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. 2017 Aug 8;7:7594. doi: 10.1038/s41598-017-07380-9

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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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Effects of HIGM2 mutant forms of AID on cell distribution and interaction with SUV4-20H enzymes. (A) Primary structure of AID. The upper part of the diagram indicates the four missense mutations related to HIGM2used in our study. The lower part indicates the two selected nonsense HIGM2 mutations. NLS, nuclear-localisation signal; CDD, cytidine deaminase domain; NES, nuclear-export sequence. (B) Western blot image showing the inducible expression of AID WT and the various HIGM2 mutants, before and after treatment with doxycycline 500 ng/ml for 48 hours. (C) Representative confocal images showing the subcellular localisation of C-terminally hemagglutinin (HA)-tagged human AID in inducible HeLa cells. A total of 20 cells from randomly selected fields were analysed in each experimental condition. The graphs next to the confocal images, show the quantification of the cellular signal of AID within the cells. Light gray section of the bar indicates the average percentage of citoplasmic AID signal. Black section of the bar indicates the average percentage of nuclear AID signal. When nuclear export was inhibited with 50 ng/ml leptomycin B (LMB) for 2 hours, most of the AID translocates from the cytoplasm to the nucleus. Protein products of missense HIGM2 mutations showed a similar response to AID WT after LMB, while truncated forms of AID lacking NES were constitutively nuclear. Scale bar: 10 μm. (D) Co-immunoprecipitation of AID WT and HIGM2 mutants with the three SUV4-20H enzymes. All the HIGM2 mutants were also able to interact with the three SUV4-20H enzymes. (E) ChIP assays showing the recruitment of wild type AID (WT) and W68X AID mutant (W68X) and H4K20me3 enrichment at the Cμ and Sμ regions in Jiyoye cells after doxycycline and LMB treatment for 24 hours. AID was immunoprecipitated using anti-HA and ChIP assays also included anti-H4K20me3 antibody and IgG as a negative control. Y-axis shows the relative enrichment of bound fraction with respect to input fraction.