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. 2018 Nov 8;47(1):283–298. doi: 10.1093/nar/gky1149

Figure 4.

Figure 4.

TNF-α decreases H3K9me3 level on the TERRA gene promoter by releasing ATF7. (A) TNF-α induces ATF7 phosphorylation in testicular germ cells. Testicular germ cells were prepared from WT mice at the indicated times after TNF-α (10 μg/kg weight) or saline single injection. Testicular germ cells were also prepared from mice treated daily with TNF-α (10 μg/kg weight) for 6 weeks. Western blotting was performed using whole cell lysates of testicular germ cells to detect the indicated proteins. (B) TNF-α induces the release of ATF7 from the TERRA gene promoter on chromosome 8q (TEERA-8q) in the testicular germ cells of F0 mice. WT and Atf7+/– male F0 mice (n = 3) were treated with TNF-α or saline as described in Figure 1A. Modified chromatin immunoprecipitation (ChIP) was performed using MNase, testicular germ cells, and anti-ATF7 antibody. Primers to amplify the region in the TERRA-8q promoter, which contains the CRE-like site or lacks the CRE site, were used for Q-PCR. Average values relative to the input ± SD are shown (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001. (C) TNF-α decreases H3K9me3 levels on the TERRA gene promoter in WT but not in Atf7+/– testicular germ cells of F0 mice. WT and Atf7+/– male F0 mice (n = 3) were treated with TNF-α or saline as described above, and ChIP was performed using testicular germ cells, anti-H3K9me3, and anti-H3 antibody. Primers in the non-CRE region were used to amplify the TERRA-8q promoter region, and the average value of the H3K9me3 signal relative to H3 ± SD is shown (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001; NS, not significant. (D) Co-immunoprecipitation of ATF7 and Suv39h1. Cell lysates of testicular germ cells were immunoprecipitated with anti-ATF7 antibody or control IgG, and the immuno-complexes were subjected to western blotting using anti-Suv39h1. Asterisk indicates IgG. (E) Amount of ATF7 on the TERRA gene promoter in Atf7+/– testicular germ cells is less than half of that in WT cells. Modified qChIP assays were used to measure the ATF7 amount on the TERRA-8q gene promoter in WT and Atf7+/– F0 testicular germ cells (n = 3) as described above.*P < 0.05; **P < 0.01; ***P < 0.001. (F) Levels of ATF7 mRNA and protein in Atf7+/– testicular germ cells. The level of ATF7 mRNA in WT and Atf7+/– testicular germ cells was examined by qRT-PCR (left). To exaine the ATF7 protein level, Western blotting was performed using serial dilution of whole cell lysates (right upper), and quantification of ATF7 band is shown as a bar graph (right lower). (G) Paternal TNF-α treatment increases the level of H3K9me3 on the TERRA gene promoter in WT but not in Atf7+/– F1 male MEFs. WT and Atf7+/– male F0 mice (n = 3) were treated with TNF-α or saline as described above, and mated with female mice. MEFs were prepared from E14.5 F1 male embryos and used for ChIP with anti-H3K9me3 and anti-H3 antibodies. Primers in the non-CRE region were used to amplify the TERRA-8q promoter region, and the average value of the H3K9me3 signal relative to H3 ± SD is shown (n = 3 from three independent pregnant mice). *P < 0.05; **P < 0.01; NS, not significant.