Supplemental Information
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Supplemental Information Supplemental Figures Supplemental Figure 1. Quantitation of bioluminescent imaging data in Figure 1B. 1 Supplemental Figure 2. Impact of Brg1 shRNAs on proliferation of immortalized murine embryonic fibroblasts (iMEFs), BCR-ABL/p19-/- B cell acute lymphoblastic leukemia (B-ALL), murine breast cancer cells (4T1), and BrafV600E melanoma. A) shRNA/GFP competition assay in indicated murine cell lines. Rpa3 shRNA targeting replication protein A3 serves as a positive control. shRNA targeting Renilla luciferase is included as a negative control. B) Western blotting for additional Brg1 shRNAs in the indicated cell lines transduced with the Ren or Brg1 LMN-shRNAs. A representative experiment of three biological replicates is shown. 2 Supplemental Figure 3. Effect of Brg1 shRNAs on proliferation of human leukemia cell lines. A) shRNA/GFP competition assay in the indicated human cell lines with shRen.713, shBrg1.4471, or shSpt16.3249. All shRNAs were evaluated in a timecourse of GFP% measurements at days 4, 8, 12, 16, 20, and 24. CML-BC indicates chronic myeloid leukemia blast crisis. B) Western blotting of SPT16 levels in whole cell lysates prepared from HeLa cells transduced with the indicated MLP shRNA constructs following puromycin selection. 3 Supplemental Figure 4. Expression and functional validation of SWI/SNF subunits in MLL-AF9/NrasG12D leukemia. A) RPKM values for genes encoding SWI/SNF complex subunits from polyA+ tail RNA-seq performed in RN2 cell line. B) shRNA/GFP competition assay with two independent shRNAs targeting indicated SWI/SNF subunits. Experiments were performed in RN2 cells as in Figure 1A. n=2-3. C) RT-qPCR was performed to test the knockdown efficiency of indicted SWI/SNF subunit shRNAs. TRMPV-Neo shRNAs transduced RN2 cell lines were used following 48 hours of dox treatment. Results are normalized to Gapdh. n=3. All error bars represent S.E.M. 4 Supplemental Figure 5. Effects of Brg1 knockdown on expression of Myc target genes. Gene Set Enrichment Analysis following Brg1 knockdown. Microarray analysis was performed comparing three independent Ren.713 shRNA RN2 lines with three independent Brg1 shRNA RN2 lines (4935, 3364, and 3232). The dox-inducible TRMPV-Neo vector was used. Dox treatment was for 96 hours. NES: normalized enrichment score. FDR: False discovery rate. The Myc target gene signature was obtained from (Schuhmacher et al. 2001) was used. 5 Supplemental Figure 6. Brg1 and Brd4 perform parallel regulatory functions in MLL- AF9/NrasG12D AML cells. A) mRNA-seq timecourse following JQ1 treatment. Shown is the relative mRNA level of 53 genes that were identified as rapidly downregulated following JQ1 treatment. Myc is labeled with a red line, as the most rapidly downregulated gene. B) GSEA of Brg1 knockdown microarray data. The gene set of 53 JQ1-sensitive genes was used, which was identified in (A). C) Brg1 or Brd4 IP-Western blotting with indicated antibodies. All IPs were performed in the nuclear extract from NOMO-1 cell line. All inputs represents 2% of the total lysate and 50% of the eluted IP material was loaded. D-E) FLAG-tag IP-Western 6 blotting of transient transfected constructs in HEK293T cell lines. All inputs represents 2% of the total lysate and 50% of the eluted IP material was loaded. In (E), BRD4S indicates the short isoform of BRD4 which retains the ET domain. BRD4(1-465) indicates a C-terminal truncation of BRD4S that lacks the ET domain. Representative experiment of three biological replicates is shown. F) ChIP-PCR with Brg1 antibody was performed in RN2 cell treated with either DMSO or 500nM JQ1 for 6h. PCR primer amplicons are indicated along the x-axis. neg refers to a negative control region found at a gene desert region. Genes were chosen that are co-occupied by Brg1/Brd4 from ChIP-seq analysis. G) same as (F), except ChIP was performed with Brd4 antibody in RN2 cells following conditional knockdown of Brg1. 7 Supplemental Figure 7. Brg1/Brd4 occupied E1-E4 regions are one of the super- enhancers in RN2 cell. Super-enhancers are based on the increasing amount of Brd4 ChiP-seq signals as described in (Loven et al. 2013). The number in parentheses indicates the ranking of the enhancer regions. 8 Supplemental Figure 8. Correlation between copy number changes at the MYC locus with MYC mRNA levels. A) Scatter plot depicting the relationship between MYC copy number (CN) and MYC mRNA expression in 5320 human tumors (data obtained from TCGA, https://tcga-data.nci.nih.gov/tcga/). Red dots indicate tumors showing a focal amplification 3’ of MYC (CN>4 between MYC and the next coding gene GSDMC) while harboring no clear MYC amplification (CN<4). MYC mRNA expression levels where categorized as high or low using the median MYC expression level in tumors harboring no MYC amplification (CN<4) as a cutoff (n = 5068 tumors, median MYC expression = 3.209, horizontal dashed line). B) Fisher exact test of tumors without amplifications (CN<4, n=5016), with MYC amplifications (CN>4, n=252), and with amplifications 3’ of MYC without MYC involvement (CN>4, n=52). These three groups were divided into MYC low or high expressing tumors, and a Fisher exact test was performed. 9 Supplemental Figure 9. Brg1 occupies the -2 kb region of Myc but not the E1-E5 elements in 4T1 breast cancer cells. ChIP-qPCR of Brg1 at the Myc -2 kb region and enhancer region in 4T1 breast cancer cells. PCR primer amplicons are indicated along the x- axis. Neg refers to a negative control region in a gene desert region. -2kb is relative to the Myc TSS. n=2. 10 Supplemental Figure 10. Low levels of H3K27 acetylation at E1-E5 in normal mouse tissues. A) ChIP-seq data from (Shen et al. 2012) highlighting the tissue specific pattern of H3K27 acetylation at the E1-E5 region. Y-axis values are reads per million. Gray box indicates the E1-E5 region. B) ChIP-seq track of E1-E5 region in different hematopoietic tissues with adjusted y-axis to highlight that low levels of histone acetylation enrichment can be found at this region. 11 Supplemental Figure 11. H3K27 acetylation and BRG1 are enriched at E3 region in K- 562 cells. ChIP-seq data were obtained from ENCODE project (http://genome.ucsc.edu/ENCODE/). 12 Supplemental Figure 12. SWI/SNF subunits BAF250a and BAF60a occupy E1-E5. ChIP- PCR in RN2 cells with Baf60a and Baf250a antibodies. Primers are indicated along the x- axis. n=3. 13 Supplemental Figure 13. Hematopoietic transcription factor expression level in RN2 cells. Illumina polyA+ tail RNA-seq RPKM value of various transcription factors expressed in RN2 cells. 14 Supplemental Figure 14. PU.1 and Cebpα can co-immunoprecipitate Brg1. FLAG-tag IP-Western blotting of transiently expressed constructs indicated. Plasmids were transfected into HEK293T cells, followed by lysate preparation at 48 hours. All inputs represents 2% of the total lysate and 50% of the eluted IP material was loaded. 15 Supplemental Figure 15. Brg1 occupies Bid, Btg1, Bmf, and Hoxa9 in RN2 cells. ChIP- seq tracks of different genomic loci showing Brg1, H3K27ac, and H3K4me3 profiles in RN2 cells. 16 Supplemental Figure 16. Topological domain boundaries that encompass the Myc gene. The top panels show Hi-C data from ES cells in chr15 around the Myc locus from (Dixon et al. 2012) with the boundaries of topological domains indicated. The bottom two panels are H3K27ac ChIP-seq track in RN2 cell or MEF cell (Shen et al. 2012). The Hi-C and 40kb domain graphs were generated from: http://chromosome.sdsc.edu/mouse/hi- c/database.php 17 Supplemental Figure 17. ChIP-seq analysis of various TFs and H3K27ac aligned with 4C-seq data in RN2 cell at the Myc locus. HPC-7 cell TF ChIP-seq data was obtained from (Wilson et al. 2010). The green box highlights intervening genomic regions where Myc and E1-E5 form physical contact. 18 Supplemental Figure 18. 4C-seq analysis at the Myc locus upon Brg1 knockdown. 4C- seq of Brg1-deficient RN2 cell line with Myc TSS anchor point. The y-axis is normalized mean value mapped reads per 10kb sliding window, which plot the relative proximity of various DNA fragments in this region to the Myc gene within the 3D nuclear space. E1 through E5 enhancers are at the located indicated. TRMPV-Neo shRNAs were induced by dox for 48h before 4C library preparation. * indicates non-coding RNA transcripts. Result shown is representative of two independent biological replicates. 19 Supplemental Figure 19. Effect of Brg1 knockdown on mRNA levels of hematopoietic TFs. RT-PCR of various genes encoding transcription factors, in Brg1-deficient RN2 cell. The experimental condition was the same as in Figure 7B-I. n=3. All error bars depict S.E.M. 20 Supplemental Figure 20. Brg1 knockdown leads to reduced eRNA production at E3. NSR and PolyA+(Illumina TruSeq) RNA-seq data obtained from RN2 cells. A) RNA expression profile is shown around the Myc gene. B) RNA expression profile in the E1-E5 enhancer region. The presence of non-polyadenylated short transcripts near E3 is consistent with the properties of eRNA (Kim et al. 2010b). C) RT-PCR of E3 eRNA in Brg1-deficient RN2 (following 48 hours dox treatment), primed with random hexamers. n=3. Error bars denote S.E.M. 21 Supplemental Figure 21. CCDC26 Comparison of focal amplifications, CCDC26, and E1- E5 region. Nine focal amplifications were derived from two prior studies (Radtke et al. 2009; Kuhn et al. 2012). 22 Supplemental Experimental Procedures Cell culture The Tet-On competent murine AML line used here (RN2) was derived from co-transduction of hematopoietic stem and progenitor cells with MSCV-rtTA3-IRES-MLL-AF9 and MSCV- Luciferase-IRES-NrasG12D followed by transplantation of infected cells into sublethally- irradiated recipient mice.