SIX1 mutations cause branchio-oto-renal syndrome by disruption of EYA1-SIX1-DNA complexes - PubMed (original) (raw)

. 2004 May 25;101(21):8090-5.

doi: 10.1073/pnas.0308475101. Epub 2004 May 12.

Pin-Xian Xu, Derek Silvius, Edgar A Otto, Frank Beekmann, Ulla T Muerb, Shrawan Kumar, Thomas J Neuhaus, Markus J Kemper, Richard M Raymond Jr, Patrick D Brophy, Jennifer Berkman, Michael Gattas, Valentine Hyland, Eva-Maria Ruf, Charles Schwartz, Eugene H Chang, Richard J H Smith, Constantine A Stratakis, Dominique Weil, Christine Petit, Friedhelm Hildebrandt

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SIX1 mutations cause branchio-oto-renal syndrome by disruption of EYA1-SIX1-DNA complexes

Rainer G Ruf et al. Proc Natl Acad Sci U S A. 2004.

Abstract

Urinary tract malformations constitute the most frequent cause of chronic renal failure in the first two decades of life. Branchio-otic (BO) syndrome is an autosomal dominant developmental disorder characterized by hearing loss. In branchio-oto-renal (BOR) syndrome, malformations of the kidney or urinary tract are associated. Haploinsufficiency for the human gene EYA1, a homologue of the Drosophila gene eyes absent (eya), causes BOR and BO syndromes. We recently mapped a locus for BOR/BO syndrome (BOS3) to human chromosome 14q23.1. Within the 33-megabase critical genetic interval, we located the SIX1, SIX4, and SIX6 genes, which act within a genetic network of EYA and PAX genes to regulate organogenesis. These genes, therefore, represented excellent candidate genes for BOS3. By direct sequencing of exons, we identified three different SIX1 mutations in four BOR/BO kindreds, thus identifying SIX1 as a gene causing BOR and BO syndromes. To elucidate how these mutations cause disease, we analyzed the functional role of these SIX1 mutations with respect to protein-protein and protein-DNA interactions. We demonstrate that all three mutations are crucial for Eya1-Six1 interaction, and the two mutations within the homeodomain region are essential for specific Six1-DNA binding. Identification of SIX1 mutations as causing BOR/BO offers insights into the molecular basis of otic and renal developmental diseases in humans.

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Figures

Fig. 1.

Fig. 1.

Loss-of-function mutations detected in exons of the SIX1 gene of BOR/BO families F1038 (A), F1120 (B), and K6/7 (C), shown in relation to normal controls.

Fig. 2.

Fig. 2.

SIX1 mutations identified in BOR/BO patients affect protein–protein and protein–DNA interactions. Alignment of the Six-domain (SD) and homeodomain (HD) regions of mouse Six1, human SIX1–6, and the fly So proteins (GenBank accession nos. XP_138167, NP_005973, and NP_476733, respectively). Note that only a partial sequence of the SIX5 cDNA has been deposited in the databases. The tetrapeptides subdividing Six family proteins are boxed (22). Three SIX1 mutations (R110W, Y129C, and delE133) identified from BOR patients are circled. Amino acid position is numbered according to human SIX1 protein sequence. R110 and E133 are common to all Six proteins isolated so far, and Y129 is also found to be highly conserved among the Six1 gene products as well as the Drosophila so gene product.

Fig. 3.

Fig. 3.

(A) Yeast two-hybrid analysis. Cotransformants were analyzed for their ability to activate lacZ expression by liquid β-gal assay. Cotransformants of Gal4AD–Eya1D prey construct with pGBT9 vector alone was used as a negative control. Strength of interactions was judged by the units of β-gal activity. A result typical of three independent experiments (each performed in triplicate), which yielded essentially the same results, is shown with the standard deviation. (B) Gel-mobility shift assay. GST-fusion proteins of Six1 wild type and its mutants were incubated with a labeled multimerized (six copies) MEF3 motif (underlined). GST alone was incubated with the same DNA probe as a negative control. DNA probe alone was loaded onto the gel (lane 1). Arrow points to the shifted complex.

Fig. 4.

Fig. 4.

SIX1 mutations abolish the activation of myogenin promoter MEF3 by coexpression of Six1 and Eya1. The myogenin luciferase reporter pGL3–6×MEF3 was cotransfected with pcDNA3-Six1, pcDNA3-Six1-R110W, pcDNA3-Six1-Y129C, pcDNA3-Six1-delE133, pFlag-Eya1, or both the Six1 and Eya1 plasmids together in HEK293 cells. Luciferase activity in the cell lysate was normalized with β-gal activity of pCMVβ-gal as an internal control. The activity at each data point is relative to that obtained by the control pCMV vector. The mean fold activation from three independent experiments (each performed in duplicate) is shown with the standard deviation.

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