DNA Methylation in the Exon 1 Region and Complex Regulation of Twist1 Expression in Gastric Cancer Cells
Fig 2
Twist1 methylation status in murine and human GC cell lines.
(A) Schematic representation of the genomic structure and the predicted CGI with MethPrimer (http://www.urogene.org/methprimer/) of the mouse Twist1 gene. Two CGIs (thin blue regions, left, -358 to -149; right, -96 to +759) were observed at the region from the promoter to entire exon 1 (Obs/Exp = 0.8, %GC> 50%). The bent arrow indicates the transcription start site (TSS). Individual CpG sites are indicated as orange vertical lines. The boxes denote the exon. The black double-headed arrows indicate the regions (S1, P1, E1-1, E1-2 and E1-3) examined by methylation specific PCR (MSP). The blue double-headed arrows indicate the regions examined by ChIP assay. The red bars indicate the regions examined by bisulfite sequencing (BS). (B) MSP analysis of MDGC cell lines and three normal gastric mucosae from Atp4b-Cre-; Cdh1loxP/loxP; Trp53 loxP/loxP mice in the five regions (left). U: unmethylated alleles; M: methylated alleles. BS of the region from +322 to +670 (BS-c) in MDGC-3 and MDGC-9 (right). BS-a and BS-b are shown in the S3 Fig. The MSP region of E1-2 and BS-c correspond to Twist1 expression. Black circles represent the methylated CpG site; white circles represent the unmethylated CpG sites. (C) Schematic representation of the genomic structure and the predicted CGI with MethPrimer of the human Twist1 gene. Two CGIs (left, -369 to -138; right, +91 to +742) were observed (Obs/Exp = 0.8, %GC> 50%). (D) MSP and BS analyses of human GC cell lines and three non-cancerous gastric mucosae from GC patients. We assessed four regions (left, P1, E1-1, E1-2 and E1-3) and exon 1 (right, +315 to +651, BS-c) in the human Twist 1 gene, which are located analogously to mouse MSP and BS regions. Dense methylation was detected in KATO-III cells lacking Twist1 expression, while MKN45 cells expressing Twist1 exhibited both methylated and unmethylated alleles.