Analysis of zebrafish periderm enhancers facilitates identification of a regulatory variant near humanKRT8/18

Abstract

Genome wide association studies for non-syndromic orofacial cleft (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 effect 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.

Two non-coding variants associated with isolated orofacial cleft promote binding of transcriptional repressors FOXE1 or ETS2 and reduce expression of IRF6

“…Interestingly, IRF6 -10kb SNP is the fourth example of a potentially functional SNP associated with orofacial cleft within the same evolutionarily conserved enhancer of IRF6, MCS9.7 6,37,53,60,61,78 . The first of these to be identified, rs642961, is associated with CL and the risk-associated allele disrupted binding of the transcription factor AP2-α (TFAP2A) in an electrophoretic mobility shift assay 60 .…”

“…S8B,C). The first candidate SNP near IRF6, rs11119348, lies within an element named "multi species conserved sequence 9.7 kb from IRF6" (MCS9.7) 60 that has enhancer activity in murine embryonic oral epithelium 61 and in zebrafish periderm 6 . This element is the site of a single-base-pair duplication that appears to cause Van der Woude syndrome 61 , and contains three other SNPs, in addition to rs11119348, that are associated with non-syndromic OFC, including rs642961, the focus of an earlier study 60 .…”

High incidence and regional distribution of cleft palate in Finns are associated with a functional variant in an IRF6 enhancer

“…H3K27Ac ChIP-Seq data from HOIEC oral epithelium cells and ATAC-Seq (Assay for Transposase-Accessible Chromatin using sequencing) data from the HOIEC oral epithelium cells and the HEPM oral mesenchyme cells were generated previously. 42 In silico transcription factor binding activities at the risk variant site were determined using HOMER 43 and the Transcription factor Affinity Prediction (TRAP) tool 44…”

“…An additional six SNPs were in strong LD (r 2 > 0.8) with the lead SNP rs570516915. To rule out the other cis-regulatory regions that may contain the causal variant in LD with rs570516915, we examined relevant chromatin marks at each of the top seven SNPs including: i) ATAC-seq peaks, which reveal open chromatin, from the HOIEC oral epithelium cells and the HEPM oral mesenchyme cells, 42 ii) H3K27Ac peaks, revealing active enhancers and promoters, from the HOIEC cells, 42 and iii) aggregate chromatin mark data from 9 principal cell types from the ENCODE project 40 and from human embryonic facial enhancers datasets 41 . Of these seven SNPs, two SNPs, rs570516915 and rs556188853, fall into regions with chromatin marks consistent with enhancer activity in epithelial cells (HOIEC and NHEK) (Figure 4A and Figure S5C).…”

TFAP2 paralogs facilitate chromatin access for MITF at pigmentation genes but inhibit expression of cell-cell adhesion genes independently of MITF

“…ATAC-seq was performed according to (Buenrostro et al, 2015;Liu et al, 2020) Library quality was assessed using a BioAnalyzer 2100 High Sensitivity DNA Chip (Agilent Technologies). All DNA libraries that exhibited a nucleosome pattern were pooled and processed for 150bp paired-end sequencing.…”