Transcriptional activation of the same locus exhibits faster kinetics during post-mitotic activation than in the previous interphase. (a) Schematic diagram of the gene expression system (Modified from Janicki et al. 2004). Binding of LacI-Fluorescent Protein to the lac operator repeats results in visualization of the gene locus. pTet-On expression in the presence of Dox induces gene expression driven by the minimal CMV promoter. MS2- Fluorescent Protein binds to the MS2 stem loop repeats, rendering visualization of the transcribed mRNA. 200 copies of the gene expression cassette are stably integrated as a transgene array at human 1p36 in U2OS 2-6-3 cells. The diagram is not drawn to scale. (b) Interphase induction: Upon Dox induction, mCherry-Pol II and MS2-YFP were recruited to the locus (arrowhead). Image stacks were acquired every 3mins. 0′ indicates the first time point where Pol II is detectable at the locus. Scale bars, 10μm, 2μm. (c) Post-mitotic re-activation: cells were induced with Dox overnight (O.N.) before imaging. Cells with an active locus were followed. Both Pol II and MS2-YFP were dissociated from the locus upon entry into mitosis. Upon exit from mitosis, both fusion proteins were recruited back to the daughter loci shortly after formation of daughter nuclei (arrowheads). Images were taken every 2mins. Scale bars, 10μm, 2μm. (d) Pol II recruitment was 13 times more rapid during post-mitotic re-activation, as shown by quantitative analysis of Pol II recruited to the locus during interphase induction and post-mitotic re-activation. The Pol II signal at the locus was quantified across the imaging session for each cell and the average curve was generated as described in Methods. The rising time of the Pol II signal for interphase induction and post-mitotic re-activation was plotted as mean±S.E.M (208.1±22.9min versus 16.5±6.0min) (n=17 for interphase induction, n=19 for post-mitotic re-activation). (e) mRNA production was 5 times more rapid during post-mitotic re-activation, as shown by quantitative analysis of mRNA production at the locus during interphase induction and post-mitotic re-activation. MS2-YFP signal was quantified across the imaging session for each cell and the average curve was generated as described in Methods. The rising time of the MS2-YFP signal for interphase induction and post-mitotic re-activation was plotted as mean±S.E.M (157.6±19.0min versus 34.3±7.6min) (n=13 for interphase induction, n=25 for post-mitotic re-activation).

Global chromatin decondensation or a second interphase induction is not sufficient to provide a more rapid transcriptional induction. (a) Schematic diagram of induction during mitosis. Cells in which the locus was inactive were synchronized with Nocodazole (50μg/ml) and, after wash-out, Dox was added and cells were allowed to progress through the cell cycle. Such cells will be referred to as mitosis induced cells. (b) Rapid chromatin decondensation upon exit from mitosis, in and of itself, is not sufficient for rapid transcriptional induction. The average curve of Pol II was generated as previously described. The rising time of Pol II signal for interphase induction (taken from , for ease of comparison) and post-mitotic induction was plotted as mean±S.E.M (208.1±22.9min versus 168±21.8min) (n=17 for interphase induction, n=7 for post-mitotic induction). (c) The average curve of MS2-YFP was generated as previously described. The rising time of MS2-YFP signal for interphase induction (taken from , for ease of comparison) and post-mitotic induction was plotted as mean±S.E.M (157.6±19.0min versus 185.2±45.5min) (n=13 for interphase induction, n=10 for post-mitotic induction). (d) Diagram of the experimental protocol for studying cells that have been transcriptionally induced in interphase twice. (e) Silencing of an active locus during interphase followed by a second transcriptional induction does not result in increased kinetics of transcriptional induction. The average curve of Pol II was generated as previously described. The rising time of Pol II signal for initial (taken from , for ease of comparison) and second interphase induction was plotted as mean±S.E.M (208.1±22.9min versus 227±41min) (n=17 for interphase induction, n=9 for interphase 2^nd induction). (f) The average curve of MS2-YFP was generated as previously described. The rising time of MS2-YFP signal for initial (taken from , for ease of comparison) and second interphase induction was plotted as mean±S.E.M (157.6±19.0min versus 169.2±49.6min) (n=13 for interphase induction, n=9 for interphase 2^nd induction).

Histone H4 lysine 5 acetylation (H4K5Ac) is a bookmark for active transcription in interphase and is maintained during mitosis. (a) Schematic diagram of primer sets used for chromatin immunoprecipitation experiments (ChIP). (b) H4K5Ac showed the largest increase on the promoter region after interphase transcriptional activation, whereas the other active histone modifications (H3K4me3, H3K36me3, H4K8Ac, H4K12Ac and H4K16Ac) did not show as large an increase. Cells stably expressing pTet-ON, were treated with or without Dox (1μg/ml) for 24hrs before being collected for ChIP experiments. Results were collected from 3 biologically independent experiments (mean±S.E.M.). (c) Increased H4K5Ac after interphase transcriptional activation was preserved in mitotic cells. U2OS 2-6-3 cells, stably expressing pTet-ON, were induced by Dox (1μg/ml) for 24hrs, followed by treatment with Nocodazole (50μg/ml) in the presence of Dox for another 16hrs for synchronization, before being collected by mechanical shake off for ChIP experiments. Results were collected from 3 biologically independent experiments. (mean±S.E.M.) (d) Association of Acetylated H4 (TH4Ac) at the locus during mitosis after Dox induction. Cells induced with Dox (1μg/ml, 24hrs) were fixed and immunolabeld with anti-H4Ac antibody (green). Association of TH4Ac with the locus (enlarged, +Dox) can be detected in mitotic cells. Scale bars, 10μm, 2 μm.

BRD4 regulates efficient post-mitotic re-activation of transcription. (a) BRD4 and Pol II were recruited to the same genetic locus, with different sequential dynamics upon interphase induction vs. post-mitotic re-activation (daughter locus of cell on right is enlarged.) (white arrowheads indicate presence of signal; yellow arrowheads indicate absence of signal). Scale bar, 10μm. (b) Pol II was recruited prior to BRD4 upon interphase induction (n=7), while BRD4 was recruited before Pol II during post-mitotic re-activation (n=7) (mean±S.E.M.) (c) siRNA knockdown of BRD4 delayed post-mitotic transcriptional re-activation. Arrowheads indicate mRNA production revealed by MS2-YFP signal. Scale bar, 10μm. (d) BRD4 regulates efficient post-mitotic transcriptional re-activation, as shown by quantitative analysis of the rising time of mRNA production at the locus in post-mitotic cells treated with either siBRD4#1 (258.75±38.0min; n=5), or siBRD4#2 (249.17±40.0min; n=6), or a control siRNA (133.3±18.7min; n=6) (mean±S.E.M.) (e) Diagram of JQ1 experiments. (f) JQ1 treatment significantly slowed down the post-mitotic transcriptional activation, as shown by quantitative analysis of the rising time of mRNA production at the locus in post-mitotic cells treated with either DMSO control (41±6.2min; n=5) or JQ1 (100.91±8.1min; n=11). The no treatment control from is re-plotted, for ease of comparison. (g) Cells were transiently transfected with LacI-mCherry and were treated as indicated with Dox or Dox + JQ1 in interphase. Cells were fixed and immunolabeled for BRD4 and metaphase cells were examined. DNA was also stained. (h) Loci are indicated by LacI-mCherry, and the area of the loci was determined (n=27, 40, 34 for −Dox, +Dox and +JQ1 groups, respectively). Scale bar: 2μm for insert and 10μm for main panel.

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).

(a) Diagram of the protein domains contained in different BRD4 deletion mutants. BD: bromodomain; ET: extraterminal; SEED: Ser/Glu/Asp-rich region; CTD: c-terminal domain. (b) BD1 but not BD2 is critical for the de-compaction function of BRD4. Cells were transiently transfected with indicated LacI-mCherry-fused BRD4 mutants. Among these mutants, 72% of the cells transfected with BRD4-722 (n=106 cells), 80% of those transfected with BRD4-Δ2 (n=108 cells), and 79% of those transfected with BRD4-NBD1 (n=104 cells), demonstrated decompacted loci; while no decompacted locus could be identified in the cells transfected with the constructs indicated (−) in (a) (> 100 cells per construct). Tethering LacI-BRD4-NBD1 in the presence of JQ1 still resulted in decompact loci (v, +JQ1) compared with control (v, −JQ1). Cells were transiently transfected with LacI-mCherry-fused BRD4-NBD1 and were treated with JQ1 for 6hrs. (c) The minimum de-compaction mutant NBD1 was sufficient to accelerate interphase transcriptional induction. Analysis of the rising time of mRNA production in Dox induced interphase cells expressing either LacI-mCherry-BRD4-Δ1 (141.25±19.4min; n=12) or LacI-mCherry-NBD1 (85.77±4.7min; n=13). (d) A model of transcriptional induction in interphase, and transcriptional re-activation in post-mitotic cells.