Figure 5.

Pc₂TF stimulates expression at a polycomb-silenced reporter gene. (A) An engineered HEK293 cell line, Gal4-EED/luc, was used for doxycycline-mediated control of H3K27me3 and PRC-mediated silencing at a Tk-luciferase reporter. Expression is partially silenced prior to dox treatment, as demonstrated previously⁴⁰ and becomes fully repressed at 96 h. (B) Fusion constructs were cloned into the MV10 vector at XbaI. Fluorescence microscopy confirmed nuclear localization of the fusion proteins in transfected cells. The same samples were used for Western blots to confirm cellular expression of full length proteins, RT-qPCR to measure mRNA levels (C), and flow cytometry to measure RFP signal (D). Changes in the expression states of Tk-luciferase in 96-h dox-treated cells were determined by RT-qPCR (C) and luc activity assays (D) (circles = replicates, described in Methods). (E) Fusion proteins were expressed in cells treated with dox for 0, 48, or 96 h to determine the activity of the fusion activators at intermediate repressed states (bars = mean values for 3 luciferase assays from one transfected sample, each scaled to mean Pc_ΔTF luc/cell; error bars = SDM).