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，已发现在淋巴细胞发育和肿瘤抑制中发挥关键作用。像许多哺乳动物基因组编码的大约800个C2H2锌指蛋白一样，Ikaros在其DNA结合结构域中含有多个串联锌指。自1992年发现Ikaros以来，人们已经从对Ikaros突变小鼠的优雅研究中了解到了它的生物学功能。然而，其精确的细胞内功能和作用机制仍然是难以捉摸的，主要是因为它似乎没有作为一个典型的转录因子的功能，因为负责其最重要的生物学功能的靶基因一直难以确定。我们已经意识到，我们可以完全理解其作用机制的唯一方法，以及我们可以评估从我们的生化研究中出现的假设的唯一方法，是在天然生理环境中研究Ikaros结构域和生化活动。为此，我们已经引入了特定的突变到内源性Ikzf1基因座，它编码Ikaros。迄今为止，已鉴定了两种突变株。这两种菌株含有编码DNA结合结构域的第一个和最后一个锌指的外显子缺失。创建这些突变小鼠是为了（1）测试这两个指调控与不同靶基因组结合的假设，（2）促进新Ikaros靶基因的发现，以及（3）通过探索锌指串联阵列存在的生物学原因，为更广泛的C2H2锌指领域做出贡献。重要的是，我们发现每个突变株都表现出先前在Ikaros null小鼠中描述的表型的选择子集，这为两个手指调控不同的靶基因和生物学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株的选择性表型，以更好地理解这两个指如何差异调节先前描述的靶基因。在目标2中，利用突变菌株的选择性表型的无偏方法将用于鉴定和表征新的Ikaros靶基因。最后，我们将表征第三种突变小鼠品系，其缺乏参与Ikaros多聚化的关键残基，并且我们将开始使用类似的方法来检查已知与Ikaros相互作用的两种辅阻遏物的功能意义。

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.