Identification of TNF-α-responsive promoters and enhancers in the intestinal epithelial cell model Caco-2.
Bottom Line: We found 520 promoters that significantly changed their usage level upon TNF-α stimulation; of these, 52% are not annotated.These enhancers share motif enrichments with similarly responding gene promoters.As a case example, we characterize an enhancer regulating the laminin-5 γ2-chain (LAMC2) gene by nuclear factor (NF)-κB binding.
Affiliation: The Bioinformatics Centre, Department of Biology & Biotech Research and Innovation Centre (BRIC), University of Copenhagen, Ole Maaloes Vej 5, Copenhagen DK-2200, Denmark.Show MeSH
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Mentions: We have previously shown that bidirectional CAGE TCs situated at most 200 nt from each other can identify enhancer RNA.19 The method has been extensively validated with >100 in vitro reporter gene assays and shown to be a more accurate prediction method for enhancer activity than untranscribed hypersensitive sites or enhancer candidates predicted by chromatin immunoprecipitation.19 We have also shown that expression correlations between the enhancer activity and that of nearby promoters can identify physical interactions. With the same approach as in ref.,19 using a combination of the CAGE data from this study supplemented by CAGE data from a wide range of tissues,14,19 we found 890 Caco-2-expressed candidate enhancers, where 62% overlap Caco-2 DNase hypersensitive sites20 (Supplementary Table S3). Of these, 443 (49.7%) responded 4-fold or more to stimulation by TNF-α compared with control and 222 (of the 443) were up-regulated as a response to TNF-α stimulation. Within the list of 443 TNF-α-responsive enhancers, we found 37 unique enhancer–promoter pairs with a maximal distance of 200 kb, where the enhancer and the promoter expression are highly correlated before and after stimulation, suggesting regulatory interaction. An interesting example of such pairings is the enhancer 60 kb downstream of the UBR4 gene,53 linked by co-expression to a novel alternative promoter within the UBR4 locus. We subsequently validated the promoter expression change by qPCR (Fig. 4A). Other examples include the TNF-α-induced candidate enhancer region located within the first intron of the TNFSF10 gene—a member of the TNF ligand superfamily, whose annotated promoter is also highly induced by TNF-α (Fig. 4B), and an enhancer candidate 2 kb upstream of the ANXA13 gene (known to increase in expression during epithelial cell differentiation54) where both the gene promoter and the enhancer expression is decreased as a response to TNF-α (Fig. 4C).Figure 4.
Affiliation: The Bioinformatics Centre, Department of Biology & Biotech Research and Innovation Centre (BRIC), University of Copenhagen, Ole Maaloes Vej 5, Copenhagen DK-2200, Denmark.