Mutations in SIX1 associated with Branchio-oto-renal Syndrome (BOR) differentially affect otic expression of putative target genes

Abstract

Single nucleotide mutations in SIX1 are causative in some individuals diagnosed with branchio-otic/branchio-oto-renal (BOR) syndrome. To test whether these mutations have differential effects on otic gene expression, we engineered four BOR mutations in Xenopus six1 and targeted mutant protein expression to the neural crest and cranial placode precursor cells in wild-type embryos. Changes in the otic expression of putative Six1 targets and/or co-factors were monitored by qRT-PCR and in situ hybridization. We found that each mutant had a different combination of effects. The V17E mutant reduced eya2, tspan13, zbtb16 and pa2g4 otic vesicle expression at a frequency indistinguishable from wild-type Six1, but reduced prdm1 more and spry1 less compared to wild-type Six1. For most of these genes, the R110W, W122R and Y129C mutants were significantly less repressive compared to wild-type Six1. Their individual effects varied according to the level at which they were expressed. The R110W, W122R and Y129C mutants also often expanded prdm1 otic expression. Since previous studies showed that all four mutants are transcriptionally deficient and differ in their ability to interact with co-factors such as Eya1, we propose that altered co-factor interactions at the mutated sites differentially interfere with their ability to drive otic gene expression.