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. 2017 Aug 2;7:7129. doi: 10.1038/s41598-017-06310-z

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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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TWIST1 suppresses IRF6 expression in vitro via its enhancer element. RTqPCR data shows that TWIST1 is not expressed in HaCaT keratinocytes, while IRF6 is highly expressed in these cells (A). Exogenous overexpression of TWIST1 using pGL3 expression vector remarkably reduced IRF6 expression by 12-fold 48 h post-transfection (A). In human embryonic kidney cells (HEK293), TWIST1 is moderately expressed compared to IRF6 that is expressed 2-fold more (B). Knockdown of TWIST1 expression with siRNA slightly increased the expression level of IRF6 in HEK293 cells (B). However, knockdown of TWIST1 expression significantly increased luciferase activity of a luciferase gene that is under direct control of IRF6 enhancer element (C). ChIP-seq data showed that TWIST1 binds to IRF6 enhancer element accompanied by enrichment of RNA PolII protein at the enhancer and promoter element of IRF6 gene, but the enrichment of PolII at the enhancer and promoter regions of IRF6 may indicate an active transcriptional state. However, the amount of PolII at the IRF6 enhancer and promoter compared to IgG was not significant and the enrichment was moderate compared to PolII at PCNA promoter (D).