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. 2021 Dec 16;4:1405. doi: 10.1038/s42003-021-02923-3

Fig. 2. The activity of IQ-Switch is not influenced by QUAS methylation.

Fig. 2

a The prepared genomic DNA from F2 offspring of Tg(10xUAS:ZsGreen-P2A-brafV610E) and F4 offspring of Tg(5xQUAS:ZsGreen-P2A-BRAFV600E) was subjected to bisulfite sequencing analysis. Both element regions became heavily methylated through successive generations. Open circles indicated methylated CpGs and dark circles represented unmethylated CpGs. Ten individual clones from each group were independently sequenced as shown on the vertical axis. b Schematic diagram of ChIP experiments in (c). The in vitro transcribed mRNA (50 pg) encoding 6xMyc-tagged Gal4DBD-VP16*-EcR and 6xMyc-QFDBD-2xAD*-VP16*-EcR was introduced into the embryos of Tg(5xQUAS:ZsGreen-P2A-BRAFV600E). The injected embryos were exposed to 50 µM of Teb for 2 h at the early somite stage and then harvested at 20 hpf. c An amplicon encompassing the 5xQUAS element region occupies 231 bp. ChIP assay of embryo with anti-Myc and anti-Flag antibody. The anti-Flag antibody was used as a negative control. The ChIP samples were subjected to qPCR with the indicated primers (arrows). While mRNA encoding 6xMyc-Gal4DBD-VP16*-EcR did not cause any embryonic malformation when exposed to Teb, sporadic exposure of 6xMyc-QFDBD-2xAD*-VP16*-EcR mRNA-injected embryos to Teb showed developmental defects (boxes). q-PCR was carried out in triplicate, and standard errors of the mean are shown in the panel. Error bars stand for the standard deviation. d The embryos were treated with 50 µM of Teb for 2 h before the onset of gastrulation and then observed at 24 hpf. e While ZsGreen reporter under the control of QF/5xQUAS showed ubiquitous expression in the whole embryo (D1 + E1), the same reporter was activated in random tissues when driven by Gal4/10xUAS (D2 + E2). Note that 10xUAS regulated by Gal4 was progressively silenced as early as F3 generation. Abbreviations: Teb; tebufenozide. Scale bar; 200 μm.