Fig 2: Minimal p300-interaction fragment of NUT in BRD4-NUT fusion is necessary and sufficient for condensate formation. (a) Schematic of all BRD4-NUT constructs. (b) Representative micrographs showing condensate formation in stable cell lines expressing different constructs. Staining against mNeonGreen shown in red, overlay with DAPI to indicate the nucleus. Scale bars = 10 µm. (c) Quantification of the micrographs represented in (b). (d) Representative micrographs comparing expression of constructs with or without p300-interaction motif in cells. Cells stained with anti-BRD4 antibody (magenta) and anti-p300 antibody (green). Scale bars = 10 µm. (e) and (f) Quantification of the overlap between condensates via co-staining shown as Pearson correlation. Cells co-stained with NUT and p300 antibodies (left) or mNeonGreen and p300 antibodies (right); Nut antibody epitope was removed in BRD4-NUT(?MIN), see Fig. S2b for more details. (g) Immunoprecipitation against mNeonGreen; western blot with p300 antibody and BRD4(N) antibody.

Fig 3: Positive nuclear immunoreactivity for nuclear protein in testis (NUT) in an abdominal lymph node derived from the patient with NUT midline carcinoma is indicated by the dark-brown staining of tumoral nuclei from oxidized diaminobenzidine chromogen used in the method. The blue counterstain is Meyer‘s hematoxylin, which stained nontumoral nuclei dark blue. Some cytoplasmic staining is also seen in lighter blue due to the long incubation time with the hematoxylin. (A) 40× (scale bar 200 μm), (B) 100× (scale bar 50 μm), and (C) 200× (scale bar 20 μm).

Fig 4: Brd4-p300 fusion forms condensates and recapitulates many of the transcriptional changes observed for Brd4-Nut fusion.a) Schematic of Brd4-Nut and Brd4-p300.b) Micrographs of Brd4-Nut and Brd4-p300 – expressing stable cell lines. Cells are stained with mNeonGreen antibody; scale bars = 10µm.c) Quantification of the micrographs represented in b.d) Quantification of histone H3K27Ac staining in untreated vs. C646-treated cells. Local acetylation shown as average fluorescence intensity per condensate.e) Venn diagram of genes acetylated upon expression of BRD4-NUT(MIN) (pink) and BRD4-p300 (blue), found via ChIPseq with @H3K27Ac antibody, at promoter-TSS regions: all annotated genes. Values in the diagram show the number of unique genes annotated; numbers in parentheses represent the mean overlap in 20 iterations with a randomly generated gene pool of the same size, from the human genome.f) Venn diagram of genes acetylated upon expression of BRD4-NUT(MIN) (pink) and BRD4-p300 (blue), found via ChIPseq with @H3K27Ac antibody, at promoter-TSS regions: Diagram shows only genes after applying 0.3–1 normalized signal value cutoff. Values in the diagram as in e).g) Venn diagram of genes upregulated upon expression of BRD4-NUT(MIN) (green) or BRD4-p300 (gray), found via RNAseq. Genes shown when fold change in expression was greater than two and p<0.05. Numbers in parentheses represent the mean overlap in 20 iterations with a randomly generated gene pool of the same size, from the human genome.h) Venn diagram of genes downregulated upon expression of BRD4-NUT(MIN) (green) or BRD4-p300 (gray), found via RNAseq. Statistical analyses as in g).i) RNAseq-ChIPseq data integration: heatmap showing up- and down-regulated genes found via RNAseq, that are bound by both BRD4-NUT(MIN) and BRD4-p300. Top up- and downregulated genes shown in the heatmap, based on log fold change, where log fold change was greater than 0.5.

Fig 5: Transcriptionally active, BET inhibitor-sensitive BRD4-NUT megadomains arise from seed regions. (A) BRD4-NUT megadomains are enriched for histone marks associated with active transcription. Average enrichment profiles within the megadomains (scaled to the same size) and 100-kb flanking regions are shown for TC-797 cells. (B) Average BRD4-NUT enrichment within the megadomains is reduced following JQ1 treatment of TC-797 cells. Scaled average profiles of BRD4-NUT enrichment are shown for TC-797 cells without JQ1 and 4 h following JQ1 treatment. (C) The average level of transcriptional activity is reduced within the megadomains and their flanking regions following JQ1 treatment of TC-797 cells. Similar to Figure 1F, the scaled average profile of nascent RNA-seq is shown for cells without JQ1 treatment and for cells 1 h, 2.5 h, and 4 h following JQ1 treatment. (D) Expression levels (log2[FPKM]) of all genes within the megadomain regions in TC-797 cells show broad reduction of transcriptional activity following JQ1 treatment. Each column corresponds to a gene within the TC-797 megadomain. Each row indicates JQ1 treatment condition: no JQ1 or 1 h, 2.5 h, or 4 h following treatment. (FPKM) Fragments per kilobase of exon per million reads mapped. (E) BRD4-NUT and H3K27ac enrichment in a 293T BRD4-NUT induction time course. The resulting domain calls (based on H3K27ac profiles) are shown as bars below the enrichment profiles. The domain border expansion is traced using green (expanding to the left) and red (expanding to the right) arrows. Seed regions are indicated by brown dotted circles, and an interseed region that will later “fill in” with BRD4-NUT and H3K27ac is indicated with a dotted green circle. For illustrative purposes, dotted arrows from seed and interseed regions point to corresponding regions in the violin plot (F) of all seed and interseed regions. (F) BRD4-NUT megadomain seed regions are enriched with H3K27ac. The violin plots show H3K27ac enrichment in 293T cells prior to BRD4-NUT induction for the seed regions that will show BRD4-NUT enrichment within 2 h following induction (early domains; brown) and the interseed regions that will be bound by BRD4-NUT only after 2 h (late domains; green). Colored dotted arrows pointing from the corresponding examples of these regions in Figure 1E are shown for illustrative purposes. The H3K27ac enrichment level found at regular enhancers within 293T cells is shown as a reference (gray). Genome-wide average enrichment of H3K27ac is shown with the dashed brown line. (G) Examples of the three classes of BRD4-NUT domain formation. Domains of the first class are formed by expanding from a single pre-existing H3K27ac region. Class 2 domains form by expanding from multiple pre-existing H3K27ac regions. Class 3 domains start with one or more pre-existing H3K27ac regions and activate additional enhancer-like regions during formation (see also Supplemental Fig. S2).