Leonhardt

Project Title: dTALEs as a tool for targeted transcriptional activation and recruitment of epigenetic modifiers

Specific control of gene activity is a valuable tool to study and engineer cellular functions. We assembled plant pathogen derived DNA binding modules to generate designer transcription activator-like effector (dTALE) molecules for targeted manipulation of gene expression in mammalian cells. We compared five distinct dTALEs binding at different sites within the oct4 pluripotency gene promoter and systematically analyzed their efficiency. Transcriptional activation greatly varied with the position of the binding site within the target gene promoter. We found that dTALEs efficiently upregulate transcription of the active oct4 promoter in embryonic stem cells (ESCs) but failed to activate the silenced oct4 promoter in ESC-derived neural stem cells (NSCs). These data indicate that as for endogenous transcription factors also dTALE activity is limited by repressive epigenetic mechanisms. We therefore manipulated the epigenetic state of the oct4 promoter and found that inhibition of histone deacetylases or DNA methyltransferases by valproic acid (VPA) and 5-aza-dC, respectively, greatly facilitated the activation of the silent oct4 promoter by dTALEs. These results show that dTALEs in combination with chemical manipulation of epigenetic modifiers are ideally suited for efficient activation of target genes in mammalian cells.