HBO1 is required for H3K14 acetylation and normal transcriptional activity during embryonic development.
ABSTRACT We report here that the MYST histone acetyltransferase HBO1 (histone acetyltransferase bound to ORC; MYST2/KAT7) is essential for postgastrulation mammalian development. Lack of HBO1 led to a more than 90% reduction of histone 3 lysine 14 (H3K14) acetylation, whereas no reduction of acetylation was detected at other histone residues. The decrease in H3K14 acetylation was accompanied by a decrease in expression of the majority of genes studied. However, some genes, in particular genes regulating embryonic patterning, were more severely affected than "housekeeping" genes. Development of HBO1-deficient embryos was arrested at the 10-somite stage. Blood vessels, mesenchyme, and somites were disorganized. In contrast to previous studies that reported cell cycle arrest in HBO1-depleted cultured cells, no defects in DNA replication or cell proliferation were seen in Hbo1 mutant embryo primary fibroblasts or immortalized fibroblasts. Rather, a high rate of cell death and DNA fragmentation was observed in Hbo1 mutant embryos, resulting initially in the degeneration of mesenchymal tissues and ultimately in embryonic lethality. In conclusion, the primary role of HBO1 in development is that of a transcriptional activator, which is indispensable for H3K14 acetylation and for the normal expression of essential genes regulating embryonic development.
- SourceAvailable from: Ignasi Forne[Show abstract] [Hide abstract]
ABSTRACT: Regulation of histone acetylation is fundamental to the utilization of eukaryotic genomes in chromatin. Aberrant acetylation contributes to disease and can be clinically combated by inhibiting the responsible enzymes. Our knowledge of the histone acetylation system is patchy because we so far lacked the meth-odology to describe acetylation patterns and their genesis by integrated enzyme activities. We devised a generally applicable, mass spectrometry-based strategy to precisely and accurately quantify combi-natorial modification motifs. This was applied to generate a comprehensive inventory of acetylation motifs on histones H3 and H4 in Drosophila cells. Systematic depletion of known or suspected acetyl-transferases and deacetylases revealed specific alterations of histone acetylation signatures, estab-lished enzyme-substrate relationships, and unveiled an extensive crosstalk between neighboring modifi-cations. Unexpectedly, overall histone acetylation levels remained remarkably constant upon depletion of individual acetyltransferases. Conceivably, the acetylation level is adjusted to maintain the global charge neutralization of chromatin and the stability of nuclei. INTRODUCTIONMolecular Cell 01/2015; 57(3). · 14.46 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Since the earliest observations of cells undergoing mitosis, it has been clear that there is an intimate relationship between the cell cycle and nuclear chromatin architecture. The nuclear envelope and chromatin undergo robust assembly and disassembly during the cell cycle, and transcriptional and post-transcriptional regulation of histone biogenesis and chromatin modification is controlled in a cell cycle-dependent manner. Chromatin binding proteins and chromatin modifications in turn influence the expression of critical cell cycle regulators, the accessibility of origins for DNA replication, DNA repair, and cell fate. In this review we aim to provide an integrated discussion of how the cell cycle machinery impacts nuclear architecture and vice-versa. We highlight recent advances in understanding cell cycle-dependent histone biogenesis and histone modification deposition, how cell cycle regulators control histone modifier activities, the contribution of chromatin modifications to origin firing for DNA replication, and newly identified roles for nucleoporins in regulating cell cycle gene expression, gene expression memory and differentiation. We close with a discussion of how cell cycle status may impact chromatin to influence cell fate decisions, under normal contexts of differentiation as well as in instances of cell fate reprogramming.Frontiers in Genetics 02/2015; 6.
- Cell cycle (Georgetown, Tex.) 07/2014; 13(15). · 5.24 Impact Factor
MOLECULAR AND CELLULAR BIOLOGY, Feb. 2011, p. 845–860
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 31, No. 4
HBO1 Is Required for H3K14 Acetylation and Normal Transcriptional
Activity during Embryonic Development?
Andrew J. Kueh,1,2Mathew P. Dixon,1Anne K. Voss,1,2†* and Tim Thomas1,2†*
The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3050, Australia,1and Department of Medical Biology,
The University of Melbourne, Parkville, Victoria 3050, Australia2
Received 9 February 2010/Returned for modification 15 March 2010/Accepted 3 December 2010
We report here that the MYST histone acetyltransferase HBO1 (histone acetyltransferase bound to ORC;
MYST2/KAT7) is essential for postgastrulation mammalian development. Lack of HBO1 led to a more than
90% reduction of histone 3 lysine 14 (H3K14) acetylation, whereas no reduction of acetylation was detected at
other histone residues. The decrease in H3K14 acetylation was accompanied by a decrease in expression of the
majority of genes studied. However, some genes, in particular genes regulating embryonic patterning, were
more severely affected than “housekeeping” genes. Development of HBO1-deficient embryos was arrested at the
10-somite stage. Blood vessels, mesenchyme, and somites were disorganized. In contrast to previous studies
that reported cell cycle arrest in HBO1-depleted cultured cells, no defects in DNA replication or cell prolif-
eration were seen in Hbo1 mutant embryo primary fibroblasts or immortalized fibroblasts. Rather, a high rate
of cell death and DNA fragmentation was observed in Hbo1 mutant embryos, resulting initially in the
degeneration of mesenchymal tissues and ultimately in embryonic lethality. In conclusion, the primary role of
HBO1 in development is that of a transcriptional activator, which is indispensable for H3K14 acetylation and
for the normal expression of essential genes regulating embryonic development.
In general, acetylation of histone lysine residues correlates
positively and strongly with transcriptionally active regions of
the genome (37, 49, 54); in contrast, heterochromatin is hypo-
acetylated (7, 8). It has been proposed that particular patterns
of posttranslational histone modifications represent a “code”
and that histone modifications are recognized by transcription
factors via specific chromatin-binding domains (2, 31, 60, 68).
Indeed, acetylated lysines are recognized by bromodomains
(13, 30). However, histones can be acetylated at multiple sites,
and with few exceptions, very little is known about the biolog-
ical function of acetylation at specific histone lysine residues in
multicellular organisms. Moreover, the residue specificity of
few histone acetyltransferases has been characterized in verte-
brate organisms. Two members of the MYST family of histone
acetyltransferases, MOF (MYST1/KAT8) and MOZ (MYST3/
KAT6A), have highly restricted substrate specificities in vivo.
In Drosophila melanogaster and mice in vivo, as well as in
human cells in vitro, MOF is specifically required for acetyla-
tion of histone 4 lysine 16 (H4K16) (3, 61, 64), whereas MOZ
has a specific role in the acetylation of H3K9 in Hox gene
clusters (69). Since embryonic development is dependent on
the precise regulation of temporal-spatial patterns of gene
expression, establishing the correct pattern of histone acetyla-
tion at developmentally important gene loci is critical. Accord-
ingly, MOF and MOZ, as well two other MYST family mem-
bers, QKF (MORF/MYST4/KAT6B) and TIP60 (KAT5), have
distinct and essential functions during mammalian develop-
ment (21, 24, 34, 47, 63, 64, 67, 69). We show here that the
MYST histone acetyltransferase HBO1 is required for the
expression of a broad range of genes and is essential for
the acetylation of H3K14.
HBO1 was originally identified in a screen for proteins in-
teracting with origin recognition complex protein 1 (ORC1)
(28). The assembly of ORC, along with CDC6/CDC18, CDT1,
and MCM2 to -7, onto replication origins forms the prerepli-
cation complex, which confers a license for the initiation of
DNA replication and ensures that DNA is replicated only once
per cell cycle (41). Apart from this interaction with ORC1,
HBO1 has also been shown to associate with other prerepli-
cation complex subunits. For instance, the zinc finger of HBO1
can interact with MCM2 in cervical carcinoma cells (9), and
the depletion of XHbo1 in Xenopus laevis egg extracts has been
reported to disrupt chromatin binding of Mcm2 to -7, resulting
in an abolishment of DNA replication activity (26). Further-
more, the knockdown of HBO1 using RNA interference
(RNAi) in preadipocytes appears to repress mitotic clonal ex-
pansion and adipogenesis, leading to the conclusion that
HBO1 acts together with Fad24 to promote adipogenesis by
regulating DNA replication (32). More recently, HBO1 has
been reported to interact directly with CDT1, where it en-
hances CDT1-dependent re-replication, and thus HBO1 is
proposed to act as a coactivator of CDT1 at replication origins
(44). Together, these findings strongly suggest that the primary
function of HBO1 is associated with DNA replication.
It has been reported that the acetyltransferase activity of
HBO1 is involved in many of its replication-associated func-
tions. HBO1 has been shown to acetylate prereplication com-
plex components, such as ORC2, MCM2, and CDC6, in cell-
free acetylation assays, and its histone acetyltransferase activity
is reported to be specifically upregulated during G2/M phase of
* Corresponding author. Mailing address: The Walter and Eliza
Hall Institute of Medical Research, Parkville, Victoria 3050, Australia.
Phone for Anne K. Voss: 61 3 9345 2642. Fax: 61 3 9347 0852. E-mail:
email@example.com. Phone for Tim Thomas: 61 3 9345 2642. Fax: 61 3
9347 0852. E-mail: firstname.lastname@example.org.
† These authors jointly supervised the project and contributed
?Published ahead of print on 13 December 2010.
the cell cycle in adenocarcinoma epithelial cells (26). In
cell-free histone acetyltransferase assays, HBO1 acetylates
recombinant Xenopus nucleosome core particles at H4K5,
H4K8, and H4K12 residues (14). These cell-free acetylation
assays suggest that HBO1 is a broad-spectrum acetyltrans-
ferase and may have a wide variety of both histone and
Apart from its proposed role in DNA replication within
ORC, there is evidence that HBO1 is involved in transcrip-
tional regulation. HBO1 colocalizes with a multisubunit his-
tone acetyltransferase complex consisting of ING4/5, hEaf6,
and JADE1/2/3, in which PHD fingers on ING4/5 and JADE1/
2/3 subunits confer targeting of the complex to chromatin by
binding methylated histone lysine residues (51). JADE1 can
alternatively be spliced into two splice variants, JADE1L and
JADE1S. Interestingly, JADE1S complexes contain HBO1
and hEaf6 but not ING4/5, resulting in a preferential shift from
binding methylated H3K4 to binding methylated H3K36 resi-
dues. Correspondingly, ING4/5 and JADE1S complexes have
been hypothesized to regulate transcriptional initiation and
elongation, respectively, based on the finding that HBO1 and
its associated subunits colocalize on transcriptional start sites
and gene coding regions in HeLa cells (51). Additionally, the
MYST domain of HBO1 is involved in enhancing progesterone
receptor transcriptional activity in monkey CV1 kidney cells
(18). The Drosophila homologue of HBO1, chameau, can ge-
netically interact with polycomb group proteins to mediate Hox
gene silencing, indicating a role in transcriptional repression
(20). On the other hand, chameau can also act downstream of
DFos and DJun in transcriptional activation (42). Further-
more, the serine-rich N terminus of HBO1 represses androgen
receptor- and NF-?B-mediated transcriptional activation in
CV1 and 293T cells, respectively (11, 57).
These studies suggest that HBO1 is a multifunctional pro-
tein that may be required for DNA replication as well as
promoting and repressing transcription by virtue of its activity
in acetylating a wide range of targets.
In order to examine the function of HBO1 during mamma-
lian development, we have created an Hbo1 null allele. Utiliz-
ing a series of cell proliferation and DNA replication assays,
we show in vivo and in vitro that HBO1 is not essential for
DNA replication or cell proliferation. Importantly, we report
that the primary biological function of HBO1 is to mediate
H3K14 acetylation and that HBO1 acts as an essential activa-
tor of gene expression during postgastrulation embryonic de-
MATERIALS AND METHODS
Generation of the Hbo1 mutant allele. A targeting construct was produced
using a 9.3-kb fragment from the bacterial artificial chromosome RP23-480C5,
which contains the entire Hbo1 gene. A loxP site was introduced 492 bp 5? of
exon 1, and a Neo cassette, flanked by FRT sites and also containing a single loxP
site at the 3? end, was introduced between exons 1 and 2 using the recombineer-
ing method (39). After electroporation into C57BL/6 embryonic stem (ES) cells,
screening of 272 clones yielded 8 correctly targeted cell lines. The Neo cassette
together with exon 1 was removed by crossing heterozygous mice to a cre-
recombinase deleter strain (55). This resulted in a mouse strain in which bases
687 bp 5? and bases 781 bp 3? of the start point of translation, including exon 1,
have been deleted. A single loxP site remains 5? of exon 2. PCR genotyping using
3 oligonucleotides allowed the identification of Hbo1?/?, Hbo1lox/lox, Hbo1?/?,
and Hbo1?/?genotypes. Amplification of the wild-type and floxed alleles using
oligonucleotide “1” (TAAGAGCTATTCCGTGTTCCGG) and oligonucleotide
“2” (AACTGGAAATTCTTTGGCGCTCC) resulted in products of 190 and 283
bases, respectively. Amplification of the null allele using oligonucleotide “1” and
oligonucleotide “3” (ATCAATTCTGCCTGGCTTAACCC) resulted in a prod-
uct of 358 bases (see Fig. 1B).
Mice were fed ad libitum and housed under a 12-h light/dark cycle. Experi-
mental animals were backcrossed onto an F1 hybrid background of the strains
FVB and BALB/c. For timed matings, females were housed with stud males,
checked after 2 h for the presence of a vaginal plug, and then left to mate
overnight. Mice were considered to be 0.5 days pregnant at midday if a vaginal
plug was observed in the morning of the same day but was not present the
previous evening. Experiments were undertaken with the approval of the Royal
Melbourne Research Foundation Animal Ethics Committee.
Hbo1?/?embryos were stage matched to control embryos by somite count in
experiments with results depicted in Fig. 2K to R, Fig. 3I to L, Fig. 4A to R, Fig.
5A to P, and Fig. 7A to F. Hbo1?/?embryos with excessively degenerated
somites were not used.
Antibodies. Antibodies used were raised against GCN5 (1:500, AB18381),
HBO1 (1:500, AB37289), acetylated H4K12 (1:500, AB1761), and MCM2 (1:200,
AB31159), purchased from Abcam; bromodeoxyuridine (BrdU) (1:10, M0744),
purchased from Dako; HIF-1? (1:20, MAB1935), purchased from R&D Sys-
tems; CD31 (1:250, 557355), purchased from BD Pharmingen; nidogen (1:200),
a gift from Marie Dziadek; acetylated H4K5 (1:2,000, 07-327), acetylated H4K8
(1:2,000, 07-328), acetylated H4K16 (1:2,000, 07-329), acetylated H3K9 (1:2,000,
07-352), and acetylated H3K14 (1:2,000, 070353), purchased from Upstate; and
actin (1:2,000, sc-1616 horseradish peroxidase [HRP]), purchased from Santa
Cruz. BrdU labeling, terminal deoxynucleotidyltransferase-mediated dUTP-bi-
otin nick end labeling (TUNEL) staining, immunohistochemistry, and Western
blotting were performed as described previously (67, 70).
Analysis of gene expression and chromatin immunoprecipitation (ChIP).
Whole-mount and radioactive RNA in situ hybridization experiments were
conducted as described previously (66, 72). Hbo1 mRNA was detected using
a 1-kb probe corresponding to exons 4 to 12 of the Hbo1 gene. Northern blot
analysis was performed using the same 1-kb Hbo1 probe. In situ hybridization
probes Nkx2.5 (4), Tbx1 (10), Vegfa (72), Flk-1 (62), Tie-1 (35), Tie-2 (53), Hex
(62), Notch1 (46), brachyury (74), and Hsp90ab1 (72) have been previously
described; other probes are listed in Table 1.
For reverse transcriptase quantitative PCR (RT-qPCR) analysis for gene ex-
pression, RNA was purified using RNA extraction columns (RNeasy minikit;
Qiagen) with an on-column DNase digest. For all samples, 1 ?g of the extracted
RNA was used as a template to generate cDNA. Quantitative PCR methodology
and subsequent data analysis have been described previously (69). Primer se-
quences are listed in Table 2.
For ChIP experiments, fragmented chromatin was prepared from embryos
using a chromatin prep kit (EZ-Zyme; Millipore), and subsequent immunopre-
cipitation steps were conducted using a chromatin immunoprecipitation kit (EZ-
Magna ChIP; Millipore). Quantitative PCR methodology and data analysis have
been described previously (69). Primer sequences are listed in Table 3.
Cell culture. For the embryonic day 3.5 (E3.5) outgrowth assay, uteri of
pregnant mice were flushed at E3.5, and embryos were collected and subse-
quently cultured in ES cell medium as described previously (71). Cultures were
For the MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bro-
mide] fibroblast proliferation assay, embryos were dissected at E9.5 and mechan-
ically dissociated by gentle pipetting before being cultured in fibroblast growth
medium (10% fetal calf serum [FCS], 2 mM L-glutamine, 100 IU/ml penicillin,
and 0.1 mg/ml streptomycin in high-glucose Dulbecco’s modified Eagle’s medium
TABLE 1. cDNA templates for cRNA probes
Gene SourceClone corresponds to:
Hoxa2Generated by PCR (GenBank
accession no. NM10451)
Generated by PCR (GenBank
accession no. Y11717)
Generated by PCR (GenBank
accession no. NM_177619)
490-bp cDNA fragment
Full-length coding region
1-kb cDNA fragment
775-bp cDNA fragment
640-bp cDNA fragment
846KUEH ET AL.MOL. CELL. BIOL.
[DMEM]). Derived primary embryonic fibroblasts (PEFs) were trypsinized, and
1,000 cells from each culture were plated in triplicate in 96-well plates. To
quantify cell number, 4 ?g of MTT was added to each sample, and samples were
left to incubate for 2 h. Culture medium was replaced by dimethyl sulfoxide
(DMSO), samples were mixed thoroughly, and absorbance readings were mea-
sured using a plate reader (Genios; Tecan).
For fluorescence-activated cell sorter (FACS) analysis for cell cycle distribu-
tion, E9.5-derived primary embryonic fibroblasts were fixed in 70% ethanol
overnight, washed twice in phosphate-buffered saline (PBS), stained with 20
?g/ml propidium iodide for 20 min at room temperature, and analyzed using a
FACSCalibur instrument. Cell cycle analysis was performed using FlowJo soft-
For the tamoxifen-induced-deletion assay, Hbo1lox/loxmice were crossed to
Hbo1lox/?mice carrying the Cre-ERT transgene (22). Embryos were dissected at
E10.5, cultured in fibroblast growth medium, and repeatedly passaged over 10
weeks to select for immortalized cells. Cultures were then treated with 0.5 ?M
4OH-tamoxifen (1,000? stock in 100% ethanol; Sigma) for 24 h to induce cre
recombination and Hbo1 deletion. Cell lines were continuously passaged once a
week for 15 weeks in the absence of tamoxifen and were genotyped every 5 weeks
to assess the viability of Hbo1-deleted cells.
Transplacental uptake assay. Pregnant female mice were injected intraperi-
toneally with a bolus of 100 ?l (10 ?Ci) of [2-deoxy-14C]glucose (PerkinElmer)
on day 8.0 of pregnancy. After 1 h, mice were sacrificed by cervical dislocation,
and individual deciduae, extraembryonic tissues, and embryos were collected
separately and lysed overnight in tail lysis buffer (10 mM Tris-Cl, pH 8.0, 100
mM EDTA, pH 8.0, 100 mM NaCl, 1% SDS). Radioisotope uptake was
determined from ?-emissions of samples placed in scintillation fluid (Starcint;
Packard BioScience BV) using a scintillation counter (Tri-Carb 2000; Pack-
ard BioScience BV).
Statistical analysis. All statistical analyses were conducted using Stata v9.2
(Statacorp) with one- or two-factor analysis of variance (ANOVA) and Fisher’s
post hoc test. Frequencies of genotypes recovered at various developmental
stages were analyzed by chi-square analysis, followed by Fisher’s exact test.
Hbo1 null allele and Hbo1 mRNA and protein expression. A
conditional Hbo1 mutant allele was generated as described in
Materials and Methods. We used a germ line-expressed cre-
recombinase to delete exon 1 of the Hbo1 gene together with
the neo selection cassette (Fig. 1A and B). This deletion pre-
vented both the transcription and the translation of the mutant
allele. No Hbo1 mRNA (Fig. 1C to H) or protein (Fig. 1I and
J) could be detected in embryonic or extraembryonic tissues in
Hbo1?/?embryos. HBO1 protein was present in all cells of the
developing Hbo1?/?embryo, and its localization was exclu-
sively nuclear (Fig. 1I). In Hbo1?/?embryos at E8.5 and E9.5,
Hbo1 was expressed ubiquitously in the embryonic and ex-
traembryonic tissues (Fig. 1C to F). High levels of Hbo1
mRNA were present in the chorionic plate (E8.5 and E9.5) as
well as in and around the foregut and hindgut regions (E9.5).
Hbo1 was ubiquitously expressed in all adult tissues examined,
with lower levels in the kidney and liver (Fig. 1K). Particularly
strong Hbo1 mRNA expression was detected in testes (Fig.
1K), in common with two other MYST family genes, Mof and
Tip60 (66). The observation that Hbo1 was expressed in the
adult brain, a tissue consisting almost exclusively of postmitotic
TABLE 2. RT-qPCR primers
Primer sequence Primer position
aPosition on cDNA sequence downstream of ATG.
TABLE 3. ChIP-qPCR primers
?1669 (between 4 and 5)
?360 (at 3)
?199 (between 1 and 2)
?923 (between 1 and 2)
aPosition on genomic sequence downstream of ATG. Positions relative to exons are in parentheses.
VOL. 31, 2011 HBO1 REGULATES DEVELOPMENTAL GENES 847
cells, indicated that it is likely that HBO1 possesses cellular
functions aside from its proposed role in cell proliferation and
Hbo1 deletion results in embryonic lethality at E10.5. Initial
germ layer formation and the establishment of the embryonic
axes proceeded normally in Hbo1?/?embryos, and no abnor-
malities were observed prior to E7.5 (E4.5, 15 embryos; E5.5,
19 embryos; E6.5, 32 embryos) (data not shown). At E7.5,
Hbo1?/?embryos were within the normal size range seen in
wild-type litters (Fig. 2A and B). At E8.0 to E8.5, Hbo1?/?
embryos were developmentally delayed by approximately 12 h
(Fig. 2C to F), and at E9.5, Hbo1?/?embryos were approxi-
mately 24 h developmentally delayed (Fig. 2G and H), in
comparison to Hbo1?/?embryos. At this point, Hbo1?/?em-
bryos underwent developmental arrest and did not develop
past the 10- to 11-somite stage. As Hbo1?/?embryos featured
FIG. 1. Hbo1 mutant alleles and expression patterns. (A) Schematic of wild-type and Hbo1 mutant alleles, with numbered black boxes
representing exons. (B) Three-way PCR used to genotype Hbo1?(wild-type), Hbo1lox(floxed), and Hbo1?(null) alleles, shown here in E8.5
embryos. (C to H) Radioactive RNA in situ hybridization depicting strong and ubiquitous Hbo1 expression at E8.5 and E9.5. Note the particularly
high levels of expression in the chorionic plate (CP) at E8.5 and E9.5 as well as in the foregut (FG) and hindgut (HG) regions at E9.5. No Hbo1
mRNA was detectable in Hbo1?/?embryos (G and H). SP, spongiotrophoblast; AL, allantois; H&E, hematoxylin and eosin. (I and J) HBO1
immunohistochemistry shows strong nuclear localization of HBO1 protein in wild-type controls (arrowheads, I), while no HBO1 protein is present
in Hbo1?/?embryos (J). SM, somites. (K) Northern blot showing Hbo1 mRNA expression in adult and embryonic tissues. Bar, 75 ?m (C and D),
350 ?m (E and F), 212 ?m (G and H), or 162 ?m (I and J).
848KUEH ET AL.MOL. CELL. BIOL.
a developmental delay, Hbo1?/?embryos were stage matched
to control embryos by somite count, as indicated in the relevant
figure legends and Materials and Methods. At E10.5, Hbo1?/?
embryos exhibited diminished posterior and midline struc-
tures. Nevertheless, 9 out of 18 Hbo1?/?embryos had beating
hearts at this stage (Fig. 2I and J). No Hbo1?/?embryos were
recovered at E11.5 (P ? 0.009) (for numbers of embryos ex-
amined, see Table 4), indicating that they underwent resorp-
tion shortly after E10.5. In wild-type embryos, the chorion-
allantois connection is established between E8.5 and E9.5. In
contrast, the allantois of Hbo1?/?embryos failed to grow di-
rectionally toward the chorionic plate but continued to expand
as a highly vascularized, bulbous structure (Fig. 2K and L).
Hbo1?/?embryos failed to complete axial rotation and devel-
oped enlarged blood vessels in the head region at E8.5, which
were more prominent at E9.5 (Fig. 2M to P). However, no
indication of edema or hemorrhage was observed at any stage.
The dorsal aorta and first pharyngeal arch artery were most
severely affected, showing grossly dilated lumina, while cells of
the surrounding mesenchyme appeared sparse. These vascular
defects extended along the entire length of the embryo, where
a comparable paucity of mesenchymal cells was evident (Fig.
2Q and R). Similarly, the cardiac inflow tract was dilated in
Hbo1?/?embryos in comparison to that in control embryos
(data not shown). Somites were indistinct and unstructured in
E9.5 Hbo1?/?embryos (Fig. 2R).
The initial vasculogenic stages of wild-type yolk sac devel-
opment are marked by the aggregation and fusion of blood
island endothelial cells to form the primitive capillary plexus, a
structure composed of a polygonal network of immature ves-
sels, at E8. At E9, the vascular plexus undergoes extensive
angiogenic remodeling to give rise to a hierarchical array of
small- and large-caliber vessels (50). Highly disorganized
Hbo1?/?yolk sac vascular branching and patterning were re-
vealed by anti-CD31 (PECAM) immunofluorescence staining
(Fig. 2S to V; 6 Hbo1?/?and 8 Hbo1?/?embryos). At E8.5,
blood vessels in Hbo1?/?yolk sacs were disordered, forming
thin networks with the presence of abnormal endothelial cell
clusters. These networks appeared to fuse randomly and re-
mained undeveloped at E9.5, where large vitelline vessels
failed to form and the vascular plexus persisted in a primitive
state (Fig. 2S to V).
Hbo1?/?embryos display adequate vascular and placental
function. As we observed defects in vascular and placental
morphology, we examined whether parameters of vascular and
placental function were normal in Hbo1?/?embryos. Nidogen
is important in stabilizing basement membranes and is partic-
ularly strongly expressed in vascular endothelial cells (65),
where it may also have a role in regulating angiogenesis (45).
Nidogen distribution was found to be normal in Hbo1?/?em-
bryos and expressed at a level similar to that for Hbo1?/?
controls (data not shown); together with no obvious observa-
tion of hemorrhage or edema, our data show that the mechan-
ical integrity of vessel walls remained intact in Hbo1?/?em-
To investigate the functional consequences of yolk sac vas-
cular abnormalities and the failure of chorioallantoic fusion in
Hbo1?/?embryos, we conducted transplacental nutrient up-
take assays and measured levels of hypoxia to assess if these
factors contributed to the restriction in developmental pro-
gression in these embryos. Dams were injected with radiola-
beled [14C]glucose at E8.0, at which point chorioallantoic fu-
sion had not occurred in Hbo1?/?or control embryos. We
observed no significant differences among Hbo1?/?, Hbo1?/?,
and Hbo1?/?tissues in radioisotope uptake (data not shown),
ruling out the possibility of glucose deficiency being the limit-
ing factor for normal developmental progression in Hbo1?/?
embryos, at least prior to E8.0. HIF-1? is a transcription factor
that is strongly upregulated under hypoxic conditions (25) and
that promotes vascular development by stabilizing vascular en-
dothelial growth factor (VEGF) (38). However, the absence of
HIF-1? staining in E8.5 and E9.5 Hbo1?/?embryos (data not
shown) suggested that hypoxia did not contribute to the
Hbo1?/?cells are not deficient in proliferation or DNA
replication. HBO1 has previously been reported to be required
for DNA replication and cell proliferation. To determine if
intrinsic defects in proliferation contributed to Hbo1?/?
growth retardation and eventual developmental arrest, we con-
ducted a series of in vivo and in vitro assays.
We cultured E3.5 blastocysts and examined these for a pe-
riod of 7 days to assess proliferation and differentiation. No
discernible differences were observed throughout the culture
period; all blastocysts hatched from the zona pellucida, at-
tached, and proliferated to form the two expected cell types,
the inner cell mass outgrowths and the surrounding tropho-
blasts (Fig. 3A and B; 5 Hbo1?/?, 25 Hbo1?/?, and 11 Hbo1?/?
We cultured dissociated E9.5 embryos in fibroblast enriching
medium and found that morphologically normal primary em-
bryonic fibroblasts (PEFs) could be derived from Hbo1?/?
embryos (Fig. 3C and D). MTT cell proliferation assays
showed robust proliferation of PEFs of all three genotypes
and no differences in proliferation rate between control and
Hbo1?/?PEFs throughout all 5 days of culture (Fig. 3E; P ?
0.9997; PEF isolates from 5 Hbo1?/?, 5 Hbo1?/?, and 5
Hbo1?/?embryos). Furthermore, cell cycle analyses revealed
that there were no differences in overall distribution of cells in
the G0/G1(P ? 0.302), S (P ? 0.621), and G2/M (P ? 0.245)
phases of the cell cycle within Hbo1?/?and Hbo1?/?PEF
cultures (Fig. 3F; PEF isolates from 3 Hbo1?/?and 3 Hbo1?/?
Hbo1lox/loxfibroblast lines, either positive or negative for a
Cre-ERT transgene, were treated with tamoxifen for 24 h and
passaged in culture for 15 weeks in the absence of tamoxifen.
This allowed the study of the effects of an acute as well as a
long-term loss of HBO1. We did not observe changes in cell
morphology or excessive cell death in Hbo1lox/loxCreT/?cell
lines in comparison to results for Hbo1lox/loxCre?/?control
cell lines following tamoxifen treatment. Tamoxifen-induced
cre deletion during the initial 24 h was not 100% efficient in all
cultures and so gave rise to a mixture of Hbo1 undeleted
(Hbo1lox/loxCreT/?), partially deleted (Hbo1lox/?CreT/?), and
completely deleted (Hbo1?/?CreT/?) cultures. This created a
competitive environment within some cultures, in which selec-
tive pressure against fibroblast genotypes that have subtle de-
fects in proliferation could be observed. After 3 days of culture,
no recombination was observed in Hbo1lox/loxCre?/?control
cell lines, as expected, whereas a high degree of recombination
was seen in Hbo1lox/loxCreT/?cell lines (Fig. 3G; each lane
VOL. 31, 2011 HBO1 REGULATES DEVELOPMENTAL GENES 849
FIG. 2. Gross morphology and histology of Hbo1?/?embryos. (A to J) Eight of 12 Hbo1?/?embryos are small albeit still within the size range
seen in wild-type E7.5 litters (A and B) but become progressively growth retarded in comparison to age-matched Hbo1?/?controls as development
proceeds. Note the bulbous shape of the allantois (white arrowheads, F, H, and J) and the large blood sinuses (red arrowheads, H and J) and the
failure of axial rotation in Hbo1?/?embryos. (K to R) Hematoxylin- and eosin-stained sections of E9.5 Hbo1?/?embryos and stage-matched E8.5
Hbo1?/?controls. Note the failure of chorioallantoic fusion (K and L), expanded dorsal aorta and first pharyngeal arch arteries (M to R), and
reduction of cephalic mesenchyme- and mesoderm-derived structures, such as somites (Q and R), in Hbo1?/?embryos. (S to V) Whole-mount
anti-CD31 (PECAM) staining of yolk sacs (S and T) and corresponding traces of blood vessels (U and V). Note the absence of large vitelline vessels
in Hbo1?/?mutants (arrowhead, S). AL, allantois; AM, amnion; CM, cephalic mesenchyme; CP, chorionic plate; DA, dorsal aorta; DE, decidua;
NE, neural ectoderm; PA, first pharyngeal arch artery; PHT, primitive heart tube; SM, somite; YS, yolk sac. Bar, 86 ?m (A to D), 142 ?m (E and
F), 245 ?m (G and H), 730 ?m (I and J), 160 ?m (K and L), 80 ?m (M and N), 201 ?m (O and P), 80 ?m (Q and R), or 68 ?m (S to V).
represents an independent cell line isolated from one embryo).
Notably, cell line 11 consisted purely of Hbo1?/?CreT/?cells
after 3 days of culture, continued proliferating normally, and
remained viable 15 weeks later (Fig. 3H). We observed detect-
able levels of undeleted Hbo1 alleles in cell line 7 after 3 days
of culture. However, by 15 weeks, cell line 7 consisted purely of
Hbo1?/?CreT/?cells. In the case of cell line 9, the relative
proportions of deleted and undeleted alleles remained similar
over the 15 weeks of analysis. These results show that Hbo1?/?
CreT/?cells were capable of continuous long-term prolifera-
tion and that Hbo1?/?CreT/?cells were not outcompeted by
Importantly, BrdU incorporation experiments revealed that
there was no significant difference (P ? 0.827) in the percent-
ages of cell nuclei undergoing DNA synthesis in E8.0 Hbo1?/?
control embryos (Fig. 3I; 66% ? 2.9%; 9 embryos) and E8.5
Hbo1?/?embryos (Fig. 3J; 65% ? 5.5%; 3 embryos). All
tissues in the developing Hbo1?/?embryo showed a high level
of BrdU staining, and Hbo1?/?cells exhibited staining inten-
sities similar to those of wild-type control cells, indicating that
Hbo1?/?embryos were as effective as wild-type controls in
incorporating BrdU into newly synthesized DNA and that
DNA replication ensued at normal rates in Hbo1?/?embryos.
We examined the cellular localization of MCM2 in Hbo1?/?
embryos compared to that in Hbo1?/?embryos. The MCM2 to
-7 complex exhibits DNA helicase activity and is essential in
separating the DNA double helix into single strands to facili-
tate DNA replication (5). Within this complex, MCM2 has
been demonstrated to interact with HBO1 (9), and the knock-
down of HBO1 using RNAi in HeLa cells has been reported to
result in a shift in localization of MCM2 protein from a nuclear
to a cytoplasmic cellular fraction (26). In contrast, we found
that MCM2 staining was present at normal levels and was
predominantly nuclear in Hbo1?/?embryos, as it was in
Hbo1?/?embryos, indicating that a loss of HBO1 had no
influence on MCM2 localization (Fig. 3K and L; 8 Hbo1?/?
and 13 control embryos).
We found that the lack of HBO1 affected cell survival rather
than DNA replication. TUNEL staining revealed an 8-fold
increase in the percentage of cells undergoing cell death in
Hbo1?/?embryos, resulting in a 3-fold reduction in the num-
ber of cells per section (Fig. 3M and N; P ? 0.0001 and P ?
0.0304, respectively; 3 Hbo1?/?, 6 Hbo1?/?, and 3 Hbo1?/?
E8.5 to E9.5 embryos). Collectively, with respect to the
Hbo1?/?phenotype, our data suggest that the reduction in
embryo size and cell number is primarily due to cell death,
rather than deficiencies in cell proliferation.
HBO1 is an essential regulator of gene expression. To de-
termine if the lack of HBO1 affected the expression of key
regulatory genes involved in patterning the early embryo, we
examined the expression profiles of 17 genes in a total of 102
embryos (3 Hbo1?/?and 3 Hbo1?/?embryos per probe) by
whole-mount in situ hybridization (WMISH).
We characterized the expression of essential regulators of
heart development Nkx2.5, Gata4, and Tbx1 (59). While the
hearts of Hbo1?/?embryos appeared morphologically normal,
apart from a modest dilation of the inflow tracks, and contin-
ued to beat until E9.5 to E10.5, cardiac patterning genes
Nkx2.5, Gata4, and Tbx1 were found to be underexpressed in
Hbo1?/?hearts. Interestingly, the expression of primary heart
field regulators Nkx2.5 and Gata4 was detectable in the cardiac
inflow tracts but was markedly reduced in the heart chambers
of Hbo1?/?embryos (Fig. 4A to D). Tbx1 is expressed strongly
in the pharyngeal endoderm and is important for inducing
the specification of the cardiac outflow tract in the under-
lying pharyngeal mesoderm. Tbx1 expression was reduced in
Hbo1?/?embryos (Fig. 4E and F).
As we observed abnormal vascular development in Hbo1?/?
embryos, we investigated the expression of genes involved in
blood vessel development. Key regulators of vascular develop-
ment include VegfA, Flk-1, Tie-1, and Tie-2 (50). Although
VegfA expression appeared unchanged in Hbo1?/?embryos
(Fig. 4G and H), the expression of its receptor, Flk-1, was
reduced in Hbo1?/?embryos but remained strong in the al-
lantois (Fig. 4I and J). Similarly, the expression of angiopoietin
receptors Tie-1 and Tie-2 was also downregulated, particularly
in regions outside the cardiac inflow tract, in Hbo1?/?em-
bryos, whereas the expression of Tie-1 remained strong in the
allantois (Fig. 4K to N).
Flk-1 knockout embryos die between E8.5 and E9.5 due to
defects in hematopoietic and endothelial cell development,
where yolk sac blood islands were absent and the dorsal aorta
was greatly reduced in diameter (56). Tie-1 knockout embryos
die between E13.5 and E14.5 due to severe edema and vascular
hemorrhaging (48), whereas Tie-2 knockout embryos die at
E10.5 due to endocardial defects, hemorrhaging, significant
reductions in endothelial cells, and collapsed vasculature (15).
Even though Flk-1, Tie-1, and Tie-2 expression was substan-
tially downregulated in Hbo1?/?embryos, there was no evi-
dence of hemorrhaging, heart defects, blood island abnormal-
ities, or reductions in the size of the vascular lumen. Rather,
the vascular lumina are grossly expanded in Hbo1?/?embryos
at E9.5. This suggests a failure of the vasculature to progress
from an immature and primitive state to the angiogenic re-
The level of Hex mRNA, an early marker of endothelial
precursors (62), was modestly reduced in the head region of
Hbo1?/?embryos (Fig. 4O and P). The Ets transcription factor
Erg, which is essential for hematopoiesis in mice (40), is also
expressed in the vasculature (Fig. 4Q) and was significantly
reduced in the Hbo1?/?embryo proper but was present in the
allantois at normal levels (Fig. 4R).
The Drosophila homologue of HBO1, chameau, has been
demonstrated to promote heterochromatin-mediated gene si-
lencing and to act as a silencer of bithorax group homeotic
TABLE 4. Distribution of the Hbo1?allele among offspring of
No. (%) of embryos of Hbo1?/?
Total no. of
aNine of 18 Hbo1?/?embryos had beating hearts at E10.5.
bAll Hbo1?/?embryos examined prior to E10.5 had beating hearts (P ?
VOL. 31, 2011HBO1 REGULATES DEVELOPMENTAL GENES 851
FIG. 3. Cell proliferation and DNA replication proceeded normally in the absence of HBO1. (A and B) Hbo1?/?and Hbo1?/?E3.5 cultures
proliferated and formed normal inner cell mass outgrowths (ICMO) and surrounding trophoblasts (T) by day 7 of culture. (C and D) Normal
primary embryonic fibroblasts could be derived from E9.5 Hbo1?/?and Hbo1?/?embryos. (E) Results from MTT proliferation assay, showing no
difference in proliferation between fibroblasts derived from Hbo1?/?and Hbo1?/?embryos (P ? 0.9997). OD, optical density. (F) FACS histogram
of propidium iodide (PI)-stained Hbo1?/?and Hbo1?/?PEFs, showing no significant differences in distribution of cells within G0/G1(P ? 0.302),
S (P ? 0.621), and G2/M (P ? 0.245) phases of the cell cycle. (G) PCR amplification of DNA extracted from long-term Hbo1lox/loxCre?/?fibroblast
lines (lanes 1 to 6) and Hbo1lox/loxCreT/?cell lines (lanes 7 to 11) (isolated from one embryo each) on day 3 after tamoxifen treatment. As expected,
852 KUEH ET AL.MOL. CELL. BIOL.
gene expression by genetically interacting with polycomb group
proteins (20). Based on this, we expected to see overexpression
of Hox genes in Hbo1?/?mouse embryos. However, unexpect-
edly, and in contrast to the effects of a loss of chameau in
Drosophila, Hoxa2 expression was significantly reduced in
Hbo1?/?embryos, while Hoxa3 staining appeared at an inten-
sity similar to that in wild-type controls (Fig. 5A to D). Otx2 (6)
and Sox2 (52) are essential genes required for patterning the
neural ectoderm. Both Otx2 and Sox2 were expressed at reduced
levels in Hbo1?/?embryos postgastrulation (Fig. 5E to H). Dur-
ing normal development, Otx2 is expressed throughout the epi-
blast and also in the visceral endoderm. However, Otx2 has a
dynamic pattern of expression, and as development proceeds, its
expression becomes progressively restricted to the head region
(1). Interestingly, we found that Otx2 expression in Hbo1?/?em-
bryos was normal at the onset of gastrulation in the epiblast (data
not shown), suggesting that HBO1 is necessary for the mainte-
nance of Otx2 expression in newly patterned tissue domains.
brachyury (58), Shh (12), and Notch1 (19) are important
regulatory genes expressed during gastrulation. We observed
that Notch1 expression was low in most areas of the paraxial
and presomitic mesoderm in Hbo1?/?embryos (Fig. 5I and J).
Limited areas of normal expression gave the Notch1 expression
pattern a variegated appearance in the presomitic mesoderm
of Hbo1?/?embryos, whereas no Notch1 expression was de-
tected in the somitic region of Hbo1?/?embryos. On the other
hand, the floorplate and notochord marker Shh and the essen-
tial regulator of mesoderm development brachyury were ex-
pressed at relatively normal levels in Hbo1?/?embryos in
comparison to wild-type controls as assessed by WMISH (Fig.
5K to N). In addition, similar expression levels of the consti-
tutively expressed chaperone Hsp90ab1 (Hsp90? ) in
Hbo1?/?and Hbo1?/?embryos (Fig. 5O and P) further con-
firmed that the reduction in expression of multiple genes seen
in Hbo1?/?embryos was not simply an inevitable consequence
of tissue degeneration and cell death.
Overall, there was a significant reduction in expression of
the majority of genes examined in Hbo1?/?embryos, and in no
case was an increase in gene expression observed in Hbo1?/?
embryos, indicating that HBO1 functions as a general activator
of gene expression.
H3K14 acetylation displays a nonredundant requirement
for HBO1. To identify specific histone residues acetylated by
HBO1, we examined differences in histone acetylation patterns
between Hbo1?/?and Hbo1?/?PEFs isolated from E9.5 em-
bryos. Hbo1?/?PEFs had more than a 10-fold reduction in
H3K14 acetylation (Fig. 6A and B; P ? 0.0001; 3 independent
PEF isolates from 3 embryos per genotype, with one PEF
isolate per lane). Interestingly, there was a significant 3-fold
increase in H4K16 acetylation (P ? 0.0098) in Hbo1?/?cells.
In addition, H3K9 (P ? 0.0029) and H4K5 (P ? 0.024) acet-
ylation levels were significantly increased, 46% and 20%, re-
spectively, while H4K8 and H4K12 acetylation levels were
largely unchanged, in Hbo1?/?cells.
We considered the possibility that HBO1 could directly reg-
ulate other histone acetyltransferases at the transcriptional
level and that the deregulation of these histone acetyltrans-
ferases, as a consequence of a loss of HBO1, could account for
the observed changes in histone acetylation patterns. Thus, we
assessed the expression of essential histone acetyltransferases
in Hbo1?/?PEFs by RT-qPCR, normalizing against Pgk1,
Rpl13a, Hsp90ab1, Psmb2, and B-actin. We found a 37% in-
crease in Mof transcripts in Hbo1?/?PEFs (P ? 0.016),
whereas Moz (P ? 0.970) and Gcn5 (P ? 0.930) expression
levels were not significantly changed (Fig. 6C; 3 independent
PEF isolates from 3 embryos per genotype). In addition, there
were no changes in GCN5 (KAT2A) protein levels in Hbo1?/?
PEFs (Fig. 6D and E; P ? 0.426; 3 independent PEF isolates
from 3 embryos per genotype).
Hbo1?/?embryos feature reduced H3K14 acetylation at
gene coding regions and a general depression of transcrip-
tional activity. To quantify gene expression levels in Hbo1?/?
embryos at E8.5 and E9.5, we conducted a series of RT-qPCR
experiments (Fig. 7A to D). The expression levels of a range of
regulatory genes were then determined relative to those of the
housekeeping genes Hsp90ab1, Pgk1, and Rpl13a. We observed
that the amount of total RNA isolated per embryo at E9.5 was
2-fold lower in the Hbo1?/?embryos than in the controls
(2.1 ? 0.2 versus 4.0 ? 0.6 ?g/embryo; 5 Hbo1?/?and 5
stage-matched wild-type embryos; P ? 0.019). This discrep-
ancy was observed despite that fact that the Hbo1?/?embryos
were similar in size to the control embryos, although, as de-
scribed above, mutant embryos display a lack of mesenchyme.
Nevertheless, this reduction in total RNA in Hbo1?/?embryos
is consistent with a global effect of HBO1 on the level of gene
transcription. For subsequent cDNA synthesis, equal amounts
of RNA from Hbo1?/?and stage-matched wild-type control
embryos were used, and qPCR results were normalized to
levels for housekeeping genes. Therefore, the large difference
observed in the initial amounts of total RNA was not rep-
resented in the RT-qPCR results, and this needs to be taken
into consideration when comparing WMISH and RT-qPCR
At E8.5, we observed significant reductions in expression of
only Hbo1lox/loxCreT/?cells displayed cre deletion and the presence of the Hbo1?(null) band, whereas Hbo1lox/loxCre?/?cells remained undeleted,
with the presence of only the Hbo1lox(lox) band. Top lanes represent DNA samples amplified for the Hbo1loxand Hbo1?allele concurrently, while
bottom lanes represent the same samples amplified for the Hbo1loxallele alone. Note that sample 11 showed the presence of the Hbo1?allele but
not the Hbo1loxallele, which indicates the presence of a pure population of Hbo1?/?cells. (H) PCR amplification of DNA extracted from the same
samples 15 weeks and 15 passages later. Sample 11 was still proliferating normally and remained comprised entirely of deleted cells. In addition,
sample 7 consisted purely of Hbo1?/?cells at this stage. (?), Hbo1?/?positive DNA control; (?), no-DNA negative control; wt, wild-type band.
(I and J) BrdU immunohistochemistry, showing similar levels of BrdU incorporation in stage-matched Hbo1?/?and Hbo1?/?embryos. (K and L)
MCM2 immunofluorescence counterstained with bisbenzimide, showing similar levels and nuclear localization of MCM2 in stage-matched
Hbo1?/?and Hbo1?/?embryos. NE, neural ectoderm. (M) Percentages of TUNEL-positive cells undergoing cell death. (N) Total cell number per
section. Data are presented as means ? standard errors of the means (SEM) and were analyzed as described in Materials and Methods. Bar, 94
?m (A to D) or 46 ?m (I to L).
VOL. 31, 2011 HBO1 REGULATES DEVELOPMENTAL GENES853
brachyury in Hbo1?/?embryos (Fig. 7A). While the mean
expression levels of other genes were lower, the differences
were not statistically significant. At E9.5, expression of
brachyury, Sox1, Sox2, Hoxa3, and Tie-2 was significantly re-
duced, while the mean expression levels of other genes were
reduced but not significantly so (Fig. 7B and C). In addition,
the expression of genes encoding histone acetyltransferases,
Moz, Gcn5, and Mof, was significantly reduced in Hbo1?/?
embryos (Fig. 7D).
These results show that, relative to those of housekeeping
FIG. 4. Whole-mount RNA in situ hybridization detecting mRNA (purple staining) of Nkx2.5, Gata4, Tbx1, VegfA, Flk-1, Tie-1, Tie-2, Hex, and
Erg in stage-matched Hbo1?/?and Hbo1?/?embryos. Note the reduced expression of most genes, whereas VegfA expression was less affected, in
Hbo1?/?embryos. AL, allantois; HC, heart chamber; IFT, inflow track; PE, pharyngeal endoderm. Bar, 70 ?m (A and B), 130 ?m (C and D), 70
?m (E and F), or 185 ?m (G to R).
854 KUEH ET AL.MOL. CELL. BIOL.
genes, the expression levels of the majority of genes pat-
terning the postgastrulation embryo show a decrease, as
seen in our WMISH experiments. Importantly, using RT-
qPCR or WMISH, we did not detect significant increases in
expression of any of the genes analyzed. This indicates that, at
least for these particular loci, HBO1 functions as a transcrip-
tional activator rather than having a previously reported role in
transcriptional repression (11, 20, 57).
Chromatin immunoprecipitation-quantitative PCR (ChIP-
qPCR) experiments using an anti-acetylated H3K14 antibody
revealed significant decreases in H3K14 acetylation in the cod-
ing regions of Hoxa3 (58% decrease; P ? 0.001), Tbx1 (50%
decrease; P ? 0.001), and Otx2 (53% decrease; P ? 0.001) in
chromatin material derived from E8.5 Hbo1?/?embryos (Fig.
7E; 6 Hbo1?/?and 6 Hbo1?/?embryos). Significant reductions
in acetylation were observed for Hoxa3 (46% decrease; P ?
0.001), Tbx1 (37% decrease; P ? 0.002), and Otx2 (42% de-
crease; P ? 0.001) even after normalization to acetylation
levels at the Hsp90ab1 locus (Fig. 7F).
In this study, we have shown that HBO1 is an essential
activator of patterning genes required for the normal develop-
ment of postgastrulation embryos. Our data suggest that
H3K14 is the primary target of HBO1 acetyltransferase activ-
ity. Contrary to previously published in vitro observations, we
found that HBO1 is not essential for cell proliferation or DNA
replication in vivo or in vitro.
As HBO1 has been implicated in DNA replication (26, 32,
44), we expected a peri-implantation lethal phenotype of
Hbo1?/?embryos at or prior to E4.5, since our previous work
FIG. 5. Whole-mount RNA in situ hybridization detecting mRNA (purple staining) of Hoxa2, Hoxa3, Otx2, Sox2, Notch1, Shh, brachyury, and
Hsp90ab1 in stage-matched Hbo1?/?and Hbo1?/?embryos. Note the reduced expression of most genes, whereas brachyury and Hsp90ab1
expression was less affected, in Hbo1?/?embryos. PSM, presomitic mesoderm; SR, somite region. Bar, 70 ?m (A and B), 135 ?m (C and D), or
185 ?m (E to P).
VOL. 31, 2011 HBO1 REGULATES DEVELOPMENTAL GENES 855
has shown that the quantity of similar essential transcriptional
regulators translated from maternally encoded mRNA is in-
sufficient to support the development of embryos beyond the
blastocyst stage (64, 73). The observations that Hbo1?/?em-
bryos developed well past E4.5 to the 10-somite stage, along
with normal proliferation of the inner cell mass outgrowths and
fibroblasts, show that HBO1 is not essential for cell prolifera-
tion. Similarly, the survival of HBO1-deficient flies to the pupal
stage (20) suggests that proliferation can also occur in the
absence of HBO1 in flies. In addition, we have shown that both
DNA replication and cell cycle progression proceed normally
in Hbo1?/?cells. Nevertheless, these results do not rule out a
nonessential role for HBO1 in DNA replication.
Since the majority of genes that we have examined showed
moderate to severe reductions in expression, we propose that
the fundamental cause of growth retardation in Hbo1?/?em-
bryos stems from deficient gene expression affecting multiple
loci. Interestingly, the initial formation of neuroectoderm and
mesoderm was normal until E8, but the expression of genes
required for subsequent patterning of the somites, heart, and
vasculature as well as the neural tube was generally reduced.
We note that there are discrepancies between our WMISH
data (Fig. 4 and 5) and RT-qPCR data (Fig. 7A to D). In
general, the large reductions in gene expression observed in
our WMISH experiments were either less pronounced or di-
minished in our RT-qPCR experiments, possibly due to the
following complications. First, we used the same amount of
RNA per sample to prepare cDNA. This approach normalized
any global reduction in RNA synthesis in the mutant embryos
compared to the control embryos and so underrepresents the
transcriptional deficiency of the Hbo1 mutant embryos. It
should be noted that substantially less total RNA could be
FIG. 6. Acetylation status analysis of H3 and H4 lysine residues in Hbo1?/?and Hbo1?/?PEFs. (A) Western blots of total PEF lysate probed
for acetylated H3K9, H3K14, H4K5, H4K8, H4K12, and H4K16 residues. Blots were stained with Ponceau S as a loading control. (B) Mean
densitometry readings of exposed film. Note the significant decrease in H3K14 acetylation (H3K14ac) (P ? 0.0001), whereas H3K9ac (P ? 0.0029),
H4K5ac (P ? 0.024), and H4K16ac (P ? 0.0098) were significantly increased, in Hbo1?/?cells. There were no significant differences in H4K8ac
(P ? 0.071) and H4K12ac (P ? 0.23) between Hbo1?/?and Hbo1?/?samples. (C) RT-qPCR analysis of histone acetyltransferase (HAT) gene
mRNA expression in Hbo1?/?and Hbo1?/?PEFs. (D) Western blots of total PEF lysate probed for GCN5 and actin. (E) Mean densitometry
readings of GCN5 protein levels normalized to actin, showing no significant changes in GCN5 protein in Hbo1?/?PEFs. Data are presented as
means ? SEM and were analyzed as described in Materials and Methods.
856 KUEH ET AL.MOL. CELL. BIOL.
isolated from the Hbo1 mutant embryos than from carefully
stage-matched controls. Second, as is customary, we have nor-
malized our RT-qPCR data against data for housekeeping
genes, including Hsp90ab1, Pgk1, and Rpl13a. As the expres-
sion levels of these housekeeping genes were lower in Hbo1?/?
embryos, normalizations made against housekeeping genes
tended to underrepresent differences in gene expression be-
tween Hbo1?/?embryos and control embryos. Our data sug-
gest that there is a global reduction in gene expression in
Hbo1?/?embryos. Despite the global reduction in gene ex-
pression and despite the fact that this reduction was normal-
ized both at the cDNA synthesis step and during analysis of the
qPCR data, certain genes are significantly reduced in the ab-
sence of HBO1, showing that they have a greater dependence
on HBO1 for normal levels of expression than other genes.
Third, the propensity for mesodermal and mesenchymal cell
FIG. 7. Hbo1?/?embryos feature a general decrease in gene expression at E8.5 and E9.5, along with a reduction of H3K14 acetylation in gene
coding regions. (A to D) RT-qPCR analysis of mRNA expression of patterning genes (A and B), vascular genes (C), and HAT genes (D) in
stage-matched Hbo1?/?and Hbo1?/?embryos at E8.5 (A) and E9.5 (B to D). (E and F) ChIP-qPCR analysis of H3K14 acetylation levels in the
coding regions of Hoxa3, Tbx1, and Otx2 in stage-matched Hbo1?/?and Hbo1?/?embryos at E8.5 (E) and normalized to H3K14 acetylation levels
at Hsp90ab1 (F).
VOL. 31, 2011 HBO1 REGULATES DEVELOPMENTAL GENES857
types to undergo cell death before other cell types (Fig. 2Q and
R) skews the cell type composition in Hbo1?/?embryos com-
pared to that in controls, resulting in a reduction of mesoderm-
and mesenchyme-associated genes over that of other genes.
Indeed, this is reflected in our RT-qPCR data, where brachyury
expression was drastically reduced in comparison to that of the
neuronal genes Sox1, Sox2, and Otx2. In contrast, our WMISH
experiments showed normal staining intensity for brachyury per
cell in Hbo1?/?embryos, possibly due to the fact that these
embryos were less developed than those used in our RT-qPCR
experiments. Similarly, while Flk-1 expression appeared to be
reduced in Hbo1?/?embryos at E8 in the WMISH data, it is
possible that the apparent increase in Flk-1 expression seen in
the RT-qPCR data could reflect the relative enrichment of
vasculature tissue compared to other tissue types in the E9.5
Hbo1?/?embryo. We propose that skewed cell type composi-
tion in Hbo1?/?embryos combined with normalization meth-
ods during cDNA synthesis and qPCR data analysis generates
the observed discrepancies between WMISH and RT-qPCR
results. However, it is important to note that the overall anal-
yses of gene expression in Hbo1?/?embryos by WMISH and
RT-qPCR are in agreement, with both techniques showing
that, in the absence of HBO1, gene expression was generally
reduced and no significant increase in expression was detect-
able for any of the genes examined.
We found that increased apoptosis, particularly affecting
mesodermal structures, ultimately led to growth arrest of Hbo1
null embryos, possibly because the postgastrulation develop-
mental programs of gene expression were unable to support
the survival of newly differentiated tissues. In contrast, fibro-
blasts isolated from embryos lacking HBO1 thrive in culture,
indicating that key subsets of genes are expressed at adequate
levels in Hbo1?/?cells. We conclude from our phenotypic and
gene expression analyses that transcriptional activity is suffi-
cient in Hbo1?/?embryos until the completion of gastrulation,
at which point the maintenance of expression of many genes
becomes dependent on HBO1 and a reduction in transcription
at multiple essential loci eventually results in cell death and
HBO1 has been implicated in the acetylation of H3, H4K5,
H4K8, and H4K12 (14, 17, 51). In contrast, we found surpris-
ingly specific changes in histone acetylation in PEFs isolated
from Hbo1 null embryos. Acetylation of H3K14 was 10-fold
lower in Hbo1?/?PEFs than in wild-type PEFs, whereas
H3K9, H4K5, and H4K16 acetylation increased by various
degrees. As H4K16 acetylation is entirely dependent on the
presence of MOF in early embryos (21, 64) and in cultured
HeLa cells (61), the 3-fold increase in H4K16 acetylation in
Hbo1?/?PEFs is likely to be attributed to the corresponding
37% increase in Mof transcripts. Indeed, we did not expect
decreases in H4K16 acetylation in Hbo1?/?PEFs, as HBO1
does not acetylate H4K16 on recombinant nucleosomes (14).
We have shown previously that MOZ displays acetylation ac-
tivity specific to H3K9, at least at Hox loci (69). The fact that
Moz expression is normal in Hbo1?/?PEFs leads us to propose
that the observed increases in H3K9 and possibly H4K5 acet-
ylation may be secondary effects arising as a consequence of a
primary loss of H3K14 acetylation in Hbo1?/?PEFs at the
histone level, rather than underlying aberrations in the expres-
sion of histone modifiers at the transcriptional level. Our data
suggest that acetylated H3K14 may act as a negative regulator
of the acetylation of H3K9, H4K5, and H4K16, either directly
via steric hindrance or by acting as a docking site for proteins
that may ultimately catalyze the removal of these acetylation
marks. In all, this supports the theory that extensive cross talk
occurs between histone modification marks (16, 23, 31, 36) and
highlights the essential contribution of HBO1 in maintaining
proper chromatin states.
Taking into account that the only histone residue in
Hbo1?/?PEFs to display compromised acetylation levels was
H3K14, our data strongly suggest that HBO1 is the principal
acetyltransferase required for the acetylation of H3K14. In
support of this, a 50% or greater reduction in the level of
H3K14 acetylation was detected in the coding regions of Tbx1,
Otx2, and Hoxa3 in E8 Hbo1?/?embryos, concomitant with a
general depression in the expression of these genes. On the
other hand, Hsp90ab1 acetylation levels were reduced by 24%,
and this corresponded to a 30% reduction in Hsp90ab1 expres-
sion in E8 Hbo1?/?embryos (data not shown). Overall, these
findings suggest that HBO1 and its associated H3K14 acet-
ylation activity are necessary for the normal expression of
most genes in a genome-wide context. Evidently, certain
genes feature a greater inherent dependency on HBO1-
mediated H3K14 acetylation than others.
Although H3K14 acetylation was reduced by more than 10-
fold in Hbo1?/?PEFs, we observed some persistence of im-
munoreactivity to acetylated H3K14. This may reflect residual
H3K14 acetylation, possibly by mammalian GCN5. Schizosac-
charomyces pombe Gcn5 (33) and mammalian GCN5 (2) can
acetylate H3K14 at gene coding regions to promote transcrip-
tional elongation. However, as GCN5 protein levels were not
significantly changed in Hbo1?/?PEFs, it is apparent that
GCN5-mediated H3K14 acetylation is insufficient to compen-
sate for the absence of HBO1, which further demonstrates the
significance of HBO1 for H3K14 acetylation.
Based on the findings that ING4/ING5-containing HBO1
complexes preferentially acetylate histones at transcriptional
promoters, whereas HBO1 complexes without ING4/ING5 are
targeted by JADE1S to predominantly acetylate histones at
downstream gene coding regions, HBO1 acetylation of pro-
moters has been proposed to facilitate transcriptional activa-
tion, whereas acetylation of downstream coding regions would
promote transcriptional elongation (51). In this respect, it is
likely that the primary requirement of most H3K14 acetylation
is regulation of gene expression patterns during the complex
process of differentiation and lineage specification that occurs
after gastrulation, whereas a basal level of H3K14 acetylation
is sufficient for basic cell survival and proliferation.
With regard to the existing literature on HBO1 function, our
data show discordances associated with cell proliferation, cell
cycle progression, DNA replication, and histone acetylation
specificity. We note that others have utilized predominantly
human cancer cell lines, including HeLa cells, C33A cells,
MCF7 cells, Saos2 cells, and A549 cells, to conduct their ex-
periments (26, 27, 29, 43, 44, 75). Evidently, these cell lines are
highly abnormal and not ideal for examining normal cell pro-
liferation, cell cycle progression, or DNA replication, due to
multiple chromosomal duplications, deletions, frameshift mu-
tations, and inversions within key cell cycle regulatory genes.
Furthermore, Iizuka and others have demonstrated that HBO1
858 KUEH ET AL.MOL. CELL. BIOL.
is highly upregulated in cancerous cells (29), suggesting that
these cells may have acquired an abnormal and complete de-
pendency on HBO1 for basic cellular processes, such as cell
proliferation and DNA replication. Lastly, it is also possible
that functional redundancy exists in our mouse model and not
in human cell lines. However, this is less likely, as HBO1 does
not coexist as a highly homologous pair of proteins, as do MOF
and TIP60 as well as QKF and MOZ.
In conclusion, we have provided the first in vivo character-
ization of the loss-of-function phenotype of Hbo1 in mice,
revealing a requirement for HBO1 in postgastrulation embry-
onic development. We show that HBO1 is not required for cell
proliferation or DNA replication but instead is an indispens-
able activator of multiple genes during postgastrulation devel-
opment. Importantly, we have identified a novel and nonre-
dundant role of HBO1 in promoting the acetylation of H3K14.
This work was funded by the Walter and Eliza Hall Institute and the
We thank C. Gatt, T. McLennan, and N. Downer for excellent
technical support. We appreciate the gifts of the recombineering re-
agents from N. Copeland and of cDNA clones from L. Robb, V.
Papaioannou, and T. Willson.
We have no conflicting interests.
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