Combination of Tdg and Tet1 for Targeted Demethylation of CXCL11 Promoter

Targeted demethylation of gene promoters is an exciting new area in epigenetic engineering (editing); the effect of custom-designed demethylases depends in great part on the choice of enzymatic effectors and their placement on the target promoter. Here we report a successful use of a combination of TDG and Tet1 to enhance transcriptional responsiveness of CXCL11 gene in a murine fibroblast line. Multiple fusion constructs aimed to bind in relative vicinity of each other have allowed a decrease of CpG methylation by 20-40 percentage points which was associated with a nearly 5-fold increase in transcriptional responsiveness. Specifically, we tested 5 CpG loci via pyrosequencing and observed 66.7±0.1 (vs. 84.7±0.2 in catalytically inactive mutant controls), 67.7±0.9 vs. 43.3±0.6, 81.3±0.7 vs. 56.5±0.4, 95.4±1.3 vs. 67.3±0.9 and 86.1±0.3 vs. 68.3±1.6, p<0.05 for each. The maximum transcriptional responsiveness of the target (via mRNA expression) measured upon an IFNγ+LPS stimulus was 2334 times over baseline, vs. 400 times in the control.

We conclude that a combination of TDG and Tet1 is a promising approach in targeted demethylation, that CXCL11 is a rewarding target for future experimentation, and we will elaborate in the presentation on the relevance to lung disease applications.