797Trex megadomain(s) Per403 megadomain(s) chr1: chr1: chr12: chr12:

Transcription

1 Table S1. Megadomains that overlap in all BRD4-NUT NMC cell lines and tissue. chromosome union region start union region end 797 megadomain(s) chr chr1: chr chr12: ; chr12: chr chr2: Trex megadomain(s) chr1: chr12: chr2: ; chr2: Per43 megadomain(s) chr1: chr12: chr2: c tissue megadomain chr1: chr12: chr2: c cell line megadomain chr1: chr12: chr2: genes within/overlap with the union region DNM3; DNM3OS; MIR214; MIR199A2; RNU6-157P; PIGC; C1orf15 TSPAN19; LRRIQ1; RP11-193M21.1; ALX1; RP11-48B11.2; RP11-629O19.1; AC ; RP13-619I2.2; RASSF9; NTS; MGAT4C; RP11-18J9.3 SNORD78; CTD- 226C7.1; VRK2; AC ; FANCL; RP11-334G22.1; EIF3FP3; AC725.4; LINC1122; AC7238.1; AC7367.1

2 chr chr3: chr chr3: ; chr3: chr3: chr3: chr3: chr3: ; chr3: chr3: chr3: chr3: chr3: RBMS3-AS3; RBMS3; AC9865.1; RPS12P5; RBMS3-AS2; AC ; RBMS3-AS1; AC ; AC ; ACEA_U3; AC ; AC ; TGFBR2 SOX2-OT; RNU6-4P; FAUP2; RP11-275H4.1; RPL7AP25; RP11-4B14.3; SOX2OT_exon1; SOX2OT_exon3; SOX2; SOX2OT_exon4; RN7SL73P; RNA5SP15; RP11-252O18.2; RP11-252O18.3; LINC126; RN7SKP265; RP11-416O18.2; RP11-338L18.1; FLJ4666; RP11-139K4.4; RP11-139K4.2; RPL7L1P8; RP11-646E18.2; RP11-646E18.3; RP11-646E18.4; RP11-225N1.1;

3 ATP11B chr chr3: chr chr8: chr3: chr8: chr3: chr8: chr3: chr8: chr3: chr8: TPRG1; TPRG1- AS2; TP63; RP11-6G3.1; MIR944; LEPREL1; MTAPP2 CTD-221A23.1; RNU6-295P; 5S_rRNA; RPL29P19; RP11-513O13.1; RP11-567J2.3; RP11-77E5.1; RP11-567J2.2; AC2694.1; RP11-567J2.1; RP11-77E5.3; RP11-77E5.2; EFCAB1; RP11-22C8.1; SNAI2

4 chr chr8: chr8: chr8: chr8: chr8: CASC19; CCAT1; CASC8; RP11-382A18.3; CASC21; POU5F1B; AC ; CASC11; MYC

5 Table S2. Oligonucleotides used for qrt-pcr and sirnas. qrt-pcr Oligos MYC-F MYC-R 5 -AATGAAAAGGCCCCCAAGGTAGTTATCC-3 5 -GTCGTTTCCGCAACAAGTCCTCTTC-3 PVT1-916-F PVT1-97-R 5 -TGAGAACTGTCCTTACGTGACC-3 5 -AGAGCACCAAGACTGGCTCT-3 TP F TP R 5 -TGGAAACCAGAGATGGGCAA-3 5 -CGGGCGCTTCGTACCATC-3 QMed24-F QMed24-F 5 -CCAAGTTTCAAGAGAGAATGCTC-3 5 -AGCTTGCCATGTGCCTT-3 sirna Oligos sipvt1-1 Sense: 5 -CCCAACAGGAGGACAGCUUUU-3 Antisense: 5'-P.AAGCUGUCCUCCUGUUGGGUU-3 sipvt1-2 Sense: 5 -CCAGCAUCACUGUGGGAAAUU-3 Antisense: 5 -PUUUCCCACAGUGAUGCUGGUU-3 sitp63-1 Sense: 5 -ACACAGACCACGCGCAGAAUU-3 Antisense: 5 -UUCUGCGCGUGGUCUGUGUU-3 sitp63-2 Sense: 5 -GGGAUGAACCGCCGUCCAAUU-3 Antisense: 5 -UUGGACGGCGGUUCAUCCCUU-3 simed24-3 simed24-4 Dharmacon Cat# D Target Sequence: 5 -CAACCUGGCAUGCGUUA-3 Darmacon Cat# D Target Sequence: 5 -GUACGAGGAGCUCAAGUG-3

6 Figure S1 B N- BIO BRD4-NUT enrichment H3K27ac enrichment BRD4-NUT BRD4-NUT BIO TAP H3K27Ac megadomains C- TAP megadomains A 293TRex TC-797TRex Peroxidase anti-peroxidase 1Kb megadomain +1Kb C 1Kb megadomain +1Kb log2 enrichment ratio 293Trex

7 no JQ1 megadomain D 6 overepresentation tests: enhancer p value =.3 super enhancer p value = no JQ1 4h JQ1 22 Kb domain rank P value 1 E 15 1Kb 2.5h JQ1 megadomain +1Kb % Class I % Class II % Class III % Altogether.5 1h JQ h JQ1.6 8 seed enhancers inflection point domain size (Kb) % of all megadomains all pre existing enhancers.4 C 1h JQ1 +1Kb.2 1Kb no JQ1 1h JQ1 2.5h JQ1 4h JQ no induction no JQ1 1h JQ1 2.5h JQ1 4h JQ1 2h induction intergenic transcription 4 3h induction genic transcription 2 15 RNA-Seq tag density 7h induction 1 RNA-Seq tag density 2 B RNA-Seq tag density Figure S2 A fold standard deviation 3 4h JQ1

8 BRD3 NUT (1326) 192 BRD3 NUT (1326) (797) (797Trex) (Per43) 44 (115 tissue) (115 cell) (293Trex) 41 (797) (797Trex) (Per43) (115 tissue) (115 cell) (293Trex) % megadomains overlapped

9 1 O/E Hi C inte raction 5 Figure S4 chr11:9,318,975-1,444,975 chr12:14,8,998-15,14,998

10 1.8 sictrl 1.6 sipvt1-1 sipvt Involucrin GADPH MED24 C TP63 189, kb IP NUT, 293Trex IP NUT, 797 megadomain, 797 IP NUT, 797Trex megadomain, 797Trex IP NUT, 115 tissue megadomain, 115 tissue IP NUT, 115 cell line megadomain, 115 cell line IP NUT, PER43 megadomain, PER43 IP BRD3-NUT, 1326 BRD3-NUT megadomain RNA-Seq no JQ1 (+ strand) RNA-Seq 1h JQ1 (+ strand) RNA-Seq 2.5h JQ1 (+ strand) RNA-Seq 4h JQ1 (+ strand) RNA-Seq no JQ1 (- strand) RNA-Seq 1h JQ1 (- strand) RNA-Seq 2.5h JQ1 (- strand) RNA-Seq 4h JQ1 (- strand) Gene PVT1 189,2 kb 189,4 kb 48h 72h 189,6 kb 189,8 kb 19, kb sitp63-2 6h 5nM JQ1 D sictrl 6h DMSO Fold Change of MYC RNA B Fold Change RNA A sitp63-1 Figure S5

11 Supplemental Figure Legends Figure S1: A. Expression of BioTAP-tagged BRD4-NUT. TRex Flp-in cells were induced to express C- or N-terminal tagged BRD4-NUT. Immunoblots were stained with peroxidase anti-peroxidase antibody, which recognizes the Protein A tag (TAP). B. Consistency between BRD4-NUT and H3K27 acetylation enrichment in 293T. The heatmap on the left shows BRD4-NUT enrichment for the domains identified based on the H3K27ac profiles, with each row showing the enrichment profile of a scaled megadomain and 1kb flanking regions. The heat map demonstrates that BRD4-NUT shows strong enrichment within the H3K27ac-defined megadomains. C. Venn diagram shows the number of overlapping megadomains between 293T and TC-797 cells. Most of the megadomains do not overlap between the two cell types. Figure S2: A. Average nascent transcription profiles are shown for scaled megadomains and 1kb flanking regions before BRD4-NUT induction and at different time points following induction in 293T cells. B. Comparison of the average nascent transcription magnitude at genic (left panel) and intergenic regions (right panel) within the megadomains without JQ1 and following JQ1 treatment over time. The error bars indicate standard error of the mean. C. The size distribution of the H3K27ac-enriched regions is shown for 293T cells before induction of BRD4-NUT, with seed regions (corresponding to positions where early domains will form following the induction) highlighted in red. Enhancer/ promoter regions, but not superenhancers, are over-represented in seed regions. P-values are calculated based on hypergeometric tests. D. The plot shows the fraction of megadomains categorized as Class I (blue), Class II (green) or Class III (orange) for an increasing stringency of H3K27ac enrichment calls used to detect seed enhancer/promoter positions (x-axis). The stringency is set as a number of standard deviations of H3K27ac distribution, which is then used as a mean of the emission probability in the Hidden Markov Model (see Methods) used to detect H3K27ac-enriched regions. At a value of two-fold standard deviation (indicated by the red dashed line), 46.5% of all megadomains can be explained by the three classes. E. Transcription start sites (TSSs), based on UCSC hg38 RefSeq annotation, are significantly enriched at the boundaries of the BRD4-NUT megadomains. The empirical P-values were estimated relative to the background distribution based on randomly-placed megadomain-sized regions. Megadomain positions in 293T cells were used. Figure S3: Similar to Figure 3B of the main manuscript, the clustered matrix shows pair-wise similarity of BRD3-NUT and BRD4-NUT megadomain positions detected in different cell types. Figure S4:

12 Two examples illustrate weak BRD4-NUT megadomains that do not reach TAD boundaries. The heatmaps show Hi-C observed/expected signal (from GM12878 cells) around the two megadomains. In both examples, BRD4-NUT megadomains (grey bars on the margins) occupy a portion of the local TAD, without reaching the boundaries. Figure S5: A. Effect of JQ1 treatment on TP63, MED24, and PVT1 RNA levels. B. Effects of sirna PVT-1 knockdown on MYC RNA levels in TC-797 cells. C. Example of BRD4-NUT megadomains within the TP63 locus in different cell types. Tracks shown in brown represent enrichment and detected megadomain of BRD3-NUT in this region. The plot also shows the effect of JQ1 treatment on the transcriptional activity (as measured by nascent RNA-seq) over time in TC-797 cells. D. Immunoblot of TC-797 cells lysed 96h following TP63 knockdown, showing no increase in the differentiation marker involucrin.