Fig. 1: Overexpression of Pramel15 leads to global DNA passive demethylation.

a Scheme of B2-17, a DNA demethylation-sensitive cell line⁴³. A CMV promoter-driven GFP fragment was methylated in vitro by HapII and then integrated into HEK293 cells. The clone with a low GFP background and high sensitivity to 5-aza-2′-deoxycytidine was selected as B2-17. b Left, fluorescence images show the GFP-activated cells in B2-17 transfected with Pramel15-expressing plasmids. Scale bars indicate 100 μm. Right, bar graph shows the ratio of GFP-positive cells in B2-17 transfected with Pramel15-expressing plasmids using fluorescence-activated cell sorting (FACS). Three biological replicates have been performed. c BS-seq shows the DNA methylation states of CMV promoter in B2-17 cells transfected with Pramel15-expressing plasmids. Genomic DNA was extracted 96 h later and followed by bisulfate sequence of CMV promoter. d Bar graphs show the relative level of 5-methylcytosine (mC) and 5-hydroxymethylcytosine (hmC) in genome of B2-17. Cells were harvested at 1–4 days following Pramel15 expression, and genomic DNA was extracted for nucleotide mass spectrum analysis. Normalization was conducted in reference to the 5mC level in Day 0 cells. e Genomic DNA of B2-17 was extracted for nucleotide mass spectrum analysis following transfection of plasmids expressing Pramel15-mCherry or mCherry, and cells were treated with 5 μM aphidicolin or vehicle for 2 days. For (d and e) each point indicates a biological replicate. Data are presented as mean value ± s.d.