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Primers used in ChIP-QPCR and mRNA expression analyses. This file contains primer sequences used for ChIP-QPCR verification of ChIPseq peaks located in the promoter regions of five selected genes. (PDF 21 kb)

Integrative analysis of ChIP-seq and gene-expression data. In order to investigate the gene-regulatory potential of BRD1-S and BRD1-L, ChIPseq data were integrated with expression data. A window of +/– 200 kb from BRD1 binding was constructed, fragmented in 9 kb segments, and determined the number of DEGs having TSSs in these segments. As opposed to the 10 kb segment used in the main manuscript the 9 kb segments used here span across 0 on the x-axis. A, B The analysis was performed for DEGs identified after upregulating BRD1-S or BRD1-L or (C, D) downregulating endogenous BRD1. Significantly more up- or downregulated genes were identified in segments where BRD1-S or BRD1-L binds in close proximity to TSSs compared to all segments across the window (P

Chromatin binding profiles for BRD1-S and BRD1-L in relation to identified epigenetic marks in HEK293 cell lines. ChIP-seq data (fastq files), from HEK293 cell lines and cell line derivatives, were obtained from the ENCODE project and mapped to hg19 (for further details see Methods). An in-house histone H3K9ac ChIP-seq dataset was also included in the analysis. The minimum distance from (A) BRD1-S or (B) BRD1-L ChIP-seq peak to transcription factor binding site or histone mark was identified for all 2205 and 1722 peaks, respectively. The results were summarized as histograms with a +/â 30Â kb window from BRD1 binding (distance from BRD1 peaks). The number of peaks within each of the summed bins (columns) can be read on the y-axis (number of peaks). Random ChIP-seq peak regions (Random), with the same composition of chromosomes and peak region sizes, were generated to evaluate if the results could be explained be chance. (PDF 635 kb)

Verification of five selected ChIP-seq promoter targets by ChIP-QPCR. From the 251 common promotor target genes (PTGs) of BRD1-S and BRD1-L, we selected five for validation with ChIP-QPCR. A The promoter loci at the WD repeat domain 7 (WDR7), digestive organ expansion factor homolog (C1ORF107 or DIEXF), proteasome subunit β type, 2 (PSMB2), ZC3H15, and zinc finger protein 226 (ZNF226) showed BRD1-S and BRD1-L binding, while the promoter loci of the lymphocyte expressed gene granzyme M (GZMM) showed no significant binding of the BRD1 isoforms and was subsequently selected as a negative control. The BRD1-S or BRD1-L binding is shown in black whereas the control ChIP-seq (IP with anti-HA antibody) is shown in pink. B Primers were designed to amplify a 80–150 bp sequence in the promoter region or the intron of all six genes (for primer sequences see Additional file 7). ChIP was performed with either anti-V5 antibody (V5) conjugated beads or anti-HA (HA) conjugated beads using extracts from BRD1-S, BRD1-L, or HEK293T cells (for further details see “Methods”). ChIP-QPCR showed higher enrichment of BRD1-S and BRD1-L at the promoter loci of WDR7, DIEXF, PSMB2, ZC3H15, and ZNF226 compared to controls. Also ChIP-QPCR showed less BRD1-S and BRD1-L binding at introns of WDR7, DIEXF, PSMB2, ZC3H15, and ZNF226 while ChIP-QPCR showed lower and approximately equal BRD1-S and BRD1-L binding at the GZMM promoter and intron. (PDF 433 kb)

Expression arrays from siRNA knockdown and over-expression. After RNA purification from BRD1-S, BRD1-L and HEK293T cells, and cells treated with siRNA directed against BRD1 and cells treated with scrambled siRNA, expression microarray analyses were performed on Affymentrix U219 chips. Individual probe emission intensities were normalized to the mean intensity of the entire array and differential values were calculated as log2(sample) – log2(control) and a cutoff value of +/– 1.5-fold determined if genes were considered upregulated or downregulated. The figure shows MA-plots, where M = log2(sample) – log2(control) and A = ½ × (log2(sample) – log2(control). The two horizontal lines represent 1.5 in expression fold change compared to controls. Probes regulated more than 1.5-fold are indicated by red dots. Probes that are not regulated more than 1.5-fold are colored blue. MA-plots from the top: (A) BRD1-S-V5 stable cells vs. control, BRD1-L-V5 stable cells vs. control and (B) cells treated with siRNA directed against BRD1 vs. cells treated with scrambled siRNA (2 array for method 1, M1.1, and M1.2, and 1 for method 2, M2), and control vs. control (ctrl_v_ctrl, from HEK293T cells treated with scrambled siRNA). The MA-plot of two independent siRNA knockdown control experiments (ctrl_v_ctrl) showed 0.5 % of the probes below or above a 1.5-fold threshold from a total of 49,386 probes. On average, the analysis showed 4.4 % of the probes to be within threshold limits. (PDF 225 kb)

Immunoprecipitation of PBRM1 and identification of BRD1-S and BRD1-L by western blotting. Co-immunoprecipitations of endogenous PBRM1 were performed on BRD1-S, BRD1-L, and untransfected HEK293T cells, by either including antibody against PBRM1 (AB +) or by not including antibody (AB –). BRD1-S-V5 and BRD1-L-V5 were visualized on the western blot using anti-V5 antibody (Invitrogen) and HRP-conjugated anti-mouse IgG antibody. Crude cell extracts (E) from BRD1-S-V5 and BRD1-L-V5 were included as migration markers of the epitope tagged proteins, at approximately 130 kDa and 140 kDa, respectively. (PDF 122 kb)

Gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) analysis by the web based software ChIP-Enrich of FUS and EWS ChIP-seq peaks [51]. (DOCX 19 kb)

qPCR validation of enrichment peaks identified by FUS and EWS ChIP-seq. The enrichment of DNA was quantified as percentage of the amount in the input sample and the p-values for enrichment of FUS and EWS were calculated. IFG28 was used as a negative control for enrichment. A control ChIP experiment was also performed with inclusion of pre-immune antiserum added to the AG beads used for chromatin purification (AG) instead of FUS and EWS antibodies. Data represents three independent experiments and standard deviation shown by error bars. A. C19orf48 and ACPT; B. RCC1 and SNHG3; C. HNRNPK. (PDF 23 kb)

Analysis of ChIP-seq FUS and EWS enrichment peaks in the RCC1 and SNHG3 gene complex, including SNORA73A and SNORA73B genes. A. Graphic distribution of ChIP-seq reads aligned to the RCC1 and SNHG3 genes from the input, FUS, EWS and Ac-H3K9 ChIP-seq samples. The number of reads is shown on the scale to the left of each figure. The transcripts from the genes in the UCSC hg19 genomic database are shown in the bottom. The arrows beneath peaks illustrate location of the several qPCR amplicons used. The arrows above the transcripts illustrate location of RT-qPCR amplicons. Numbers above arrows denotes target exon numbers. B-C. FUS and EWS effect for RCC1, SNHG3 and snoRNA expression. The expression levels were determined by qPCR from HEK-293 cells transfected with siRNA for FUS and EWS or control siRNA. Three independent experiments were performed and error bars indicate standard deviation. In B., simultaneous depletion of FUS and EWS was performed, whereas in C. FUS and EWS were individually depleted. (PDF 552 kb)

Annotated genes overlapping the significant enrichment peaks determined from FUS, EWS and Ac-H3K9 ChIP-seq analysis. Chromosome (CH): the peak start and end in base pairs; Length: the peak length in base pairs; Tags: the number of sequences included in the peak; p-value: p-value on log2 scale; fold-change (fc): relative to input sample; FDR: false discovery rate in percentage; ENS ID: gene ID in the ensemble database; gene location: location of the enrichment in the gene (upstream, downstream, intron, exon); and T: number of known transcript variants produced from the given gene. (PDF 1497 kb)

Cross-comparison of the genes identified to be associated with FUS, EWS or Ac-H3K9 by ChIP-seq analysis. The displayed overlap categories are FUS and EWS, FUS and Ac-H3K9, EWS and Ac-H3K9, FUS and EWS and Ac-H3K9, FUS and EWS. Chromosome (CH): the peak start and end in base pairs; Length: the peak length in base pairs; Tags: the number of sequences included in the peak; p-value: p-value on log2 scale; fold-change (fc): relative to input sample; FDR: false discovery rate in percent; ENS ID: gene ID in the ensemble database; gene location: location of the enrichment in the gene (upstream, downstream, intron, exon); and T: number of known transcript variants produced from the given gene. (DOCX 325 kb)

Upregulation of BRD1-S and BRD1-L. A QPCR measurement of BRD1 mRNA in BRD1-S and BRD1-L cells showed a ~24-fold higher BRD1 mRNA levels compared to HEK293T controls (*P

Immunofluorescence staining of HEK293T cells stably expressing V5-epitope tagged BRD1-S and BRD1-L. Stable cell lines (BRD1-S-V5 and BRD1-L-V5) and the untransfected HEK293T cell line (HEK293T) were cultured for 48 h in 2 mL slide flasks followed by fixation in 2 % freshly prepared paraformaldehyde solution. Slides were incubated with anti-V5 antibody (Invitrogen), washed, and incubated with anti-mouse immunoglobulins/FITC F(ab’)2. Cell nuclei were stained by Hoechst staining. From the left: immunoflourescence staining of cell nuclei (Hoechst, blue), imunnoflourescence staining of V5-tagged BRD1-S and BRD1-L (Anti-V5, green), and the two images merged (Merge, purple). (PDF 94 kb)

Immunofluorescence staining of HEK293T cells stably expressing V5-epitope tagged BRD1-S and BRD1-L. Stable cell lines (BRD1-S-V5 and BRD1-L-V5) and the untransfected HEK293T cell line (HEK293T) were cultured for 48 h in 2 mL slide flasks followed by fixation in 2 % freshly prepared paraformaldehyde solution. Slides were incubated with anti-V5 antibody (Invitrogen), washed, and incubated with anti-mouse immunoglobulins/FITC F(ab’)2. Cell nuclei were stained by Hoechst staining. From the left: immunoflourescence staining of cell nuclei (Hoechst, blue), imunnoflourescence staining of V5-tagged BRD1-S and BRD1-L (Anti-V5, green), and the two images merged (Merge, purple). (PDF 94 kb)

Supporting information for ChIP-seq analysis. A Fragmented DNA for ChIP-seq, with an average size of 100â 150Â bp from HEK293T cells and stable BRD1-S-V5 and BRD1-L-V5 expressing HEK293T cells. B IP-western blot of IPed BRD1-S-V5 and BRD1-L-V5 using V5-ChIP-protocol. V5-tagged BRD1-S and BRD1-L was IPed from stable expressing HEK293T cells using anti-V5 antibody conjugated agarose beads (IP-V5) or anti-HA antibody conjugated beads (IP-HA) as control IP. V5-tagged BRD1-S and BRD1-L were visualized on a western blot using an anti-V5 antibody (Invitrogen) and a HRP-conjugated anti-mouse IgG. (PDF 78 kb)

Supporting information for ChIP-seq analysis. A Fragmented DNA for ChIP-seq, with an average size of 100â 150Â bp from HEK293T cells and stable BRD1-S-V5 and BRD1-L-V5 expressing HEK293T cells. B IP-western blot of IPed BRD1-S-V5 and BRD1-L-V5 using V5-ChIP-protocol. V5-tagged BRD1-S and BRD1-L was IPed from stable expressing HEK293T cells using anti-V5 antibody conjugated agarose beads (IP-V5) or anti-HA antibody conjugated beads (IP-HA) as control IP. V5-tagged BRD1-S and BRD1-L were visualized on a western blot using an anti-V5 antibody (Invitrogen) and a HRP-conjugated anti-mouse IgG. (PDF 78 kb)

Upregulation of BRD1-S and BRD1-L. A QPCR measurement of BRD1 mRNA in BRD1-S and BRD1-L cells showed a ~24-fold higher BRD1 mRNA levels compared to HEK293T controls (*P