Analysis of zebrafish periderm enhancers facilitates identification of a regulatory variant near human KRT8/18.

Huan Liu,Patrick Breheny,Axel Visel,Camille Mumm,Jenna Colavincenzo Carlson,Robert A Cornell,Fabrice Darbellay,Albert J Erives,Yao Xiao,Kaylia Duncan,Elizabeth Leslie,Annika Helverson,Priyanka Kumari

doi:10.7554/elife.51325

Abstract

Genome-wide association studies for non-syndromic orofacial clefting (OFC) have identified single nucleotide polymorphisms (SNPs) at loci where the presumed risk-relevant gene is expressed in oral periderm. The functional subsets of such SNPs are difficult to predict because the sequence underpinnings of periderm enhancers are unknown. We applied ATAC-seq to models of human palate periderm, including zebrafish periderm, mouse embryonic palate epithelia, and a human oral epithelium cell line, and to complementary mesenchymal cell types. We identified sets of enhancers specific to the epithelial cells and trained gapped-kmer support-vector-machine classifiers on these sets. We used the classifiers to predict the effects of 14 OFC-associated SNPs at 12q13 near KRT18. All the classifiers picked the same SNP as having the strongest effect, but the significance was highest with the classifier trained on zebrafish periderm. Reporter and deletion analyses support this SNP as lying within a periderm enhancer regulating KRT18/KRT8 expression.

Highlights

Orofacial clefting (OFC), which can include cleft lip, cleft palate, or both, is among the most common of structural birth defects
We discovered a second periderm enhancer by searching the genome for elements sharing several ChIP-seq features of IRF6-9.7; we filtered on strong H3K27 acetylation (H3K27Ac) signal (ENCODE data) and peaks of IRF6 47, KLF4 48, and TP63 49 binding, all assessed in normal human epidermal keratinocytes (NHEK)
We undertook an analysis of zebrafish periderm enhancers with two objectives relevant to the genetic underpinnings of orofacial clefting

Summary

Introduction

Orofacial clefting (OFC), which can include cleft lip, cleft palate, or both, is among the most common of structural birth defects. We revisit the meta-analysis results and find an additional 13 SNPs in this locus that have at least a suggestive statistical association to OFC To prioritize these SNPs for functional tests using the deltaSVM method it would be optimal to train a classifier on a set of enhancers that are active in human palate-shelf periderm. If the binding site features of zebrafish periderm enhancers are conserved with those of human periderm enhancers a classifier trained on the former could be used to conduct a successful deltaSVM-based screen for SNPs that disrupt the latter This approach is promising in that the genetic pathways that underlie periderm development in mice and zebrafish are shared, in spite of the fact that the embryonic origins of periderm in the two species are distinct.

Results

Discussion

Materials and Methods