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

<div><p>The <i>Ferritin heavy polypeptide-like 17</i> (<i>Fthl17</i>) 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 <i>FTHL17</i> gene expression, detailed epigenetic regulation of its expression has not been investigated. To address this, we assessed the epigenetic regulation of murine <i>Fthl17</i> gene expression in cancer cells and germ cells. <i>Fthl17</i> 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 <i>Fthl17</i> 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 <i>Fthl17</i> promoter in the regulation of <i>Fthl17</i> expression. In KLN205 cells, testis, and GSCs, the <i>Fthl17</i> 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 <i>Fthl17</i> 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 <i>Fthl17</i> up-regulation in each cell type. Following treatment with 5-Azacytidine, the -0.3 kb to 0 kb region became hypo-methylated, and <i>Fthl17</i> 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 <i>Fthl17</i> 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.</p></div

Implication of DNA demethylation and bivalent histone modification for selective gene regulation in mouse primordial germ cells.

Primordial germ cells (PGCs) sequentially induce specific genes required for their development. We focused on epigenetic changes that regulate PGC-specific gene expression. mil-1, Blimp1, and Stella are preferentially expressed in PGCs, and their expression is upregulated during PGC differentiation. Here, we first determined DNA methylation status of mil-1, Blimp1, and Stella regulatory regions in epiblast and in PGCs, and found that they were hypomethylated in differentiating PGCs after E9.0, in which those genes were highly expressed. We used siRNA to inhibit a maintenance DNA methyltransferase, Dnmt1, in embryonic stem (ES) cells and found that the flanking regions of all three genes became hypomethylated and that expression of each gene increased 1.5- to 3-fold. In addition, we also found 1.5- to 5-fold increase of the PGC genes in the PGCLCs (PGC-like cells) induced form ES cells by knockdown of Dnmt1. We also obtained evidence showing that methylation of the regulatory region of mil-1 resulted in 2.5-fold decrease in expression in a reporter assay. Together, these results suggested that DNA demethylation does not play a major role on initial activation of the PGC genes in the nascent PGCs but contributed to enhancement of their expression in PGCs after E9.0. However, we also found that repression of representative somatic genes, Hoxa1 and Hoxb1, and a tissue-specific gene, Gfap, in PGCs was not dependent on DNA methylation; their flanking regions were hypomethylated, but their expression was not observed in PGCs at E13.5. Their promoter regions showed the bivalent histone modification in PGCs, that may be involved in repression of their expression. Our results indicated that epigenetic status of PGC genes and of somatic genes in PGCs were distinct, and suggested contribution of epigenetic mechanisms in regulation of the expression of a specific gene set in PGCs

<i>Fthl17</i> gene expression was induced by promoter hypo-methylation in GSCs.

<p>(A) Gene expression levels of <i>Fthl17</i> in testis and GSCs were investigated by quantitative RT-PCR analysis. The primer sequences are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172219#pone.0172219.t001" target="_blank">Table 1</a>. The expression level in testis was set as 1.0. The data were obtained from three independent experiments. ***p < 0.001. Error bars represent SEM. (B) Normalized intensity values of <i>Fthl17</i> expression in type A spermatogonia (TypeA SG), type B spermatogonia (TypeB SG), pachytene spermatocyte (PS) and round spermatids (RS). The data obtained from published microarray data (GSE4193) were normalized by using global median scaling, and were re-analyzed using GeneSpring (Agilent). Plots show average values from two independent data sets. (C) The DNA methylation status of the 5’-upstream region of <i>Fthl17</i> in GSCs was investigated by bisulfite sequencing analysis. The sequencing primers are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172219#pone.0172219.t001" target="_blank">Table 1</a>. Methylated and unmethylated CpGs are presented as closed circles and open circles, respectively. The 5’-upstream region of <i>Fthl17</i> is schematically shown at the top of the panel. TSS represents the transcription start site. Sequence data were obtained from two independent experiments. (D-E) Quantitative evaluation of DNA methylation levels of the 5’-upstream region of <i>Fthl17</i> in GSCs and testis (D), and GSCs and KLN205 cells (E). The region I, II, III and IV showed -1.4 kb to -1.0 kb, -1.0 kb to -0.6 kb, -0.6 kb to -0.3 kb and -0.3 kb to 0 kb, respectively. The central bars indicate medians, lower and upper limits of the boxes mark the 25^th and 75^th percentiles. The whiskers extend to the most extreme data point. Significant difference of methylation levels in each region was statistically evaluated by using the QUMA program and Mann-Whitney U-test. (F) The luciferase activity of the reporter vectors with either methylated or unmethylated <i>Fthl17</i> 5’-upstream regions (-1.7 kb or -0.6 kb) in GSCs. Luciferase activity was normalized to the activity of a co-transfected Renilla phRL-TK vector. Luciferase activity from the methylated vectors was set as 1.0. The data were obtained from three independent experiments. ***p < 0.001. (G) Representative images of H3K9me2 ChIP-seq read density at the <i>Fthl17</i> promoter in GSCs, (GS, blue), pachytene spermatocyte (PS, red) and round spermatids (RS, green). ChIP-seq data (GSE69946) were re-analyzed using the Integrative Genomics Viewer (IGV).</p

<i>Fthl17</i> gene expression was induced by promoter hypo-methylation in KLN205 cells.

<p>(A) Gene expression levels of <i>Fthl17</i> in normal lung tissue, KLN205 lung cancer cell line and testis, which were investigated by quantitative RT-PCR analysis. The primer sequences are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172219#pone.0172219.t001" target="_blank">Table 1</a>. The expression level in one sample of KLN205 cells was set as 1.0. The data were obtained from three independent experiments. *p < 0.05, ***p < 0.001. Error bars represent SEM. (B) DNA methylation status of the 5’-upstream region of <i>Fthl17</i> in normal lung tissue, KLN205 lung cancer cell line and testis, which was investigated by bisulfite sequencing analysis. The sequencing primers are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172219#pone.0172219.t001" target="_blank">Table 1</a>. Methylated and unmethylated CpGs are presented as closed circles and open circles, respectively. The 5’-upstream region of <i>Fthl17</i> is schematically shown at the top of the panel. TSS represents the transcription start site. The sequence data of each cell type were obtained from two independent experiments. (C-E) Quantitative evaluation of DNA methylation levels of the 5’-upstream region of <i>Fthl17</i> in normal lung tissues and KLN205 cells (C), testis and KLN205 cells (D), and normal lung tissues and testis (E). The region I, II, III and IV showed -1.4 kb to -1.0 kb, -1.0 kb to -0.6 kb, -0.6 kb to -0.3 kb and -0.3 kb to 0 kb, respectively. The central bars indicate medians, lower and upper limits of the boxes mark the 25^th and 75^th percentiles. The whiskers extend to the most extreme data point. Significant difference of methylation levels in each region was statistically evaluated by using the QUMA program and Mann-Whitney U-test. (F) The luciferase activity of the reporter vectors with either methylated or unmethylated <i>Fthl17</i> 5’-upstream regions (-1.7 kb or -0.6 kb) in KLN205 cells. Luciferase activity was normalized to the activity of a co-transfected Renilla phRL-TK vector. Luciferase activity from the methylated vectors was set as 1.0. The data were obtained from three independent experiments. *p < 0.05, **p < 0.01. Error bars represent SEM.</p

5-Azacytidine induced hypo-methylation and up-regulation of <i>Fthl17</i> gene expression in KLN205 cells.

<p>(A) Gene expression levels of <i>Fthl17</i> in KLN205 cells cultured with or without 5-Azacytidine (5-Aza) were investigated by quantitative RT-PCR analysis. The primer sequences are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172219#pone.0172219.t001" target="_blank">Table 1</a>. The expression level in one control sample (without 5-Aza) was set as 1.0. The data were obtained from three independent experiments. *p < 0.05. Error bars represent SEM. (B) DNA methylation status of the -0.3 kb to 0 kb region of the <i>Fthl17</i> promoter in KLN205 cells cultured with or without 5-Aza was investigated by bisulfite sequencing analysis. Methylated and unmethylated CpGs are presented as closed circles and open circles, respectively. The 5’-upstream region of <i>Fthl17</i> is schematically shown at the top of the panel. TSS represents the transcription start site. Sequence data were obtained from two independent experiments. (C) Quantitative evaluation of DNA methylation levels of the -0.3 kb to 0 kb region of <i>Fthl17</i> in control sample and with 5-Aza sample. The central bars indicate medians, lower and upper limits of the boxes mark the 25^th and 75^th percentiles. The whiskers extend to the most extreme data point. Significant difference of methylation levels in each region was statistically evaluated by using the QUMA program and Mann-Whitney U-test.</p

Primers used for quantitative RT-PCR, bisulfite sequencing and cloning.

<p>Primers used for quantitative RT-PCR, bisulfite sequencing and cloning.</p

Binding sites of transcription factors in the 5’-upstream region of <i>Fthl17</i>.

<p>(A) The -0.6 kb to -0.3 kb region and the -0.3 kb to 0 kb region were analyzed by ConSite and JASPAR, respectively, to identify binding sites of transcription factors. (B) Representative images of ChIP-seq read density for PLZF in the 5’-upstream region of <i>Fthl17</i> in undifferentiated spermatogonia. ChIP-seq data (GSE73390) were re-analyzed by using the Integrative Genomics Viewer (IGV).</p

Dnd1-mediated epigenetic control of teratoma formation in mouse

Spontaneous testicular teratoma develops from primordial germ cells (PGCs) in embryos; however, the molecular mechanisms underlying teratoma formation are not fully understood. Mutation of the dead-end 1 (Dnd1) gene, which encodes an RNA-binding protein, drastically enhances teratoma formation in the 129/Sv mouse strain. To elucidate the mechanism of Dnd1 mutation-induced teratoma formation, we focused on histone H3 lysine 27 (H3K27) trimethylation (me3), and found that the levels of H3K27me3 and its responsible methyltransferase, enhancer of zeste homolog 2 (Ezh2), were decreased in the teratoma-forming cells of Dnd1 mutant embryos. We also showed that Dnd1 suppressed miR-26a-mediated inhibition of Ezh2 expression, and that Dnd1 deficiency resulted in decreased H3K27me3 of a cell-cycle regulator gene, Ccnd1. In addition, Ezh2 expression or Ccnd1 deficiency repressed the reprogramming of PGCs into pluripotent stem cells, which mimicked the conversion of embryonic germ cells into teratoma-forming cells. These results revealed an epigenetic molecular linkage between Dnd1 and the suppression of testicular teratoma formation