Probing the Functions of KLF2 Downregulation

 描述（由申请人提供）：KLF 2是Kruppel样转录因子家族的成员，其控制许多不同细胞类型中的不同过程。KLF 2在多种细胞和组织中的功能已被证实，包括肺发育、血管壁细胞迁移、脂肪细胞发育和红细胞生成。在免疫系统中，KLF 2还具有多种功能，包括调节T和B细胞运输、维持骨髓细胞静止和内皮细胞中的抗炎作用。最近，KLF 2已被牵连在调节辅助性T细胞分化，但这一功能的机制尚不清楚。 KLF 2在淋巴细胞转录因子中有些独特，因为其表达在通过Ag受体（TCR和BCR）活化后迅速丧失，并且这种表达丧失是在mRNA和蛋白质水平上。类似的过程发生在骨髓细胞和内皮细胞中，其中KLF 2的条件性失活导致活化的促炎状态。KLF 2的下调是否是其他细胞类型（包括肺、血管、脂肪细胞和早期红细胞）发育或功能的重要方面尚不清楚。 Ag受体结合后KLF 2的下调在转录和翻译后水平迅速发生，通常在约8-12小时内。介导转录沉默的机制尚未确定。相反，已经确定KLF 2通过充分研究的泛素-蛋白体系统在蛋白水平下调，并且已经鉴定了在各种非造血细胞中介导这种作用的几种E3泛素连接酶。此外，已经鉴定了泛素化所必需的关键赖氨酸残基，并且将该赖氨酸突变为精氨酸完全阻断了KLF 2的蛋白水解破坏。然而，这些研究都没有解决为什么会发生这种情况的基本问题，也没有确定任何细胞类型中需要KLF 2下调的生物学功能。 我们将通过生产一种新的小鼠品系来解决这个问题，在这种小鼠品系中，KLF 2的表达可以在表达cre重组酶的任何细胞类型中被加强（Aim 1），并使用这种小鼠来理解T细胞区室中KLF 2下调的功能（Aim 2）。此外，我们生产的突变小鼠也将是在许多其他细胞类型中解决这个问题的理想选择。由于KLF 2下调是许多（如果不是大多数或所有）表达它的细胞类型的特征，因此该项目将在该领域产生重大影响。

 DESCRIPTION (provided by applicant): KLF2 is a member of the Kruppel-like family of transcription factors, which control diverse processes in many different cell types. KLF2 has documented functions in a wide variety of cells and tissues, including lung development, vascular mural cell migration, adipocyte development, and erythropoiesis. Within the immune system, KLF2 also subserves multiple functions, including regulation of T and B cell traffic, maintenance of myeloid cell quiescence, and anti-inflammatory actions in endothelial cells. More recently, KLF2 has been implicated in regulation of T helper differentiation, but mechanisms underlying this function are not yet clear. KLF2 is somewhat unique among lymphocyte transcription factors in that its expression is rapidly lost upon activation through Ag receptors (TCR and BCR), and this loss of expression is at both the mRNA and protein levels. A similar process occurs in both myeloid cells and in endothelium, where conditional inactivation of KLF2 leads to an activated, pro-inflammatory state. Whether downregulation of KLF2 is an essential aspect of the development or function of other cell types, including lung, blood vessels, adipocytes, and early erythrocytes, is unknown. Downregulation of KLF2 following Ag receptor engagement occurs rapidly at both the transcriptional and post-translational levels, generally within ~8-12 hours. Mechanisms which mediate transcriptional silencing have not been identified. In contrast, it is well established that KLF2 downregulation at the protein level s via the well studied ubiquitin-proteosome system, and several E3 ubiquitin ligases which mediate this effect in various non-hematopoietic cells have been identified. Further, the key lysine residue essential for ubiquitination has been identified, and mutation of this lysine to arginine completely blocks proteolytic destruction of KLF2. However, none of these studies have addressed the basic question of why this occurs, and no biological functions in any cell types have been identified which require KLF2 downregulation. We will address this issue by producting a novel mouse strain in which KLF2 expression can be enforced in any cell type which expresses the cre recombinase (Aim 1), and use this mouse to understand the functions of KLF2 downregulation in the T cell compartment (Aim 2). Further, the mutant mice which we produce will be ideal for approaching this question in numerous other cell types as well. As KLF2 downregulation is a feature of many, if not most or all cell types in which it is expressed, this project will make a major impact in the field.