RESUMEN
Genome-wide association studies identified numerous disease risk loci. Delineating molecular mechanisms influenced by cis-regulatory variants is essential to understand gene regulation and ultimately disease pathophysiology. Combining bioinformatics and public domain chromatin information with quantitative proteomics supports prediction of cis-regulatory variants and enabled identification of allele-dependent binding of both, transcription factors and coregulators at the type 2 diabetes associated PPARG locus. We found rs7647481A nonrisk allele binding of Yin Yang 1 (YY1), confirmed by allele-specific chromatin immunoprecipitation in primary adipocytes. Quantitative proteomics also found the coregulator RING1 and YY1 binding protein (RYBP) whose mRNA levels correlate with improved insulin sensitivity in primary adipose cells carrying the rs7647481A nonrisk allele. Our findings support a concept with diverse cis-regulatory variants contributing to disease pathophysiology at one locus. Proteome-wide identification of both, transcription factors and coregulators, can profoundly improve understanding of mechanisms underlying genetic associations.
Asunto(s)
Alelos , PPAR gamma/genética , Proteómica , Elementos Reguladores de la Transcripción , Tejido Adiposo/metabolismo , Animales , Células Cultivadas , Proteínas de Unión al ADN/metabolismo , Sitios Genéticos , Variación Genética , Humanos , Resistencia a la Insulina/genética , Ratones , Ratas , Factores de Transcripción/metabolismo , Transcripción Genética , Factor de Transcripción YY1/metabolismoRESUMEN
BACKGROUND: Genome-wide association studies of common diseases or metabolite quantitative traits often identify common variants of small effect size, which may contribute to phenotypes by modulation of gene expression. Thus, there is growing demand for cellular models enabling to assess the impact of gene regulatory variants with moderate effects on gene expression. Mitochondrial fatty acid oxidation is an important energy metabolism pathway. Common noncoding acyl-CoA dehydrogenase short chain (ACADS) gene variants are associated with plasma C4-acylcarnitine levels and allele-specific modulation of ACADS expression may contribute to the observed phenotype. METHODS AND FINDINGS: We assessed ACADS expression and intracellular acylcarnitine levels in human lymphoblastoid cell lines (LCL) genotyped for a common ACADS variant associated with plasma C4-acylcarnitine and found a significant genotype-dependent decrease of ACADS mRNA and protein. Next, we modelled gradual decrease of ACADS expression using a tetracycline-regulated shRNA-knockdown of ACADS in Huh7 hepatocytes, a cell line with high fatty acid oxidation-(FAO)-capacity. Assessing acylcarnitine flux in both models, we found increased C4-acylcarnitine levels with decreased ACADS expression levels. Moreover, assessing time-dependent changes of acylcarnitine levels in shRNA-hepatocytes with altered ACADS expression levels revealed an unexpected effect on long- and medium-chain fatty acid intermediates. CONCLUSIONS: Both, genotyped LCL and regulated shRNA-knockdown are valuable tools to model moderate, gradual gene-regulatory effects of common variants on cellular phenotypes. Decreasing ACADS expression levels modulate short and surprisingly also long/medium chain acylcarnitines, and may contribute to increased plasma acylcarnitine levels.