RESUMEN
Gene expression is controlled by the involvement of gene-proximal (promoters) and distal (enhancers) regulatory elements. Our previous results demonstrated that a subset of gene promoters, termed Epromoters, work as bona fide enhancers and regulate distal gene expression. Here, we hypothesized that Epromoters play a key role in the coordination of rapid gene induction during the inflammatory response. Using a high-throughput reporter assay we explored the function of Epromoters in response to type I interferon. We find that clusters of IFNa-induced genes are frequently associated with Epromoters and that these regulatory elements preferentially recruit the STAT1/2 and IRF transcription factors and distally regulate the activation of interferon-response genes. Consistently, we identified and validated the involvement of Epromoter-containing clusters in the regulation of LPS-stimulated macrophages. Our findings suggest that Epromoters function as a local hub recruiting the key TFs required for coordinated regulation of gene clusters during the inflammatory response.
Asunto(s)
Elementos de Facilitación Genéticos/fisiología , Inflamación/genética , Factores Reguladores del Interferón/metabolismo , Regiones Promotoras Genéticas/fisiología , Animales , Elementos de Facilitación Genéticos/efectos de los fármacos , Regulación de la Expresión Génica , Células HeLa , Humanos , Inflamación/metabolismo , Interferón Tipo I/metabolismo , Interferón-alfa/farmacología , Células K562 , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Ratones , Familia de Multigenes/efectos de los fármacos , Familia de Multigenes/genética , Regiones Promotoras Genéticas/efectos de los fármacos , Factor de Transcripción STAT1/metabolismo , Factor de Transcripción STAT2/metabolismoRESUMEN
Mapping the binding sites of DNA- or chromatin-interacting proteins is essential to understanding biological processes. DNA adenine methyltransferase identification (DamID) has emerged as a comprehensive method to map genome-wide occupancy of proteins of interest. A caveat of DamID is the specificity of Dam methyltransferase for GATC motifs that are not homogenously distributed in the genome. Here, we developed an optimized method named MadID, using proximity labeling of DNA by the methyltransferase M.EcoGII. M.EcoGII mediates N6-adenosine methylation in any DNA sequence context, resulting in deeper and unbiased coverage of the genome. We demonstrate, using m6A-specific immunoprecipitation and deep sequencing, that MadID is a robust method to identify protein-DNA interactions at the whole-genome level. Using MadID, we revealed contact sites between human telomeres, repetitive sequences devoid of GATC sites, and the nuclear envelope. Overall, MadID opens the way to identification of binding sites in genomic regions that were largely inaccessible.