Dissecting Protein Interactions for Developmental Analysis of SO-Cofactor Complexes

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Project Summary/Abstract The sine oculis (SO) gene belongs to the evolutionarily conserved ‘SIX’ family of homeobox transcription factors. Family members control cell fate, morphology, proliferation and survival in multiple tissues and organs of metazoans, including humans. In Drosophila, SO functions during development of the visual system, specific neuroblasts and glia of the central and peripheral nervous systems, endocrine glands, and in specialized somatic cells of the testis and the ovary of the adult fly. Several vertebrate SIX genes, including the SO orthologue SIX1, are required for the normal development of the brain, cranial sensory organs and the kidney; thus mutations in human homologues of SO lead to birth defects, including BOS/BOR (OMIM 601205, 600963), Holoprosencephaly 2 and Schizencephaly (OMIM 603714). SO/SIX1 functions as a transcriptional regulator together with a number of protein cofactors. We and others have shown that these cofactors modify the transcriptional activity of SO in vitro; moreover, in vivo evidence suggests that SO-cofactor complexes contribute to development in specific ways. Thus, in order to understand SO function in specific developmental contexts, we need to define the function of the various complexes. Our preliminary findings provide fundamental evidence that targeted mutagenesis of SO can be used to separately and specifically disrupt two well-known SO-cofactor interactions, one with the transcriptional activator Eya/EYA and the other with the transcriptional repressor Gro/TLE/GRG. In addition, we show that it is possible to assess the function of these SO variants in the organism. We propose here to dissect the role of these complexes in vivo as well as initiate an analysis of all known transcriptional cofactors of SO. The goal of this grant is to demonstrate that we can generate ‘designer SO proteins’ that either lack or retain only one or a few protein-protein interactions, and that we can develop assays for their functional assessment both in vitro and in vivo - in preparation for an R01 submission. Since SO is highly conserved from fly to human and the mouse orthologues, SIX1 and SIX2, can substitute for SO in the fly; most changes that affect partner binding in SO will similarly impact the vertebrate proteins. Thus, the proposed work is directly relevant to the human proteins and their linked human disorders.