The BRD4–NUT fusion protein forms nuclear foci containing hyperacetylated chromatin domains. Cos7 cells were transfected with the indicated expression vectors, (A) GFP–BRD4–NUT, (B) HA–sBRD4, (C) HA–NUT and (D) HA–sBRD4 together with GFP–NUT. Ectopically expressed proteins were visualized by GFP or indirect fluorescence after anti-HA detection (A–D) and co-detected with an anti-pan acetylated histone H4 antibody (A–C). Bar: 10 μm.

p53 inactivation in BRD4–NUT-expressing cells. (A) HCC2429 and A549 cells were treated with 20 μM etoposide for 15 h and p21, p53 and p300 were visualized as indicated. (B) A546 cells were transfected with an empty vector (−) or with a BRD4–NUT-expressing vector (+) and treated or not with etoposide as above (+ and −, respectively) and the indicated proteins were detected with the corresponding antibodies. (C) HCC2429 cells were treated with scrambled siRNAs or two specific anti-BRD4–NUT siRNAs and the indicated proteins were visualized as above. (D) HCC2429 cells were treated with the anti-BRD4–NUT siRNAs indicated above as well as with an anti-p53 siRNA and the indicated proteins were visualized using the corresponding antibodies. (E) The expression of p21, PUMA and GADD45 were monitored after the knockdown of BRD4–NUT with the two above-mentioned siRNAs in the presence (dark grey histograms) or absence (light grey histograms) of p300 inhibitor. Data represent the mean±s.d. values of at least three independent experiments. (F) The association to p21 promoter of p53, acetylated p53 (p53ac) and p300 was monitored in HCC2429 cells before and after the knockdown of BRD4–NUT by siRNA as above. The PCR amplification shown here is representative of three independent ChIPs.

Mapping of domains in NUT and p300 involved in a direct interaction between the two proteins. (A) Schematic representation of domains and fragments used in both p300 and NUT. (B) Cos7 cells were transfected with Myc-tagged Δ870 p300 and the indicated fragments of HA-tagged NUT-expressing vectors and, after anti-HA immunoprecipitation, the immunoprecipitated proteins were visualized using the indicated antibodies. (C) The indicated HA-tagged NUT fragments were expressed in Cos7 cells and, after anti-HA immunoprecipitation (anti-HA panel), the association of the endogenous p300 was visualized (anti-p300 panel). The input panel indicates the presence of p300 in the different extracts. (D) Glutathione-bound GST or the GST–F1c fragment of NUT were incubated in a nuclear extract from Cos7 cells. Fractions of the bound proteins were either used to detect the presence of endogenous p300 (bottom panel) or to run a HAT assay using purified histones, as described previously. The Coomassie-stained gel (upper panel) shows GST and GST–F1c NUT before and after the pull down. The gel was then dried and used to visualize acetylated proteins after autoradiography (middle panel). (E) GST or GST–F1c were used to pull down either Myc-tagged p300 WT or the HA-tagged CH3 domain of p300. Extracts and proteins used in the pull down are shown after the Coomassie staining of the gel (upper panel). The proteins pulled down were visualized using the indicated antibodies. (F) In an experiment similar to (E), the interaction of GST or GST–F1c with the indicated fragments of p300 was monitored. Please note that as the HA-tagged proteins were of different sizes (B, C), only the corresponding areas of the films are shown and separated by lines. A full-colour version of this figure is available at The EMBO Journal Online.

Direct enhancement of p300 HAT activity by NUT. (A) p300L (amino acids 324–2094 fragment) or p300S, a deletion mutant lacking its CH3 domain (amino acid 1045–1666 fragment) were expressed using a baculovirus system and purified. In parallel, GST and GST–F1c were expressed in bacteria and purified and used in a pull-down assay. The gel shows proteins present in the input (left panel) and after the pull down (right panel). (B) Cos7 cells were transfected with vectors expressing the indicated tagged proteins alone or together. After an immunoprecipitation using anti-Myc (p300) and anti-HA (NUT) antibodies, a fraction was used to monitor protein immunoprecipitations (upper panels). Another fraction was used for an in vitro HAT assay as described in Figure 2C. Coomassie-stained histones and the corresponding autoradiography are shown (middle and lower panels, respectively). (C) Purified p300 was incubated with histone H3 in the absence (0) or presence of increasing amounts of purified GST–F1c fragment of NUT (Coomassie panel). Aliquots corresponding to each incubation conditions were used to monitor site-specific histone H3 acetylation using antibodies recognizing the indicated acetylated sites. Anti-acetylated lysine and anti-H3 antibodies were used to show p300 autoacetylation and H3 loading, respectively. A full-colour version of this figure is available at The EMBO Journal Online.

Propagation of chromatin acetylation through a feed-forward mechanism induced by BRD4–NUT. (A) The first BRD4 bromodomain was inactivated by site-directed mutagenesis (BRD4–NUTΔbr1) and the capacity of this mutant to form nuclear foci and acetylated chromatin domains enriched in p300 was analysed after the transfection of Cos7 cells. (B) Co-transfection of p300 together with GFP–BRD4–NUT leads to the enlargement of BRD4–NUT foci. The inset shows a cell expressing Myc–p300 alone. The histogram shows the results of quantitative evaluations (ratios of the values observed in p300-transfected cells to those observed in non-transfected cells) of the mean foci area (left), mean foci number (middle) and mean total foci area per cell (right), obtained as described in Materials and methods section. (C) HCC2429 were transfected either with the NUT interaction domain of p300, CH3 (upper panel), or the p300-interacting domain of NUT, F1c (lower panel), and the formation of foci by the endogenous p300 was monitored. NUT–F1c and p300–CH3 were detected with an anti-HA antibody, endogenous p300 with an anti-p300 antibody and BRD4–NUT with an anti-NUT antibody. Please note that the anti-NUT antibody also recognizes the NUT–F1c fragment. The images are representative of nearly 100% of the cells expressing either fragments. (D) HCC2429 cells were treated with the small p300 inhibitor, C646 (+ p300 inhibitor) or its inactive analogue (+ control inhibitor) and BRD4–NUT and p300 were visualized with the corresponding antibodies. The images are representative of nearly 100% of the cells. Bar: 10 μm.

Specific recruitment of cellular p300 by the NUT moiety of BRD4–NUT to the nuclear foci. (A) Cos7 cells were transfected with GFP–BRD4–NUT and the endogenous cellular p300 was detected by immunofluorescence using an anti-p300 antibody. Both GFP and p300-related fluorescence were recorded and shown individually or together (merge panel). (B) Cos7 cells were transfected with the indicated HA-tagged vectors and, after anti-HA immunoprecipitation, HA-tagged proteins and associated endogenous p300 were visualized using the corresponding antibodies. The ‘input' panel shows the amount of cellular p300 in each extract. (C) Cos7 cells were transfected with vectors expressing HA-tagged GCN5 or NUT and the corresponding empty vector (φ) and extracts were used to immunoprecipitate HA-tagged proteins. A fraction of immunoprecipitated materials was used to visualize proteins by an anti-HA western blot (anti-HA panel). Another fraction was used to monitor the HAT activity present in the immunoprecipitates using purified histones (lower panel, showing the corresponding autoradiography), which were visualized on a Coomassie-stained gel (middle panel). (D) Detection of endogenous BRD4–NUT with anti-NUT antibody in the HCC2429 cells compared with two other lung cancer cell lines (indicated). As a control, NUT was also detected in a rat total testis extract (lane testis). (E) Endogenous BRD4–NUT foci and p300 were detected in HCC2429 cells using the corresponding antibodies. (F) BRD4–NUT was co-detected with histone H4K8ac. The specific in vivo signatures of p300 activity, histones H3K56ac and H3K18ac, were also co-detected with endogenous p300 in HCC2429 cells (indicated). (G) BrU incorporation in nascent RNAs and BRD4–NUT foci were visualized in HCC2429 and A549 cells after a pulse labelling with BrUTP. Bar, 10 μm.