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. 2012 Nov 2;7(11):e47545. doi: 10.1371/journal.pone.0047545

Figure 2. REG4 promoter analysis.

Figure 2

Localization of regulatory elements and CDX2 binding sites in the 5′-flanking region of the REG4 gene. A: Schematic representation of the 5′-flanking region of the REG4 gene. The location and sequence of 4 consensus CDX2-binding sites in the 5′-flanking region of REG4 (i.e., sites A, B, C, and D) is indicated. B: Schematic representation of REG4 reporter gene constructs. The REG4 genomic DNA sequences present in the reporter gene vectors are indicated. Key sequences for REG4 transcription reside between base pairs −634 and −116. Reporter assays with the series of REG4 deletion constructs were performed in the CDX2-expressing GC cell line, HSC-39. The luciferase activity of the empty pGL4.10 basic vector was assigned a value of 1. The reporter assays were performed in triplicate, and mean and SD values of luciferase activity are shown. C: Localized mutations in the candidate CDX2-binding sites (i.e., sites A, B, C, and D) were introduced into the −2019/+58 construct, and the series of constructs generated is shown. The CDX2 candidate binding site designated as “C” plays critical roles in REG4 transcription. Reporter assays were performed in CDX2-expressing GC cell line, HSC-39. The activity of the pGL4.10 basic vector was assigned a value of 1. Assays were performed in triplicate. Mean and SD luciferase activity values are shown.