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（由适用提供）：一个基本的未解决的问题是，谱系承诺控制不同脂肪决策的主要监管者的能力是主要来自独特的功能能力还是来自表达的细胞上下文的能力。 THPOK和LRF是转录因子的POK家族的相关成员，在造血的不同阶段具有重要作用。 THPOK控制CD4 T细胞的承诺，而LRF控制B细胞承诺。这些因素在造血的其他阶段也起着关键作用，包括用于造血干细胞维持（请参阅初步结果）。尽管它们具有多个关键作用，但这些因素的基本特定功能仍然待完全阐明，即使是基本问题，即它们的独特作用是由独特的功能能力还是由独特的功能能力或FRM表达模式差异产生的基本问题。在这里，我们建议根据两个具体的目的来阐明这些问题：目标1。造血期间Thpok和LRF媒体固有的不同功能是否存在？ THPOK和LRF的非冗余作用可能反映出固有的功能差异，该功能差异由其独特的氨基酸序列或替代表达模式决定。为了解决这个问题，我们产生了新颖的敲蛋白小鼠，其中ThpoK编码外显子被LRF（LRF敲击蛋白或LKI小鼠）所取代。 LKI小鼠已经设计为维持基因表达的DNA和mRNA依赖性控制机制，因为所有DNA顺式调节元件以及5'和3'未翻译（UT）mRNA区域均来自THPOK基因座。因此，LKI等位基因与WT THPOK等位基因完全支持基因表达的水平和动力学。我们将使用纯合LKI/LKI小鼠评估LRF在没有THPOK的情况下是否可以挽救T细胞发育和早期造血的缺陷，以定义LRF和THPOK编码序列是否编码不同的或冗余的功能。我们提供了强烈建议功能专业化的初步证据。目标2。评估功能特异性 使用嵌合基因方法的ThPOK BTB结构域的构造。如果THPOK在功能上与LRF不同，那么专业功能可能至少部分由其BTB/POZ结构域编码，这对于与核共抑制剂和Hisstone脱乙酰基酶二聚化至关重要。尽管THPOK和LRF BTB结构域具有重要的作用，但它们的功能特异性/冗余从未在体内进行检查。在这里，我们将使用敲蛋白方法选择性地用LRF的BTB结构域，反之亦然，并测试在没有WT THPOK的情况下，所得嵌合因子是否可以恢复CD4和InkT细胞的胸腺开发。这将确定THPOK BTB域是否需要和/或足以复制Thpok在这些过程中的基本作用。这些小鼠在造血的其他方面也暗示了THPOK的其他方面，包括HSC维护和髓样发育，这些小鼠也将很有价值。