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Identification of TNF-α-responsive promoters and enhancers in the intestinal epithelial cell model Caco-2.

Boyd M, Coskun M, Lilje B, Andersson R, Hoof I, Bornholdt J, Dahlgaard K, Olsen J, Vitezic M, Bjerrum JT, Seidelin JB, Nielsen OH, Troelsen JT, Sandelin A - DNA Res. (2014)

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.

View Article: PubMed Central - PubMed

Affiliation: The Bioinformatics Centre, Department of Biology & Biotech Research and Innovation Centre (BRIC), University of Copenhagen, Ole Maaloes Vej 5, Copenhagen DK-2200, Denmark.

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Examples of CAGE-inferred TNF-α responsive enhancers and their potential targets. Each panel consists of sub-panels: top, an UCSC browser43 overview of the gene landscape around the enhancer and promoter of interest; bottom, zoom-in version(s) of the above. (A) An enhancer is predicted downstream of the UBR4 gene identified by bidirectional CAGE tag pairs (left panel shows a zoom-in). It has support from multiple data types from the ENCODE project,21 including ChIP-seq for transcription factors, and histone marks typical of enhancers, as well as Caco-2-specific DNase sensitivity site peaks. CAGE data are shown as in Fig. 2. The enhancer is induced by TNF-α, and is predicted to interact with a CAGE-defined alternative promoter within the gene (middle panel), which is also induced by TNF-α (verified by qPCR as in Fig. 2). Conversely, the annotated promoter is not responding highly to TNF-α (right panel). (B) A TNF-α-induced enhancer is predicted within the first intron of the TNFSF10 gene, and predicted to interact with the annotated TSS, which also is highly TNF-α-induced. The enhancer has support by ENCODE histone marks and multiple transcription factor ChIP peaks. (C) An enhancer, which is only used in non-induced cells, is predicted 2 kb upstream of the annotated TSS of the ANXA13 gene, which also has much higher expression in the non-induced state. The enhancer has support by multiple ENCODE transcription factor ChIP peaks. See main text for further discussion. This figure appears in colour in the online version of DNA Research.
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DSU022F4: Examples of CAGE-inferred TNF-α responsive enhancers and their potential targets. Each panel consists of sub-panels: top, an UCSC browser43 overview of the gene landscape around the enhancer and promoter of interest; bottom, zoom-in version(s) of the above. (A) An enhancer is predicted downstream of the UBR4 gene identified by bidirectional CAGE tag pairs (left panel shows a zoom-in). It has support from multiple data types from the ENCODE project,21 including ChIP-seq for transcription factors, and histone marks typical of enhancers, as well as Caco-2-specific DNase sensitivity site peaks. CAGE data are shown as in Fig. 2. The enhancer is induced by TNF-α, and is predicted to interact with a CAGE-defined alternative promoter within the gene (middle panel), which is also induced by TNF-α (verified by qPCR as in Fig. 2). Conversely, the annotated promoter is not responding highly to TNF-α (right panel). (B) A TNF-α-induced enhancer is predicted within the first intron of the TNFSF10 gene, and predicted to interact with the annotated TSS, which also is highly TNF-α-induced. The enhancer has support by ENCODE histone marks and multiple transcription factor ChIP peaks. (C) An enhancer, which is only used in non-induced cells, is predicted 2 kb upstream of the annotated TSS of the ANXA13 gene, which also has much higher expression in the non-induced state. The enhancer has support by multiple ENCODE transcription factor ChIP peaks. See main text for further discussion. This figure appears in colour in the online version of DNA Research.

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.


Identification of TNF-α-responsive promoters and enhancers in the intestinal epithelial cell model Caco-2.

Boyd M, Coskun M, Lilje B, Andersson R, Hoof I, Bornholdt J, Dahlgaard K, Olsen J, Vitezic M, Bjerrum JT, Seidelin JB, Nielsen OH, Troelsen JT, Sandelin A - DNA Res. (2014)

Examples of CAGE-inferred TNF-α responsive enhancers and their potential targets. Each panel consists of sub-panels: top, an UCSC browser43 overview of the gene landscape around the enhancer and promoter of interest; bottom, zoom-in version(s) of the above. (A) An enhancer is predicted downstream of the UBR4 gene identified by bidirectional CAGE tag pairs (left panel shows a zoom-in). It has support from multiple data types from the ENCODE project,21 including ChIP-seq for transcription factors, and histone marks typical of enhancers, as well as Caco-2-specific DNase sensitivity site peaks. CAGE data are shown as in Fig. 2. The enhancer is induced by TNF-α, and is predicted to interact with a CAGE-defined alternative promoter within the gene (middle panel), which is also induced by TNF-α (verified by qPCR as in Fig. 2). Conversely, the annotated promoter is not responding highly to TNF-α (right panel). (B) A TNF-α-induced enhancer is predicted within the first intron of the TNFSF10 gene, and predicted to interact with the annotated TSS, which also is highly TNF-α-induced. The enhancer has support by ENCODE histone marks and multiple transcription factor ChIP peaks. (C) An enhancer, which is only used in non-induced cells, is predicted 2 kb upstream of the annotated TSS of the ANXA13 gene, which also has much higher expression in the non-induced state. The enhancer has support by multiple ENCODE transcription factor ChIP peaks. See main text for further discussion. This figure appears in colour in the online version of DNA Research.
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Related In: Results  -  Collection

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DSU022F4: Examples of CAGE-inferred TNF-α responsive enhancers and their potential targets. Each panel consists of sub-panels: top, an UCSC browser43 overview of the gene landscape around the enhancer and promoter of interest; bottom, zoom-in version(s) of the above. (A) An enhancer is predicted downstream of the UBR4 gene identified by bidirectional CAGE tag pairs (left panel shows a zoom-in). It has support from multiple data types from the ENCODE project,21 including ChIP-seq for transcription factors, and histone marks typical of enhancers, as well as Caco-2-specific DNase sensitivity site peaks. CAGE data are shown as in Fig. 2. The enhancer is induced by TNF-α, and is predicted to interact with a CAGE-defined alternative promoter within the gene (middle panel), which is also induced by TNF-α (verified by qPCR as in Fig. 2). Conversely, the annotated promoter is not responding highly to TNF-α (right panel). (B) A TNF-α-induced enhancer is predicted within the first intron of the TNFSF10 gene, and predicted to interact with the annotated TSS, which also is highly TNF-α-induced. The enhancer has support by ENCODE histone marks and multiple transcription factor ChIP peaks. (C) An enhancer, which is only used in non-induced cells, is predicted 2 kb upstream of the annotated TSS of the ANXA13 gene, which also has much higher expression in the non-induced state. The enhancer has support by multiple ENCODE transcription factor ChIP peaks. See main text for further discussion. This figure appears in colour in the online version of DNA Research.
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.

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.

View Article: PubMed Central - PubMed

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
Related in: MedlinePlus