HEMATOPOIESISAND STEM CELLS
*Yuta Mishima,1-3*Satoru Miyagi,1,3*Atsunori Saraya,1,3Masamitsu Negishi,1,3Mitsuhiro Endoh,3,4TakahoA. Endo,5
Tetsuro Toyoda,5Jun Shinga,4Takuo Katsumoto,6Tetsuhiro Chiba,1,3Naoto Yamaguchi,2Issay Kitabayashi,6
Haruhiko Koseki,3,4and Atsushi Iwama1,3
1Department of Cellular and Molecular Medicine, Graduate School of Medicine, and2Department of Molecular Cell Biology, Graduate School of Pharmaceutical
Sciences, Chiba University, Chiba, Japan;3JST, CREST, Tokyo, Japan;4RIKEN Research Center forAllergy and Immunology, Yokohama, Japan;5RIKEN
Genomic Sciences Center, Yokohama, Japan; and6Molecular Oncology Division, National Cancer Center Research Institute, Tokyo, Japan
The histone acetyltransferases (HATs)
of the MYST family include TIP60,
HBO1, MOZ/MORF, and MOF and func-
tion in multisubunit protein complexes.
Bromodomain-containing protein 1 (BRD1),
also known as BRPF2, has been consid-
ered a subunit of the MOZ/MORF H3 HAT
complex based on analogy with BRPF1
tion remains obscure. Here we show that
BRD1 forms a novel HAT complex with
deficient embryos showed severe anemia
because of impaired fetal liver erythropoi-
esis. Biochemical analyses revealed that
BRD1 bridges HBO1 and its activator
protein, ING4. Genome-wide mapping in
erythroblasts demonstrated that BRD1
and HBO1 largely colocalize in the ge-
nome and target key developmental regu-
lator genes. Of note, levels of global acet-
ylation of histone H3 at lysine 14 (H3K14)
were profoundly decreased in Brd1-
deficient erythroblasts and depletion of
Hbo1 similarly affected H3K14 acetyla-
tion. Impaired erythropoiesis in the ab-
sence of Brd1 accompanied reduced ex-
including Gata1, and was partially re-
stored by forced expression of Gata1.
Our findings suggest that the Hbo1-Brd1
complex is the major H3K14 HAT required
for transcriptional activation of erythroid
developmental regulator genes. (Blood.
The histone acetyltransferases (HATs) of the MYST family, which
include TIP60, HBO1, MOZ/MORF, and MOF, are highly con-
served in eukaryotes and perform a significant proportion of all
nuclear acetylation. They share a highly conserved MYST domain
composed of an acetyl-CoA binding motif and a zinc finger and
function in multisubunit protein complexes.1,2Among the MYST
family members, HBO1 and MOZ/MORF form complexes of very
similar composition: JADE family proteins bridge HBO1 with
inhibitor of growth 4 and 5 (ING4/5) and Esa1-associated factor 6
ortholog (EAF6), whereas BRPF family proteins bridge MOZ/
MORF with ING5 and EAF6, respectively.1,3,4The plant homology
domain (PHD) fingers in JADE1/2/3, BRPF1/2/3, and ING4/5
interact with histones and are thought to define the substrate-
specificity of the HBO1 and MOZ/MORF complexes.1HBO1 is
considered responsible for the bulk of the acetylation of histone H4
at lysines 5, 8, and 12 (H4K5, K8, and K12), and the interaction
augments activity of HBO1 to acetylate histone H3.5Furthermore,
the HBO1 complexes are enriched throughout the coding regions
of genes, suggestive of a role in transcriptional elongation.6By
contrast, MOZ and MORF are HATs specific for histone H3.
Binding of Yng1, a yeast ortholog of the ING family, to H3K4me3
has been shown to promote Sas3 (yeast ortholog of MOZ) HAT
activity at H3K14.7The mammalian MOZ complex also showed
specificity for H3K14 acetylation in vitro.3
Moz-deficient mice have a severe defect in the maintenance of
HSCs.8,9During zebrafish development, both moz and brpf1 are
required for maintenance of cranial Hox gene expression and
proper determination of pharyngeal segmental identities.10,11Simi-
lar findings were reported from analyses of Moz-deficient mice and
medaka fish in which brpf1 was mutated.12The genetic interaction
between Moz and Brpf1 supports that Brpf1 is the major bridging
protein of the MOZ HAT complex. In contrast to Brpf1, however,
distinctive functions of other BRPF family members have not been
BRD1 (initially named BR140-LIKE; BRL) was originally
cloned as a protein containing a cysteine-rich region related to that
of AF10 and AF17, which are leukemic fusion partners of MLL.13
BRD1 contains a bromodomain, 2 PHD zinc fingers, and a
proline-tryptophan-tryptophan-proline (PWWP) domain, 3 types
of modules characteristic of chromatin regulators. Recently, BRD1
was reported to belong to a small family of BRPF proteins that
includes BRPF1, BRD1/BRPF2, and BRPF3.1,3BRD1 has been
considered a subunit of the MOZ/MORF H3 HAT complex on the
basis of analogy with BRPF1 and BRPF3.3,4However, no detailed
analysis of BRD1 has been reported. In this study, we found that
BRD1 forms a novel HAT complex with HBO1 and is responsible
for the bulk of the acetylation of H3K14. We confirmed a drastic
reduction in levels of acetylated H3K14 in Brd1-deficient mice and
found that the Hbo1-Brd1 HAT complex is required for full
Submitted January 20, 2011; accepted June 20, 2011. Prepublished online as
Blood First Edition paper, July 13, 2011; DOI 10.1182/blood-2011-01-331892.
*Y.M., S.M., andA.S. contributed equally to this work.
The online version of this article contains a data supplement.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
© 2011 by TheAmerican Society of Hematology
2443 BLOOD, 1 SEPTEMBER 2011?VOLUME 118, NUMBER 9
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transcriptional activation of the erythroid-specific regulator genes
essential for terminal differentiation and survival of erythroblasts
in fetal liver.
Gene targeting of Brd1
Brd1-deficient mice were generated by the use of R1 embryonic stem cells
according to the conventional protocol. Brd1-deficient mice were back-
crossed to the C57BL/6 background ? 5 times. All experiments in which
mice were used received approval from the Chiba University Administra-
tive Panel forAnimal Care.
To prepare the retrovirus, pMC-ires-GFP was used as a vector.14The
production and concentration of the recombinant retrovirus have been
described previously.15To prepare the lentivirus, pCSII-EF1-MCS-IRESII-
Venus and pCS-H1-shRNA-EF-1?-EGFP were used as vectors.16The
viruses were produced as described previously.16Target sequences were as
follows; Sh-mHbo1#2; GAGGGAAGCAACATGATTA, Sh-mHbo1#3;
GATAGAAGA, and Sh-hHBO1#3; CTCAAATACTGGAAGGGAA.
Purification of BRD1-containing protein complex
Protein purification, trypsin digestion, and liquid chromatography tandem
mass spectrometry (LC/MS/MS) were performed as described previously.17
In brief, K562 cells expressing Flag-Brd1 (2.5 ? 108cells) were suspended
in 15 mL of lysis buffer (20mM sodium phosphate, pH 7.0; 350mM NaCl;
30mM sodium pyrophosphate; 0.1% NP-40; 5mM EDTA; 10mM NaF;
0.1mM Na3VO4; and 1mM phenylmethylsulfonyl fluoride) containing
protease inhibitors (cOmplete mini; Roche) and sonicated for 20 minutes.
The lysates were cleared by centrifugation and incubated with 100 ?L of
anti-FLAG M2 affinity gel (Sigma-Aldirch) with rotation at 4°C for
16 hours. The beads were extensively washed 6 times with 15 mL of lysis
buffer. The complexes were eluted by incubating twice with 0.2 mg/mL of
FLAG peptide in 300 ?Lof lysis buffer for 2.5 hours. This purification was
repeated 10 times.Then, eluents were pooled and concentrated by the use of
a filtration device (Vivaspin 10K-PES; Sartorius) and separated by 7.5%-
Immunoprecipitation and extraction of histones
Transfected 293T cells were lysed in lysis buffer containing 250mM NaCl
and then immunoprecipitation was performed. Immunocomplexes were
eluted with FLAG peptide as describe previously. Histone proteins were
extracted following the method described previously.18
ChIP-on-chip analyses of BRD1 and HBO1 binding were performed by use
of the Human Promoter ChIP-on-chip Microarray Set (G4489A; Agilent
Technologies). The assignment of IP regions and calculations were
performed as described.19K562 cells were fixed with 1% formaldehyde in
PBS for 10 minutes at room temperature and washed twice with PBS. Fixed
cells swelled in the buffer (20mM HEPES, pH 7.8; 1.5mM MgCl2; 10mM
KCl; 0.1% NP-40; and 1mM DTT) for 10 minutes on ice and nuclei were
prepared by Dounce homogenizer. Nuclei were then lysed with RIPA
(10mM Tris, pH 8.0; 0.5% SDS; 140mM NaCl; 1mM EDTA; 1% TritonX-
100; 0.1% SDS; 0.1% sodium deoxycholate; and a proteinase inhibitor
cocktail [cOmplete mini]), and sonicated for 30 minutes with a Bioruptor
(Cosmobio Co Ltd). After centrifugation, the soluble chromatin fraction
was precleared with a mixture of protein A and G-conjugated Dynabeads
(Invitrogen) blocked with BSA and salmon sperm DNA. Three hundred
micrograms of chromatin was immunoprecipitated overnight at 4°C with
the use of 25 ?L of antibody-conjugated Dynabeads. The immunoprecipi-
tates were washed extensively and subjected to a quantitative PCR analysis
with SYBR Premix Ex TaqTM II (Takara). For the ChIP of erythroblasts,
the steps to prepare nuclei were omitted, and fixed cells were directly lysed
by RIPA. Primer sequences used are listed in supplemental Methods
(available on the Blood Web site; see the Supplemental Materials link at the
top of the online article).
Expression vectors and antibodies
Other methods, the expression vectors, and antibodies used are described in
Brd1?/?embryos die at mid-gestation because of anemia
To clarify the physiologic function of Brd1, we generated Brd1-
deficient mice in which exon 2 containing the firstATG of the Brd1
gene was deleted (Figure 1A). Northern blot analysis detected no
Brd1 mRNA in Brd1?/?embryos (data not shown). The Brd1?/?
embryos were recovered at nearly the expected Mendelian ratio at
12.5 days postcoitum (dpc) but most had died by 15.5 dpc (Table
1). Brd1?/?embryos showed growth retardation (92 of 99 embryos
at 12.5 dpc), failure to fuse the neural tube (30 of 135 embryos at
8.5-12.5 dpc), and abnormal lenses with disoriented optic cups (74
of 122 embryos at 10.5-12.5 dpc; Figure 1B and Table 1). These
results indicated Brd1 as having pivotal roles in embryonic
development in multiple tissues and organs, but none of them was
considered to be the cause of death.
We then analyzed hematopoiesis in the absence of Brd1.
Numbers of total yolk sac cells and Ter119?erythroblasts were
rather increased in Brd1?/?yolk sac compared with those in
wild-type yolk sac (Figure 1C-D). This trend was more apparent at
later stages. At 12.5 dpc, erythropoiesis was still active in Brd1?/?
yolk sac, whereas erythropoiesis tended to decline in wild-type
yolk sac (supplemental Figure 1A). Together, our findings suggest
that primitive erythropoiesis in the Brd1?/?yolk sac was not
affected but rather enhanced. Nevertheless, Brd1?/?embryos at
12.5 dpc were pale and the fetal liver, in which fetal hematopoiesis
mainly occurs, was significantly smaller than that of littermate
controls (Figure 2A-D). Cytologic analysis revealed that Brd1?/?
roblast stage than did wild-type fetal livers (Figure 2E-F).
Brd1 is required for erythropoiesis in fetal liver
Among the phenotypes associated with Brd1 deficiency, we
focused on anemia, which is a major causative defect for lethality at
this stage of development. Flow cytometric analysis of fetal livers
at 12.5 dpc revealed a 2-fold reduction in theTer119?erythroid cell
fraction and a 2-fold increase in the c-Kit?hematopoietic progeni-
tor fraction (Figure 2G). Because the total number of Brd1?/?fetal
liver cells was decreased to 22% of the control, the absolute
number of Ter119?erythroid cells was decreased by 91% in
Brd1?/?fetal livers compared with wild-type fetal livers, whereas
that of c-Kit?hematopoietic progenitors was not profoundly
changed (Figure 2H). The number of Dlk1?hepatoblasts was
reduced to 57% of the control, but the differentiation of hepato-
blasts into hepatocytes and cholangiocytes was grossly normal in
the Brd1?/?fetal liver (Figure 2G-H; and data not shown). These
results indicated that the fetal liver hypoplasia in Brd1?/?embryos
Detailed flow cytometric analyses revealed a significant in-
crease in the CD71?Ter119?fraction and a drastic reduction in the
2444MISHIMAet alBLOOD, 1 SEPTEMBER 2011?VOLUME 118, NUMBER 9
For personal use only.on November 5, 2015. by guest
in Immune System Regulation and Treatment), MEXT, Japan, a
Grant-in-Aid for the Core Research for Evolutional Science and
Technology (CREST) from the Japan Science and Technology
Contribution: Y.M., S.M., and A.S. performed the experiments,
analyzed results, made the figures, and wrote the manuscript; M.N.
cloned Brd1 cDNA; M.E., T.A.E., and T.T. performed the ChIP-
chip assay; J.S. and H.K. generated Brd1-deficient mice; T.K. and
I.K. provided Moz-deficient mice and prepared Moz-deficient
MEFs; T.C. performed phenotypic analysis of Brd1-deficient mice;
N.Y. gave a critical suggestion to the project; andA.I. conceived of
Conflict-of-interest disclosure: The authors declare no compet-
ing financial interests.
Correspondence: Atsushi Iwama, MD, PhD, 1-8-1 Inohana,
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HBO1-BRD1 REGULATES ERYTHROPOIESIS 2453 BLOOD, 1 SEPTEMBER 2011?VOLUME 118, NUMBER 9
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online July 13, 2011
2011 118: 2443-2453
Kitabayashi, Haruhiko Koseki and Atsushi Iwama
Endo, Tetsuro Toyoda, Jun Shinga, Takuo Katsumoto, Tetsuhiro Chiba, Naoto Yamaguchi, Issay
Yuta Mishima, Satoru Miyagi, Atsunori Saraya, Masamitsu Negishi, Mitsuhiro Endoh, Takaho A.
H3K14 and required for fetal liver erythropoiesis
The Hbo1-Brd1/Brpf2 complex is responsible for global acetylation of
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