SUPPORTING INFORMATION
SI Materials and Methods
Generation of BDI/II ESCs. For stable expression of the Brd4 dominantnegative inhibitor BDI/II in ESCs, the BDI/II coding DNA was PCR-amplified from
pCMV2-FLAG-mBrd4 plasmid (kindly provided by Dr. Keiko Ozato, NICHD) using
primers that introduce the coding sequence for a nuclear localization signal (NLS). The
resulting NLS-BDI/II fragment was subcloned using Xho I and Mlu I sites into pTRIPZXpress vector, which was modified from pTRIPZ vector (Open biosystems) by adding
an Xpress-coding sequence between AgeI and Mlu I sites. To package lentiviruses,
HEK 293T cells were cultured in 10 cm dishes to 90% confluency. pTRIPZ-XpressNLS-BDI/II was transfected into HEK 293T cells together with psPAX2 and pMD.2G
using Lipofectamine 2000 (Invitrogen). At 6 h post-transfection, the culture medium was
changed to fresh medium. Twenty-four hours later, lentiviruses were harvested from
the supernatant and filtered through a 0.45 m filter. ESCs were transduced with the
purified lentiviruses supplemented with polybrene. Starting on day 2 after transduction,
cells were selected using 1 g/ml puromycin for 4 days.
Immunoprecipitation and Western Blot Analysis. To prepare whole cell lysates, cells
were lysed in lysis buffer (10 mM HEPES, pH 7.9, 500 mM NaCl, 3 mM MgCl2, 1 mM
DTT, 1 mM PMSF, 0.5% Triton X-100 supplemented with protease inhibitors). After 60
min incubation on ice, whole cell lysates were centrifuged at 15,000 g for 10 min at 4°C
to remove the debris. Protein concentration of whole cell lysates was determined using
the Bradford assay.
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For preparing nuclear extracts, cells were first gently resuspended in buffer A (10
mM HEPES, pH 7.9, 10 mM KCl, 0.1mM EDTA, 0.1mM EGTA, 1 mM DTT, 0.2 mM
PMSF, 0.6% NP-40 supplemented with protease inhibitors). After 10 min incubation on
ice, nuclear pellets were obtained following centrifugation at 1,000 g for 5 min in 4°C.
The pellets were resuspended in buffer B (20 mM HEPES, pH 7.9, 400 mM NaCl, 1 mM
EDTA, 1 mM EGTA, 1 mM DTT, 0.2 mM PMSF supplemented with protease inhibitors).
After rotating in cold room for 1 h, the nuclear extracts were centrifuged at 15,000 g for
10 min in 4°C. Protein concentration of nuclear extracts was determined using the
Bradford assay. For BRD4 IP, the lysates were immunoprecipitated using protein A
beads (blocked with 1% BSA in PBS at 4 °C for 1 h) mixed with 2 g of either normal
rabbit IgG or the polyclonal BRD4C antibody at 4°C for 2h. The beads were washed
and eluted as described previously 1.
For Western blot analysis, the protein samples were resolved on SDS-PAGE
gels, transferred onto PVDF membranes, and immunoblotted with specific primary
antibodies as indicated in the figure legends. The primary antibodies used in this study
include anti-BRD4C (recognizes BRD4 aa 1313–1362, 1:40,000), anti-SOX2 (1:1,000,
MAB2018, R&D), anti-POU5F1 (1:1,000, SC-5279, Santa Cruz), anti-NANOG (1:1,000,
ab80892, Abcam), anti-BRG1 (1:500, sc-17796, Santa Cruz), anti-NANOG (1:2000,
4903, Cell Signaling Technology), and anti-ACTIN (1:150,000, MAB1501, Millipore).
The secondary antibodies used were HRP-linked anti-rabbit IgG (1:4,000, 7074S, Cell
Signaling) and HRP-linked anti-mouse IgG (1:4,000, 7076S, Cell Signaling). Western
blots were developed using Western Lightning ECL solution (PerkinElmer) and images
were captured using a Fuji imaging system.
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Protein Expression and Purification. Recombinant GST-Brd4CTD carrying Brd4 aa
1313-1362 was expressed in E. coli BL21(DE3)plyss. GST-Brd4CTD expression was
induced using 0.4 mM isopropyl--D-1-thiogalactopyranoside (IPTG) in E. Coli overnight
at 16C. Cells were then lysed by sonication in buffer containing 50 mM Tris-Cl pH 7.6,
0.1 mM EDTA, 2 mM dithiothreitol (DTT), 0.2 mM PMSF, 0.4mg/ml lysozyme, and
protease inhibitors. GST-Brd4CTD was purified using glutathione agarose (Sigma)
according to the manufacturer's protocol and concentrated using a Microcon column
(Millipore). Expressed proteins were analyzed using sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie brilliant blue staining 2.
Gene expression microarrays. TL1 ESCs were transfected with control siRNA, or
Brd4 siRNA 1. At 48 h post-transfection, total RNAs were isolated using the RNeasy
minikit (Qiagen). Expression microarray analyses were performed on mRNA samples
isolated from three independent experiments using a mouse Gene 2.0ST array
(Affymetrix) at the University of Pennsylvania Microarray Core Facility. RMA from
Affymetrix package3 was applied to the data for preprocessing and normalization.
Limma 4 was used for detection of differentially expressed genes. Benjamini &
Hochberg correction was applied for the multiple comparisons. The differentially
expressed genes in Brd4 knockdown data (p-value≤0.05) were extracted and compared
with the Nanog knockdown data (GEO accession number: GSE26520)5. To simplify the
analysis and to avoid the interference of different platforms, GeneOverlap package of
Bioconductor (http://www.bioconductor.org/packages/release/bioc/html
/GeneOverlap.html) was applied to compare the significance of the overlap of the
differentially expressed genes between the Brd4 knockdown and Nanog knockdown
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transcriptomes. GeneOverlap was also used to perform the Fisher’s exact test and to
calculate the odds ratio of the overlap of the gene lists.
Mass spectrometry analysis of BRD4-binding proteins. The human Brd4 gene was
subcloned in-frame with the coding sequence for the highly specific Xpress tag into the
pcDNA4C vector 6. HEK 293T cells were transfected with the pcDNA4C-Xpress-Brd4
construct or an empty vector using FuGENE 6 Transfection Reagent (Promega).
Nuclear extracts were prepared as described previously 6. BRD4 and associated
proteins were affinity purified from nuclear lysates using Xpress antibody conjugated to
Protein A Sepharose (GE Healthcare Life Sciences). Purified proteins were separated
by SDS-PAGE and stained with Coommassie brilliant blue. The protein bands present
in the BRD4 sample but not in the vector control sample were excised and analyzed by
mass spectrometry at the BIDMC Mass Spectrometry Facility, Beth Israel Deaconess
Medical Center. Protein samples were digested with trypsin. Digested peptides were
analyzed by linear ion trap microcapillary LC/MS/MS with a LTQ Mass Spectrometer
(Thermo Scientific).
Supporting Figures
Figure S1. JQ1(+) impairs ESC maintenance. (A) R1 ESCs were either untreated
(UN), or treated with 300 nM of JQ1(-) or JQ1(+) for 48 h. Whole cell lysates were
immunoblotted with the indicated antibodies. ACTIN was used as a loading control.
Protein signal intensity was quantified using ImageJ. This experiment was performed at
least three times with similar results. (B) H9 human ESCs were either untreated (UN),
or treated with 300 nM of JQ1(-) or JQ1(+) for 48 h. Whole cell lysates were
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immunoblotted with the indicated antibodies. This experiment was performed three
times with similar results. (C) R1 ESCs were either untreated, or treated with 300 nM of
JQ1(-) or JQ1(+) for 24 h. The mRNA levels of Brd4, Pou5f1, Sox2, and Nanog were
measured by qRT-PCR and normalized to Gapdh mRNA levels. (D) R1 ESCs were
either untreated, or treated with 300 nM of JQ1(-) or JQ1(+) for 48 h. The mRNA levels
of the indicated cell lineage markers were measured by qRT-PCR and normalized to
Gapdh mRNA levels. For both (C) and (D), the mRNA levels in untreated cells are set
as 1. Error bars represent s.e.m. of three independent experiments. * P<0.05, **
P<0.01.
Figure S2. BRD4 knockdown decreases Nanog expression in ESCs. (A) R1 ESCs
were transfected with control siRNA, Brd4 siRNA 1, or Brd4 siRNA 2. At 48 h posttransfection, the mRNA levels of Brd4, Pou5f1, Sox2, and Nanog were measured by
qRT-PCR and normalized to Gapdh mRNA levels. The mRNA levels in control cells
were set as 1. Error bars represent s.e.m. of three independent experiments. (B) R1
ESCs were either untreated or transfected as in (A). At 48 h post-transfection, whole
cell lysates were extracted and immunoblotted with the indicated antibodies. Protein
signal intensity was quantified using ImageJ. This experiment was performed at least
three times with similar results. (C) TL1 ESCs were transfected as in (A). At 48 h posttransfection, the cells were co-stained with BRD4 and NANOG antibodies. Nuclei were
counterstained with DAPI. Bar, 10 m. (D) TL1 and R1 ESCs were transfected with
control siRNA, Brd4 siRNA 1, or Brd4 siRNA 2 in the 2i/LIF medium. At 48 h posttransfection, whole cell lysates were extracted and immunoblotted with the indicated
antibodies. Protein signal intensity was quantified using ImageJ. This experiment was
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performed three times with similar results. (E) R1 ESCs were transfected as in (A).
The number of colonies generated from 500 seeded cells was quantified at day 6 posttransfection. (F) R1 ESCs were transfected as in (A). At 72 h post-transfection, the
mRNA levels of the indicated cell lineage markers were measured by RT-qPCR and
normalized to Gapdh mRNA levels. The mRNA levels in control cells were set as 1.
Error bars represent s.e.m. of three independent experiments. *P<0.05, ** P<0.01.
Figure S3. Partial BRD4 knockdown only moderately inhibits ESC proliferation.
TL1 (A) and R1 (B) ESCs were transfected with control siRNA, Brd4 siRNA 1, or Brd4
siRNA 2, and 150,000 transfected cells were seeded on day 0. Cell numbers were
counted on day 1-3 after seeding. Values represent the average of three independent
experiments with error bars indicating s.e.m.
Figure S4. Gene ontology analysis of BRD4 regulated genes in mouse ESCs. The
gene sets differentially expressed in control and Brd4 siRNA 1-treated samples (with pvalue <0.05) were identified in the microarray analysis and analyzed using Panther
Biological Process Analysis in DAVID (Database for Annotation, Visualization and
Integrated Discovery) v6.7.
Figure S5. The pair-wise comparison of the overlap of the differentially expressed
genes in Brd4 knockdown (KD) and Nanog knockdown transcriptomes. The darkness
of the color represents the odds ratio, and the statistical significances were labeled in
the comparisons. N.S.: Not significant.
Figure S6. Brd4 knockdown does not dramatically affect Sox2 expression in ESCs.
TL1 (A) and R1 (B) ESCs were transfected with control siRNA, Brd4 siRNA 1, or Brd4
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siRNA 2. At 48 h post-transfection, the cells were co-stained with BRD4 and SOX2
antibodies. Nuclei were counterstained with DAPI. Bar, 10 m.
Figure S7. Brd4 expression and knockdown during early embryo development.
(A) Representative images of the temporal and spatial pattern of BRD4 expression
during preimplantation development. Full-grown mouse oocytes, metaphase II eggs, 1cell, 2-cell, 4-cell, 8-cell embryos, and blastocysts were stained with a Brd4 antibody.
DNA was labeled with Sytox Green. All confocal images were taken with the same
laser power. The experiment was performed twice and at least 20 oocytes/embryos
were analyzed for each stage. (B) Brd4 knockdown does not significantly affect
POU5F1 levels in preimplantation embryos. Two-cell embryos were microinjected with
control siRNA or Brd4 siRNA 1 and cultured to the late morula stage.
Immunofluorescence was performed for POU5F1 (red). (C) Two-cell embryos were
microinjected with control siRNA or Brd4 siRNA 1 and cultured to the blastocyst stage.
Pictures of live blastocysts were taken on the stage of a Nikon Eclipse TE2000
microscope equipped with Hoffman optics. Images were captured with a MicroMAX
camera (Roper Scientific) driven by MetaMorph image analysis software (Molecular
Devices). Magnification: 100x. The experiment was performed three times using at
least 20 embryos per group. Representative images are shown.
Figure S8. Short-term JQ1(+) treatment has little effect on ESC proliferation. TL1
(A) and R1 (B) ESCs were treated with either DMSO or JQ1(+) at the indicated
concentrations. Cell numbers were counted at 6, 24, and 48 h post-treatment. The cell
numbers of JQ1(+)-treated cells were normalized to that of DMSO treated cells. The
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cell numbers of DMSO-treated cells were set as 100. Values represent the average of
three independent experiments with error bars indicating s.e.m.
Figure S9. BRD4 occupies the regulatory regions of the Nanog gene in ESCs and
the occupancy is inhibited by JQ1(+). TL1 cells were treated with 500 nM JQ1(-) or
JQ1(+) for 6 h and subjected to the ChIP assay with normal rabbit IgG (IgG, as a
negative control) or BRD4C antibody recognizing BRD4 aa 1313–1362. ChIP samples
were analyzed by qPCR using primers targeting the regions indicated in Fig 5A. Values
represent the average of three independent experiments with error bars indicating
s.e.m.
Figure S10. Identification of the BRD4 and BRG1 interaction. (A) Purification of the
BRD4 functional complex using a proteomic approach. HEK 293T cells were transfected
with the pcDNA4C-Xpress-Brd4 construct (BRD4) or an empty vector (V). BRD4 and
associated proteins were affinity purified from nuclear lysates using Xpress antibodyconjugated sepharose. Proteins samples were resolved on SDS-PAGE and stained
with Coommassie brilliant blue. The protein bands present in the BRD4 sample but not
in the vector control sample were excised and identified by mass spectrometry. BRG1
peptides were identified in P1 band. (B) Brd4 interacts with Brg1 in P19 cells. P19 cells
nuclear extracts immunoprecipitated with normal rabbit IgG (IgG) or the polyclonal
BRD4C antibody (BRD4) were immunoblotted with the indicated antibodies. IN: input.
Figure S11. BRD4 regulates Nanog expression in P19 and NT2 embryonic cancer
stem cells. (A) P19 cells 7 were treated with JQ1(-) or JQ1(+) at the indicated
concentrations. Whole cell lysates were extracted at 24 h and immunoblotted with the
indicated antibodies. ACTIN was used as a loading control. (B) P19 cells were either
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untreated or transfected with control siRNA, Brd4 siRNA 1, or Brd4 siRNA 2. At 48 h
post-transfection, whole cell lysates were extracted and immunoblotted with the
indicated antibodies. (C) NTERA-2 (NT2) cells 8 were treated with JQ1(-) or JQ1(+) at
the indicated concentrations. Whole cell lysates were extracted at 24 h post-treatment
and immunoblotted with the indicated antibodies. NANOG signal intensity in (A-C) was
quantified using ImageJ. (D) P19 cells were treated with 500 nM JQ1(-) or JQ1(+) for 6
h and subjected to the ChIP assay with normal rabbit IgG (IgG, as a negative control)
and the BRD4C antibody. ChIP samples were analyzed by qPCR using primers
targeting the Nanog regulatory regions as indicated in Fig. 5A. Values represent the
average of three independent experiments with error bars indicating s.e.m.
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