1. Volume 58, Issue 6, Pages 1028-1039 (June 2015)
BET Bromodomain Inhibition Suppresses the Function of Hematopoietic Transcription Factors in Acute Myeloid Leukemia 
Jae-Seok Roe, Fatih Mercan, Keith Rivera, Darryl J. Pappin, Christopher R. Vakoc 
Molecular Cell 
Volume 58, Issue 6, Pages (June 2015)
DOI: /j.molcel
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2. Molecular Cell 2015 58, 1028-1039DOI: (10.1016/j.molcel.2015.04.011)
Copyright © 2015 Elsevier Inc. Terms and Conditions

3. Figure 1
BRD4 Occupancy in AML Correlates with Hematopoietic TFs Flanked by Histone Acetylation
(A) ChIP-seq meta-profiles for BRD4, H3K27ac, H4K8ac, and H3 representing the average read counts per 20-bp bin for 5,135 BRD4-occupied regions. Data were normalized to max signal.
(B) Density plot of different ChIP-seq datasets centered on 1,950 BRD4-occupied promoters and 3,185 BRD4-occupied enhancers. Each row represents a single peak.
(C) MEME suite motif analysis performed on BRD4-occupied sites. A 400-bp region centered on BRD4 peaks was used for motif discovery. The distribution of each motif relative to the BRD4 peak summit is indicated.
(D) ChIP-seq occupancy profiles for BRD4, p300, H3K27ac, H4K8ac, and hematopoietic TFs at the Myc locus.
(E) ChIP-seq occupancy profiles at Myc enhancer E5. See also Figure S1.
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4. Figure 2
Hematopoietic TFs Can Facilitate BRD4 Recruitment to Their Occupied Sites
(A) Western blotting and light microscopy of TF-transduced NIH 3T3 fibroblasts 72 hr following retroviral transduction. Cells were transduced at ∼100% efficiency for each of the individual TF-expressing retroviral vectors.
(B) qRT-PCR analysis of RNA prepared from TF-transduced fibroblasts. A 6-hr exposure to DMSO or 500 nM of JQ1 was performed following 72 hr of TF retroviral transduction. Each mRNA level was normalized to Gapdh. Data are represented as mean ± SEM, and n = 3.
(C–I) ChIP-qPCR with indicated antibodies after 72 hr of TF transduction in fibroblasts. Neg refers to a negative control region in a gene desert region. ChIP-qPCR primers for Btk, Ccl4, Fcgr2b, and Pecam1 were designed based on BRD4 ChIP-seq profile from RN2 cells. Data are represented as mean ± SEM, and n = 3. See also Figure S2.
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5. Figure 3
The KAT p300 Is Recruited by Hematopoietic TFs to Support BRD4 Occupancy in Leukemia
(A) Summary of competition-based negative selection shRNA experiments targeting the indicated KAT enzymes performed in RN2 cells. The average fold decrease in GFP percentage over 10 days for three to six independent shRNAs is plotted, which represents the relative degree of growth inhibition conferred by the indicated shRNA. Red bars indicate KATs having a greater than 2-fold average decrease in the percentage of GFP-positive cells. Data are represented as mean ± SEM.
(B) Heatmap of unsupervised hierarchical clustering of RNA-seq data performed using the GENE-E software. A total of two independent shRNAs against each candidate KAT, BRD4, or Renilla were induced with doxycycline for 48 hr using the TRMPV-Neo vector in RN2 cells. Each row represents the normalized expression value of an individual gene.
(C) p300 ChIP-qPCR following 72 hr of TF transduction in fibroblasts as described in Figure 2.
(D and E) BRD4 ChIP-seq meta-profiles for 1,950 BRD4-occupied promoters and 3,185 BRD4-occupied enhancers following 10 μM C646 treatment for 2 hr.
(F–H) ChIP-seq occupancy profiles of BRD4 at Myc, Cdk6, and Pecam1 loci, in the presence or absence of C646.
(I) qRT-PCR following 6 hr of JQ1 or C646 exposure in RN2 cells.
(J) Western blotting of RN2 lysates following JQ1 or C646 exposure.
(K) qRT-PCR following 6 hr of JQ1 or C646 exposure in MM1.S cells.
(L) Gene set enrichment analysis (GSEA) evaluating a JQ1-sensitive gene signature in the RNA-seq analysis of 6-hr C646 exposure in RN2. NES, normalized enrichment score; FDR, false-discovery rate. For (C), (I), and (K), data are represented as mean ± SEM, and n = 3. See also Figure S3.
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6. Figure 4
p300/CBP Maintains Local Histone Acetylation near BRD4-Occupied Sites
(A–D) H3K27ac and H4K8ac ChIP-seq meta-profiles of BRD4-occupied promoters or enhancers following 2 hr of C646 exposure.
(E–G) ChIP-seq occupancy profile of H3K27ac and H4K8ac at Myc, Cdk6, and Pecam1 loci following 2 hr of C646 exposure.
(H and I) Comparison of fold change for H3K27ac, H4K8ac, and BRD4 tag counts at BRD4-enriched regions following C646 treatment. A R2 value was calculated using linear regression analysis.
(J and K) ChIP-qPCR analysis of H3K27ac and H4K8ac following 72 hr of TF transduction in fibroblasts as described in Figure 2. See also Figure S4.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 5 BRD4 Interactions with Hematopoietic TFs
(A) FLAG-TF immunoprecipitation performed in nuclear extracts prepared from HEK293T cells, transfected with the indicated pcDNA3 expression plasmids.
(B and C) Immunoprecipitation experiments, as in (A), with 10 μM JQ1 or vehicle included in the lysate prior to IP.
(D) Schematic depiction of the protein domains of ERG and fragments utilized for KAT assays.
(E) In vitro KAT assay with purified p300 KAT domain (amino acids 1,135–1,810) and indicated ERG protein fragments. Purified p53 C terminus (309–393) was used as a positive control. Acetylated products were detected by pan-acetyl lysine antibody in western blots.
(F) Mapping of ERG (1–208) acetylation sites by mass spectrometry using reaction conditions as shown in (E). Relative abundance of acetylation was calculated by acetylation signal intensity of Acetyl-CoA (+) / Acetyl-CoA (−) from the same peptide.
(G) Sequence alignment of histone H4, ERG, TWIST, and GATA-1.
(H) Peptide-pull-down assay was carried out by mixing FLAG-BRD4 (1–722) purified from E. coli with indicated biotinylated peptides using streptavidin beads. The bound FLAG-BRD4 was analyzed by anti-FLAG western blotting.
(I) FLAG-ERG (wild-type or K96R/K99R mutant) IP-western blotting as shown in (A).
(J) qRT-PCR analysis in NIH 3T3 fibroblasts following transduction with the indicated retroviral constructs. Data are represented as mean ± SEM, and n = 3. See also Figure S5.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 6
Hematopoietic TFs Are Essential in AML and Are Functionally Suppressed by BET Inhibitors
(A) Western blotting in whole-cell lysates prepared from RN2 cells following TRMPV-shRNA induction with dox for 48 hr.
(B) Competition-based negative selection experiments that track the relative abundance of shRNA+/GFP+ cells over time. Percentages were normalized to day 2 values. Data are represented as mean ± SEM, and n = 3.
(C) TF gene signatures, defined by RNA-seq analysis following shRNA-based knockdown for 48 hr with two independent shRNAs. The 100 top downregulated genes were identified by comparison to shRen.713 control. Each row represents row-normalized unit based on linkage analysis-mediated hierarchical clustering using Cluster 3.0 software.
(D) GSEA of each TF signature following JQ1 treatment for 6 hr. RNA-seq was performed following 500 nM of JQ1 exposure for 6 hr in RN2 cells.
(E and F) Western blotting in RN2 lysates following indicated intervals of 500 nM JQ1 exposure.
(G) qRT-PCR analysis of primary transcripts of three representative TF signature genes following JQ1 exposure. See also Figure S6.
Molecular Cell  , DOI: ( /j.molcel )
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