Fig. 4: Sensitivity of various human promoters to the m6dA modification.

a Expression constructs used to generate stable HEK293 cell lines containing a dox-inducible TRE3G promoter, which allow co-expression of a CcrM variant (WT or D39A) and dsRed over an internal ribosomal entry site (IRES). The same HEK293 cells also contain different versions of m6dA-sensitive promoters upstream of a GFP reporter gene. ➀ After addition of dox, CcrM is expressed. ➁ CcrM methylates adenine residues within GANTC motifs of the investigated promoters in an untargeted manner. ➂ The m6dA-dependent changes in GFP expression are then tracked by flow cytometry. b Median GFP signals of various CcrM (WT or D39A) cell lines expressing GFP under the control of different human promoters. GFP signals were obtained by flow cytometry before dox treatment. c Ratio of the median GFP signals of CcrM and CcrM D39A, before and after dox treatment, showing the effects of m6dA on GFP expression. d Flow cytometry data showing the dynamics of GFP expression under the control of the m6dA-sensitive EMILIN2 promoter. Cells were either constantly treated with dox (orange line, black data points), or (starting at day 10) cultivated in the absence of dox (red line and data points).