Figure 3.

Exposure to phenobarbital results in precise changes in the mouse liver 5hmC genomic profile. (a) Heat map of average reproducible 5hmC levels over all PPRs on chromosome 7. PPRs are clustered by both 'Euclidian' and 'Ward' methods. Green boxes and arrows denote PPRs showing an increase in 5hmC levels in multiple PB animals. (b) Induction of gene expression is linked to a 5hmC increase and 5mC decrease over promoter regions. Scatter graph plots indicate average changes in 5hmC, 5mC, H3K4me2, H3K27me3 and H3K36me3 against fold change (>1.5) in expression for all genes upon PB treatment. Trend lines are displayed in red with associated Pearson correlation ('cor') values. (c) Top: plot showing reciprocal PB driven changes to 5hmC and 5mC patterns; most of which occur outside of the core promoters of PB-induced genes. Average changes in 5hmC levels upon PB treatment are shown in purple and changes in 5mC in red. Overlap in the changes of both marks results in darker regions. Bottom: plot of 30 genes showing no change in expression. (d) Patterns of DNA and histone modification change over the Cyp2b10 gene upon PB exposure. Genes unaffected by PB (for example, Pgf) do not display dynamic changes. Patterns of changes in 5mC (red), 5hmC (purple), H3K36me3 (green), H3K27me3 (orange) and H3K4me2 (blue) are plotted. ChIP-Seq samples were plotted on a scale of +70 to -70 reads; promoter arrays (5hmC and 5mC) plotted from +1.5 log₂ to -1.5 log₂. Gene structure with TSSs denoted by black arrows is shown below with scale bars. Dynamic and reciprocal changes in 5hmC and 5mC levels occur over regions flanking the TSS (+230 bp to +1,920 bp and -1,470 bp to -1,760 bp) and a long-range upstream element (-7,720 to -5,915 bp). (e) Average changes in epigenetic marks over the Cyp2b/2c gene family compared to genes unaffected by 28-day PB exposure. Average changes in the log₂ scores (DNA modifications) or fold change in number of reads (histone modifications are plotted against regions outlined in Figure 1). Error bars are standard error and points showing significant deviation from unaffected genes (Willcox test, P-value < 0.005) are denoted by the asterisk. The red dashed line represents zero change in epigenetic marks upon PB exposure.