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 prepared CAGE libraries from total RNA from Caco-2 cells pre-treated with or without TNF-α in biological triplicates. CAGE tags were mapped to the human genome, and nearby CAGE tags were grouped into 52,451 TCs. As in previous work, we will for simplicity refer to these TCs as candidate ‘promoters’. Using an expression cut-off of four TPM to focus on strong initiation events, 13,970 TCs were retained, of which 92% overlapped an annotated RefSeq gene model. The remaining 8% were labelled novel intergenic promoter candidates. A substantial fraction of genes (23%) had two or more overlapping TCs on the same strand, suggesting alternative initiation events that would not be detected by, for example, microarray techniques. As in the ENSEMBL annotation pipeline35 we defined the canonical TSS as the TSS that is the most upstream in annotated gene models. Of the TCs overlapping RefSeq genes, 61% (7.814 TCs) were within 100 nt from the canonical annotated TSS, while 7% (902 TCs) were within 100 nt of annotated alternative promoters, and the final 32% (4,101) did not overlap any annotated TSS; we labelled these candidate novel alternative promoters (Fig. 1A and B). In order to compare with a more comprehensive, but less conservative, gene annotation, the same TCs were overlapped with the Gencode V19 basic gene annotation.36 While the number of TCs mapping to gene models and canonical TSSs did not change substantially (94 and 53%, respectively) compared with the RefSeq results, a larger fraction of TCs within genes were overlapping Gencode-annotated TSSs (25%), indicating that many of the intragenic TCs correspond to real mRNA TSSs (Fig. 1C).Figure 1.
Affiliation: The Bioinformatics Centre, Department of Biology & Biotech Research and Innovation Centre (BRIC), University of Copenhagen, Ole Maaloes Vej 5, Copenhagen DK-2200, Denmark.