Abstract
Twist1 overexpression is frequently observed in various cancers including gastric cancer (GC). Although DNA methylation of the Twist1 gene has been reported in cancer cells, the mechanisms underlying transcriptional activation remain uncertain. In this study, we first examined epigenetic alterations of the Twist1 using Twist1 transcription-positive and -negative cell lines that are derived from our established diffuse-type GC mouse model. Treatment with a DNA demethylation agent 5-aza-dC re-activated Twist1 expression in Twist1 expression-negative GC cells. According to methylation-specific PCR and bisulfite sequencing analysis, methylation at the CpG-rich region within Twist1 coding exon 1, rather than its promoter region, was tightly linked to transcriptional silencing of the Twist1 expression in mouse GC cells. Chromatin immunoprecipitation assays revealed that active histone mark H3K4me3 was enriched in Twist1 expression-positive cells, and inactive histone mark H3K9me3 was enriched in Twist1 expression-negative cells. The expression levels of Suv39h1 and Suv39h2, histone methyltransferases for H3K9me3, were inversely correlated with Twist1 expression, and knockdown of Suv39h1 or Suv39h2 induced Twist1 expression. Moreover, Sp1 transcription factor bound to the exon 1 CpG-rich region in Twist1 expression-positive cell lines, and Twist1 expression was diminished by mithramycin, which that interferes with Sp1 binding to CpG-rich regulatory sequences. Our studies suggested that the Twist1 transcription in GC cells might be regulated through potential cooperation of DNA methylation, histone modification in complex with Sp1 binding to CpG-rich regions within the exon 1 region.
Highlights
Twist1, acting as a basic helix-loop-helix transcription factor, directly binds to E-box elements (NCANNTGN) on specific target genes [1]
In the eight human gastric cancer (GC) cell lines examined, Twist1 expression was downregulated in four cell lines (Fig 1E, left), three (KATO-III, GCIY and AGS) of which demonstrated the re-activation of Twist1 expression after treatment with 5-aza-dC by Reverse transcription-polymerase chain reaction (RT-PCR)
As for Twist1 expression-positive GC cell lines, after 5-aza-dC treatment, 2.1- and 3.2-fold up-regulation of Twist1 was detected in MKN45 and MKN7, respectively, by quantitative Reverse transcription (RT)-PCR (qRT-PCR), whereas Twist1 expression was not changed in MDGC-9 cells (Fig 1F)
Summary
Twist, acting as a basic helix-loop-helix transcription factor, directly binds to E-box elements (NCANNTGN) on specific target genes [1]. Twist is widely known to be essential for mesoderm formation during early embryonic development of drosophila and mouse [2, 3]. PLOS ONE | DOI:10.1371/journal.pone.0145630 December 22, 2015
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