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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 promoters and their response to TNF-α stimulation. Each panel describes a validated gene locus: (A) CFLAR, (B) NCK1, (C) PLD1, (D) the RP11-283G6.5 lncRNA, (E) novel putative lncRNA, (F) IFIH1 and a putative long distal promoter of GCA. Each panel consists of three sub-panels: top, an UCSC browser43 overview of the gene landscape around the promoter(s) of interest; middle, a zoom-in version of the above; bottom, qPCR validations of respective RNA isoforms emanating from the promoters of interests as a function of TNF-α concentration. UCSC browser sub-panels show (when relevant and available) RefSeq genes, expressed sequenced tags (ESTs), GenCode36 annotation and mean normalized CAGE signal per nucleotide on relevant strand over three replicates. Arrows indicate TSS clusters (core promoters) of interest and their direction of transcription. Sizes of arrows correspond to the CAGE signal strength. Locations of qPCR primer pairs are shown also in Supplementary Table S1. Respective examples are commented in detail in the main text. For qPCR bar plots, the vertical axis shows the mean fold change vs. un-stimulated cells (four replicates), error bars indicate standard error of mean. The dotted line indicates a fold change of 1. The statistical significance (t-test) is indicated above the bars: *P < 0.05, **P < 0.01, ***P < 0.001. This figure appears in colour in the online version of DNA Research.
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DSU022F2: Examples of CAGE-inferred promoters and their response to TNF-α stimulation. Each panel describes a validated gene locus: (A) CFLAR, (B) NCK1, (C) PLD1, (D) the RP11-283G6.5 lncRNA, (E) novel putative lncRNA, (F) IFIH1 and a putative long distal promoter of GCA. Each panel consists of three sub-panels: top, an UCSC browser43 overview of the gene landscape around the promoter(s) of interest; middle, a zoom-in version of the above; bottom, qPCR validations of respective RNA isoforms emanating from the promoters of interests as a function of TNF-α concentration. UCSC browser sub-panels show (when relevant and available) RefSeq genes, expressed sequenced tags (ESTs), GenCode36 annotation and mean normalized CAGE signal per nucleotide on relevant strand over three replicates. Arrows indicate TSS clusters (core promoters) of interest and their direction of transcription. Sizes of arrows correspond to the CAGE signal strength. Locations of qPCR primer pairs are shown also in Supplementary Table S1. Respective examples are commented in detail in the main text. For qPCR bar plots, the vertical axis shows the mean fold change vs. un-stimulated cells (four replicates), error bars indicate standard error of mean. The dotted line indicates a fold change of 1. The statistical significance (t-test) is indicated above the bars: *P < 0.05, **P < 0.01, ***P < 0.001. This figure appears in colour in the online version of DNA Research.

Mentions: To verify the findings, we randomly selected 18 TNF-α(+) and 2 TNF-α(−) TCs within different classes: known promoters, novel alternative promoters and novel intergenic promoters. We validated their response to TNF-α by qPCR in quadruplicates and four different TNF-α concentrations. For validating alternative promoters, we used two primer pairs: one pair located immediately downstream of the TC of interest and the other pair just downstream of the most upstream annotated promoter. In the large majority (16 of 20) of cases, the qPCR results validated the CAGE data as well as the response to TNF-α (Fig. 2 and Supplementary Figure S1). We highlight a few examples below; fold changes and P-values refer to qPCR validations at 10 nM TNF-α, two-sided t-tests.Figure 2.


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 promoters and their response to TNF-α stimulation. Each panel describes a validated gene locus: (A) CFLAR, (B) NCK1, (C) PLD1, (D) the RP11-283G6.5 lncRNA, (E) novel putative lncRNA, (F) IFIH1 and a putative long distal promoter of GCA. Each panel consists of three sub-panels: top, an UCSC browser43 overview of the gene landscape around the promoter(s) of interest; middle, a zoom-in version of the above; bottom, qPCR validations of respective RNA isoforms emanating from the promoters of interests as a function of TNF-α concentration. UCSC browser sub-panels show (when relevant and available) RefSeq genes, expressed sequenced tags (ESTs), GenCode36 annotation and mean normalized CAGE signal per nucleotide on relevant strand over three replicates. Arrows indicate TSS clusters (core promoters) of interest and their direction of transcription. Sizes of arrows correspond to the CAGE signal strength. Locations of qPCR primer pairs are shown also in Supplementary Table S1. Respective examples are commented in detail in the main text. For qPCR bar plots, the vertical axis shows the mean fold change vs. un-stimulated cells (four replicates), error bars indicate standard error of mean. The dotted line indicates a fold change of 1. The statistical significance (t-test) is indicated above the bars: *P < 0.05, **P < 0.01, ***P < 0.001. This figure appears in colour in the online version of DNA Research.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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DSU022F2: Examples of CAGE-inferred promoters and their response to TNF-α stimulation. Each panel describes a validated gene locus: (A) CFLAR, (B) NCK1, (C) PLD1, (D) the RP11-283G6.5 lncRNA, (E) novel putative lncRNA, (F) IFIH1 and a putative long distal promoter of GCA. Each panel consists of three sub-panels: top, an UCSC browser43 overview of the gene landscape around the promoter(s) of interest; middle, a zoom-in version of the above; bottom, qPCR validations of respective RNA isoforms emanating from the promoters of interests as a function of TNF-α concentration. UCSC browser sub-panels show (when relevant and available) RefSeq genes, expressed sequenced tags (ESTs), GenCode36 annotation and mean normalized CAGE signal per nucleotide on relevant strand over three replicates. Arrows indicate TSS clusters (core promoters) of interest and their direction of transcription. Sizes of arrows correspond to the CAGE signal strength. Locations of qPCR primer pairs are shown also in Supplementary Table S1. Respective examples are commented in detail in the main text. For qPCR bar plots, the vertical axis shows the mean fold change vs. un-stimulated cells (four replicates), error bars indicate standard error of mean. The dotted line indicates a fold change of 1. The statistical significance (t-test) is indicated above the bars: *P < 0.05, **P < 0.01, ***P < 0.001. This figure appears in colour in the online version of DNA Research.
Mentions: To verify the findings, we randomly selected 18 TNF-α(+) and 2 TNF-α(−) TCs within different classes: known promoters, novel alternative promoters and novel intergenic promoters. We validated their response to TNF-α by qPCR in quadruplicates and four different TNF-α concentrations. For validating alternative promoters, we used two primer pairs: one pair located immediately downstream of the TC of interest and the other pair just downstream of the most upstream annotated promoter. In the large majority (16 of 20) of cases, the qPCR results validated the CAGE data as well as the response to TNF-α (Fig. 2 and Supplementary Figure S1). We highlight a few examples below; fold changes and P-values refer to qPCR validations at 10 nM TNF-α, two-sided t-tests.Figure 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.

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