Summary While the Notch signaling pathway is one of the most intensely studied intracellular signaling pathways, the mechanisms by which Notch signaling regulates transcription remain incompletely understood. Here we report that B-cell leukemia/lymphoma 6 (BCL6), a transcriptional repressor, is a Notch-associated factor. BCL6 is necessary to maintain the expression of Pitx2 in the left lateral plate mesoderm during the patterning of left-right asymmetry in Xenopus embryos. For this process, BCL6 forms a complex with BCL6 co-repressor (BCoR) on the promoters of selected Notch target genes such as enhancer of split related 1. BCL6 also inhibits the transcription of these genes by competing for the Notch1 intracellular domain, preventing the co-activator Mastermind-like1 (MAM1) from binding. These results define a mechanism restricting Notch-activated transcription to cell-type-appropriate subsets of target genes, and elucidate its relevance in vivo, during left-right asymmetric development.

Ocular developmental anomalies (ODA) such as anophthalmia/microphthalmia (AM) or anterior segment dysgenesis (ASD) have an estimated combined prevalence of 3.7 in 10,000 births. Mutations in SOX2 are the most frequent contributors to severe ODA, yet account for a minority of the genetic drivers. To identify novel ODA loci, we conducted targeted highthroughput sequencing of 407 candidate genes in an initial cohort of 22 sporadic ODA patients. Patched 1 (PTCH1), an inhibitor of sonic hedgehog (SHH) signaling, harbored an enrichment of rare heterozygous variants in comparison to either controls, or to the other candidate genes (four missense and one frameshift); targeted resequencing of PTCH1 in a second cohort of 48 ODA patients identified two additional rare nonsynonymous changes. Using multiple transient models and a CRISPR/Cas9-generated mutant, we show physiologically relevant phenotypes altering SHH signaling and eye development upon abrogation of ptch1 in zebrafish for which in vivo complementation assays using these models showed that all six patient missense mutations affect SHH signaling. Finally, through transcriptomic and ChIP analyses, we show that SOX2 binds to an intronic domain of the PTCH1 locus to regulate PTCH1 expression, findings that were validated both in vitro and in vivo. Together, these results demonstrate that PTCH1 mutations contribute to as much as 10% of ODA, identify the SHH signaling pathway as a novel effector of SOX2 activity during human ocular development, and indicate that ODA is likely the result of overactive SHH signaling in humans harboring mutations in either PTCH1 or SOX2.

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