An Evolutionarily Conserved TNF-α–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.

IFN-α-driven CCL2 production recruits inflammatory monocytes to infection site in mice

“…Additionally, CCL2 production has been shown to be regulated by both TNF-α and STAT-2 signals. 37, 38 However, we show the CCL2 upstream promoter contains an IFN-responsive element, and cells treated with recombinant IFN alone up-regulate CCL2 mRNA. Such results imply multiple host signals elicit CCL2 expression dependent upon the initiating stimulus.…”

Inflammatory Monocyte Recruitment Is Regulated by Interleukin-23 during Systemic Bacterial Infection

“…This could be attributed to the infection-induced production of proinflammatory cytokines that may act to enhance CCL2 production during L. monocytogenes infection. Genome analysis has identified a TNF-␣-responsive enhancer region in the CCL2 locus, suggesting that this proinflammatory cytokine can cause the upregulation of CCL2 expression (3). In a rheumatoid arthritis model, IL-17 mediates monocyte recruitment both directly, by acting as a chemoattractant (36), and indirectly, by inducing the production of CCL2 (37), further supporting the notion of an IL-23/IL-17/CCL2/monocyte axis.…”

The rs1024611 Regulatory Region Polymorphism Is Associated with CCL2 Allelic Expression Imbalance

“…Reporter based assays do not account for the chromatin context of the regulatory region in question and cannot address the three dimensional interactions (“looping”) between distal and proximal regulatory sequences that play an important role in CCL2 gene regulation [49], [50]. As AEI captures allele-specific differences in the context of the chromatin as well as genetic polymorphisms it provides a powerful alternative to the reporter based studies for highly polymorphic genes with complex and extended regulatory regions.…”