Genomic approaches offer a comprehensive and unbiased strategy toward generating relevant data towards determining the function of novel proteins. If, for example, genetic modifier screens, yeast two hybrid, and gene expression analyses, all identify subsets of proteins involved in a particular cellular process, then this would provide strong evidence that the novel protein indeed functioned in that particular pathway or process. Through positional cloning, we identified the novel Tub gene which when mutated in mice causes obesity, and retinal and cochlear degeneration, diseases that cause significant morbidity in the human population. The tubby mouse is also a good model for human sensory loss/obesity syndromes such as Alstrom and Bardet-Biedi. Identifying the function of TUB may, therefore, lead to further understanding of pathways leading to these diseases. TUB belongs to a family of proteins (TULPs) that share a high degree of sequence similarity, and are highly conserved through evolution. TULPs have been found in vertebrates, invertebrates, and plants. Mutations in EULP1 have been shown to cause retinitis pigmentosa in humans and mice, and a disrupted allele of TULP3 in mouse leads to embryonic lethality. The strong sequence conservation and the fact that each family member is found in a different nuclear compartment indicate that this gene family has an important basic cellular function. Although several starkly differing hypotheses about tubby's function have been recently suggested, its biological function is still unknown. In this application, as a first step towards elucidating their biological role, we propose to use an unbiased genomic approach to identify pathways in which TULPs function. We will: a) Identify genes that modify TULP phenotypes in mice. Specifically, we will clone the genes whose resistance alleles protect tubby mice from hearing loss and obesity, respectively, and we will map the gene(s) that prevent embryonic lethality in hererozygous TULP3 knockout mice; and b) identify genes whose products physically interact with TULPs. Specifically, we will carry out yeast two hybrid analyses to identify proteins that interact with TUB and TULP3, and gene expression array analysis to identify expression patterns that are associated with tubby phenotypes. At the successful conclusion of this work, we will have established a framework of interacting genes and proteins and of expression patterns that will allow us to place the TULPs in a biological context and in pathways that they act in. In addition, we will identify two new modifier genes that suppress TULP phenotypes, as well as proteins that directly bind to TUB and TULP3 to mediate their biological function and genes whose expression is regulated by TUB in specific phenotypic contexts such as suppression or induction of obesity. Cumulatively these studies will also provide ready targets for testing specific hypotheses suggested by the genomic data.
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