Figure 3.

Determining negative and positive RNA controls for EZH2 interactions. EZH2 reportedly binds many RNAs without well-defined protein-binding motifs. As a negative control, a nonrelated Renilla luciferase transcript was tested that did show some affinity for the EZH2 protein in the (A) Alphascreen assay (n = 3) as well as in the (B) RNA EMSA assay. This finding is consistent with reports of EZH2 promiscuity, including the ability of this enzyme to bind nonmammalian transcripts with low affinity.¹⁶ (C) The ability of biotinylated lncRNAs BDNF-AS and HOTAIR to interact with EZH2 is measured in the AlphaScreen assay. Both lncRNAs are titrated in EZH2 (4 nM). EZH2 interacts with both lncRNAs in a concentration-dependent manner (n = 3). HOTAIR serves as a positive RNA control and biologically important screen.