Biological functions and mechanisms of action of Ikaros proteins

描述(由申请人提供)：Ikaros是C2H2锌指DNA结合蛋白小家族的创始成员，已被发现在淋巴细胞发育和肿瘤抑制中发挥关键作用。与哺乳动物基因组编码的大约800个C2H2锌指蛋白中的许多蛋白一样，Ikaros在其DNA结合域中包含多个串联的锌指蛋白。自从1992年发现Ikaros以来，人们从对Ikaros突变小鼠的优雅研究中了解到了许多关于它的生物学功能。然而，其确切的细胞内功能和作用机制仍不清楚，这主要是因为它似乎不是一个典型的转录因子，而且负责其最重要生物学功能的靶基因一直很难识别。我们已经认识到，我们完全了解其作用机制的唯一方法，以及我们评估从我们的生化研究中出现的假说的唯一方法，就是在自然的生理环境中研究Ikaros结构域和生化活性。为此，我们将特定的突变引入了内源IKZF1基因座，该基因座编码Ikaros。到目前为止，已经确定了两个突变菌株的特征。这两个菌株含有编码DNA结合域的第一个和最后一个锌指的外显子的缺失。这些突变小鼠被创造出来(1)测试这两个手指调节与不同目标基因组结合的假设，(2)促进发现新的Ikaros目标基因，(3)通过探索锌手指串联阵列存在的生物学原因，为更广泛的C2H2锌手指领域做出贡献。重要的是，我们发现每个突变株都表现出先前在Ikaros缺失小鼠中描述的表型的一个精选子集，这为两个手指调节不同的靶基因和生物功能提供了强有力的证据。在目标1中，我们将进一步表征突变菌株的选择性表型，以更好地了解这两个手指如何差异调控先前描述的靶基因。在目标2中，将使用一种利用突变菌株的选择性表型的无偏见的方法来识别和表征新的Ikaros目标基因。最后，我们将鉴定第三个突变小鼠品系，该品系缺乏参与Ikaros多聚体的关键残基，我们将开始使用类似的方法来检查已知与Ikaros相互作用的两个共抑制因子的功能意义。

DESCRIPTION (provided by applicant): Ikaros is the founding member of a small family of C2H2 zinc finger DNA-binding proteins and has been found to play critical roles in lymphocyte development and tumor suppression. Like many of the approximately 800 C2H2 zinc finger proteins encoded by mammalian genomes, Ikaros contains multiple tandem zinc fingers within its DNA-binding domain. Since the discovery of Ikaros in 1992, much has been learned about its biological functions from elegant studies of Ikaros mutant mice. However, its precise intracellular functions and mechanisms of action have remained elusive, largely because it does not appear to function as a typical transcription factor and because target genes responsible for its most important biological functions have been difficult to identify. We have come to realize that the only way we can fully understand its mechanisms of action, and the only way we can evaluate hypotheses that have emerged from our biochemical studies, is to study Ikaros domains and biochemical activities in a native physiological setting. Toward this end, we have introduced specific mutations into the endogenous Ikzf1 locus, which encodes Ikaros. Two mutant strains have been characterized to date. These two strains contain deletions of exons encoding the first and last zinc fingers of the DNA-binding domain. These mutant mice were created (1) to test a hypothesis that these two fingers regulate binding to distinct sets of target genes, (2) to facilitate the discovery of new Ikaros target genes, and (3) to contribute to the broader C2H2 zinc finger field by exploring the biological reason for the existence of tandem arrays of zinc fingers. Importantly, we have found that each mutant strain exhibits a select subset of the phenotypes previously described in Ikaros null mice, providing strong evidence that the two fingers regulate different target genes and biological functions. In Aim 1, we will further characterize selective phenotypes of the mutant strains to better understand how these two fingers differentially regulate previously described target genes. In Aim 2, an unbiased approach that takes advantage of the selective phenotypes of the mutant strains will be used to identify and characterize new Ikaros target genes. Finally, we will characterize a third mutant mouse strains that lacks a key residue involved in Ikaros multimerization, and we will begin to use a similar approach to examine the functional significance of two co-repressors that are known to interact with Ikaros.