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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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CAGE-defined promoters in Caco-2 cells. (A) Schematic illustration showing classification of CAGE-defined promoters based on RefSeq annotation. The upper panel shows the number of CAGE tag clusters (TCs) mapping around an example gene loci (the CFLAR gene) on the plus strand. The lower panel shows the RefSeq gene model exon–intron structure of the gene (blocks are exons, lines are introns). CAGE peaks are commented as belonging to specific classes based on their location. CAGE tags falling outside of gene loci will be classified as ‘novel intergenic’ (grey areas). Box labels indicate names used for respective classes in (B) and (C). RefSeq-annotated alternative promoters that are not hit by CAGE are also indicated. (B) Distribution of Caco-2 TCs (TPM > 4) according to the TC classes defined above using RefSeq annotation. (C) Distribution of Caco-2 TCs (TPM > 4) according to the TC classes defined above using Gencode annotation. (D and E) Per cent of TCs falling into each of the classes from (B) and (C) split up by TNF-α response, using RefSeq (D) or Gencode annotation (E). Bars shaded from black to light grey represent canonical, known alternative, novel intragenic and novel intergenic TCs, respectively. Numbers above bars indicate the number of promoters in respective category.
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DSU022F1: CAGE-defined promoters in Caco-2 cells. (A) Schematic illustration showing classification of CAGE-defined promoters based on RefSeq annotation. The upper panel shows the number of CAGE tag clusters (TCs) mapping around an example gene loci (the CFLAR gene) on the plus strand. The lower panel shows the RefSeq gene model exon–intron structure of the gene (blocks are exons, lines are introns). CAGE peaks are commented as belonging to specific classes based on their location. CAGE tags falling outside of gene loci will be classified as ‘novel intergenic’ (grey areas). Box labels indicate names used for respective classes in (B) and (C). RefSeq-annotated alternative promoters that are not hit by CAGE are also indicated. (B) Distribution of Caco-2 TCs (TPM > 4) according to the TC classes defined above using RefSeq annotation. (C) Distribution of Caco-2 TCs (TPM > 4) according to the TC classes defined above using Gencode annotation. (D and E) Per cent of TCs falling into each of the classes from (B) and (C) split up by TNF-α response, using RefSeq (D) or Gencode annotation (E). Bars shaded from black to light grey represent canonical, known alternative, novel intragenic and novel intergenic TCs, respectively. Numbers above bars indicate the number of promoters in respective category.

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.


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)

CAGE-defined promoters in Caco-2 cells. (A) Schematic illustration showing classification of CAGE-defined promoters based on RefSeq annotation. The upper panel shows the number of CAGE tag clusters (TCs) mapping around an example gene loci (the CFLAR gene) on the plus strand. The lower panel shows the RefSeq gene model exon–intron structure of the gene (blocks are exons, lines are introns). CAGE peaks are commented as belonging to specific classes based on their location. CAGE tags falling outside of gene loci will be classified as ‘novel intergenic’ (grey areas). Box labels indicate names used for respective classes in (B) and (C). RefSeq-annotated alternative promoters that are not hit by CAGE are also indicated. (B) Distribution of Caco-2 TCs (TPM > 4) according to the TC classes defined above using RefSeq annotation. (C) Distribution of Caco-2 TCs (TPM > 4) according to the TC classes defined above using Gencode annotation. (D and E) Per cent of TCs falling into each of the classes from (B) and (C) split up by TNF-α response, using RefSeq (D) or Gencode annotation (E). Bars shaded from black to light grey represent canonical, known alternative, novel intragenic and novel intergenic TCs, respectively. Numbers above bars indicate the number of promoters in respective category.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4263293&req=5

DSU022F1: CAGE-defined promoters in Caco-2 cells. (A) Schematic illustration showing classification of CAGE-defined promoters based on RefSeq annotation. The upper panel shows the number of CAGE tag clusters (TCs) mapping around an example gene loci (the CFLAR gene) on the plus strand. The lower panel shows the RefSeq gene model exon–intron structure of the gene (blocks are exons, lines are introns). CAGE peaks are commented as belonging to specific classes based on their location. CAGE tags falling outside of gene loci will be classified as ‘novel intergenic’ (grey areas). Box labels indicate names used for respective classes in (B) and (C). RefSeq-annotated alternative promoters that are not hit by CAGE are also indicated. (B) Distribution of Caco-2 TCs (TPM > 4) according to the TC classes defined above using RefSeq annotation. (C) Distribution of Caco-2 TCs (TPM > 4) according to the TC classes defined above using Gencode annotation. (D and E) Per cent of TCs falling into each of the classes from (B) and (C) split up by TNF-α response, using RefSeq (D) or Gencode annotation (E). Bars shaded from black to light grey represent canonical, known alternative, novel intragenic and novel intergenic TCs, respectively. Numbers above bars indicate the number of promoters in respective category.
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.

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