In an effort to understand the role of Distal-less 3 (Dlx3) in cutaneous biology and pathophysiology, we generated and characterized a mouse model with epidermal ablation of Dlx3. K14cre;Dlx3 Kin/f mice exhibited epidermal hyperproliferation and abnormal differentiation of keratinocytes. Results from subsequent analyses revealed cutaneous inflammation that featured accumulation of IL-17-producing CD4 + T, CD8 + T, and γδ T cells in the skin and lymph nodes of K14cre;Dlx3 Kin/f mice. The gene expression signature of K14cre;Dlx3 Kin/f skin shared features with lesional psoriatic skin, and Dlx3 expression was markedly and selectively decreased in psoriatic skin. Interestingly, cultured Dlx3 null keratinocytes triggered cytokine production that is potentially linked to inflammatory responses in K14cre;Dlx3 Kin/f mice. Thus, Dlx3 ablation in epidermis is linked to altered epidermal differentiation, barrier development, and IL-17-associated skin inflammation. This model provides a platform that will allow the systematic exploration of the contributions of keratinocytes to cutaneous inflammation.barrier function | psoriasis | inflammatory diseases | homeobox transcription factor | mouse model

Genetic variants affecting gene-expression levels are a major source of phenotypic variation. The approximate locations of these variants can be mapped as expression quantitative trait loci (eQTLs); however, a major limitation of eQTLs is their low resolution, which precludes investigation of the causal variants and their molecular mechanisms. Here we report RNA-seq and full genome sequences for 85 diverse isolates of the yeast -including wild, domesticated, and human clinical strains-which allowed us to perform eQTL mapping with 50-fold higher resolution than previously possible. In addition to variants in promoters, we uncovered an important role for variants in 3'UTRs, especially those affecting binding of the PUF family of RNA-binding proteins. The eQTLs are predominantly under negative selection, particularly those affecting essential genes and conserved genes. However, applying the sign test for lineage-specific selection revealed the polygenic up-regulation of dozens of biofilm suppressor genes in strains isolated from human patients, consistent with the key role of biofilms in fungal pathogenicity. In addition, a single variant in the promoter of a biofilm suppressor,, showed the strongest genome-wide association with clinical origin. Altogether, our results demonstrate the power of high-resolution eQTL mapping in understanding the molecular mechanisms of regulatory variation, as well as the natural selection acting on this variation that drives adaptation to environments, ranging from laboratories to vineyards to the human body.

Despite recent advances in our ability to detect adaptive evolution involving the cis-regulation of gene expression, our knowledge of the molecular mechanisms underlying these adaptations has lagged far behind. Across all model organisms, the causal mutations have been discovered for only a handful of gene expression adaptations, and even for these, mechanistic details (e.g. the trans-regulatory factors involved) have not been determined. We previously reported a polygenic gene expression adaptation involving down-regulation of the ergosterol biosynthesis pathway in the budding yeast Saccharomyces cerevisiae. Here we investigate the molecular mechanism of a cis-acting mutation affecting a member of this pathway, ERG28. We show that the causal mutation is a two-base deletion in the promoter of ERG28 that strongly reduces the binding of two transcription factors, Sok2 and Mot3, thus abolishing their regulation of ERG28. This down-regulation increases resistance to a widely used antifungal drug targeting ergosterol, similar to mutations disrupting this pathway in clinical yeast isolates. The identification of the causal genetic variant revealed that the selection likely occurred after the deletion was already present at high frequency in the population, rather than when it was a new mutation. These results provide a detailed view of the molecular mechanism of a cis-regulatory adaptation, and underscore the importance of this view to our understanding of evolution at the molecular level.

Genetic variants affecting gene expression levels are a major source of phenotypic variation. The approximate locations of these variants can be mapped as expression quantitative trait loci (eQTLs); however a major limitation of eQTLs is their low resolution, which precludes investigation of the causal variants and their molecular mechanisms. Here we report RNA-seq and full genome sequences for 85 diverse isolates of the yeast Saccharomyces cerevisiae-including wild, domesticated, and human clinical strains-which allowed us to perform eQTL mapping with 50-fold higher resolution than previously possible. In addition to variants in promoters, we uncovered an important role for variants in 3' untranslated regions, especially those affecting binding of the PUF family of RNA-binding proteins. The eQTLs are predominantly under purifying selection, particularly those affecting essential genes and conserved genes. However, applying the sign test for lineage-specific selection revealed the polygenic upregulation of dozens of biofilm suppressor genes in strains isolated from human patients, consistent with the key role of biofilms in fungal pathogenicity. In addition, a single variant in the promoter of a biofilm suppressor, NIT3, showed the strongest genome-wide association with clinical origin. Altogether our results demonstrate the power of high-resolution eQTL mapping in understanding the molecular mechanisms of regulatory variation, as well as the natural selection acting on this variation that drives adaptation to environments ranging from laboratories to vineyards to the human body.

Sun-exposure is a key environmental variable in the study of human evolution. Several skin-pigmentation genes serve as classical examples of positive selection, suggesting that sun-exposure has significantly shaped worldwide genomic variation. Here we investigate the interaction between genetic variation and sun-exposure, and how this impacts gene expression regulation. Using RNA-Seq data from 607 human skin samples, we identified thousands of transcripts that are differentially expressed between sun-exposed skin and non-sun-exposed skin. We then tested whether genetic variants may influence each individual’s gene expression response to sun-exposure. Our analysis revealed 10 sun-exposure-dependent gene expression quantitative trait loci (se-eQTLs), including genes involved in skin pigmentation (SLC45A2) and epidermal differentiation (RASSF9). The allele frequencies of the RASSF9 se-eQTL across diverse populations correlate with the magnitude of solar radiation experienced by these populations, suggesting local adaptation to varying levels of sunlight. These results provide the first examples of sun-exposure-dependent regulatory variation and suggest that this variation has contributed to recent human adaptation.

With the rise in the prevalence of type 2 diabetes (T2D), as well as undiagnosed cases of T2D and prediabetes (25% and 90%, respectively), early detection is imperative to minimize individual and societal burden. T2D is highly heritable, and personal genetic information is increasingly available to the general public. Studies have suggested that T2D risk reduction strategies may be more effective for individuals with high T2D genetic risk, supporting the use of genetics as a screening tool to inform cost-effective interventions. We trained a polygenic risk score (PRS) for T2D based on >1,200 genotyped variants in >600,000 European consented research participants from a consumer genetic database who self-reported if they had been diagnosed with T2D. We tested the PRS' performance in separate sets of participants covering five different ancestries (African-American, East-Asian, European, Latino, and South-Asian; ~ 600,000 participants). The area under the receiver-operator curve of this PRS varied from 0.65 to 0.57, performing best in European and worst in African ancestries. The PRS was calibrated separately in each ancestry to account for differences in T2D prevalence. European participants with a PRS in the top 5% of the distribution have a T2D odds ratio of more than 3, and lifetime risk for this group exceeds 65%. Our PRS is strongly correlated with an independently derived T2D PRS from (Scott et al. 2017 GWAS, Spearman rho=0.44, p < 1x10^-200) but is more predictive in our dataset (AUC 0.65 vs. 0.59). Lastly, we defined an "increased likelihood" result based on the PRS threshold at which risk of T2D from genetics alone exceeds the risk of T2D due to being overweight. In our database, 19% of individuals met this criterion. We find that personalized PRS' have the potential to identify large numbers of individuals with increased T2D susceptibility equal to or greater than known risk factors and could prove useful in evaluating T2D risk at individual as well as population levels. Disclosure M.L. Multhaup: Employee; Self; 23andMe. R. Kita: Employee; Self; 23andMe. N. Eriksson: None. S. Aslibekyan: Employee; Self; 23andMe. J. Shelton: None. R.I. Tennen: Employee; Self; 23andMe. E. Kim: Employee; Self; 23andMe, inc. B. Koelsch: Employee; Self; 23andMe.

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and 0.1 mg/injection in vivo. Cytotoxicity was assessed by cell proliferation and colony formation assays. Apoptosis was analyzed using the Annexin V-FITC Apoptosis Detection Kit. Cell cycle analysis was performed using flow cytometry. Markers of DNA damage response was performed using Western blot analysis. Tumor growth delay experiments were performed in mice using orthotopic UM-SCC1 xenografts and heterotopic UM-SCC47 xenografts. Results: We found that the addition of CHKi to the EGFR inhibitor cetuximab with and without radiotherapy significantly decreased cell proliferation and survival fraction in HPV-positive and HPV-negative HNSCC cell lines. Reduced proliferation was accompanied by decreased checkpoint activation, induced S-phase accumulation, persistent DNA damage, and increased caspase cleavage and apoptosis. Importantly, a significant tumor growth delay was observed in vivo in both HPV-positive and HPV-negative cell line xenografts receiving triple combination therapy with CHKi, cetuximab, and radiotherapy without a concomitant increase in toxicity as assessed by mouse body weight. Conclusion: The combination of CHKi with cetuximab plus irradiation displayed significant antitumor effects to HNSCCs both in vitro and in vivo, suggesting this combination therapy may increase clinical benefit. A clinical trial to test this treatment for patients with head and neck cancer is currently ongoing (NCT02555644).