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Intestinal DMBT1 expression is modulated by Crohn's disease-associated IL23R variants and by a DMBT1 variant which influences binding of the transcription factors CREB1 and ATF-2.

Diegelmann J, Czamara D, Le Bras E, Zimmermann E, Olszak T, Bedynek A, Göke B, Franke A, Glas J, Brand S - PLoS ONE (2013)

Bottom Line: Several DMBT1 SNPs were associated with CD susceptibility.All haplotype groups tested showed highly significant associations with CD (including omnibus P-values as low as 6.1 × 10(-18)).Intestinal DMBT1 expression is decreased in CD patients carrying the rs2981804 CD risk allele.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine II - Grosshadern, Ludwig-Maximilians-University (LMU), Munich, Germany ; Department of Preventive Dentistry and Periodontology, Ludwig-Maximilians-University (LMU), Munich, Germany.

ABSTRACT

Objectives: DMBT is an antibacterial pattern recognition and scavenger receptor. In this study, we analyzed the role of DMBT1 single nucleotide polymorphisms (SNPs) regarding inflammatory bowel disease (IBD) susceptibility and examined their functional impact on transcription factor binding and downstream gene expression.

Methods: Seven SNPs in the DMBT1 gene region were analyzed in 2073 individuals including 818 Crohn's disease (CD) patients and 972 healthy controls in two independent case-control panels. Comprehensive epistasis analyses for the known CD susceptibility genes NOD2, IL23R and IL27 were performed. The influence of IL23R variants on DMBT1 expression was analyzed. Functional analysis included siRNA transfection, quantitative PCR, western blot, electrophoretic mobility shift and luciferase assays.

Results: IL-22 induces DMBT1 protein expression in intestinal epithelial cells dependent on STAT3, ATF-2 and CREB1. IL-22 expression-modulating, CD risk-associated IL23R variants influence DMBT1 expression in CD patients and DMBT1 levels are increased in the inflamed intestinal mucosa of CD patients. Several DMBT1 SNPs were associated with CD susceptibility. SNP rs2981804 was most strongly associated with CD in the combined panel (p = 3.0 × 10(-7), OR 1.42; 95% CI 1.24-1.63). All haplotype groups tested showed highly significant associations with CD (including omnibus P-values as low as 6.1 × 10(-18)). The most strongly CD risk-associated, non-coding DMBT1 SNP rs2981804 modifies the DNA binding sites for the transcription factors CREB1 and ATF-2 and the respective genomic region comprising rs2981804 is able to act as a transcriptional regulator in vitro. Intestinal DMBT1 expression is decreased in CD patients carrying the rs2981804 CD risk allele.

Conclusion: We identified novel associations of DMBT1 variants with CD susceptibility and discovered a novel functional role of rs2981804 in regulating DMBT1 expression. Our data suggest an important role of DMBT1 in CD pathogenesis.

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Related in: MedlinePlus

Variants in rs2981804 alter binding of the transcription factors CREB1 and ATF-2 to the respective genomic DNA sequence.(A) EMSA analysis was performed with biotinylated probes and nuclear extracts from the intestinal epithelial cell lines HT-29 (lanes 2, 6), DLD-1 (lanes 3, 7), SW480 (lanes 4, 8) and HCT116 (lanes 5, 9). A DNA probe containing the IBD risk allele A of rs2981804 is much stronger bound by nuclear proteins than a probe with the G allele. Lane 1 contains the DNA probe only with no nuclear extract added. (B) Two specific protein complexes binding to the DMBT1 probe can be detected. The addition of a ATF-2 (lane 3) or CREB1 (lane 4) antibody to the EMSA binding reactions inhibit protein binding to the DMBT1 probe with the risk allele A while a non-specific isotype control antibody had no effect (lane 9). Addition of 50-fold excess of unlabelled CREB1, ATF-2 or DMBT1 probe (G allele) reduced protein binding (lanes 5, 6, 8). A DMBT1 probe with the A allele completely abolished protein binding (lane 7) while an unlabelled non-specific DNA probe did not inhibit DNA-protein complex formation confirming specificity of binding (lane 10); ns = non-specific. (C) In EMSAs with a labelled CREB1 consensus probe (lanes 1–7), the addition of 50-fold excess of unlabelled DMBT1 probe inhibited protein binding (lane 7) as well as did unlabelled CREB1 probe or a CREB1 antibody (lanes 4, 6). The protein complex bound to a labelled DMBT1 probe (lanes 8–9) is migrating slower (and therefore larger) than that of the CREB1 probe. (D) Silencing of ATF-2 or CREB1 expression diminished protein binding to the DMBT1 probe. DLD-1 cells were transfected with siRNA against CREB1, ATF-2 or a non-specific control siRNA 48 h prior to nuclear protein isolation. EMSA was performed as in Fig. 2A with a DMBT1 probe containing the A allele. While expression silencing of CREB1 completely abolished protein binding to the DMBT1 probe, silencing of ATF-2 had a weaker but still detectable effect.
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pone-0077773-g004: Variants in rs2981804 alter binding of the transcription factors CREB1 and ATF-2 to the respective genomic DNA sequence.(A) EMSA analysis was performed with biotinylated probes and nuclear extracts from the intestinal epithelial cell lines HT-29 (lanes 2, 6), DLD-1 (lanes 3, 7), SW480 (lanes 4, 8) and HCT116 (lanes 5, 9). A DNA probe containing the IBD risk allele A of rs2981804 is much stronger bound by nuclear proteins than a probe with the G allele. Lane 1 contains the DNA probe only with no nuclear extract added. (B) Two specific protein complexes binding to the DMBT1 probe can be detected. The addition of a ATF-2 (lane 3) or CREB1 (lane 4) antibody to the EMSA binding reactions inhibit protein binding to the DMBT1 probe with the risk allele A while a non-specific isotype control antibody had no effect (lane 9). Addition of 50-fold excess of unlabelled CREB1, ATF-2 or DMBT1 probe (G allele) reduced protein binding (lanes 5, 6, 8). A DMBT1 probe with the A allele completely abolished protein binding (lane 7) while an unlabelled non-specific DNA probe did not inhibit DNA-protein complex formation confirming specificity of binding (lane 10); ns = non-specific. (C) In EMSAs with a labelled CREB1 consensus probe (lanes 1–7), the addition of 50-fold excess of unlabelled DMBT1 probe inhibited protein binding (lane 7) as well as did unlabelled CREB1 probe or a CREB1 antibody (lanes 4, 6). The protein complex bound to a labelled DMBT1 probe (lanes 8–9) is migrating slower (and therefore larger) than that of the CREB1 probe. (D) Silencing of ATF-2 or CREB1 expression diminished protein binding to the DMBT1 probe. DLD-1 cells were transfected with siRNA against CREB1, ATF-2 or a non-specific control siRNA 48 h prior to nuclear protein isolation. EMSA was performed as in Fig. 2A with a DMBT1 probe containing the A allele. While expression silencing of CREB1 completely abolished protein binding to the DMBT1 probe, silencing of ATF-2 had a weaker but still detectable effect.

Mentions: In EMSA experiments with probes containing either the IBD risk allele A or the protective allele G of rs2981804 and the surrounding genomic sequences, nuclear extracts from IEC lines HT-29, DLD-1, HCT116 and SW480 bound strongly to the probe containing the risk allele A. The probe with the IBD-protective allele G showed clearly weaker protein binding (Fig. 4A). To analyze which proteins were bound to the probes, antibodies against phosphorylated cAMP responsive element binding protein 1 (CREB1) or activating transcription factor 2 (ATF-2) were incubated together with the DNA binding reaction with nuclear extracts from DLD-1 cells. For both transcription factors, higher binding scores were predicted for the A allele in comparison to the G allele (table 6). The addition of an ATF-2 or a CREB1 antibody diminished but did not abolish DNA binding of protein to the DMBT1 probe (Fig. 4B, lanes 3, 4) as well as did unlabelled competitor probes with binding sequences for ATF-2 or CREB1 (Fig. 4B, lanes 5, 8). Addition of 50-fold excess of an unlabelled DMBT1 probe with the A allele completely inhibited binding to the labelled probe while a DMBT1 probe with the G allele had a weaker effect (Fig. 4B, lanes 6, 7). Control reactions (Fig. 4B, lanes 9, 10) with a non-specific antibody or a non-specific DNA probe which did not inhibit specific DNA-protein complex formation demonstrated that two specific protein complexes with two distinct sizes are bound to the DMBT1 probe (Fig. 4B, see arrows).


Intestinal DMBT1 expression is modulated by Crohn's disease-associated IL23R variants and by a DMBT1 variant which influences binding of the transcription factors CREB1 and ATF-2.

Diegelmann J, Czamara D, Le Bras E, Zimmermann E, Olszak T, Bedynek A, Göke B, Franke A, Glas J, Brand S - PLoS ONE (2013)

Variants in rs2981804 alter binding of the transcription factors CREB1 and ATF-2 to the respective genomic DNA sequence.(A) EMSA analysis was performed with biotinylated probes and nuclear extracts from the intestinal epithelial cell lines HT-29 (lanes 2, 6), DLD-1 (lanes 3, 7), SW480 (lanes 4, 8) and HCT116 (lanes 5, 9). A DNA probe containing the IBD risk allele A of rs2981804 is much stronger bound by nuclear proteins than a probe with the G allele. Lane 1 contains the DNA probe only with no nuclear extract added. (B) Two specific protein complexes binding to the DMBT1 probe can be detected. The addition of a ATF-2 (lane 3) or CREB1 (lane 4) antibody to the EMSA binding reactions inhibit protein binding to the DMBT1 probe with the risk allele A while a non-specific isotype control antibody had no effect (lane 9). Addition of 50-fold excess of unlabelled CREB1, ATF-2 or DMBT1 probe (G allele) reduced protein binding (lanes 5, 6, 8). A DMBT1 probe with the A allele completely abolished protein binding (lane 7) while an unlabelled non-specific DNA probe did not inhibit DNA-protein complex formation confirming specificity of binding (lane 10); ns = non-specific. (C) In EMSAs with a labelled CREB1 consensus probe (lanes 1–7), the addition of 50-fold excess of unlabelled DMBT1 probe inhibited protein binding (lane 7) as well as did unlabelled CREB1 probe or a CREB1 antibody (lanes 4, 6). The protein complex bound to a labelled DMBT1 probe (lanes 8–9) is migrating slower (and therefore larger) than that of the CREB1 probe. (D) Silencing of ATF-2 or CREB1 expression diminished protein binding to the DMBT1 probe. DLD-1 cells were transfected with siRNA against CREB1, ATF-2 or a non-specific control siRNA 48 h prior to nuclear protein isolation. EMSA was performed as in Fig. 2A with a DMBT1 probe containing the A allele. While expression silencing of CREB1 completely abolished protein binding to the DMBT1 probe, silencing of ATF-2 had a weaker but still detectable effect.
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Related In: Results  -  Collection

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pone-0077773-g004: Variants in rs2981804 alter binding of the transcription factors CREB1 and ATF-2 to the respective genomic DNA sequence.(A) EMSA analysis was performed with biotinylated probes and nuclear extracts from the intestinal epithelial cell lines HT-29 (lanes 2, 6), DLD-1 (lanes 3, 7), SW480 (lanes 4, 8) and HCT116 (lanes 5, 9). A DNA probe containing the IBD risk allele A of rs2981804 is much stronger bound by nuclear proteins than a probe with the G allele. Lane 1 contains the DNA probe only with no nuclear extract added. (B) Two specific protein complexes binding to the DMBT1 probe can be detected. The addition of a ATF-2 (lane 3) or CREB1 (lane 4) antibody to the EMSA binding reactions inhibit protein binding to the DMBT1 probe with the risk allele A while a non-specific isotype control antibody had no effect (lane 9). Addition of 50-fold excess of unlabelled CREB1, ATF-2 or DMBT1 probe (G allele) reduced protein binding (lanes 5, 6, 8). A DMBT1 probe with the A allele completely abolished protein binding (lane 7) while an unlabelled non-specific DNA probe did not inhibit DNA-protein complex formation confirming specificity of binding (lane 10); ns = non-specific. (C) In EMSAs with a labelled CREB1 consensus probe (lanes 1–7), the addition of 50-fold excess of unlabelled DMBT1 probe inhibited protein binding (lane 7) as well as did unlabelled CREB1 probe or a CREB1 antibody (lanes 4, 6). The protein complex bound to a labelled DMBT1 probe (lanes 8–9) is migrating slower (and therefore larger) than that of the CREB1 probe. (D) Silencing of ATF-2 or CREB1 expression diminished protein binding to the DMBT1 probe. DLD-1 cells were transfected with siRNA against CREB1, ATF-2 or a non-specific control siRNA 48 h prior to nuclear protein isolation. EMSA was performed as in Fig. 2A with a DMBT1 probe containing the A allele. While expression silencing of CREB1 completely abolished protein binding to the DMBT1 probe, silencing of ATF-2 had a weaker but still detectable effect.
Mentions: In EMSA experiments with probes containing either the IBD risk allele A or the protective allele G of rs2981804 and the surrounding genomic sequences, nuclear extracts from IEC lines HT-29, DLD-1, HCT116 and SW480 bound strongly to the probe containing the risk allele A. The probe with the IBD-protective allele G showed clearly weaker protein binding (Fig. 4A). To analyze which proteins were bound to the probes, antibodies against phosphorylated cAMP responsive element binding protein 1 (CREB1) or activating transcription factor 2 (ATF-2) were incubated together with the DNA binding reaction with nuclear extracts from DLD-1 cells. For both transcription factors, higher binding scores were predicted for the A allele in comparison to the G allele (table 6). The addition of an ATF-2 or a CREB1 antibody diminished but did not abolish DNA binding of protein to the DMBT1 probe (Fig. 4B, lanes 3, 4) as well as did unlabelled competitor probes with binding sequences for ATF-2 or CREB1 (Fig. 4B, lanes 5, 8). Addition of 50-fold excess of an unlabelled DMBT1 probe with the A allele completely inhibited binding to the labelled probe while a DMBT1 probe with the G allele had a weaker effect (Fig. 4B, lanes 6, 7). Control reactions (Fig. 4B, lanes 9, 10) with a non-specific antibody or a non-specific DNA probe which did not inhibit specific DNA-protein complex formation demonstrated that two specific protein complexes with two distinct sizes are bound to the DMBT1 probe (Fig. 4B, see arrows).

Bottom Line: Several DMBT1 SNPs were associated with CD susceptibility.All haplotype groups tested showed highly significant associations with CD (including omnibus P-values as low as 6.1 × 10(-18)).Intestinal DMBT1 expression is decreased in CD patients carrying the rs2981804 CD risk allele.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine II - Grosshadern, Ludwig-Maximilians-University (LMU), Munich, Germany ; Department of Preventive Dentistry and Periodontology, Ludwig-Maximilians-University (LMU), Munich, Germany.

ABSTRACT

Objectives: DMBT is an antibacterial pattern recognition and scavenger receptor. In this study, we analyzed the role of DMBT1 single nucleotide polymorphisms (SNPs) regarding inflammatory bowel disease (IBD) susceptibility and examined their functional impact on transcription factor binding and downstream gene expression.

Methods: Seven SNPs in the DMBT1 gene region were analyzed in 2073 individuals including 818 Crohn's disease (CD) patients and 972 healthy controls in two independent case-control panels. Comprehensive epistasis analyses for the known CD susceptibility genes NOD2, IL23R and IL27 were performed. The influence of IL23R variants on DMBT1 expression was analyzed. Functional analysis included siRNA transfection, quantitative PCR, western blot, electrophoretic mobility shift and luciferase assays.

Results: IL-22 induces DMBT1 protein expression in intestinal epithelial cells dependent on STAT3, ATF-2 and CREB1. IL-22 expression-modulating, CD risk-associated IL23R variants influence DMBT1 expression in CD patients and DMBT1 levels are increased in the inflamed intestinal mucosa of CD patients. Several DMBT1 SNPs were associated with CD susceptibility. SNP rs2981804 was most strongly associated with CD in the combined panel (p = 3.0 × 10(-7), OR 1.42; 95% CI 1.24-1.63). All haplotype groups tested showed highly significant associations with CD (including omnibus P-values as low as 6.1 × 10(-18)). The most strongly CD risk-associated, non-coding DMBT1 SNP rs2981804 modifies the DNA binding sites for the transcription factors CREB1 and ATF-2 and the respective genomic region comprising rs2981804 is able to act as a transcriptional regulator in vitro. Intestinal DMBT1 expression is decreased in CD patients carrying the rs2981804 CD risk allele.

Conclusion: We identified novel associations of DMBT1 variants with CD susceptibility and discovered a novel functional role of rs2981804 in regulating DMBT1 expression. Our data suggest an important role of DMBT1 in CD pathogenesis.

Show MeSH
Related in: MedlinePlus