| Branchio-oto-renal syndrome (BOR) is an autosomal dominant developmental disorder characterized by hearing loss, branchial arch defects, and renal anomalies. Recently, eight different mutations in the Six1 homeobox gene have been described in BOR patients. In addition, mutations in the Six1 co-factor, Eya1, are also observed in BOR patients, suggesting that the Six1-Eya1 transcriptional complex is critical for normal development of the ears, facial structure and kidney, consistent with mouse knockout studies. I demonstrate using size exclusion chromatography that only the most N-terminal mutation in Six1 (V17E) completely abolishes Six1-Eya complex formation while all of the other mutants are able to form a stable complex. While none of the remaining five Six1 mutations abolish Eya binding, they are all deficient in DNA binding. Taken together, our experiments demonstrate that the Six1 BOR mutations contribute to the pathology of the disease through at least two different mechanisms that involve: (1) abolishing the formation of the Six1-Eya complex or (2) diminishing the ability of Six1 to bind DNA.;The Six1/Eya transcriptional complex plays a role in normal embryonic development by stimulating the proliferation and survival of progenitor cells, as well as by promoting the migration of certain populations of cells. Both Six and Eya genes are turned off in most adult tissues, and are re-expressed in a variety of cancers. It is believed that they contribute to malignant disease by reinitiating developmental pathways that should not be active in the adult, resulting in increased cellular proliferation and survival as well as increased cellular migration. Here we show, by analyzing existing microarray data, that the coordinated over-expression of Six1 and Eya2 correlates with a shortened time to metastasis and significantly decreases overall survival. We also attempt to obtain the 3-dimentional structure of the Six1/Eya complex using X-ray crystallography as a first step towards "rational" structure based drug design. Initial crystallization trials have resulted in diamond shaped crystals that have diffracted to 4 angstroms, and we anticipate that we will be able to determine the structure after further optimization of the crystals. |
