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. 2017 Jun 20;7:3896. doi: 10.1038/s41598-017-04248-w

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Epigenetic silencing of downstream genes located in tandem orientation. (A) Outline of the organization of tandem oriented genes with a CpG island at the transcriptional end site (TES) of the upstream gene and the transcriptional start site (TSS) of the downstream gene. (B) Methylation level of TSS-associated CGI in A549 cells was plotted depending on the distance between TES and TSS in kb (n = 258 to 297 CGI). Methylation as detected using 450 K bisulfite bead chip arrays. “All CpGs” and “all TSS” indicate the methylation level of CpGs located at CGI and TSS-associated CGI, respectively. (C) Methylation levels of tandem oriented downstream CGI in H322 cells. (D) RASSF1A expression levels were analyzed in lung cancer cell lines (LuCa: A427, A459, H322 and H358) and normal lung cells (NC: PAF and PASMC) by qRT-PCR. RASSF1A expression was normalized to the GAPDH level. (E) Methylation of RASSF1A was analyzed in 69 non small cell lung cancer (NSCLC) cell lines and 20 normal lung tissues (NL). For details see also Fig. 3. (F) ZMYND10 expression levels were analyzed in LuCa and NC as measured by qRT-PCR and normalized to GAPDH levels. (G) Methylation of ZMYND10 was analyzed in 69 NSCLC cell lines and 20 NL. *p < 0.05, **p < 0.01 and ***p < 0.001.