Targeted DNA Demethylation: Vectors, Effectors and Perspectives

Abstract

Aberrant DNA hypermethylation at regulatory cis-elements of particular genes is seen in a plethora of pathological conditions including cardiovascular, neurological, immunological, gastrointestinal and renal diseases, as well as in cancer, diabetes and others. Thus, approaches for experimental and therapeutic DNA demethylation have a great potential to demonstrate mechanistic importance, and even causality of epigenetic alterations, and may open novel avenues to epigenetic cures. However, existing methods based on DNA methyltransferase inhibitors that elicit genome-wide demethylation are not suitable for treatment of diseases with specific epimutations and provide a limited experimental value. Therefore, gene-specific epigenetic editing is a critical approach for epigenetic re-activation of silenced genes. Site-specific demethylation can be achieved by utilizing sequence-dependent DNA-binding molecules such as zinc finger protein array (ZFA), transcription activator-like effector (TALE) and clustered regularly interspaced short palindromic repeat-associated dead Cas9 (CRISPR/dCas9). Synthetic proteins, where these DNA-binding domains are fused with the DNA demethylases such as ten-eleven translocation (Tet) and thymine DNA glycosylase (TDG) enzymes, successfully induced or enhanced transcriptional responsiveness at targeted loci. However, a number of challenges, including the dependence on transgenesis for delivery of the fusion constructs, remain issues to be solved. In this review, we detail current and potential approaches to gene-specific DNA demethylation as a novel epigenetic editing-based therapeutic strategy.

Keywords: DNA demethylase; DNA-binding domains; epigenetic therapy; gene targeting; vectors.

Figure 1

ZFA-TETs fusion protein (A) and CRISPR-dCas9-TET based (B) site-specific demethylation.

Figure 2

(A). Structure and size comparison of ZFA-TDG and ZFA-TET1 constructs. (B). Anticipated 3D structural drawings of ZFA-TET1CD and dCAS9-TET1CD constructs. The images were generated with PHYRE2 Protein Fold Recognition Server. (http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index accessed on 20 April 2023). (C). Sequence-accurate to-scale 3D rendering of ZFA-TDGCD and dCAS9-TDGCD bound to a target gene promoter. The image was derived from ICM Browser using custom PDB files.