Fig. 7: Faster elongation correlates with reduced AID occupancy in DOT1L-deficient cells. | Nature Communications

Fig. 7: Faster elongation correlates with reduced AID occupancy in DOT1L-deficient cells.

From: DOT1L activity limits transcription elongation velocity and favors RNAPII pausing to facilitate mutagenesis by AID

Fig. 7: Faster elongation correlates with reduced AID occupancy in DOT1L-deficient cells.

a TT-seq log2 fold changes (Dot1l−/− over WT) at AID target genes categorized according to their differential expression by RNA-seq in CH12F3 cells. P values by one-sample two-sided Wilcoxon test, comparing the mean of distributions to 0. b Metaplots showing average elongation velocities for AID target genes in WT and Dot1l−/− CH12F3 cells. Box plots show quantification of average elongation velocities in the 3 kb region from TSS + 1 kb to TSS + 4 kb for AID target genes. P-values by paired two-sided Wilcoxon rank sum test. c Metaplots overlaying elongation velocity changes in Dot1l−/− compared to WT CH12F3 cells, with H3K79me3 distribution in WT for AID target genes. d Metagene plots showing average ChIP-seq coverage of RNAPII, S2P-RNAPII, and S5P-RNAPII signals in WT and Dot1l−/− CH12F3 cells for AID target genes. Box plots show quantification of total RNAPII, S2P-RNAPII, and S5P-RNAPII ChIP-seq signals at the TSS (from TSS-0.1 kb to TSS + 0.3 kb) and at the peak of H3K79me3 mark (TSS + 0.5 kb to TSS + 3 kb) in AID target genes (n = 63). P values by paired two-sided Wilcoxon rank sum test. b–d Metaplots show 5% trimmed means. a, b, d In box and whisker plots, line inside box represents median; top and bottom edges of the box indicate 25th and 75th percentiles; and whiskers extend the largest value up to 1.5 times the inter-quartile range. e UCSC snapshot of H3K79me2, H3K79me3, RNAPII elongation velocity, TT-seq, RNAPII, S2P-RNAPII, and S5P-RNAPII ChIP-seq in WT and Dot1l−/− CH12F3 cells, as well as signal difference between Dot1l−/− and WT at the Sμ and Sα GLTUs of the Igh. f AID occupancy by ChIP-qPCR at the indicated Igh positions for WT and Dot1l−/− CH12F3 cells at 16 h post-CIT. Mean (bars) and individual values (symbols) for n = 3 biological replicates. g As in f for selected AID off targets. Mean (bars) and individual values (symbols) for n = 3 biological replicates. h Model for DOT1L function in limiting RNAPII elongation velocity, RNAPII pausing, and AID occupancy. f, g P values for significant differences by one-way ANOVA with Dunnett’s multiple comparison test.

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