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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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TNF-α regulates LAMC2 transcriptional activity by an NF-κB-bound enhancer. (A) An overview of the LAMC2 gene and its predicted enhancer location upstream of the LAMC2 TSS. The RELA ChIP peak overlapping the predicted enhancer is taken from the ENCODE UCSC ChIP track.21 Genome browser zoom-in shows CAGE data around the annotated TSS as shown in Fig. 2. (B) Dose-dependent increase of LAMC2 mRNA expression in cells treated with increasing concentrations of TNF-α (0, 0.1, 1 or 10 nM). (C) Western blot analysis of LAMC2 protein levels in TNF-α-treated cells and GAPDH used as an internal loading control. The shown blot represents three independent experiments. The effect of (D) FR180204, an ERK inhibitor, and (E) TPCK, an NF-κB inhibitor, on the TNF-α-mediated up-regulation of LAMC2 mRNA expression and (F) LAMC2 and GAPDH protein levels. Cells were pre-treated with inhibitors or with vehicle DMSO (0.4%) for 1 h and then incubated with TNF-α (10 nM) for 24 h. (G) The effect of TNF-α and overexpression of NF-κB subunits on the LAMC2 promoter activity with and without the enhancer shown in (A). Cells were transiently transfected with the human LAMC2 promoter (pGL3-LAMC2) (grey bars) or LAMC2 promoter/enhancer construct (pGL3-LAMC2 + enhancer) (black bars) and were unstimulated, stimulated with TNF-α for 24 h (black bars), co-transfected with plasmids overexpressing NFKB1(p50) and RELA NF-κB subunits, or co-transfected with plasmids overexpressing NFKB2 and RELA NF-κB subunits. Asterisks indicate levels of significance: *P < 0.05, **P < 0.01, ***P < 0.001 (t-test): error bars indicate the standard error of the mean. This figure appears in colour in the online version of DNA Research.
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DSU022F5: TNF-α regulates LAMC2 transcriptional activity by an NF-κB-bound enhancer. (A) An overview of the LAMC2 gene and its predicted enhancer location upstream of the LAMC2 TSS. The RELA ChIP peak overlapping the predicted enhancer is taken from the ENCODE UCSC ChIP track.21 Genome browser zoom-in shows CAGE data around the annotated TSS as shown in Fig. 2. (B) Dose-dependent increase of LAMC2 mRNA expression in cells treated with increasing concentrations of TNF-α (0, 0.1, 1 or 10 nM). (C) Western blot analysis of LAMC2 protein levels in TNF-α-treated cells and GAPDH used as an internal loading control. The shown blot represents three independent experiments. The effect of (D) FR180204, an ERK inhibitor, and (E) TPCK, an NF-κB inhibitor, on the TNF-α-mediated up-regulation of LAMC2 mRNA expression and (F) LAMC2 and GAPDH protein levels. Cells were pre-treated with inhibitors or with vehicle DMSO (0.4%) for 1 h and then incubated with TNF-α (10 nM) for 24 h. (G) The effect of TNF-α and overexpression of NF-κB subunits on the LAMC2 promoter activity with and without the enhancer shown in (A). Cells were transiently transfected with the human LAMC2 promoter (pGL3-LAMC2) (grey bars) or LAMC2 promoter/enhancer construct (pGL3-LAMC2 + enhancer) (black bars) and were unstimulated, stimulated with TNF-α for 24 h (black bars), co-transfected with plasmids overexpressing NFKB1(p50) and RELA NF-κB subunits, or co-transfected with plasmids overexpressing NFKB2 and RELA NF-κB subunits. Asterisks indicate levels of significance: *P < 0.05, **P < 0.01, ***P < 0.001 (t-test): error bars indicate the standard error of the mean. This figure appears in colour in the online version of DNA Research.

Mentions: As a case example, we focused on the LAMC2 gene (Fig. 5A) whose main promoter is >8-fold up-regulated in TNF-α-stimulated Caco-2 cells as measured by CAGE. LAMC2 has previously been shown to be induced by TNF-α in other cell lines.58 Indeed, we validated that TNF-α stimulation (P < 0.05, t-test) increased the expression of LAMC2 mRNA and protein levels in a dose-dependent manner with a maximal effect observed at 10 nM (Fig. 5B and C). TNF-α can initiate pro-inflammatory signalling by activating the NF-κB pathway and the mitogen-activated protein kinase (MAPK) pathway. In mammals there are three major constituents of the MAPK superfamily: ERKs (ERK1/2 or p42/p44), c-Jun N-terminal kinases (JNK1/2/3) and the p38 MAPK family.56 The NF-κB family of transcription factors consists of five mammalian members [NFKB1(p50), NFKB2(p52), RELA(p65) and RELB]59,60 that can form either homodimers or heterodimers. Therefore, the role of these signalling pathways in TNF-α-mediated up-regulation of endogenous LAMC2 expression in Caco-2 cells was investigated. Interestingly, we noticed a ∼400 nt long candidate enhancer region (chr1: 183,149,939–183,150,336, hg19 assembly) upstream of the LAMC2 TSS, which is predicted to have a single NF-κB-binding site and is also bound by RELA as indicated by ENCODE ChIP-seq21 data (Fig. 5A). Consistent with the hypothesis of NF-κB-mediated regulation, the NF-κB inhibitor TPCK effectively diminished the effect of TNF-α on LAMC2 mRNA and protein levels (Fig. 5E and F). Moreover, it has previously been demonstrated that an active ERK pathway is involved in the regulation of LAMC2 in squamous cell carcinoma.61 However, inhibition of ERK1/2 with FR180204 had only a minor effect on the TNF-α-induced LAMC2 mRNA expression (Fig. 5D) and almost no effect on the LAMC2 protein levels (Fig. 5F) in our experiments. Since these findings indicate a transcriptional up-regulation by TNF-α and NF-κB, we measured the effect of TNF-α on the activity of an LAMC2 promoter construct32 with and without the predicted enhancer; the enhancerless construct did not respond to TNF-α, but the enhancer–promoter construct gave a 3-fold increase in reporter signal (P < 0.001 (t-test)) (Fig. 5G). Validating the causal role of NF-κB, co-transfection of LAMC2 reporter constructs with RELA in combination with NFKB1 or NFKB2-expression plasmids could significantly (P < 0.001 (t-test)) up-regulate the enhancer–promoter construct but not the enhancerless construct. This effect was highest in Caco-2 cells overexpressing both RELA and NFKB2 subunits (P < 0.001) (Fig. 5G).Figure 5.


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)

TNF-α regulates LAMC2 transcriptional activity by an NF-κB-bound enhancer. (A) An overview of the LAMC2 gene and its predicted enhancer location upstream of the LAMC2 TSS. The RELA ChIP peak overlapping the predicted enhancer is taken from the ENCODE UCSC ChIP track.21 Genome browser zoom-in shows CAGE data around the annotated TSS as shown in Fig. 2. (B) Dose-dependent increase of LAMC2 mRNA expression in cells treated with increasing concentrations of TNF-α (0, 0.1, 1 or 10 nM). (C) Western blot analysis of LAMC2 protein levels in TNF-α-treated cells and GAPDH used as an internal loading control. The shown blot represents three independent experiments. The effect of (D) FR180204, an ERK inhibitor, and (E) TPCK, an NF-κB inhibitor, on the TNF-α-mediated up-regulation of LAMC2 mRNA expression and (F) LAMC2 and GAPDH protein levels. Cells were pre-treated with inhibitors or with vehicle DMSO (0.4%) for 1 h and then incubated with TNF-α (10 nM) for 24 h. (G) The effect of TNF-α and overexpression of NF-κB subunits on the LAMC2 promoter activity with and without the enhancer shown in (A). Cells were transiently transfected with the human LAMC2 promoter (pGL3-LAMC2) (grey bars) or LAMC2 promoter/enhancer construct (pGL3-LAMC2 + enhancer) (black bars) and were unstimulated, stimulated with TNF-α for 24 h (black bars), co-transfected with plasmids overexpressing NFKB1(p50) and RELA NF-κB subunits, or co-transfected with plasmids overexpressing NFKB2 and RELA NF-κB subunits. Asterisks indicate levels of significance: *P < 0.05, **P < 0.01, ***P < 0.001 (t-test): error bars indicate the standard error of the mean. This figure appears in colour in the online version of DNA Research.
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DSU022F5: TNF-α regulates LAMC2 transcriptional activity by an NF-κB-bound enhancer. (A) An overview of the LAMC2 gene and its predicted enhancer location upstream of the LAMC2 TSS. The RELA ChIP peak overlapping the predicted enhancer is taken from the ENCODE UCSC ChIP track.21 Genome browser zoom-in shows CAGE data around the annotated TSS as shown in Fig. 2. (B) Dose-dependent increase of LAMC2 mRNA expression in cells treated with increasing concentrations of TNF-α (0, 0.1, 1 or 10 nM). (C) Western blot analysis of LAMC2 protein levels in TNF-α-treated cells and GAPDH used as an internal loading control. The shown blot represents three independent experiments. The effect of (D) FR180204, an ERK inhibitor, and (E) TPCK, an NF-κB inhibitor, on the TNF-α-mediated up-regulation of LAMC2 mRNA expression and (F) LAMC2 and GAPDH protein levels. Cells were pre-treated with inhibitors or with vehicle DMSO (0.4%) for 1 h and then incubated with TNF-α (10 nM) for 24 h. (G) The effect of TNF-α and overexpression of NF-κB subunits on the LAMC2 promoter activity with and without the enhancer shown in (A). Cells were transiently transfected with the human LAMC2 promoter (pGL3-LAMC2) (grey bars) or LAMC2 promoter/enhancer construct (pGL3-LAMC2 + enhancer) (black bars) and were unstimulated, stimulated with TNF-α for 24 h (black bars), co-transfected with plasmids overexpressing NFKB1(p50) and RELA NF-κB subunits, or co-transfected with plasmids overexpressing NFKB2 and RELA NF-κB subunits. Asterisks indicate levels of significance: *P < 0.05, **P < 0.01, ***P < 0.001 (t-test): error bars indicate the standard error of the mean. This figure appears in colour in the online version of DNA Research.
Mentions: As a case example, we focused on the LAMC2 gene (Fig. 5A) whose main promoter is >8-fold up-regulated in TNF-α-stimulated Caco-2 cells as measured by CAGE. LAMC2 has previously been shown to be induced by TNF-α in other cell lines.58 Indeed, we validated that TNF-α stimulation (P < 0.05, t-test) increased the expression of LAMC2 mRNA and protein levels in a dose-dependent manner with a maximal effect observed at 10 nM (Fig. 5B and C). TNF-α can initiate pro-inflammatory signalling by activating the NF-κB pathway and the mitogen-activated protein kinase (MAPK) pathway. In mammals there are three major constituents of the MAPK superfamily: ERKs (ERK1/2 or p42/p44), c-Jun N-terminal kinases (JNK1/2/3) and the p38 MAPK family.56 The NF-κB family of transcription factors consists of five mammalian members [NFKB1(p50), NFKB2(p52), RELA(p65) and RELB]59,60 that can form either homodimers or heterodimers. Therefore, the role of these signalling pathways in TNF-α-mediated up-regulation of endogenous LAMC2 expression in Caco-2 cells was investigated. Interestingly, we noticed a ∼400 nt long candidate enhancer region (chr1: 183,149,939–183,150,336, hg19 assembly) upstream of the LAMC2 TSS, which is predicted to have a single NF-κB-binding site and is also bound by RELA as indicated by ENCODE ChIP-seq21 data (Fig. 5A). Consistent with the hypothesis of NF-κB-mediated regulation, the NF-κB inhibitor TPCK effectively diminished the effect of TNF-α on LAMC2 mRNA and protein levels (Fig. 5E and F). Moreover, it has previously been demonstrated that an active ERK pathway is involved in the regulation of LAMC2 in squamous cell carcinoma.61 However, inhibition of ERK1/2 with FR180204 had only a minor effect on the TNF-α-induced LAMC2 mRNA expression (Fig. 5D) and almost no effect on the LAMC2 protein levels (Fig. 5F) in our experiments. Since these findings indicate a transcriptional up-regulation by TNF-α and NF-κB, we measured the effect of TNF-α on the activity of an LAMC2 promoter construct32 with and without the predicted enhancer; the enhancerless construct did not respond to TNF-α, but the enhancer–promoter construct gave a 3-fold increase in reporter signal (P < 0.001 (t-test)) (Fig. 5G). Validating the causal role of NF-κB, co-transfection of LAMC2 reporter constructs with RELA in combination with NFKB1 or NFKB2-expression plasmids could significantly (P < 0.001 (t-test)) up-regulate the enhancer–promoter construct but not the enhancerless construct. This effect was highest in Caco-2 cells overexpressing both RELA and NFKB2 subunits (P < 0.001) (Fig. 5G).Figure 5.

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