Induced DNA demethylation by targeting Ten-Eleven Translocation 2 to the human ICAM-1 promoter

Abstract

Increasing evidence indicates that active DNA demethylation is involved in several processes in mammals, resulting in developmental stage-specificity and cell lineage-specificity. The recently discovered Ten-Eleven Translocation (TET) dioxygenases are accepted to be involved in DNA demethylation by initiating 5-mC oxidation. Aberrant DNA methylation profiles are associated with many diseases. For example in cancer, hypermethylation results in silencing of tumor suppressor genes. Such silenced genes can be re-expressed by epigenetic drugs, but this approach has genome-wide effects. In this study, fusions of designer DNA binding domains to TET dioxygenase family members (TET1, -2 or -3) were engineered to target epigenetically silenced genes (ICAM-1, EpCAM). The effects on targeted CpGs’ methylation and on expression levels of the target genes were assessed. The results indicated demethylation of targeted CpG sites in both promoters for targeted TET2 and to a lesser extent for TET1, but not for TET3. Interestingly, we observed re-activation of transcription of ICAM-1. Thus, our work suggests that we provided a mechanism to induce targeted DNA demethylation, which facilitates re-activation of expression of the target genes. Furthermore, this Epigenetic Editing approach is a powerful tool to investigate functions of epigenetic writers and erasers and to elucidate consequences of epigenetic marks.

Local chromatin microenvironment determines DNMT activity: from DNA methyltransferase to DNA demethylase or DNA dehydroxymethylase

“…Further cellular studies are now required to investigate the physiological relevance of DNMT-3A and -3B as players in active DNA demethylation. Epigenetic editing tools [51][52][53][54][55] offer exciting possibilities to address differential effects of epigenetic enzymes in various chromatin contexts in living cells. 60 …”

“…To identify such situations in physiological situations, the targeting of DNMTs to pre-determined genomic loci by epigenetic editing, as described by us and others previously [51][52][53][54][55] provides an interesting tool to test and to eventually exploit DNA demethylation activities of DNMTs in vivo.…”

Targeted DNA demethylation in human cells by fusion of a plant 5-methylcytosine DNA glycosylase to a sequence-specific DNA binding domain

“…18,19,21,22 TET enzymes mediate DNA demethylation indirectly through oxidation of 5mC to 5hmC, and further oxidation of 5hmC to 5fC and 5caC. 29,30 However, bisulfite sequencing does not distinguish between unmethylated C and either 5fC or 5caC, since these two derivatives are not resistant to deamination.…”

“…20 Furthermore, TET2 fused to a ZFP (CD54) that binds ICAM-1 promoter, partially demethylates the CpG sites in the surrounding region of the ZF binding site in A2780 cells, leading to a 2-fold increase in gene expression. 18 Conversely, there is also evidence showing that increasing methylation of a single CpG site can be sufficient to repress gene expression. 32 In conclusion, this work demonstrates that targeted expression of ROS1 catalytic domain in human cells reactivates a methylation-silenced gene in a replication-independent manner.…”

“…16,17 Recent studies have shown targeted DNA demethylation and gene reactivation in mammalian cells by fusing different enzymes involved in DNA demethylation to diverse DBDs. [18][19][20][21][22] However, no efforts have been reported so far to express or target plant 5mC DNA glycosylases, which directly excise 5mC from DNA.…”