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OP019. Hypoxia responsive elements methylation status governs adherent-invasive Escherichia coli-receptor CEACAM6 expression in Crohn's disease

J. Denizot1, A. Agus1, A. Desrichard2, N. Uhrhammer2, A. Darfeuille-Michaud1, N. Barnich1, 1Université d'Auvergne, M2iSH, UMR INSERM/Université d'Auvergne U1071; Unité Sous Contrat Institut National de la Recherche Agronomique 2018, Clermont-Ferrand, France, 2Université d'Auvergne, Department of Oncogenetics, Department of Breast Oncology, Centre Jean Perrin, Clermont-Ferrand, France

Background

Abnormal expression of CEACAM6 is observed at the apical surface of the ileal epithelium in Crohn's disease (CD) patients. This allows Adherent-Invasive Escherichia coli (AIEC) to colonize gut mucosa, leading to development of inflammation. Our aim was to understand the molecular mechanisms involved in the abnormal expression of CEACAM6 in the mucosa of CD patients. Since changes in DNA methylation patterns were reported in CD patients, we analyzed whether epigenetic mechanisms are involved in the up-regulation of CEACAM6 expression in intestinal epithelial cells.

Methods

Global methylation status of CEACAM6 promoter was analyzed using bisulfite sequencing and site-specific methylation level was quantified using snap-shot approach in intestinal epithelial cells. Protein expression and localization were performed using immunofluorescence staining and Western-blot analysis. Chromatin immunoprecipitation was used to determine HIF-1 transcription factor binding on CEACAM6 gene promoter. pCpG free SEAP reporter system was used to analyze CEACAM6 promoter activity. Transgenic CEABAC10 mice expressing human CEACAM6 fed either standard food (Normal Diet) or a Low-Methyl-Diet (LMD) composed with lower quantities of B12 vitamins, folic acid and choline, and were orally challenged with 109 AIEC LF82 bacteria. At 3 days post-infection, gut-associated AIEC and pro-inflammatory cytokines were quantified.

Results

Analysis of CEACAM6 gene promoter revealed potentially methylated dinucleotide CpGs within Hypoxia Inducible Factor-1 (HIF-1) Responsive Elements (HREs). Methylation levels of these CpG were inversely correlated with CEACAM6 expression in intestinal epithelial cells (IEC) expressing various levels of CEACAM6. We also demonstrated the importance of HRE methylation levels and transcription factor HIF-1 in the regulation of CEACAM6 gene in IEC using pCpG free SEAP promoter reporter system. This was confirmed in vivo in CEABAC10 mouse model under a methyl donor-deficient diet. Low methyl diet-dependent HRE demethylation led to abnormal gut expression of CEACAM6, favoring AIEC colonization and subsequent inflammation.

Conclusion

A CEACAM6 promoter hypomethylation on HRE-containing CpG correlates with higher CEACAM6 expression in IEC. Our findings suggest that abnormal DNA methylation leading to CEACAM6 increased expression and AIEC-mediated gut inflammation can be related to changes in nutritional habits, such as low intake in methyl donor molecules, leading to abnormal epigenetic marks in Crohn's disease patients.