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. 2020 Aug 6;10:1172. doi: 10.3389/fonc.2020.01172

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Copyright © 2020 Zhao, Liang and Sun.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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DLX6-AS1 promotes methylation of LARGE promoter by recruiting DNMT1. (A) The condition of CpG islands in LARGE promoter region predicted by the MethPrimer website; (B) the binding of DLX6-AS1 and LARGE analyzed by Blast online comparative analysis; (C) MSP assessment of methylation status in LARGE promoter region under different treatments; IVD: in vitro methylated DNA, positive control; NL, normal lymphocytes, unmethylated positive control; Blank, blank control; U, unmethylation; M, methylation; sh-NC, cells transduced with shRNA negative control; sh-DLX6-AS1, cells transduced with shRNA targeting DLX6-AS1; sh-DLX6-AS1 + DMSO, cells that received shRNA targeting DLX6-AS1 and DMSO treatment; sh-DLX6-AS1 + Aza-dC, cells that received shRNA targeting DLX6-AS1 and methyltransferase inhibitor Aza-dC. (D) the expression of LARGE in cells after Aza-dC treatment measured by RT-qPCR and western blot assay; (E) the enrichment of DNMT1, DNMT3a, and DNMT3b in LARGE promoter region, and the enrichment of DNMT1 after sh-DLX6-AS1 transduction assessed by ChIP; (F) the enrichment of DLX6-AS1 pulled down by IgG and DNMT1 measured by RIP assay; (G) the binding of DLX6-AS1 to DNMT1 assessed by RNA pull-down assay. *p < 0.05. Measurement data were summarized as mean ± standard deviation. Unpaired t-test was used for data comparison between two groups, and comparisons among multiple groups were analyzed by the one-way ANOVA with Tukey's post-hoc test. Cell experiment was repeated three times.