ERA-Chemistry: New approaches for oligonucleotide-targeted enzymatic DNA methylation
Final Report Abstract
DNA methylation in combination with histone modifications plays important roles in regulating gene expression in mammalian cells and methylation of promotor sequences is often associated with transcriptional inactivation (gene silencing). Silencing mammalian genes by DNA methylation of selected CpG sites in the genome would be a powerful technique to analyse epigenomic information and to study the roles of DNA methylation in health and disease. The concept of targeted DNA methylation is based on covalently linking a DNA (cytosine‐5) methyltransferase (DNA MTase) to a targeting device, which can bind to DNA with high sequence specificity and serves to anchor the DNA MTase in the vicinity of the addressed CpG site(s). Initially we concentrated on using triple helix‐forming oligodeoxynucleotides (TFOs) for sequence‐ specific recognition and programmable targeting of double‐stranded DNA (dsDNA). TFOs bind preferentially to homopurine∙homopyrimidine sequences, wind around the double helix in the major groove and form specific Hoogsteen hydrogen bonds with the Watson‐Crick base pairs. A TFO with a long flexible linker was coupled to the CpG‐specific DNA MTase M.MpeI using SNAP‐tag technology. The M.MpeI‐TFO conjugate was obtained in good yield and showed high activity. With this approach previous problems associated with maleimide chemistry to couple the TFO to the CpG‐specific DNA MTase M.SssI were solved because no low activity C141S mutant is required to protect the enzyme from maleimide inactivation in the coupling step. Targeted methylation of plasmid DNA with the M.MpeI‐TFO conjugate in vitro revealed some degree of methylation specificity but the methylation level remained low. Interestingly, methylation specificity strongly depends on the topological state of the plasmid DNA, with linear DNA showing enhanced specificity compared to supercoiled DNA. This observation indicates that the compact nature of supercoiled DNA allows the plasmid‐bound M.MpeI not only to methylate nearby sites but also to reach over and additionally methylate sites which are close in space but far away in sequence. This is not possible with the linear DNA and targeted methylation is more specific. In addition, we used dCas9, an endonuclease deficient variant of CRISPR/Cas9, as targeting device. The M.MpeI‐TFO conjugate was bound to dCas9 by duplex formation with a 3’‐extended single‐guide RNA (sgRNA) and much higher specificity for targeted methylation and higher methylation levels, compared to targeted methylation by triple helix‐formation, were obtained in vitro. The combination of dCas9 targeting with linear DNA substrate led to the highest specificity and targeted methylation is estimated to be at least 50‐fold faster than non‐targeted methylation. Taken together our results are important and lead to a better understanding of the underlying principles of targeted DNA methylation. Furthermore, linking dCas9 with effector domains by non‐ covalent interactions with enlarged sgRNA for targeting specific DNA sequences has been demonstrated before, but joining the targeting and effector domains by RNA‐DNA hybridization could open up new possibilities for epigenetic editing, provided that these complexes can be efficiently delivered to the cell.
Publications
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A Circularly permuted variants of two CG‐specific prokaryotic DNA methyltransferases. PLoS ONE 2018, 13, e0197232
P. Albert, B. Varga, N. Zsibrita, A. Kiss
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Triple helix‐targeted DNA methylation with DNA methyltransferase‐oligodeoxy‐ nucleotide conjugates, Dissertation, RWTH Aachen University, 2018
A. Giesbertz
