Figure 4.

Prolonged phenobarbital exposure results in depletion of both 5mC and 5hmC over the Cyp2b10 promoter. (a) Continuous exposure to PB leads to reciprocal perturbations of 5hmC and 5mC patterns over the Cyp2b10 promoter (TSS ±3 kb). Mice receiving PB for 1, 7, 28 or 91 days display dynamic changes to their 5mC (red) and 5hmC (purple) profiles at Cyp2b10. Plots display changes in log2 score for either 5hmC or 5mC between PB-treated and control mice. 5hmC levels increased following 1 day of drug exposure whilst 5mC levels decreased with prolonged exposure. The region around the TSS lost both marks at around 7 days, which is most pronounced in mice that have received PB for 91 days. This may represent a transition to unmodified cytosine through a 5hmC intermediate. The ActB promoter exhibits no such dynamic change. (b) Models describing maintenance of 5hmC levels at expressed and non-expressed genes with example profiles for 5hmC displayed below (purple). Typical gene promoters (CGI) lack 5hmC- and 5mC-modified DNA (i). Demethylation is likely maintained by the Tet1 protein (5mC > 5hmC) and an unknown factor, possibly TDG, as part of the base excision repair pathway (5hmC > > C). 5hmC may prevent re-methylation occurring by inhibiting DNA methyltransferases (for example, Dnmt1). As 5hmC levels are high in the bodies of actively transcribing genes, Tet proteins must access this DNA, possibly in tandem with the elongating polymerase complex. A few promoter regions are enriched for 5hmC (ii), and associated genes tend to be inactive or lowly expressed (lacking the binding of RNAPII). Whether a unique factor is required to attract the Tets or repel TDG at these loci is unknown. TF, transcription factor.