RESUMO
A causative role for single nucleotide polymorphisms (SNPs) in many genetic disorders has become evident through numerous genome-wide association studies. However, identification of these common causal variants and the molecular mechanisms underlying these associations remains a major challenge. Differential transcription factor binding at a SNP resulting in altered gene expression is one possible mechanism. Here we apply PWAS ("proteome-wide analysis of SNPs"), a methodology based on quantitative mass spectrometry that enables rapid screening of SNPs for differential transcription factor binding, to 12 SNPs that are highly associated with type 1 diabetes at the IL2RA locus, encoding the interleukin-2 receptor CD25. We report differential, allele-specific binding of the transcription factors RUNX1, LEF1, CREB, and TFAP4 to IL2RA SNPs rs12722508*A, rs12722522*C, rs41295061*A, and rs2104286*A and demonstrate the functional influence of RUNX1 at rs12722508 by reporter gene assay. Thus, PWAS may be able to contribute to our understanding of the molecular consequences of human genetic variability underpinning susceptibility to multi-factorial disease.
Assuntos
Diabetes Mellitus Tipo 1/genética , Subunidade alfa de Receptor de Interleucina-2/genética , Polimorfismo de Nucleotídeo Único/genética , Proteoma , Alelos , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Regulação da Expressão Gênica , Predisposição Genética para Doença , Genoma Humano , Ensaios de Triagem em Larga Escala , Humanos , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Fator 1 de Ligação ao Facilitador Linfoide/metabolismo , Ligação Proteica , Proteoma/análise , Proteoma/genéticaRESUMO
Ultraconserved elements (UCEs) have been the subject of great interest because of their extreme sequence identity and their seemingly cryptic and largely uncharacterized functions. Although in vivo studies of UCE sequences have demonstrated regulatory activity, protein interactors at UCEs have not been systematically identified. Here, we combined high-throughput affinity purification, high-resolution mass spectrometry, and SILAC quantification to map intrinsic protein interactions for 193 UCE sequences. The interactome contains over 400 proteins, including transcription factors with known developmental roles. We demonstrate based on our data that UCEs consist of strongly conserved overlapping binding sites. We also generated a fine-resolution interactome of a UCE, confirming the hub-like nature of the element. The intrinsic interactions mapped here are reflected in open chromatin, as indicated by comparison with existing ChIP data. Our study argues for a strong contribution of protein-DNA interactions to UCE conservation and provides a basis for further functional characterization of UCEs.