Deep mutational scanning quantifies DNA binding and predicts clinical outcomes of PAX6 variants
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 2,700 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 Ile71 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 to suggest that 1,306 variants are likely benign, and 977, likely pathogenic. 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. Overall design: To investigate the effects on DNA-binding of amino acid substitutions in paired domain of the human transcription factor PAX6, we established a deep mutational scanning assay that employed a yeast one-hybrid functional readout and a competitive growth assay. Each yeast cell contained one of two genomically integrated PAX6-binding sequences (one assay per binding sequence; LE9 and BLX) upstream of an antibotic resistance gene, and expressed, from plasmid DNA, a single PAX6 variant fused to GAL4 activation domain. Binding of PAX6 to the binding sequence drove expression of the antibiotic resitance gene, growth rate in the presence of antibotic served as a proxy for PAX6:DNA perturbation. Each PAX6 variant was phased with a 30-nt barcode. Enrichment or depletion of each variant in the culture was quanitified using deep sequencing of barcodes before (timepoint 0) and after (timepoints 1-3) the competitive growth assay. Libraries were prepared from plasmids isolated from aliquots of each timepoint and PCR-amplified to append illumina-compatible adapters and dual indexes.