Functional characterization of the NF-kappaB binding site in the human NOD2 promoter.
Nucleotide-binding and oligomerization domain 2 (NOD2), a member of the NOD protein family, plays an important role in innate immunity. In response to pathogen attack, NOD2 stimulates cytokine and defensin production by activating nuclear factor (NF)-kappaB, a key transcription factor responsible for mediating downstream reactions. However, the mechanism linking NOD2 regulation and NF-kappaB activation is poorly understood. Using bioinformatics, we found a completely preserved canonical NF-kappaB binding site in the NOD2 core promoter (-16 to -25 bp) in both humans and chimpanzees. The functional role of this NF-kappaB binding site was investigated using the enhanced green fluorescent protein (EGFP) reporter system, site-directed mutagenesis, the NF-kappaB activation inhibitor (JSH-23) and the chromatin immunoprecipitation (ChIP) assay. The results show that the NF-kappaB binding site is critical for regulation of the NOD2 gene. Either deletion of the NF-kappaB binding elements within the NOD2 promoter or treatment with an NF-kappaB activation inhibitor could lead to a significant loss of NOD2 promoter activity as detected by reporter gene assay. The canonical NF-kappaB binding site was bound by NF-kappaB as determined by the ChIP method. Based on these results, we suggest a positive feedback regulation between NF-kappaB and NOD2, which may represent an efficient mechanism in response to pathogen invasion.
Hu
Chaofeng
C
Department of Pathophysiology, Medical College, Jinan University, Guangzhou, China.
Sun
Liping
L
Hu
Yiling
Y
Lu
Daxiang
D
Wang
Huadong
H
Tang
Suisheng
S
D020029:Base Pairing / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D047369:Chromatin Immunoprecipitation; D017124:Conserved Sequence / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000378:metabolism*; D053473:Nod2 Signaling Adaptor Protein / Q000235:genetics* / Q000378:metabolism; D010655:Phenylenediamines / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D011485:Protein Binding / Q000187:drug effects; D015203:Reproducibility of Results; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017384:Sequence Deletion / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid
C549066:4-methyl-N1-(3-phenylpropyl)benzene-1,2-diamine; D016328:NF-kappa B; C429018:NOD2 protein, human; D053473:Nod2 Signaling Adaptor Protein; D010655:Phenylenediamines; D049452:Green Fluorescent Proteins