RESUMEN
DNA variants that alter gene expression contribute to variation in many phenotypic traits. In particular, trans-acting variants, which are often located on different chromosomes from the genes they affect, are an important source of heritable gene expression variation. However, our knowledge about the identity and mechanism of causal trans-acting variants remains limited. Here, we developed a fine-mapping strategy called CRISPR-Swap and dissected three expression quantitative trait locus (eQTL) hotspots known to alter the expression of numerous genes in trans in the yeast Saccharomyces cerevisiae. Causal variants were identified by engineering recombinant alleles and quantifying the effects of these alleles on the expression of a green fluorescent protein-tagged gene affected by the given locus in trans. We validated the effect of each variant on the expression of multiple genes by RNA-sequencing. The three variants differed in their molecular mechanism, the type of genes they reside in, and their distribution in natural populations. While a missense leucine-to-serine variant at position 63 in the transcription factor Oaf1 (L63S) was almost exclusively present in the reference laboratory strain, the two other variants were frequent among S. cerevisiae isolates. A causal missense variant in the glucose receptor Rgt2 (V539I) occurred at a poorly conserved amino acid residue and its effect was strongly dependent on the concentration of glucose in the culture medium. A noncoding variant in the conserved fatty acid regulated (FAR) element of the OLE1 promoter influenced the expression of the fatty acid desaturase Ole1 in cis and, by modulating the level of this essential enzyme, other genes in trans. The OAF1 and OLE1 variants showed a non-additive genetic interaction, and affected cellular lipid metabolism. These results demonstrate that the molecular basis of trans-regulatory variation is diverse, highlighting the challenges in predicting which natural genetic variants affect gene expression.
Asunto(s)
Proteínas de Unión al ADN/genética , Evolución Molecular , Secuencias Reguladoras de Ácidos Nucleicos/genética , Proteínas de Saccharomyces cerevisiae/genética , Estearoil-CoA Desaturasa/genética , Factores de Transcripción/genética , Sistemas CRISPR-Cas/genética , Ácidos Grasos/genética , Ácidos Grasos/metabolismo , Regulación Fúngica de la Expresión Génica/genética , Proteínas Fluorescentes Verdes/genética , Metabolismo de los Lípidos/genética , Proteínas de Transporte de Monosacáridos/genética , Mutación Missense/genética , Fenotipo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMEN
Purpose: The prevalence of childhood caries in urban Chicago, compared with national and state data, indicates that neighborhood context influences oral health. Our objective was to delineate the influence of a child's neighborhood on oral health outcomes that are predictive of caries (toothbrushing frequency and plaque levels). Methods: Our study population represents urban, Medicaid-enrolled families in the metropolitan Chicago area. Data were obtained from a cohort of participants (child-parent dyads) who participated in the Coordinated Oral Health Promotion (CO-OP) trial at 12 months of study participation (N = 362). Oral health outcomes included toothbrushing frequency and plaque levels. Participants' neighborhood resource levels were measured by the Area Deprivation Index (ADI). Linear and logistic regression models were used to measure the influence of ADI on plaque scores and toothbrushing frequency, respectively. Results: Data from 362 child-parent dyads were analyzed. The mean child age was 33.6 months (SD 6.8). The majority of children were reported to brush at least twice daily (n = 228, 63%), but the mean plaque score was 1.9 (SD 0.7), classified as "poor." In covariate-adjusted analyses, ADI was not associated with brushing frequency (0.94, 95% CI 0.84-1.06). ADI was associated with plaque scores (0.05, 95% CI 0.01-0.09, p value = 0.007). Conclusions: Findings support the hypothesis that neighborhood-level factors influence children's plaque levels. Because excessive plaque places a child at high risk for cavities, we recommend the inclusion of neighborhood context in interventions and policies to reduce children's oral health disparities. Existing programs and clinics that serve disadvantaged communities are well-positioned to support caregivers of young children in maintaining recommended oral health behaviors.