The Ikaros family of transcription factors includes 4 related zinc finger DNA-binding proteins (Ikaros, Aiolos, Helios, Eos) that are expressed mainly in hematopoietic cells, where they are essential for immune cell differentiation and function. Studies in mouse and man have demonstrated the absolute requirement for these proteins in coordinating gene expression during B and T cell development, and as tumor suppressors, whose loss-of-function mutations promote leukemia expansion. These results have revealed with surprising clarity that Ikaros proteins function in a non-redundant fashion in different cell types and stages of differentiation. Yet their underlying mechanisms of action remain unclear. Some studies have shown that Ikaros family proteins can act as transcriptional activators and repressors, through binding to a common GGGAA motif that is present in both proximal and distal regions of target genes. Other studies have indicated that Ikaros family proteins can function as homo- and heterodimers, each with potentially different mechanisms that depend at least in part on the overall chromatin environment and interacting partners, including the NuRD nucleosome remodeling and deacetylase complex, the SWI-SNF ATPase-dependent chromatin remodeling complex, and the PRC2 polycomb repressive complex. These complex scenarios, investigated in different systems, have been difficult to interpret, and they have often led to generalized conclusions about the Ikaros family as a whole. We hypothesize that Ikaros family proteins are unique in their binding specificity and protein interaction, and that these differences underlie their disparate biological activities. Understanding the differences is important because Ikaros family proteins are associated with diverse diseases, and it is critical to gain a clear view of their biological and molecular functions in order to design better ways to block or promote their activities. In this grant, we propose to evaluate the specificity of Ikaros family members at 3 levels: at the physiological level, by investigating a novel requirement for Ikaros family proteins in the DNA damage response of hematopoietic stem cells; at the molecular level, by investigating the regulation of gene expression, DNA binding specificities and protein interaction of Ikaros family homo- and heterodimers; and at the atomic level, by solving the 3-dimensional structure of human Ikaros. Specifically, we will: Aim 1 - Determine the physiological role of Ikaros family proteins in hematopoietic stem cells (HSC). We will study the DNA damage response of Helios-deficient HSCs and immediate progeny (Task 1). We will investigate the mechanism of Helios function (direct gene regulation vs. cooperation with p53 and NuRD complexes) (Task 2). We will determine how Helios differs from Ikaros and Eos in these cells (Task 3). Aim 2 - Define the molecular specificity of the Ikaros family. We will generate novel cellular systems to study homo- and heterodimer function (Task 4). We will investigate the ability of Ikaros family homodimers to modulate gene expression and differentiation in vitro (Task 5). We will determine the target gene repertoire of Ikaros family homo- and heterodimers (Task 6). We will identify their protein partners (Task 7). Aim 3 - Investigate the structure-function relationship of human Ikaros with DNA. We will solve the 3D structure of full-length Ikaros in the presence or absence of target DNA (Task 8). We will predict the consequences of pathological IKZF1 mutations on protein function (Task 9). Our proposed project will thus address fundamental questions of gene regulation by the Ikaros family. We expect that our results will have enormous biomedical potential for immunity and cancer.