼠已经被改造成既维持DNA依赖的基因表达调控机制，又保持mRNA依赖的基因表达调控机制。因此，lki等位基因支持与wt ThPOK等位基因完全相同的基因表达水平和动力学。我们将使用纯合子Lki/Lki小鼠来评估LRF在缺乏ThPOK的情况下是否能够挽救T细胞发育和早期造血的缺陷，以确定LRF和ThPOK编码序列编码的是不同的还是冗余的功能。我们提出的初步证据强烈表明功能专门化。目标2.评估功能特异性 使用嵌合基因方法对ThPOK BTB结构域进行分析。如果ThPOK在功能上不同于LRF，那么特定的功能很可能至少部分由它们的BTB/POZ结构域编码，这些结构域对于与核共抑制物和组蛋白去乙酰基酶的二聚化至关重要。尽管ThPOK和LRF BTB结构域具有重要作用，但它们的功能特异性/冗余性从未在体内得到检验。在这里，我们将使用敲击法选择性地用LRF替换ThPOK的BTB结构域，或者反之亦然，并测试在没有wt ThPOK的情况下，所产生的嵌合因子是否能够恢复CD4和iNKT细胞的胸腺发育。这将确定ThPOK BTB域是否必要和/或足以复制ThPOK在这些进程中的基本作用。这些小鼠还将有价值地剖析ThPOK参与的其他造血方面对ThPOK BTB结构域的特殊需求，包括HSC的维持和髓系发育。

 DESCRIPTION (provided by applicant): A fundamental unanswered question is whether the ability of master regulators of lineage commitment to control disparate fate decisions results primarily from unique functional capabilities or from the cellular context in which they are expressed. ThPOK and LRF are related members of the POK family of transcription factors, with essential roles at distinct stages of hematopoiesis. Thus ThPOK controls CD4 T cell commitment, while LRF controls B cell commitment. These factors also play critical roles at other stages of hematopoiesis, including for hematopoietic stem cell maintenance (see preliminary results). Despite their multiple critical roles, the mechanism/s underlying specific functions of these factors remains to be fully elucidated, including even the fundamental question of whether their distinct roles result primarily from unique functional capabilities or frm expression pattern differences. Here we propose to elucidate these questions according to two specific aims: Aim 1. Do ThPOK and LRF mediate inherently distinct functions during hematopoiesis? Non- redundant roles of ThPOK and LRF may reflect inherent functional differences, dictated by their distinct amino acid sequences, or alternative expression patterns. To resolve this question we have generated novel knockin mice in which ThPOK coding exons are precisely replaced by those of LRF (LRF knockin, or LKI mice). LKI mice have been engineered to maintain both DNA- and mRNA-dependent control mechanisms of gene expression, as all DNA cis regulatory elements as well as 5' and 3' untranslated (UT) mRNA regions are derived from the ThPOK locus. Therefore, the LKI allele supports precisely the same level and kinetics of gene expression as the wt ThPOK allele. We will use homozygous LKI/LKI mice to assess whether LRF can rescue defects in T cell development and early hematopoiesis in the absence of ThPOK, in order to definitively establish whether LRF and ThPOK coding sequences encode distinct or redundant functions. We present preliminary evidence that strongly suggests functional specialization. Aim 2. Assessing functional specificity of ThPOK BTB domain using a chimeric gene approach. If ThPOK is functionally distinct from LRF, specialized functions are likely to be encoded at least in part by their BTB/POZ domains, which are critical for dimerization with nuclear co-repressors and histone deacetylases. Despite the essential roles of the ThPOK and LRF BTB domains, their functional specificity/redundancy has never been examined in vivo. Here we will use a knockin approach to selectively replace the BTB domain of ThPOK with that of LRF, or vice versa, and test whether the resulting chimeric factors can restore thymic development of CD4 and iNKT cells in the absence of wt ThPOK. This will establish whether the ThPOK BTB domain is necessary and/or sufficient to replicate the essential role of ThPOK in these processes. These mice will also be valuable for dissecting the specific requirement for the ThPOK BTB domain in other aspects of hematopoiesis in which ThPOK has been implicated, including HSC maintenance and myeloid development.