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. 2017 Jul 3;7:4523. doi: 10.1038/s41598-017-04776-5

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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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Effect of DNA methylation change at homeobox gene loci on gene expression. (A) The correlation between DNA methylation change between first and third trimester (y-axis) and RNA expression change between first and third trimester (x-axis) for each homeobox gene. Eleven out of 13 genes showed lower expression in third trimester compared to first (clustering left of centre). Only 3 genes - DLX5, HOXA10 and TLX1 showed a concomitant increase in methylation (clustering in top left corner). (B) Shows the magnified view of the boxed area in panel A. DNA methylation data points represent n = 18 first trimester and n = 10 third trimester samples, while gene expression data points represent n = 5 first trimester and n = 2 third trimester samples. (C) Change in DNA methylation in second and third trimester compared to first trimester for DLX5 (HM27 probe cg00503840), HOXA10 (HM27 probe cg00518911), TLX1 (HM27 probe cg11498156). The second trimester shows an intermediate level of methylation change compared to third trimester, which indicates that DNA methylation is changing progressively over gestation. The change in methylation at second or third trimester is based on the difference between the mean methylation of these time-points relative to the first trimester time-point (delta Beta). (D) Quantitative RT-PCR confirms the reduction in TLX1, HOXA10 and DLX5 expression during gestation. N = 12 first trimester, n = 4 second trimester, and n = 6 third trimester, data are presented as relative expression change (mean+/− SD).