title: An evolutionarily conserved TNF-alpha-responsive enhancer in the far upstream region of human CCL2 locus influences its gene expression.
abstract: Comparative cross-species genomic analysis has served as a powerful tool to discover novel noncoding regulatory regions that influence gene expression in several cytokine loci. In this study, we have identified several evolutionarily conserved regions (ECRs) that are shared between human, rhesus monkey, dog, and horse and that are upstream of the promoter regions that have been previously shown to play a role in regulating CCL2 gene expression. Of these, an ECR that was ~16.5 kb (-16.5 ECR) upstream of its coding sequence contained a highly conserved NF-κB site. The region encompassing the -16.5 ECR conferred TNF-α responsiveness to homologous and heterologous promoters. In vivo footprinting demonstrated that specific nucleotide residues in the -16.5 ECR were protected or became hypersensitive after TNF-α treatment. The footprinted regions were found to bind NF-κB subunits in vitro and in vivo. Mutation/deletion of the conserved NF-κB binding site in the -16.5 ECR led to loss of TNF-α responsiveness. After TNF-α stimulation, the -16.5 ECR showed increased sensitivity to nuclease digestion and loss of histone signatures that are characteristic of a repressive chromatin. Chromosome conformation capture assays indicated that -16.5 ECR physically interacts with the CCL2 proximal promoter after TNF-α stimulation. Taken together, these results suggest that the -16.5 ECR may play a critical role in the regulation of CCL2.
authors.affiliation: Center for Personalized Medicine, South Texas Veterans Health Care System, San Antonio, TX 78229, USA.
authors.forename: Grégory B
authors.initials: GB
authors.lastname: Bonello
authors.forename: Minh-Hieu
authors.initials: MH
authors.lastname: Pham
authors.forename: Kazi
authors.initials: K
authors.lastname: Begum
authors.forename: Jose
authors.initials: J
authors.lastname: Sigala
authors.forename: Kavithalakshmi
authors.initials: K
authors.lastname: Sataranatarajan
authors.forename: Srinivas
authors.initials: S
authors.lastname: Mummidi
mesh_terms: D000818:Animals; D001665:Binding Sites; D018932:Chemokine CCL2 / Q000235:genetics*; D017124:Conserved Sequence; D018983:DNA Footprinting; D004742:Enhancer Elements, Genetic / Q000235:genetics*; D019143:Evolution, Molecular; D005786:Gene Expression Regulation; D006801:Humans; D016328:NF-kappa B / Q000378:metabolism; D011401:Promoter Regions, Genetic; D013045:Species Specificity; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics*
chemical_list: C111835:CCL2 protein, human; D018932:Chemokine CCL2; D016328:NF-kappa B; D014409:Tumor Necrosis Factor-alpha