Fig. 6: Mutation of the MBD2 methyl-CpG binding domain impairs HBG repression.
From: Removal of promoter CpG methylation by epigenome editing reverses HBG silencing
a Schematic of the MBD2 and MBD3 protein sequences showing the methyl-CpG binding domains (MBD) and the location of the MBD2 Y178F mutation to create an MBD3-like domain with impaired methyl-CpG binding. Created in BioRender. Bell, H. (2025) https://BioRender.com/ztcmiqi. b Diagram of components used in EMSA analysis. The MBD2 single construct (MBD2sc) contains the MBD (red), intrinsically disordered region (IDR, blue), and coiled-coil (CC, dark green) of MBD2 linked to the CC of GATAD2A (light green). Target probes for the BCAT1 and HBG promoters are shown with CpG sites (gray pins) indicated. Created in BioRender. Bell, H. (2025) https://BioRender.com/ztcmiqi. c Electrophoretic mobility Shift assay (EMSA) of probes representing BCAT1 or HBG promoter CpG sites complexed with WT and Y178F MBD2sc expressed protein from b. + or - signs represent the presence or absence of each component in the lane. Arrows indicate the positions of free probe, MBD2:probe complexes, and antibody:MBD2:probe (ab) complex supershifts. d MA plot of differentially expressed genes in MBD2 Y178F mutant vs. WT HUDEP2 cells (five clones per group). Colored points represent genes identified as upregulated (red, 21 genes) or downregulated (blue, 12 genes) with a false discovery rate (FDR) cutoff of 1% (Benjamini-Hochberg method). HBG represents transcripts mapping to both HBG1 and HBG2. e Principal component (PC) analysis of transcript expression in MBD2 Y178F mutant and WT HUDEP2 lines. Source data are provided as a Source Data file.
