BRD4 facilitates post-mitotic transcriptional re-activation through chromatin decompaction. (a) Locus size change under different treatments. The locus is compacted in the absence of Dox, and there was no CFP protein production. Upon Dox addition the locus was de-compacted, and there was CFP protein production. The locus was even more de-compacted with LacI-mCherry-BRD4 tethering in the absence of Dox, but no CFP was produced. (n=24, 27, 36 for LacI−Dox, LacI+Dox and LacI−BRD4−Dox respectively). (b) Tethering BRD4 protein, by fusing BRD4 with LacI-mCherry, to the locus in the absence of Dox de-compacted the locus without the recruitment of Pol II or mRNA production (arrowheads), in cells stably expressing YFP-Pol II or MS2-YFP. BRD4 tethering did not result in significant increase of histone acetylation on the locus, nor did it result in decreased association of heterochromatin marker HP1α. Cells were transiently transfected with LacI-mCherry-BRD4 and were fixed at interphase. Cells were immunostained for TH4 or HP1α. (Scale bar, 10μm) (c) Tethering BRD4 protein to the locus significantly accelerated Pol II recruitment during interphase induction (75.8±10.6min) as compared to control interphase induction (taken from , for ease of comparison) without BRD4 tethering (208.1±22.9min). However, Pol II kinetics was still significantly slower than that in post-mitotic re-activation (16.5±6.0min) (mean±S.E.M.). (n=17 for interphase induction, n=13 for LacI-BRD4 interphase induction, n=19 for post-mitotic re-activation). (d) Tethering BRD4 protein to the locus significantly accelerated mRNA synthesis during interphase induction (71.5±13.2min) as compared to control interphase induction (re-plotted from for ease of comparison) without BRD4 tethering (157.6±19.0min). Tethering BRD4 results in kinetics that are closer to that observed in post-mitotic re-activation (34.3±7.6 min). (mean±S.E.M.) (n=13 for interphase induction, n=17 for LacI-BRD4 interphase induction, n=25 for post-mitotic re-activation).