Fig. 3: TET3-dependent 5hmC formation stabilizes SETD2-Pol II recruitment, facilitating intragenic H3K36me3 deposition within highly transcribed genes.
From: Spurious transcription causing innate immune responses is prevented by 5-hydroxymethylcytosine
a, Co-IP of WT (TET3WT) or catalytically inactive (TET3CD) HA-tagged human TET3 after expression in HEK293T cells followed by western blot analysis (n = 3 independent experiments). Quantification of coprecipitated Pol II (pan), Pol II (pSer2) and SETD2 is shown on the right (n = 3; two-tailed unpaired t-test: **P = 0.0059, *P = 0.0276, *P = 0.0172). b, PLA to visualize interactions of 5hmC and NSD3, TET3 and Pol II (pan), and 5hmC and SETD2 in control SMCs (n = 2 independent animals). Positive PLA signals are indicated by white arrows. Quantification of average PLA signal per nuclei (%) in control SMCs is shown in the right panel (n = 2). One-way ANOVA with Tukeyʼs post hoc test: ***P < 0.001. Scale bar, 50 μm. c, Co-IP to detect interactions of Pol II with SETD2 in HEK293T cells following mock, TET3WT and TET3CD transfections (n = 3 independent experiments). Quantification of coprecipitated Pol II is shown on the right (n = 3, one-way ANOVA with Tukey’s post hoc test: *P = 0.0156, **P = 0.005). d, Analysis of H3K36me3 signals within gene bodies of quartiles defined by RNA-seq analysis of transcriptional activity (n = 2). P values were calculated with the one-tailed likelihood-ratio test: ***P < 0.001. e, Distribution of H3K36me3 ChIP–seq signals within gene bodies of different subgroups of genes as defined in Fig. 2b (n = 2). Data in a–c are presented as mean values ± s.e.m.
