GADD45A Does Not Promote DNA Demethylation (original) (raw)
Figure 2
Overexpression of GADD45A does not promote demethylation of a methylated pOct4-EGFP plasmid.
A. Schematic diagram of the pOct4-EGFP plasmid (not to scale). The 2.4 kb Oct4 promoter sequences are upstream of the EGFP gene. DE is the distal element, PE is the proximal element, and P is the minimal promoter of Oct4. This plasmid was methylated in vitro with HpaII or HpaII + HhaI. Black circles indicate HpaII sites and black triangles indicate HhaI sites. B. Confirmation of the methylation status of the six HpaII sites (black circles in panel A) by sodium bisulfite sequencing. Closed circles are methylated sites and open circles are unmethylated sites. C. Southern blot assay of plasmids recovered from HEK293 cells after transient transfection of the methylated and unmethylated pOct4-EGFP plasmid with and without GADD45A overexpression. The plasmids were digested with HincII and HpaII after in vitro methylation of the plasmid with HpaII (left panel) or HpaII and HhaI (right panel) prior to transfection. Digestion of the recovered methylated plasmid with HpaII does not lead to cleavage indicating that demethylation did not occur in HEK293 cells that overexpress GADD45A. D. Confirmation of GADD45A expression in transfected HEK293 cells by Western blotting with anti-GADD45A antibody. Beta-tubulin was probed as a loading control.