Prolonged re-expression of the hypermethylated gene <i>EPB41L3</i> using artificial transcription factors and epigenetic drugs

<div><p></p><p>Epigenetic silencing of tumor suppressor genes (TSGs) is considered a significant event in the progression of cancer. For example, <i>EPB41L3</i>, a potential biomarker in cervical cancer, is often silenced by cancer-specific promoter methylation. Artificial transcription factors (ATFs) are unique tools to re-express such silenced TSGs to functional levels; however, the induced effects are considered transient. Here, we aimed to improve the efficiency and sustainability of gene re-expression using engineered zinc fingers fused to VP64 (ZF-ATFs) or DNA methylation modifiers (ZF-Tet2 or ZF-TDG) and/or by co-treatment with epigenetic drugs [5-aza-dC2'-deoxycytidine or Trichostatin A (TSA)]. <i>EPB41L3</i>-ZF effectively bound its methylated endogenous locus, as also confirmed by ChIP-seq. ZF-ATFs reactivated the epigenetically silenced target gene <i>EPB41L3</i> (˜10-fold) in breast, ovarian, and cervical cancer cell lines. Prolonged high levels of <i>EPB41L3</i> (˜150-fold) induction could be achieved by short-term co-treatment with epigenetic drugs. Interestingly, for otherwise ineffective ZF-Tet2 or ZF-TDG treatments, TSA facilitated re-expression of <i>EPB41L3</i> by up to twofold. ATF-mediated re-expression demonstrated a tumor suppressive role for <i>EPB41L3</i> in cervical cancer cell lines. In conclusion, epigenetic reprogramming provides a novel way to improve sustainability of re-expression of epigenetically silenced promoters.</p>