Figure 4.

TAB-seq shows the differential distribution of 5-mC and 5-hmC along the GAD1 promoter. The GAD1 promoter includes regulatory elements proximal to both sides of the transcriptional start site (TSS).³² Pyrosequencing was used to determine the % 5-hmC or 5-mC (y axis) at five CpG sites proximal to the GAD1 TSS. CpGs (x axis) are numbered relative to the TSS (NCBI Ref: NM_000817) and Cs correspond to the following positions: −2=−5 bp, −1=−1 bp, 1=+8 bp, 2=+27 bp and 3=+48 bp. Bisulfite-treated DNA from −54 bp to +69 bp of the GAD1 gene was amplified. While the fragment assayed is shorter, it contains the amplicon used to immunoprecipitate the 5-hmC and 5-mC containing GAD1 fragments (−55 bp to +123 bp, see Supplementary Figure 1). Each bar represents the mean of three sample measurements. Total 5-hmC and 5-mC content is determined by the number of Cs at each position following bisulfite conversion and amplification of the same region. Based on evaluations of the 5-hmC (pGEM1) and the 5-mC (λDNA), we estimate the efficiency of β-glucosylation (protection) to be >95%, while the bisulfite conversion efficiency was greater than 99%. CON, control; GAD1, glutamic acid decarboxylase 67 GAD1.