Targeted regulation of plasmid DNA expression in eukaryotic cells with a methylated-DNA-binding activator
PurposeTargeted regulation of transfected extra-chromosomal plasmid DNA typically requires the integration of 9 - 20 bp docking sites into the plasmid. Here, we report an elegant approach, The Dpn Adaptor Linked Effector (DAL-E) system, to target fusion proteins to 6-methyladenosine in GATC, which appears frequently in popular eukaryotic expression vectors and is absent from endogenous genomic DNA. Methods: The DNA-binding region from the DpnI endonuclease binds 6-methyladenosine within the GATC motif. We used a Dpn-transcriptional activator (DPN7-TA) fusion to induce gene expression from transiently transfected pDNAs. ResultsWe validated methylation-dependent activity of DPN7-TA with a panel of target pDNAs. We observed stronger transactivation when GATC targets were located upstream of the transcriptional start site in the target pDNA. Conclusion: DAL-E, consisting of a 108 aa, 12 kD DNA-binding adaptor and a 4 bp recognition site, offers a genetically-tractable, tunable system that can potentially be redesigned to recruit a variety of regulators (e.g. activators, silencers, epigenome editors) to transfected plasmid DNA. LAY SUMMARYTransfection of plasmid DNA (pDNA) is a commonly used method for introducing exogenous genetic material into mammalian cells. Once introduced into cells not all pDNAs express this genetic material at sufficient levels. Current techniques to improve transgene expression are limited and are not always feasible for all plasmids. This report presents a new method to improve gene expression from pDNA. The Dpn Adaptor Linked Effector (DAL-E) binds to methylated adenines in the pDNA resulting in increased expression. This technique has exciting implications for improved genetic engineering of mammalian cells. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=124 HEIGHT=200 SRC="FIGDIR/small/466616v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@1116db0org.highwire.dtl.DTLVardef@1386f99org.highwire.dtl.DTLVardef@26c63corg.highwire.dtl.DTLVardef@1a09673_HPS_FORMAT_FIGEXP M_FIG C_FIG