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

剖析造血关键调节因子 ThPOK 和 LRF 的不同和冗余作用

• 批准号: 9130273
• 负责人: Dietmar J Kappes
• 金额: $ 26.24万
• 依托单位: RESEARCH INST OF FOX CHASE CAN CTR
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-08-20 至 2018-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9130273
• 关键词: Address; Affinity; Alleles; Amino Acid Sequence; B-Lymphocytes; BTB/POZ Domain; Biology; CD4 Positive T Lymphocytes; Cell Maintenance; Cells; Code; DNA; Defect; Development; Dimerization; Engineering; Exons; Family; Gene Expression; Gene Family; Gene Targeting; Genes; Health; Hematopoiesis; Hematopoietic stem cells; Histones; Kinetics; Knock-in; Knock-in Mouse; Maintenance; Maps; Mediating; Messenger RNA; Mus; Myelogenous; Nuclear; Pattern; Play; Post-Transcriptional Regulation; Process; Proteins; Recruitment Activity; Regulatory Element; Role; Specificity; Staging; T-Cell Development; Testing; base; chimeric gene; in vivo; member; novel; transcription factor

 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.

 描述（由申请人证明）：基本的未解决的是，谱系comitmetm的主监管机构的能力是主要来自独特的功能cabilities的命运命运命运还是来自蜂窝上下文的能力。造血的不同阶段关键角色，这些因素的特定功能是完全清晰的，即使是资金的角色，也主要是由于独特的功能能力或FRMP Ressy模式差异所致。在SIS中，固有的函数是THPOK和LRF的非冗余作用，可能反映出固有的功能差异，它们的不同氨基酸序列表达模式。 LKI小鼠已被设计为维持基因表达的DNA和mRNA依赖性控制机制，所有DNA顺式调节元件为5'和3'未翻译（UT）mRNA区域。基因表达的水平和动力学与WT THPOK等位基因相同。冗余功能。 thpok btb域基因方法。 t有一段时间的敲击方法可以选择性地使用LRF的BTB结构域，反之亦然，并在没有WT的情况下测试CD4和Inkt细胞的重新融合嵌合Factore的胸腺发育是必要的。 /或足以复制THPOK在这些过程中的基本作用。