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. 2018 Mar 6;37(20):2714–2727. doi: 10.1038/s41388-018-0133-3

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© The Author(s) 2018

Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License, which permits any non-commercial 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. If you remix, transform, or build upon this article or a part thereof, you must distribute your contributions under the same license as the original. 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-nc-sa/4.0/.

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Fig. 6 — Complex epigenetic regulation of NTRK1 promoters by EZH2 and/or DNA methylation. a The positions of NTRK1 variants were displayed in the UCSC genome browser (Human GRCh38/hg38, https://genome.ucsc.edu). ChIP qPCR site positions in the two promoters of NTRK1, Promoter 1 (P1) and Promoter 2 (P2). b qChIP assay for EZH2 in NTRK1 P1 and P2 promoters in NB-39-nu cells. Immunoprecipitation was performed by an anti-EZH2 antibody and control rabbit IgG. Primer locations are indicated in the diagrams in a. Primer sequences are shown in Supplementary Table S4. Results are presented as fold enrichment and are representative of at least three independent experiments. c qChIP assay for H3K27me3 in NTRK1 P1 and P2 promoters in mock- and EZH2 knocked down NB-39-nu cells by the shEZH2-1 lentivirus. Immunoprecipitation was performed by an anti-H3K27me3 antibody and control rabbit IgG. d qChIP assay for H3K27me3 and EZH2 in the NTRK1 P1 core promoter region in mock- and EPZ6438-treated NB-39-nu cells. Immunoprecipitation was performed with an anti-EZH2 antibody, an anti-H3K27me3 antibody, and control rabbit IgG. e RT-PCR for NTRK1 variants 1/2 and 3 in EZH2 knockdown NB cells. f Bisulfite-sequence analysis of NTRK1 P1 and P2 promoters. Red and gray asterisks indicate methylated and unmethylated CpG sites, respectively. CpG methylase-treated DNA was used as a positive control. g RT-PCR for NTRK1 variants 1/2 and 3 in 5 aza-dC (5 μM)-treated NB cells