Fig. 3.

LPS stimulation of bone marrow-derived macrophages induces 5hmC deposition at enhancers. A Flowchart of experiments. B Number of differentially hydroxymethylated regions (DhmRs) comparing unstimulated vs. LPS-stimulated BMDMs. C LPS induces 5hmC deposition in BMDMs at an intergenic region between the Jdp2 and Batf genes (for genome browser views of the Il1b and Il6 loci, see Fig. S3). D For all the de novo 5hmC peaks in LPS-stimulated BMDMs, Log2 fold change in 5hmC is plotted against Log2 fold change in RNA expression. 5hmC peaks overlapping with latent enhancers (regions that acquire H3K4me1 and H3K27Ac only after stimulation) are shown in red; of these, peaks in the vicinity of the Batf, Ptgs2, Stat1, Alcam, Mdfic, Il1b, and Il6 genes are shown in light blue. E Bar graphs show the RNA expression levels of the Batf, Ptgs2, Stat1, Alcam, Mdfic, Il1b, and Il6 genes in WT vs. TET iTKO (left), or WT vs. TDG iKO (right), as determined by RNA-seq. BMDMs stimulated with (+) or without (−) LPS were used. F Heat maps depicting the percentage of (5mC+5hmC)/total Cs for each CpG in enhancers close to the Batf, Ptgs2, Stat1, Alcam, Mdfic, Il1b, and Il6 genes, as determined by BS-seq. All seven selected latent enhancers show 5hmC deposition upon LPS stimulation (see Fig. S3E), but none undergoes DNA demethylation (loss of 5mC+5hmC)