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. 2020 Aug 6;10:13275. doi: 10.1038/s41598-020-70289-3

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© The Author(s) 2020

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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IR induces PRMT1-dependent methylation of BRCA1. (A) Methylation status of BRCA1 in breast cancer cells after IR treatments (10 Gy). One milligram of cytosolic extracts was immunoprecipitated using an anti-BRCA1 antibody (clone 6B4), separated using SDS-PAGE and immunoblotted with the indicated antibodies. The histogram represents methylated BRCA1 (IP ASYM24) with respect to immunoprecipitated BRCA1 (IP BRCA1) at different post-IR times and relative to nonirradiated cells after correction by the amount of β-actin. The IR-dependent increase in BRCA1 methylation was statistically significant when compared with nonirradiated cells (*P < 0.005). (B) Effect of PRMT1 silencing on BRCA1 methylation in breast cancer cells. The histogram represents methylated BRCA1 (IP ASYM24) with respect to immunoprecipitated BRCA1 (IP BRCA1) relative to nonirradiated cells and β-actin. The IR-dependent increase in BRCA1 methylation was statistically significant when compared with nonirradiated cells in siControl cells (*P < 0.005) but not significant (NS) in siPRMT1 cells. (C) PRMT1 gene knockout using the CRISPR/Cas9 system in 4T1 cells. The schematic diagram shows the guide RNA (gRNA) targeting site on exon 1 of the PRMT1 gene. Protospacer adjacent motif (PAM) sequences are also presented. The figure also shows Sanger sequencing analysis of PCR fragments amplified from gRNA target regions (the inserted nucleotide is in blue) and protein sequence in wild type (WT) and knockout (KO) cells (PRMT1 protein expression in WT and two selected clones was assayed by western blot). (D) Methylation status of BRCA1 in 4T1 and 4T1-KO-PRMT1 cells. Conditions were as indicated in section A of this figure. (E) Time- and dose-dependent effects of IR on PRMT1 expression at the protein (western blots) and mRNA (histogram) levels in MDA-MB-231 and MCF7 cells. The groupings blots in this figure were cropped from different gels. Full blots are shown in the Supplementary Information, Fig. S5.