Figure 2: Engineering a synthetic initiator to establish N6-methyladenine (m6A) DNA modifications at target reporter loci in human cells.

(A) Design of synthetic initiator module (synI). synI is a fusion of a Dam (DNA adenine methyltransferase) “writer” domain and an engineered zinc finger (ZF), which specifically binds a 20-bp synthetic binding sequence (BS). synI enables de novo placement of m6A marks at designer reporters integrated into 293FT cells. For these experiments, we used stable cell lines harboring a singly-integrated Clustered Reporter, with ZF BS and GATC arrays upstream of a pMinCMV driving expression of destabilized GFP (d2EGFP), as the background strain., (B) Screening Dam variants for synI factors that induce sequence-specific enrichment of m6A at target sites. Quantification of m6A enrichment at target reporter (red) and off-target, GATC-containing endogenous loci (grey shades) by transfected synI constructs composed of different Dam mutants. Off-target loci were chosen to represent different chromosomal locations. m6A enrichment is obtained by measuring fraction methylation at a single GATC probe site in the locus of interest using m6A-qPCR, and normalizing to basal methylation induced by the Dam variant not fused to ZF (see Methods, Figure S2). (n=3; error bars, SD)., (C) Expression of synI has minimal effect on the transcriptome. Correlation of transcriptome from RNA-seq measurements for reporter cells transfected with synI vs. empty plasmid. Correlation coefficient of endogenous genes between samples was calculated using log₂ transformed expression values. mRNA corresponding to synI is labeled. The data are representative of two biological replicates., See also Figure S2.