Figure 2.

Targeted recruitment of GFP-tagged effector proteins. (A) GBP-dCas9-mRFP recruits GFP-TET1CD to chromocenters. Recruited GFP-TET1CD oxidizes 5mC to 5hmC at CCs in transfected ESCs. In untransfected cells, no 5hmC signal was detected. (B) When targeted to CCs, GFP-DNMT3ACD mediates de novo DNA methylation in TKO cells, which was not observed in untransfected control cells. Line plots represent the signal intensity of the different channels along the indicated chromocenter (solid white line). White dashed lines indicate the nuclear border. Scale bar: 10 µm. (C) Schematic representation of the inducible vector system. A bi-directional promoter drives the expression of GBP-dCas9-mRFP as well as GFP. The vector additionally encodes a transcriptional repressor (tTR) and a transcriptional activator (rtTA). In the absence of doxycycline (Dox), tTR binds to a tetracycline response element (TRE) within the promoter sequence and represses transcription. Upon addition of Dox to the culture medium, rtTA replaces tTR and induces gene transcription.