Epigenetic Control of the S100A6 (Calcyclin) Gene Expression

Abstract

S100A6 (calcyclin) is a calcium-binding protein of cell-specific expression whose gene is clustered with other S100 genes within the epidermal differentiation complex, on human chromosome 1q21. Many S100 proteins, including S100A6, are expressed in human epidermis at various stages of differentiation and their expression is often deregulated in skin and epithelial cancers. To gain insight into the mechanism of regulation of S100A6 expression, we examined epigenetic marks, that is DNA methylation and histone modifications along the S100A6 gene. Sequencing of bisulfite-modified DNA within a 3,247 bp long genomic region encompassing the promoter/first exon CpG island, the coding sequence of the S100A6 gene and a downstream region showed that it is almost entirely methylation-free in S100A6 expressing human epidermoid carcinoma (Hep-2) cells and lymphocytes and methylated in S100A6-negative embryonic epithelial (HEK293) cells. Chromatin immunoprecipitation revealed profound differences in the level of histone H3 acetylation and methylation and in the in vivo binding of upstream regulatory factor (USF), to the S100A6 gene promoter in S100A6-negative and -positive cells. These data demonstrate that cell-specific S100A6 expression is under control of epigenetic mechanisms.

S100A6 Overexpression Is Associated with Poor Prognosis and Is Epigenetically Up-Regulated in Gastric Cancer

“…The primers were as reported. 23 Then the PCR products of cell lines were subcloned into the pMD18-T vector and sequenced, while the tissue PCR products were sequenced directly. The methylated percentages of the CpG sites in primary gastric cancer tissues and adjacent nonneoplastic gastric mucosa were calculated separately.…”

“…The subsequent PCR was performed with the forward: 5Ј-CTGGCTC-GAGGGTCA-3Ј and backward: 5Ј-GGCTGTTCCGGGT-TAGGA-3Ј primers. 23 Then the average percentage of input signal of the primary gastric cancer and nonneoplastic mucosa were compared.…”

“…Because previous studies have shown that epigenetic factors may regulate S100A6 gene expression, 23 to further explore the mechanism of the up-regulation of S100A6, epigenetic regulation was investigated. First, we chose the S100A6 high-and low-expressing cell lines by measuring S100A6 expression levels in seven gastric cancer cell lines.…”

“…com, last accessed May, 2010), and the proximal S100A6 gene promoter fragment, the first exon and a part of the first intron, are in a CpG-rich region that fulfills all of the criteria for a CpG island as previously reported. 23 CpG islands are defined here as sequences longer than 500 bp, with an average GC level higher than 50% and a frequency of CpG greater than 60% of the statistically expected value. 31 Methylation status of S100A6 in above seven gastric cancer cell lines was investigated by clone sequencing of two regions amplified from bisulfite-modified DNA, the promoter/first exon (P/E1) region, which contains the upstream promoter (11 CpG) and first exon region (31 CpG), and the second exon/second intron (E2/I2) region, which contains the second exon (3 CpG) and second intron (2 CpG).…”

“…31 Methylation status of S100A6 in above seven gastric cancer cell lines was investigated by clone sequencing of two regions amplified from bisulfite-modified DNA, the promoter/first exon (P/E1) region, which contains the upstream promoter (11 CpG) and first exon region (31 CpG), and the second exon/second intron (E2/I2) region, which contains the second exon (3 CpG) and second intron (2 CpG). HEK293 cells were used as a positive control, 23 with 43 of 47 CpG sites were fully methylated, while the sixth, 13th, and 14th CpG pairs in the P/E1 and first in the E2/I2 incompletely methylated in this study. The difference of methylation status between S100A6 high-expressing cells (AGS, MKN45, KATO3, BGC823, and SGC7901) and low-expressing cells (RF1 and RF48) was obvious.…”