Figure 1. TET enzymes mediate the inflammatory transcriptional memory of a methylated CMV reporter.
(A) Schematic of the EGFP reporter gene stably inserted into the HEK293F genome. The CMV promoter is highly modified and silenced by DNA methylation. (B) Experiment scheme. (C) Flow cytometry analysis of EGFP fluorescent intensity for the cells treated with TNF-α for 0 day, 2 days, 4 days, 8 days, and 12 days. (D) Flow cytometry analysis of EGFP fluorescent intensity for the cells with various pretreatments that received a 12-hr second TNF-α stimulation. (E) RT-qPCR results show the EGFP mRNA level in cells that received a first TNF-α induction and cells that received a second TNF-α induction after 12 hr or 12-day TNF-α treatment. The cells were cultured in TNF-α-free media for 10 days before receiving a second TNF-α induction. GAPDH is used as the internal control. Data are shown as mean ± SD from three independent experiments. Note: although not observable due to the scale of Y-axis, at 0 hr, EGFP mRNA level in cells with 12-day TNF-α pretreatment is fivefold higher than that in naïve cells. (F) Locus-specific bisulfite sequencing results of the CMV promoter for the cells treated with TNF-α for 0 day, 2 days, 4 days, 8 days, and 12 days. Filled circles, methylated CpGs; open circles, unmethylated CpGs. (G) The CMV promoter DNA methylation level of the top 10% of EGFP-positive cells sorted from 12-day-TNF-α-treated WT, TET1 KO, TET2 KO, TET3 KO, and TET TKO cells by flow cytometry. (H) Flow cytometry analysis of EGFP fluorescent intensity for 12-day-TNF-α-treated WT, TET1 KO, TET2 KO, TET3 KO, and TET TKO cells. (I) Flow cytometry analysis of the inflammatory transcriptional memory of the EGFP reporter in WT, TET2 KO, and TET TKO cells.
Figure 1—figure supplement 1. TET enzymes mediate the inflammatory transcriptional memory of a methylated CMV reporter.
