转录因子PAX6的无义和错义突变引起广泛的眼部发育缺陷,包括无虹膜,小眼症和结肠瘤。为了了解PAX6:DNA结合的变化如何导致这些表型,我们将PAX6配对结构域的饱和诱变与酵母单杂交(Y1H)分析相结合,其中PAX6-GAL4融合基因的表达驱动抗生素抗性.我们定量了超过2700个单一氨基酸变体与两个DNA序列元件的结合。N端亚结构域和接头区的面向DNA残基的突变是最有害的,脯氨酸和带负电荷的残基的突变。许多变体引起序列特异性分子功能增益效应,包括在71位的变体,其增加与LE9增强子的结合但降低与SELEX衍生的结合位点的结合。在没有抗生素选择的情况下,保留DNA结合的变体减缓了酵母的生长,可能是因为这些变异扰乱了酵母转录组。针对已知患者变异进行基准测试,并将ACMG/AMP指南应用于变异分类,我们获得了支持性到中度证据,即977个变异体可能是致病性的,1306个变异体可能是良性的.我们的分析表明,PAX6配对结构域中的大多数致病突变可以简单地通过这些突变对PAX6的影响来解释:DNA关联,并建立了Y1H作为解释转录因子变异效应的通用测定法。
Nonsense and missense mutations in the transcription factor PAX6 cause a wide range of eye development defects, including aniridia, microphthalmia and coloboma. To understand how changes of PAX6:DNA binding cause these phenotypes, we combined saturation mutagenesis of the paired domain of PAX6 with a yeast one-hybrid (Y1H) assay in which expression of a PAX6-GAL4 fusion gene drives antibiotic resistance. We quantified binding of more than 2700 single amino-acid variants to two DNA sequence elements. Mutations in DNA-facing residues of the N-terminal subdomain and linker region were most detrimental, as were mutations to prolines and to negatively charged residues. Many variants caused sequence-specific molecular gain-of-function effects, including variants in position 71 that increased binding to the LE9 enhancer but decreased binding to a SELEX-derived binding site. In the absence of antibiotic selection, variants that retained DNA binding slowed yeast growth, likely because such variants perturbed the yeast transcriptome. Benchmarking against known patient variants and applying ACMG/AMP guidelines to variant classification, we obtained supporting-to-moderate evidence that 977 variants are likely pathogenic and 1306 are likely benign. Our analysis shows that most pathogenic mutations in the paired domain of PAX6 can be explained simply by the effects of these mutations on PAX6:DNA association, and establishes Y1H as a generalisable assay for the interpretation of variant effects in transcription factors.