Supplemental Information

Supplemental Figures

Supplemental Figure 1. Quantitation of bioluminescent imaging data in Figure 1B.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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/).

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.

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.

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.

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.

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

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.

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.

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.

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.

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. The RN2 line was derived from bone marrow and spleen of these terminally-diseased mice (Zuber et al. 2011). B-ALL is BCR-ABL1+/p19Arf-/- (Williams et al. 2006), melanoma cell line is from BrafV600E/wt, pten-/-, cdkn2a-/- animals (Dankort et al. 2009), breast cancer is the 4T1 line (Aslakson and Miller 1992), Tet-On iMEF are murine embryonic fibroblasts immortalized with polyoma middle T (Zuber et al. 2011). RN2 and B-ALL cells were cultured in RPMI1640 supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin. iMEF, 4T1, and melanoma cells were grown in DMEM with 10% FBS and 1% glutamine (GIBCO). NOMO-1, MV4-11, MOLM13, JURKAT, REH, NB-4, HEL, CMK, THP-1, NB-4, HL-60, and K562 human AML cell lines were cultured in RPMI1640/10% FBS. KASUMI-1 cell line was culture in RPMI1640/20%FBS. HEK293T, HCC1143 and Ecotropic Phoenix cells were cultured in DMEM/10% FBS. All retroviral packaging was performed using Ecotropic Phoenix cells according to established protocols.

Plasmids

For conditional RNAi experiments, shRNAs were expressed from the TRMPV-Neo vector (Zuber et al. 2011). For shRNA/GFP competition assays in murine cells, the LMN shRNA retroviral vector was used (MSCV-miR30-shRNA-PGKp-NeoR-IRES-GFP). For shRNA/GFP competition assays in human cells, the MLS vector was used (MSCV-miR30-shRNA-SV40p- GFP). For Myc cDNA experiments, the wild-type mouse Myc cDNA was subcloned into MSCV-PGK-Puro-IRES-GFP. Halo-BRD4L expression plasmid was purchased from Promega. FLAG-BRD4, FLAG-Pu.1 and FLAG-CEBPα were cloned into PCDNA3 or MSCV vectors using standard techniques. BRG1 cDNA (isoform F) was purchased from addgene (#19143) and was subcloned into MSCV-IRES-GFP vector using standard techniques. Point mutation constructs were made using overlap PCR.

Competition assay to measure cell proliferation

For evaluating the impact of shRNAs or cDNAs on cell proliferation, cultures were retrovirally transduced with individual LMN-shRNA or MSCV-cDNA-IRES-GFP constructs, followed by measurement of the GFP percentage at various days post-infection using a Guava Easycyte (Millipore). The rate at which the GFP% declines over time was used to infer the relative growth disadvantage conferred by a given shRNA or cDNA construct relative to the uninfected cells in the same culture. For human AML cell line experiments, we used the MLS vector, which allows higher retroviral transduction efficiency in these lines. Brg1 mutations used in Figure 7J were cloned using overlap PCR.

In vivo shRNA studies in AML

For in vivo RNAi experiments, RN2 leukemia cultures were retrovirally transduced with TRPMV-Neo constructs (pSIN-TRE-dsRed-miR30-shRNA-PGK-Venus-IRES-NeoR) followed by G418 selection (1 mg/ml for 6 days). Clonal lines were then derived by limiting 23 dilution. Transduced leukemia cells were transplanted by tail-vein injection of 1 x 106 cells into sublethally (5.5 Gy) irradiated B6/SJL(CD45.1) recipient mice. For whole body bioluminescent imaging mice were intraperitoneally injected with 50 mg/kg D-Luciferin (Goldbio), and after 10 min. analyzed using an IVIS Spectrum system (Caliper LifeSciences). Quantification was performed using Living Image software (Caliper LifeSciences) and standardized rectangular region of interests covering the mouse trunk and extremities. For shRNA induction, animals were treated with dox in both drinking water (2 mg/ml with 2% sucrose; Sigma-Aldrich) and food (625 mg/kg, Harlan laboratories). All mouse experiments included in this work were approved by The Cold Spring Harbor Animal Care and Use Committee.

BrdU incorporation assay

BrdU incorporation assays were performed according to the manufacturer’s protocol (BD, APC BrdU flow kit). Cells were pulsed with BrdU for 30min. Cells were co-stained with DAPI for DNA content measurement. For all conditional shRNA experiments, the analysis was gated on Venus+/dsRed+ double positive cell populations, which indicates shRNA expression. All analyses were performed using FlowJo software (Tree Star).

Protein lysate preparation and Western blotting For all immunoblots, whole cell lysates were prepared by resuspending cell pellets in SDS- PAGE sample buffer and about 50,000 cell-equivalents were loaded into each lane. Protein extracts were resolved by SDS polyacrylamide gel electrophoresis and transferred to nitrocellulose for immunoblotting.

May-Grunwald-Giemsa Cytospin staining RN2 cells transduced with TRMPV-Neo constructs cells were treated with 0.1 ug/ml dox for 3 days to induce shRNA expression. 50,000 cells were resuspended in 100 µl FACS buffer (5% FBS, 0.05%NaN3 in PBS) and cytospun onto glass slides using Shandon Cytospin 2 Centrifuge at 500 rpm for 5 min. May-Grunwald (Sigma #019K4368) and Giemsa (Sigma #010M4338) stainings were performed according to manufacturer’s protocols. Images were collected using a Zeiss Observer Microscope with a 40x objective.

ckit/mac1 staining and flow cytometry Cells were incubated with ckit(1:100) or Mac-1(1:100) in FACS buffer (5% FBS, 0.05% NaN3 in PBS) for 1 hour. Stained cells were analyzed on an LSRII flow cytometer. Data analysis and graphs were performed using Flowjo software.

Expression microarrays

Microarrays were performed using the CSHL microarray shared resource. RNA was isolated using RNeasy Mini Kit (QIAGEN, #74104) and quality was assessed on an Agilent 2100 Bioanalyzer, RNA 6000 Pico Series II Chips (Agilent, Palo Alto, CA, USA). RN2 carrying three different Brg1 shRNAs (4935, 5466, and 3364) were treated with dox for four days. RNA was amplified by a modified Eberwine Technique, aRNA was then cDNA converted, using an WT Expression kit (Ambion, Austin, TX). The cDNA was then fragmented and terminally labeled with biotin, using the Affymetrix GeneChip WT Terminal Labeling kit 24 (Affymetrix, Santa Clara, CA). Samples were then prepared for hybridization, hybridized, washed, and scanned according to the manufacturer's instructions on Mouse Gene ST 1.0 GeneChips (Affymetrix, Santa Clara, CA). Affymetrix Expression Console QC metrics were used to pass the image data. Raw data was processed by Affymetrix and Limma package in R based Bioconductor. Heatmap was generated by using the Java Tree View software.

Gene set enrichment analysis was performed using GSEA v2.07 software with 1000 phenotype permutations (Subramanian et al. 2005). The leukemia stem cell (Somervaille et al. 2009), Myc gene sets (Schuhmacher et al. 2001), Myc network gene sets (Schuhmacher et al. 2001; Kim et al. 2010a) and Hoxa9_Meis1 gene sets (Hess et al. 2006) were obtained from prior studies. The macrophage development gene set was obtained from the Ingenuity Pathway Analysis (IPA) software (Ingenuity, Redwood City, CA). The apoptosis_GO gene sets was download from MSigDB. All the gene sets used here are provided in a supplemental spreadsheet.

ChIP-seq

Protocols for performing ChIP have been previously described (Boyer et al. 2006). In brief, cells were crosslinked using 1% formaldehyde for 20 min at room temperature. Cells were resuspended, lysed in lysis buffers and sonicated with a Misonix Sonicator 3000 to solubilize and shear crosslinked DNA. The resulting cell extract was incubated overnight at 4°C with 100ul of Dynal Protein G magnetic beads that had been pre-incubated with approximately 10 µg of the appropriate antibody. Beads were washed followed by elution from the beads (50 mM Tris-Hcl, pH 8.0, 10 mM EDTA and 1% SDS) and by heating at 65°C for 1 hr with vortexing. Crosslinking was reversed by overnight incubation at 65°C. Following purification of IP DNA, Illumina libraries were prepared essentially as described (http://www.illumina.com/pages.ilmn?ID=203). All ChIP-seq data sets were aligned using Bowtie (version 0.12.2) to build version mm8 of the murine genome. We used the MACS version 1.4.1 (Model based analysis of ChIP-seq) (Zhang et al. 2008) peak finding algorithm to identify regions of ChIP-seq enrichment over background. The GEO accession number and background used for each dataset will be provided pending approval. Super-enhancers were defined as in (Loven et al. 2013). Briefly, enhancers were first defined as regions of promoter distal Brd4 enrichment. To identify regions of clustered enhancers, groups of nearby Brd4 defined enhancers (within 12.5kb of one another) were stitched together. For each enhancer, the overall Brd4 signal in units of reads per million mapped reads was determined. Enhancers were then plotted ranked by increasing amounts of overall Brd4 signal. Finally a graphical algorithm was used to identify a signal cutoff for super-enhancers (the vertex in the plotted curve).

Copy number analysis

‘Start’ and ‘End’ coordinates for somatic 8q24.21 copy number alterations were obtained from published studies (Table S2a of Radtke et al. 2009; Table S2 of Kuhn et al. 2012). Coordinates from (Radtke et al. 2009) were converted from hg17 assembly to hg19 for browser visualization. Coordinates from (Kuhn et al. 2012) were converted from hg18 assembly to hg19 for visualization as well. Amplified regions are represented as solid horizontal red lines in human hg19 reference genome. Four instances of 8q24.21 25 amplifications were identified in a total of 111 samples from (Radtke et al. 2009) and five instances of 8q24.21 amplification were identified in 164 unique samples from (Kuhn et al. 2012). Note: eleven patient samples were analyzed in both studies, which we only counted once in our analysis. Thus, we estimate a frequency of 3% (9 out of 275 samples) across these two studies. CCDC26 transcript (ENST00000446592) was mapped to human hg19 based on the coordinates chr8:130363940-130692485.

ChIP-qPCR ChIP-qPCR assays were performed as described (Steger et al. 2008). All results were quantified by qPCR performed using SYBR green (ABI) on an ABI 7900HT. Each IP signal was referenced to an input standard curve dilution series (IP/Input) to normalize for differences in starting cell number and for primer amplification efficiency.

Enhancer reporter assay

The enhancer regions were cloned into the 5’ end of pGL4.23 luc2 vector with minimum TK promoter (Promega). The luciferase enhancer constructs were co-transfected with Renilla luciferase control vector in 10:1 ratio into K562 or HEK293T cells using Lipofectamine LTX reagent (Invitrogen) according to the manufacturer's instructions. Luciferase assay was performed using Dual Luciferase Luciferase Reporter Assay System (Promega) 48 hours after transfection following the manufacturer's instructions. All the data shown were normalized to the internal Renilla luciferase activity.

4C-seq assay

107 RN2 cells were harvested and crosslinked with 1% formaldehyde at room temperature for 20 minutes, followed by nuclear isolation. Nuclei were digested for 4 hrs with 200U HindIII (New England Biolabs) at 37°C, followed by overnight incubation with 400U HindIII, and a final 4hr incubation with 200U HindIII. HindIII was heat inactivated for 20 min at 65°C, and samples were ligated overnight in 7ml using 50U T4 DNA ligase (Roche) at 16°C. Ligation circles were phenol-chloroform extracted and ethanol precipitated with glycogen as a carrier (Roche, 20mg/ml). These were further digested with 50U of DpnII (New England Biolabs) overnight at 37°C, followed by heat inactivation for 25 min at 65°C, and ligation in 14ml with 100U T4 DNA ligase at 16°C. These trimmed circles (4C template) were phenol- chloroform extracted and ethanol precipitated with glycogen as a carrier.

Inverse PCR was performed using primers designed to target two primary restriction fragments (viewpoints) to amplify ligated interacting sequences. Viewpoints MycTSS-4C (chr15:61812989-61813775) falls within the ~2kb upstream region of the Myc transcription start site, and viewpoint Enh3_4C (chr15:63481224-63482043) is ~800bp downstream of the Enh3 region. Sixteen identical 50ul PCR reactions were performed per viewpoint using the Expand Long Template PCR system with 200 ng 4C template per reaction. The PCR program is as follows: 94°C for 2min, 30 cycles of 94°C for 15 s, 55°C for 1 min, and 68°C for 3 min, followed by a final step of 68°C for 7 min. The 16 reactions were pooled and purified for next-generation sequencing using the Roche High Pure PCR Products Purification Kit. 4C primers carried 5' overhangs composed of adaptor sequences for Illumina paired-end sequencing, and were sequenced on a MiSeq machine (Illumina).

26 Mapping of 4C data was performed using 4Cseqpipe, as described in (van de Werken et al. 2012). 4Cseqpipe analyzes 4C-seq experiments by including packages that allow sequence extraction, mapping, normalization, and plotting of cis-contact profiles around viewpoints. Custom restriction site tracks were built using the -build_re_db option of 4Cseqpipe for the mm8 mouse genomic version (UCSC) and HindIII and DpnII as first and second cutters, respectively. 4C primers were removed from reads and the first 30bp were mapped to the custom mm8 tracks with the in-built 4Cseqpipe mapper. A total of 1,544,253 reads were mapped for the MycTSS_4C viewpoint, and 1,648,978 for Enh3_4C. 85% and 90% of these reads are in cis-, respectively. Near-cis domainograms were generated for the whole chr15 for both viewpoints using the median stat_type and plotting the 80th quantile of the distribution of normalized contact intensities for 10Kb windows in MycTSS_4C and in Enh3_4C. These values were put into wig files for visualization using the UCSC genome browser mm8 version, where data range of the vertical axis was set to the ~7% and 8% of the maximal normalization value for MycTSS_4C and Enh3_4C, respectively, to deal with the fact that the majority of data are very close to the viewpoint.

For calculating the mean value of the 4C-seq data, reads were mapped against the mm8 mouse genome and binned into 1kb windows for the whole of chromosome 15 with less than two mismatches. And then normalized using TSS viewpoint read number as reference. Sliding windows of 10kb were made for the whole chromosome 15 sequence. For each window, the highest value in the window was substituted for the second highest value to avoid outlier bias for mean calculation. 2,958,300 reads were obtained for Myc TSS shBrg1 and 4,860,980 for Myc TSS shRen viewpoints.

Chromosome conformation capture (3C)

The 3C assay was performed essentially as described (Jing et al. 2008) with the following modifications: 107 cells were crosslinked with formaldehyde (1%) for 20 min at room temperature, followed by glycine quenching, cell lysis, Bgl II digestion, and T4 ligation. 3C ligation products were quantified in triplicates by quantitative TaqMan real-time PCR. Probes and primers were designed using Primer3 software and tested by serial dilution to ensure specific and linear amplification. Taqman probe and primer set sequences are in the supplemental spreadsheet file. Digestion efficiencies were monitored by SybrGreen qPCR with primer pairs that amplify genomic regions containing or devoid of BglII digestion sites. Two different bacterial artificial chromosome (BAC) containing the human E1-E5 region (RP11-770K21) and the MYC gene (CTD-2034C18) were digested with BglII and religated to generate random ligation products of BglII fragments. The DNA was serially diluted and used to generate a standard curve to which all 3C products were normalized.

RNA-seq

Total RNA was prepared using TRIzol reagent (Invitrogen) according to the manufacturer’s protocol. 1 ug of total RNA was subjected to “not-so-random” (NSR) primer-based RNA-seq library preparation according to protocols described previously (Armour et al. 2009). For the polyA+ tail RNA-seq, the library was made following the instructions of illumina Truseq RNA sample prep kit v2. The prepared DNA was sequenced using an Illumina HiSEq 2000. The obtained reads were trimmed into 28 base reads corresponding to 9th to 36th position from the 5′ ends of the reads. The trimmed reads were mapped to the mouse genome (mm9) using Bowtie with no mismatch. Gene expression was analyzed by using Cufflinks software (http://cufflinks.cbcb.umd.edu/). 27

RNA-SEQ timecourse following JQ1 treatment

The RNA-seq evaluation was performed using a custom method. Briefly, amplified cDNA (>2ng) was used to construct Illumina sequencing libraries following the Illumina’s Ultra Low Input mRNA-Seq Guide. The amplified cDNA was fragmented into 200bp using Covaris acoustic shearing instrument. The resulting fragments were end-repaired, single A base added, followed by ligation with PE adaptor and amplification by 15 cycles of PCR. The resulting libraries are expected to show a peak at around 300bp on Bioanalyzer (Agilent). In order to enrich the reads containing variental tag and sample barcode, we using Dynabeads® mRNA Purification Kit for mRNA Purification (Invitrogen Cat. No. 610-06). The libraries were heat at 95C for 2min before mixing with polyT magnetic beads. Then, we incubated the beads at 47C for overnight and run 6-8 cycles of PCR from the elution the next day. As a result, approximately 50% of the sequencing raw reads will contain variental tags and sample barcodes.

Illumina sequences were split into separate files based on sample barcodes and mapped to the mouse mm9 reference genome using tophat2 software. Read counts per gene are computed by counting reads that overlap the mm9 refseq gene annotations downloaded from the UCSC Genome Browser ftp site. For each series of time points for each gene a best continuous piecewise linear fit is computed. The intent is to model the response of each gene by a latency period of no response followed by a response reaching a new stable level after a period of time. The form of this curve is three linear segments with the first and last segments slope zero and the middle segment connecting the two end segments. The curves are defined by the pair of endpoints of the middle segment. All pairs of points are tested to find the pair of points minimizing the sum of the squared difference to the data. Rapidly downregulated genes were identified based on two criteria: a three-fold decrease in mRNA level at 6 hours together with a good fit to two-step linear model of consistent decreases over consecutive timepoints.

Immunoprecipitations

Cell pellets were washed with PBS followed by resuspension in Buffer A2 (10 mM Hepes- KOH pH 7.9, 1.5 mM MgCl2, 10 mM KCl) and incubated on ice for 30 minutes. Cells were then vortexed vigorously, followed by a spin at 4100 RPM for 5 minutes. Nuclei were then resuspended in Buffer C2 (20 mM Hepes-KOH pH 7.9, 25% glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA) and incubated on ice for 30 minutes. All buffers contained protease inhibitors (Roche) and 1 mM DTT. Material was then spun at max speed in a table-top centrifuge for 10 min at 4 degrees C. The resulting supernatant was then diluted in C2 buffer lacking NaCl to reduce the salt concentration to 150 mM. All IPs were performed with 1 mg of nuclear extract and 0.5 to 2 ug of antibody incubated overnight. For endogenous IPs, 25 ul of Protein A or G Dynabeads were added for 2 hours, followed by three 1 ml washes (300 mM NaCl, Tris-HCl pH 7.4, 0.1% NP40). Material was eluted from beads by addition of SDS page loading buffer and boiling for 5 min.

For IP experiments in HEK293T, cells were harvested 48 h after transfection and nuclear extracts prepared as described above. Immunoprecipitation was performed with 25 ul of anti- FLAG beads (#A2220; Sigma-Aldrich). Wash conditions are as described above. Elution was carried out with 3X FLAG peptide (#F4799; Sigma-Aldrich). 28

Antibodies anti ß-actin HRP (Sigma, #A3854), anti-H3K4me3 (Millipore, 07-473), H3K4me1 (ab8895; abcam), H3K27ac (ab4729; abcam), FLAG (F1804; Sigma-Aldrich), SPT16 (Santa Cruz; sc- 165989), BRG1 (sc-10768; Santa Cruz and 2822-1, 5716-1,2776-1; Epitomics), BAF155 (A301-021A; Bethyl), Brd4 (A301-985A; Bethyl and 5716-1;Epitomics), control IgG (I8140; Sigma), Myc (1472-1; Epitomics), Baf60a (A301-595A(for WB) A301-594A(for ChIP); Bethyl), Baf250a (04-080; Millipore); Cebpα(Santa Cruz; SC-61x) Cebpß (Santa Cruz; sc- 150x); Pu.1(Santa Cruz; sc-352x); Erg (Santa Cruz; sc-354x); Lmo2 (abcam, ab81988). Some of the Brg1 western blots were performed with a polyclonal antibody made in house raised against the peptide: SRSVKVKIKLGRKEKA. For flow cytometry, APC-labeled ckit (#105811) and mac-1 (#101211) were purchased from biolegend.

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