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.

In developing melanocytes and in melanoma cells, multiple paralogs of the Activating-enhancer-binding Protein 2 family of transcription factors (TFAP2) contribute to expression of genes encoding pigmentation regulators, but their interaction with Microphthalmia transcription factor (MITF), a master regulator of these cells, is unclear. Supporting the model that Tfap2 facilitates MITF’s ability to activate expression of pigmentation genes, single-cell seq analysis of zebrafish embryos revealed that pigmentation genes are only expressed in the subset of mitfa-expressing cells that also express Tfap2 paralogs. To test this model in SK-MEL-28 melanoma cells we deleted the two TFAP2 paralogs with highest expression, TFAP2A and TFAP2C, creating TFAP2 knockout (TFAP2-KO) cells. We then assessed gene expression, chromatin accessibility, binding of TFAP2A and of MITF, and the chromatin marks H3K27Ac and H3K27Me3 which are characteristic of active enhancers and silenced chromatin, respectively. Integrated analyses of these datasets indicate TFAP2 paralogs directly activate enhancers near genes enriched for roles in pigmentation and proliferation, and directly repress enhancers near genes enriched for roles in cell adhesion. Consistently, compared to WT cells, TFAP2-KO cells proliferate less and adhere to one another more. TFAP2 paralogs and MITF co-operatively activate a subset of enhancers, with the latter necessary for MITF binding and chromatin accessibility. By contrast, TFAP2 paralogs and MITF do not appear to co-operatively inhibit enhancers. These studies reveal a mechanism by which TFAP2 profoundly influences the set of genes activated by MITF, and thereby the phenotype of pigment cells and melanoma cells.

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.

Introduction Genome wide association studies (GWAS) have identified loci associated with risk for non‐syndromic cleft lip with or without cleft palate (NSCL/P). At every locus there are multiple single nucleotide polymorphisms (SNPs) associated with disease and it is a challenge to distinguish functional SNPs from those merely in linkage disequilibrium with functional SNPs. Objective At many of the associated loci, the presumed risk‐relevant gene is expressed in oral epithelium. We hypothesize that at such loci functional SNPs have allele‐specific effects on the activity of oral epithelium enhancers. Our objective is to identify the candidate functional SNPs for NSCL/P at each of the loci identified by GWAS. Materials and Methods To test SNPs for their effects on enhancer activity we carry out massively parallel reporter assays (MPRAs) on 890 SNPs from 8 loci in the GMSM‐K human fetal oral epithelium cell line. To test the SNP‐target gene association we use the Activity‐by‐Contact method, which incorporates open chromatin, H3K27Ac, and HiC data. To confirm MPRA results on top‐scoring SNPs we perform luciferase reporter assays. We use CRISPR‐mediated homology directed repair to engineer GMSM‐K cells to be homozygous for risk or non‐risk alleles for promising SNPs and assess the expression level of the target gene in engineered cells of each genotype. Assays that pass these assays are tested in allele‐specific reporter assays in zebrafish and mouse embryos. Results Using the described methods we tested 608 NSCL/P‐associated SNPs in the IRF6 locus and 11 such SNPs in the FOXE1 locus. Two SNPs in the first group and one in the second had consistent allele‐specific effects in the MPRA and in luciferase assays. Importantly, for one SNP in the IRF6 locus, we have engineered cells to be homozygous for the non‐risk‐associated or risk‐associated allele and found expression of IRF6 is lower in the latter. In vivo enhancer reporter assays in mouse and zebrafish embryos are ongoing. Conclusion Out of more than 600 NSCL/P‐risk‐associated SNPs in the IRF6 locus, we have evidence that a particular one directly contributes to pathogenesis for this disorder. Significance This study illustrates a method to screen among multiple non‐coding SNPs identified by GWAS to identify candidates for those that are functional. Moreover, identifying the functional SNP near IRF6 is a step towards illuminating how a common variant contributes risk for this disorder.

Introduction: Strong evidence suggests physical activity may prevent cognitive decline. Less research has explored potential associations of physical activity with brain structure. This is an important research gap given that age related alterations in brain structure are thought to occur prior to signs of cognitive decline. Objective: To determine if accelerometer measured sedentary time (SED), light-intensity physical activity (LPA), and moderate-to-vigorous intensity physical activity (MVPA) at ages 38-50 years are prospectively associated with brain structure. Methods: We studied 562 Black and White men and women enrolled in CARDIA who participated in the 2005-06 exam (baseline) and exams 5 (2010-11) and/or 10 years later (2015-16). SED, LPA, and MVPA were measured by the ActiGraph 7164 accelerometer at baseline. Magnetic resonance imaging (MRI) was used to quantify whole brain, white matter, gray matter, and abnormal tissue volume of white matter at the 5- and/or 10-year follow-up. Compositional isotemporal substitution analysis examined associations of SED, LPA, and MVPA at baseline with repeated measures (unstructured covariance) of MRI measures at follow-up after accounting for intracranial volume and adjusting for confounders ( Table ). Results: Consistent with our hypothesis, substituting 30 minutes of SED to LPA was associated with 0.044% greater total white matter volume (95% CI: 0.001, 0.086). The effect size was larger when substituting SED to MVPA, but this association was not statistically significant. There were no associations observed between accelerometer measures and total brain, total gray matter, or abnormal white matter tissue volumes. Associations did not differ by sex or age. Conclusions: Statistical substitution of time from SED to higher intensity physical activity (LPA or MVPA) was associated with greater total white matter volume. It is unclear why this association was observed in white matter only. Additional research with longer follow-up is needed to confirm these findings.