The broad objective of this proposal is to determine how the lineage-determining factors encoded by Ikaros, play a key role in T cell differentiation and function, by mediating changes in chromatin structure. The regulation of chromatin structure via ATP-dependent remodelers, histone deacetylases and possibly through DNA methylases, is important in reprogramming gene expression during differentiation, for maintenance of cellular memory, and for the stable propagation of genetic material. The proposed studies on the function and regulation of the Ikaros- NURD complex, which combines the aforementioned chromatin modifying properties, and its effects on T cell differentiation, will provide us with a paradigm for a basic developmental mechanism. For the first specific aim we will delineate the interaction between Ikaros and the ATPase Mi-2beta of the NURD complex and examine whether their association is regulated through postranslational modifications. Mutations that disable this interaction will be tested for their ability to effect T cell differentiation and activation. For the second specific aim we will examine whether Ikaros partitioning with NURD or other complexes changes during T cell differentiation. Changes in Ikaros-complex partitioning may be responsible for its functional diversity in the hemo-lymphoid system. For the third specific aim we will study targeting of the NURD complex by Ikaros proteins. Sequence specific targeting of the Ikaros-NURD complex will be examined in chromatin remodeling and binding assays in vitro. Natural gene targets for the Ikaros-NURD complex, will be pursued by a CHromatin ImmunoPrecipitation assay, in T cells. In addition, mutations in Ikaros that interfere with its ability to relocate into heterochromatin during the T cell cycle will be examined. For the fourth specific aim we will examine the activity of the Ikaros-NURD complex on chromatin-based in vitro transcription assays and its potential modulation during T cell differentiation. For the fifth specific aim, we will determine the role of the NURD chromatin remodeling complex in T cell differentiation and function using an allelic series of mutations in the Mi-2beta locus. These studies will provide a link between the function of the NURD complex and that of Ikaros in T cell differentiation, and will permit an examination of the effects of the selective loss of the Ikaros-NURD complexes without the disruption of other Ikaros-complexes. Taken together, these studies will establish new molecular underpinnings for the production and regulation of the immune system and will provide insights into their malfestations i.e immuno-deficiency, autoimmunity and neoplastic transformation. An important outcome of these studies will be an increase in our ability to apply molecular intervention to these differentiation processes.
