Biography: Alexey V Fedulov
Statement of the Problem: Epigenetic engineering (editing) is an exciting path to novel therapeutics. Custom-designed
demethylases allow gene-specific reactivation of epigenetically silenced genes. Progress in this area depends in great part on
the choice of enzymatic effectors and their targeted binding to promoters. 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.
Methodology & Theoretical Orientation: We designed multiple fusion constructs (n=6) aimed to bind in relative vicinity of
each other in the key regulatory areas of mCXCL11. Zinc-finger protein arrays served as DNA-binding domains; murine TDG
isotype 2 and human Tet1CD were the enzymatic effectors. Constructs with catalytically inactive single aminoacid mutant
enzymes served as controls. The constructs were delivered into 3T3 fibroblasts via lentiviral transduction.
Findings: After 2 weeks of constitutive expression the pyrosequencing analysis demonstrated a decrease in CpG methylation by
up to 40 percentage points in several loci in the targeted area. This was associated with a nearly 5-fold increase in transcriptional
responsiveness of CXCL11 after stimulation with a combination of IFNγ and LPS. The maximum transcriptional responsiveness
measured ~X 2000 times over baseline, vs. ~X 400 times in the control.
Conclusion & Significance: We conclude that a combination of multiple TDG and Tet1 complexes with zinc-finger arrays is a
promising approach in targeted demethylation and that CXCL11 is a rewarding target for future experimentation.