DNA methylation of the Fthl17 5’-upstream region regulates differential Fthl17 expression in lung cancer cells and germline stem cells

Abstract

The Ferritin heavy polypeptide-like 17 (Fthl17) gene is a member of the cancer/testis antigen gene family, and is preferentially expressed in cancer cells and in testis. Although DNA methylation has been linked to the regulation of human FTHL17 gene expression, detailed epigenetic regulation of its expression has not been investigated. To address this, we assessed the epigenetic regulation of murine Fthl17 gene expression in cancer cells and germ cells. Fthl17 was more highly expressed in testis, a murine lung cancer cell line, KLN205, and in germline stem cells (GSCs) than in normal lung tissues. Furthermore, the Fthl17 expression level in GSCs was significantly higher than in KLN205 cells. We performed bisulfite-sequencing and luciferase (luc) reporter assays to examine the role of DNA methylation of the Fthl17 promoter in the regulation of Fthl17 expression. In KLN205 cells, testis, and GSCs, the Fthl17 5’-upstream region was hypo-methylated compared with normal lung tissues. Luc reporter assays indicated that hypo-methylation of the -0.6 kb to 0 kb region upstream from the transcription start site (TSS) was involved in the up-regulation of Fthl17 expression in KLN205 cells and GSCs. Because the -0.6 kb to -0.3 kb or the -0.3 kb to 0 kb region were relatively more hypo-methylated in KLN205 cells and in GSCs, respectively, compared with other regions between -0.6 kb to 0 kb, those regions may contribute to Fthl17 up-regulation in each cell type. Following treatment with 5-Azacytidine, the -0.3 kb to 0 kb region became hypo-methylated, and Fthl17 expression was up-regulated in KLN205 cells to a level comparable to that in GSCs. Together, the results suggest that hypo-methylation of different but adjacent regions immediately upstream of the Fthl17 gene contribute to differential expression levels in lung cancer cells and GSCs, and hypo-methylation of the TSS-proximal region may be critical for high level expression.

Novel mutation in FTHL17 gene in pedigree with 46,XY pure gonadal dysgenesis

“…DNA hypomethylation of the À0.6 to 0 kb region upstream of the transcription start site (TSS) was involved in the up-regulation of FTHL17 expression: the À0.6 to À0.3 kb and the À0.3 to 0 kb regions were relatively more hypomethylated in tumor cells and in germline stem cells, respectively. In addition, induced hypomethylation of the TSS-proximal region by 5-azacytidine up-regulated FTHL17 gene expression (24,31). Histone H3 lysine 9 dimethylation is a repressive epigenetic modification of FTHL17 expression in advanced testicular germ cells.…”

“…Most X-linked genes are considered to be likely regulated by histone modifications during meiotic sex chromosome inactivation (MSCI), and these genes are silenced from the pachytene stage of meiosis to postmeiosis. It is possible that differential expression of FTHL17 in more advanced testicular germ cells may be repressed by MSCI-related mechanisms (31). The origination of 46,XY PGD is the failure of testicular development during embryogenesis.…”

“…The expression of FTHL17 shows a significant correlation with the expression of the MAGE gene family. DNA methylation and histone modifications cooperatively regulate FTHL17 gene expression, and methylation, which generally represses gene expression, plays a fundamental role in the process (31). DNA hypomethylation of the À0.6 to 0 kb region upstream of the transcription start site (TSS) was involved in the up-regulation of FTHL17 expression: the À0.6 to À0.3 kb and the À0.3 to 0 kb regions were relatively more hypomethylated in tumor cells and in germline stem cells, respectively.…”

Regulation and signaling pathways in cancer stem cells: implications for targeted therapy for cancer

“…Furthermore, DNA demethylation plays a role in regulating the sphere-formation ability of CSCs and CSC-associated therapy resistance. Hyun-Mi Kwon et al demonstrated that DNMT1 inhibitors affect the sphere-formation ability of CSCs by effectively suppressing the expression of several TFs in pancreatic cancer, as well as in ovarian cancer and lung cancer [ 193 – 195 ]. In another study, DNMT1 inhibitor-mediated demethylation contributes to CSC resistance to sorafenib treatment [ 196 ].…”