MOLECULAR AND CELLULAR BIOLOGY, Jan. 2006, p. 592–604
Vol. 26, No. 2
Down-Regulation of Nucleosomal Binding Protein HMGN1
Expression during Embryogenesis Modulates Sox9
Expression in Chondrocytes†
Takashi Furusawa,1Jae-Hwan Lim,1,3Fre ´de ´ric Catez,1Yehudit Birger,1
Susan Mackem,2and Michael Bustin1*
Protein Section, Laboratory of Metabolism,1and Laboratory of Pathology,2Center for Cancer Research,
National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, and
Department of Biology, Andong National University, 388 Seongcheon-dong,
Andong, Gyungsangbuk-do 760-749, South Korea3
Received 14 September 2005/Returned for modification 14 October 2005/Accepted 25 October 2005
We find that during embryogenesis the expression of HMGN1, a nuclear protein that binds to nucleosomes
and reduces the compaction of the chromatin fiber, is progressively down-regulated throughout the entire
embryo, except in committed but continuously renewing cell types, such as the basal layer of the epithelium.
In the developing limb bud, the expression of HMGN1 is complementary to Sox9, a master regulator of the
chondrocyte lineage. In limb bud micromass cultures, which faithfully mimic in vivo chondrogenic differen-
tiation, loss of HMGN1 accelerates differentiation. Expression of wild-type HMGN1, but not of a mutant
HMGN1 that does not bind to chromatin, in Hmgn1?/?micromass cultures inhibits Sox9 expression and
retards differentiation. Chromatin immunoprecipitation analysis reveals that HMGN1 binds to Sox9 chroma-
tin in cells that are poised to express Sox9. Loss of HMGN1 elevates the amount of HMGN2 bound to Sox9,
suggesting functional redundancy among these proteins. These findings suggest a role for HMGN1 in chro-
matin remodeling during embryogenesis and in the activation of Sox9 during chondrogenesis.
During organogenesis of the vertebrate embryo, multipotent
progenitor cells undergo a complex process of differentiation
according to their fate. Cellular differentiation involves the
execution of a preprogrammed, orderly process that involves
multiple changes in gene expression. It is well documented that
chromatin structure plays a key role in regulating gene expres-
sion and that the chromatin structure of specific genes is re-
modeled during the differentiation. It is therefore possible that
nuclear proteins such as the high mobility group N (HMGN),
which affect chromatin structure (4), histone modifications (17,
18) and transcription rates (11, 30), may play a role in differ-
The HMG superfamily of proteins consists of three families,
HMGA, HMGB, and HMGN, all of which have been shown to
modulate the structure and activity of the chromatin fiber (5).
Members of the HMGN family bind specifically to the building
block of the chromatin fiber, the nucleosome core particle,
without any known specificity for the underlying DNA se-
quence (28). The interaction of the proteins with chromatin is
dynamic, and HMGN proteins continuously exchange among
nucleosomes (7, 25). The binding of HMGN to nucleosomes
reduces the compaction of chromatin fiber and enhances tran-
scription from chromatin templates (11, 22, 30). HMGNs mod-
ulate the levels of posttranslational modifications in the his-
tone tails (17), most likely because their presence on
nucleosomes affects the ability of nucleosome remodeling com-
plexes to reach their targets. These findings and additional
studies suggest that HMGN proteins modulate chromatin-re-
lated activities, including transcription (4).
The expression level of Hmgn genes is related to cellular
differentiation processes, such as erythropoiesis, myogenesis,
osteoblast differentiation, kidney organogenesis, preimplanta-
tion development of early mouse embryo, and Xenopus embry-
ogenesis (1, 9, 15, 16, 20, 23, 27). Transient depletion of
HMGN proteins from one- or two-cell mouse embryos slowed
the progression of preimplantation development (20). Overex-
pression of HMGN1 in myoblasts inhibited their differentia-
tion into myotubes (23). HMGN2 plays a role in the activation
of genes regulating kidney organogenesis (16). During Xeno-
pus embryogenesis, either enhancement or depletion of
HMGN protein levels led to malformed tadpole embryos (15).
These results suggest a link between regulated expression of
HMGN proteins and cellular differentiation during embryo
Here we use in situ hybridization and immunohistochemistry
to examine the expression of HMGN1, a major member of the
HMGN protein family, during mouse embryogenesis. We find
a differentiation-related, global down-regulation of HMGN1
expression throughout the entire embryo; however, in specific
cell types the levels of this protein remain high, even in fully
differentiated organs. We focus on the developmental down-
regulation of HMGN protein during limb bud development
and demonstrate that expression of the protein is related to
chondrocyte differentiation. Chondrocyte differentiation is a
multistep genetic program, regulated by combinatorial signal-
ing of various growth and differentiation factor networks (29).
* Corresponding author. Mailing address: National Cancer Institute,
National Institutes of Health, Building 37, Room 3122, 9000 Rockville
Pike, Bethesda, MD 20892. Phone: (301) 496-5234. Fax: (301) 496-
8419. E-mail: firstname.lastname@example.org.
† Supplemental material for this article may be found at http://mcb
Sox9 is a transactivator and master regulator of chondrogenic
fate that is required for initiating chondrogenesis and subse-
quent chondrocyte differentiation (10). We find that the ex-
pression patterns of Hmgn1 and Sox9 are complementary both
in vivo and in vitro. We demonstrate that in micromass cultures
of differentiating limb bud cells, the expression of Sox9 and the
rate of differentiation are modulated by the levels of HMGN1
protein and that HMGN1 is present in Sox9 chromatin of
chondrogenic cells and absent from this gene in nonchondro-
genic cells. We demonstrate that the close homologue
HMGN2 is also associated with Sox9 chromatin and that loss
of HMGN1 increases the amount of HMGN2 on the Sox9
gene. Taken together with previous studies, our results suggest
that HMGN proteins affect chromatin remodeling and cellular
differentiation during embryogenesis.
MATERIALS AND METHODS
Mouse strains. Hmgn1?/?mice were previously described (3).
Whole-mount in situ hybridization. Whole-mount in situ hybridizations were
done according to the method of Wilkinson (34) with minor modifications:
punctured, and proteinase K-digested embryos were rinsed with phosphate-
buffered saline (PBS) containing 0.1% Tween 20 and immediately fixed with 4%
paraformaldehyde (PFA). Blocking reagent (Roche Diagnostics) was used at a
concentration of 1.5%. For the Hmgn1 in situ hybridization probe, the 1.2-kb
mouse Hmgn1 cDNA cloned in pBluescriptII KS (Stratagene) was linearized by
HindIII and XmaI to synthesize sense and antisense probes, respectively. For the
Sox9 probe, 500 bp of sequence 3? to the DNA binding domain of mouse Sox9
cDNA was generated by reverse transcription-PCR using the oligonucleotides
5?-ACCAATACTTGCCACCCAAC-3? and 5?-TAGGAGCCGGAGTTCTGAT
G-3?, cloned in pCR2.1-TOPO (Invitrogen), and linearized with BamH I to
synthesize the antisense probe. Sense and antisense RNA probes were prepared
by transcription of the linearized plasmids using T3 and T7 RNA polymerases
(Stratagene) with digoxigenin-11-UTP (Roche Diagnostics).
Cell culture and transfections. Embryonic day 10.5 (E10.5) limb buds were
collected in Dulbecco’s modified PBS (GIBCO) at 4°C. Mesenchymal cells were
dissociated in Dulbecco’s modified PBS containing 0.1% trypsin, 0.4 mM EDTA,
and 0.1% collagenase at 37°C for 10 min, resuspended in Dulbecco’s modified
Eagle’s medium-F12 medium (GIBCO) with 10% fetal bovine serum, 50 U/ml
penicillin, and 50 mg/ml streptomycin, at 2 ? 107cells/ml, and a 10-?l drop of cell
suspension was placed in the center of a well in a standard 24-well polystyrene
tissue culture dish or on Labteck chamber slides. Cells were allowed to adhere
for 1 h at 37°C and 5% CO2, and 1 ml of medium was added to the culture.
Medium was changed every 2 days. Alcian blue staining and quantification were
performed as previously described (12). For transient expression of the DNA in
micromass cultures, 8 ? 106limb bud mesenchymal cells were resuspended in the
0.4 ml of ice-cold PBS with 20 ?g of plasmid DNA in 4-mm wide electroporation
cuvettes and electroporated with a BTX T820 electroporator (Genetronics Inc.)
using a single 225-V square pulse at 50 ms. HMGN1-YFP and HMNG1(S20,
24E)_YFP (where YFP is yellow fluorescent protein) expression vectors were as
previously described (26). Transfection efficiency was assessed by YFP after 2
days of culture, and chondrocyte differentiation was assessed by Alcian blue
staining (12) or immunostaining for Sox9 after another 4 days of culture. For
Alcian blue quantification, the experiments were repeated three times. Mouse
embryonic fibroblasts (MEFs) were prepared as previously described (3).
Confocal microscopy. Micromass cultures were grown on Lab-Tek chambered
cover slides (Nalgen), and immunostaining was performed as previously de-
scribed (26) except that the micromass cultures were fixed with 4% PFA in PBS
for 10 min, washed with PBS, and permeabilized for 40 min in PBS containing
1% Triton X-100 and blocked overnight in PBS containing 1% fetal bovine
serum. Micromass cultures were incubated 6 h with the primary antibody (anti-
Sox9) (H-90; Santa Cruz) at a 1:100 dilution and with anti-HMGN1 at a 1:200
dilution. The micromass was washed in PBS three times for 20 min each time,
incubated for 2 h with the secondary antibody labeled with either AlexaFluor 488
or AlexaFluor 594 (Molecular Probes). DNA was stained with Hoechst 33258 at
0.5 ?g/ml in PBS for 10 min.
Microscopy was performed on a Zeiss LSM 510 confocal setting, using a 63?
differential interference contrast objective (1.4 numeric aperture). Stacks (57 ?m
thick) were collected through the entire micromass.
Histology. Embryos were fixed in 4% PFA, dehydrated and embedded in
paraffin, and sectioned at a thickness of 5 ?m. Sections were deparaffinized and
subjected to antigen retrieval by microwaving for 10 min in 10 mM citric acid
buffer (pH 6.0). After endogenous peroxidase activities were quenched by treat-
ment with 1% H2O2in PBS for 30 min, sections were blocked with 5% goat
serum-PBS for 30 min, incubated overnight with primary antibody at 4°C, washed
three times with PBS, incubated with biotinylated secondary antibodies for 1 h at
room temperature, and stained with the Vectastain ABC Elite kit (Vector Lab-
oratories) using diaminobenzidine as substrate. Sections were counter-stained
with hematoxylin and mounted in Permount (Fisher Scientific). For immunoflu-
orescence, the procedure was identical except that AlexaFluor 488-labeled goat
anti-rabbit (Invitrogen) was used to visualize the primary antibody.
ChIP assay, RNA analysis, and real-time reverse transcription-PCR. Chro-
matin was prepared from E10.5 mouse limb buds and MEFs and immunopre-
cipitated with a chromatin immunoprecipitation (ChIP) assay kit (Upstate
Biotechnology) (17) using either anti-HMGN1, mouse immunoglobulin G
(Santa Cruz) or no antibody as a negative control. Primer sets numbered 1 to 21
(see Fig. 7) used for PCR amplification of the Sox9 gene are described in Table
S1 of the supplemental material. Differences in specific DNA enrichment were
determined in duplicate by real-time quantitative PCR using an ABI Prism
7900HT sequence detector (Applied Biosystems). Beta-globin served as a refer-
ence gene. For RNA quantification, 50 ng of purified RNA isolated from limb
bud cells or culture cells with TRIZOL (Invitrogen) was subjected to reverse
transcription with SuperscriptII (Invitrogen) and amplified with primers specific
for either Hmgn1 (forward, 5?-TTGTAGCCATGAGTGACGTTGAA-3?; re-
verse, 5?-ATGACCCAGAACATTAGCCAGG-3?) or Sox9 (forward, 5?-CGGC
TCCAGCAAGAACAAG-3?; reverse, 5?-TTGTGCAGATGCGGGTACTG-3?).
The level was normalized using glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) primers (Applied Biosystems).
RNA dot blot analysis was performed as previously described (33) with a
mouse RNA master dot blot (BD-Clonetech). In these blots the amount of RNA
spotted from each tissue is normalized to that of eight housekeeping genes (see
DNase I digestion assay. To prepare nuclei, E10.5 limb bud cells or MEFs
were suspended and swollen in ice-cold lysis buffer (10 mM Tris [pH 7.4], 3 mM
CaCl2, 2 mM Mg Cl2); the swollen cells were resuspended in an equal volume of
buffer containing 10 mM Tris, pH 7.4, 10 mM NaCl, 3 mM Mg Cl2, and 0.5%
NP-40 and then homogenized with a Dounce homogenizer and centrifuged at
800 ? g for 10 min at 4°C. Nuclear pellets were stored in 25% glycerol, 5 mM Mg
acetate, 50 mM Tris (pH 8.0), 0.1 mM EDTA, and 12 mM 2-mercaptoethanol at
?70°C. Nuclei (1 ?g of DNA) were digested with DNase I in buffer containing
50 mM Tris (pH 7.4), 100 mM NaCl, 10 mM MgCl2, 5% glycerol, and 1 mM
dithiothreitol at 37°C for 15 min. Purified genomic DNA was digested as con-
trols. Digestion was terminated by adding proteinase K buffer (20 mM Tris [pH
8.0], 100 mM KCl, 50 mM Mg Cl2, 1% Tween 20, 1% NP-40, 1 mg/ml proteinase
K) at 55°C for 3 h. The control primer for beta-globin spans exon 2 of the
beta-globin gene: forward, 5?-TGAAGGCCCATGGCAAGA-3?; reverse, 5?-GC
Progressive down-regulation of Hmgn1 expression during
mouse embryogenesis. Whole-mount in situ hybridization and
immunostaining analyses revealed that the expression of
Hmgn1 is selectively down-regulated during mouse embryo-
genesis. In 7.5-day-old mouse embryos (E7.5), the expression
of Hmgn1 was high in the epiblast, weak in the extra-embryonic
ectoderm, and absent from the ectoplacental cone (Fig. 1A,
frame a). At E8.5 and E9.5, high Hmgn1 expression was ob-
served throughout the entire embryo, except in the heart (Fig.
1A, frames b and c), an organ known to begin differentiating
early (14). At E10.5, fairly strong expression of Hmgn1 was
widespread through the embryo and was prominent in the
forelimb and hind limb buds, branchial arches, and tail bud
(Fig. 1A, frames d to f) but was totally absent from the heart.
At subsequent developmental stages, the expression level of
Hmgn1 selectively declines, coincident with the onset of differ-
VOL. 26, 2006A ROLE FOR HMGN1 IN CHONDROGENESIS593
entiation in many organs, as more easily seen in tissue sections
Quantitative RNA dot blot analysis fully supports the con-
clusion of the in situ analyses (Fig. 1C). In these blots, the
amount of RNA in each spot is normalized to the transcription
levels of eight housekeeping genes; therefore, the intensity of
the spot indicates the relative mRNA abundance in a tissue.
The relative levels of Hmgn1 RNA were highest in 7-day-old
embryos and then gradually decreased; the RNA levels in
17-day-old embryo were only 50% of those of the 7-day-old
embryos. In differentiated tissues such as the adult heart and
spleen, the levels of Hmgn1 RNA are only about 10% of the
level present in young embryos (Fig. 1C).
To discern the developmental pattern of Hmgn1 expression
in the internal organs of the embryos, we analyzed the pres-
ence of HMGN1 protein in multiple serial sections by immu-
nohistochemistry using affinity-purified anti-mouse HMGN1
antibody. Representative results from several tissues (Fig. 2)
(images are best viewed at large magnifications; see Fig. S1 to
S3 in the supplemental material) are described below.
Neural tube. In E10.5 transverse sections, HMGN1 protein
is visible throughout the embryo, including the surface ecto-
derm, neural tube, dorsal root ganglia, and the mesenchymal
cells (Fig. 2A). The neural tube is already differentiated into an
outer marginal layer, an intermediate mantle layer, and inner
ependymal layer; HMGN1 is strongly expressed in all the three
layers. By E14.5 HMGN1 expression is localized primarily to
the dorsal half of the mantle layer (Fig. 2A, frames c), which
expresses higher HMGN1 levels than the ventral half of the
mantle layer (Fig. 2A, frames d), an indication that HMGN1
expression correlates with neural tube differentiation (19, 36).
Stomach. At E12.5, HMGN1 was detected in both the epi-
thelial and mesenchymal layers of the developing stomach
(Fig. 2B), most prominently in the outermost mesothelium. At
E16.5, when the stomach underwent additional differentiation,
HMGN1 expression in the epithelium was most prominent in
the relatively undifferentiated basal layer (Fig. 2B, bl in frames
Lung. At E12.5 HMGN1 was detected throughout the de-
veloping lung, both in the epithelial and in the dense mesen-
chymal cells (Fig. 2C, me) surrounding the bronchioles (Fig.
FIG. 1. Expression patterns of Hmgn1 during mouse embryogene-
sis. (A) Whole-mount in situ hybridization with digoxigenen-labeled
Hmgn1 antisense probes. At E7.5 strong expression is observed in the
embryonic region (arrowhead). No signal is observed in the ectopla-
cental cone (arrow). At E8.5 and E9.5, strong expression of Hmgn1 is
visible throughout the embryo except in the heart (arrowhead). At
E10.5, HMGN1 is most strongly expressed in the distal portion of
forelimb and hind limb mesenchyme (filled arrowhead), in branchial
arches (arrow), and in tail bud (open arrowhead). After stage E11.5
Hmgn1 expression remains strong mostly in the developing forelimbs
and hind limbs and in the tail. As the embryos develops, Hmgn1
becomes progressively more localized to the interdigit mesenchyme of
the limbs and to the tail. (B) Whole-mount in situ hybridization with
Hmgn1 sense probes. (C) Bar graph depicting the relative expression
levels of Hmgn1 transcripts at the developmental stage indicated or in
the tissue indicated, determined by quantitative dot blot analysis with
RNA master blots (Clonetech), relative to 7-day embryos, which are
considered as 100%.
594FURUSAWA ET AL.MOL. CELL. BIOL.
2C). However at E16.5, when the bronchioles are fully differ-
entiated but the distal epithelium Fig. 2C, de still forms alveoli,
expression of HMGN1 is localized to the developing distal
epithelium and is greatly decreased in the bronchiols (Fig. 2C,
br in frames d).
To examine whether the HMGN1 expression levels are re-
lated to cellular proliferation, we compared the expression
patterns of HMGN1 with that of PCNA. Comparison of the
PCNA-stained panels with the HMGN1-stained panels reveals
that the expression of HMGN1 and PCNA do not always
overlap, which is an indication that in the developing embryo
the levels of HMGN1 are not linked to cellular proliferation
per se, a finding consistent with previous observations in other
experimental systems (16, 24).
FIG. 2. HMGN1 expression correlates with cellular differentiation rather than cell proliferation. The localization of HMGN1 and PCNA in
developing mouse tissues was detected by immunofluorescence. Images at higher magnifications are presented in Fig. S1 to S3 of the supplemental
material. Green staining visualizes either HMGN1 or PCNA, as indicated in the figure. Cell nuclei are counterstained blue with DAPI
(4?,6?-diamidino-2-phenylindole). (A) Sections through the neural tube. (a) At E10.5, HMGN1 was detected in the neural tube (nt), dorsal root
ganglia (drg), and the mesenchymal cells. nl, neural lumen. (b) At E14.5, HMGN1 was localized to the roof plate (rp) and ependymal layer (el)
of the neural tube but down-regulated in the mantle layer (ml) and absent from marginal layer (mg). cc, central canal. (c and d) Higher
magnification of dorsal and ventral regions of E14.5 neural tube indicated in the boxed areas of frames b are shown. (B) Sections through the
developing mouse stomach at E12.5 (a and e) and E16.5 (b and f). At E12.5, HMGN1 was detected both in the epithelial (ep) and mesenchymal
layer (me); however at E16.5, its expression was localized to basal layer (bl) of gastric mucosa and longitudinal muscle layer (lm). mt, mesothelium;
ls, lumen of stomach; pc, peritoneal cavity; lv, liver; cm, circular muscle layer. Frames c and d show higher magnifications of E16.5 stomach wall
lumenal side and peritoneal cavity side indicated by boxed areas in frames b. (C) Section through the lung. (a) At E12.5, HMGN1 was detected
in both of the bronchi (br) and surrounding mesenchymal cells (me). (b) At E16.5, bronchial expression of HMGN1 was down-regulated in the
proximal region (br) and decreased in the mesenchymal region (me) but remained strong in the distal region (de). (c and d) Higher magnification
of distal bronchi and proximal bronchi of E16.5 lung section indicated by boxed areas in frames b. Corresponding PCNA staining is shown in
adjacent sections of all panels.
VOL. 26, 2006A ROLE FOR HMGN1 IN CHONDROGENESIS 595
Our analyses revealed that in every tissue examined, the
expression of HMGN1 was down-regulated as organ differen-
tiation proceeded during development. However, the levels of
the protein remained high in progenitor cells, such as the basal
skin layer cells that are committed and poised for further
differentiation and renewal. Thus, the dot blot RNA analysis,
the in situ hybridization, and the immunofluorescence analyses
indicate a differentiation-related down-regulation of HMGN1
Reciprocal expression patterns of Hmgn1 and Sox9 in the
developing limb bud. To gain additional insights into the role
of HMGN1 in developmental processes, we focused on its
possible involvement in the differentiation of the limb bud,
where Hmgn1 expression correlated inversely with mesenchy-
mal chondrogenic differentiation. Whole-mount in situ hybrid-
ization demonstrates clearly that the expression of Hmgn1 is
down-regulated during development and is complementary to
that of Sox9, a major transactivator involved in the initiation
FIG. 3. Reciprocal expression patterns of Hmgn1 and Sox9 during limb bud development. (A to J) Whole-mount in situ hybridization analysis
of Hmgn1 and Sox9 expression in E10.5 to E14.5 forelimbs. At E10.5 Hmgn1 is strongly expressed in the distal region of limb bud (arrowhead in
A) while weak Sox9 expression commences in the proximal region where Hmgn1 is down-regulated (arrowhead in B). In the E11.5 limb bud, the
expression of Hmgn1 starts to be restricted to the interdigit mesenchyme (arrowheads in C), whereas Sox9 expression is observed in the early digit
condensation (arrowheads in D). At E12.5 Hmgn1 is expressed in the interdigit mesenchyme (arrowheads in E), while Sox9 is expressed in digit
chondrogenic condensations (arrowheads F). In the E13.5 forelimb bud the expression of Hmgn1 in the interdigit mesenchyme begins to weaken
(arrowheads in G) and is restricted to perichondrium while the expression of Sox9 is strongest in the distal regions of the digit cartilage (arrowheads
in H). At E14.5 Hmgn1 remains localized to the perichondrium while Sox9 is expressed mainly in the distal digit cartilage (I and J). (K to R)
Immunohistochemical staining of HMGN1 and Sox9 protein in the developing forelimb bud. Higher magnifications are shown in supplemental Fig.
S4. At E10.5, HMGN1 is strongly expressed in distal mesenchyme (K, arrow) and in the epithelium (K, arrowhead), while Sox9 is strongly expressed
in the proximal mesenchyme (L, arrow). At E12.5, HMGN1 protein expression is seen through most of the limb bud but is relatively weak in the
differentiating digit chondrocytes (M, arrows) where Sox9 protein is strongly expressed (N). At E14.5, the relative levels of HMGN1 protein (Q) are
highest in the skin (sk) and perichondrium (pc) and lowest in the digit cartilage (dc). Conversely, Sox9 protein is strongly expressed in the digit
596 FURUSAWA ET AL.MOL. CELL. BIOL.
and propagation of chondrogenesis (Fig. 3). Thus, in the E10.5
forelimb bud (and hind bud; data not shown), Hmgn1 was
strongly expressed in a broad anterior and distal region and
down-regulated in the proximal region (Fig. 3A), where mes-
enchymal cells condense and differentiate into chondrocytes,
as evidenced by the onset of Sox9 expression (Fig. 3B). As
chondrogenesis initiated in the more distal digit region over
time, expression of Hmgn1 was localized to the future inter-
digit region and down-regulated in the future digit region (Fig.
3C), coinciding with activation of Sox9 expression (Fig. 3D). In
the E12.5 forelimb bud, Hmgn1 expression is most prominent
in the interdigit mesenchyme (Fig. 3E), while Sox9 transcripts
are most abundant in future digit regions (Fig. 3F). In the
E13.5 and E14.5 forelimb buds, Hmgn1 expression is most
prominent in the interdigital mesenchyme (Fig. 3G and I),
while Sox9 transcripts are most abundant in the cartilage of
phalanges (Fig. 3H and J). A similar pattern of reciprocal
Hmgn1 and Sox9 expression was observed during hind limb
bud development (data not shown). Thus, during the develop-
ment of both limbs, the decline of Hmgn1 expression preceded
that of Sox9, the latter being expressed in regions with signif-
icantly diminished levels of HMGN1 (Fig. 3E to J).
More detailed immunohistochemical analyses of sections
from the developing limb bud region verified the reciprocal
expression of Hmgn1 and Sox9. The expression of Hmgn1 de-
creased while that of Sox9 increased as the prechondrogenic
mesenchyme condensed in the cartilage primordia. Thus, in
the E10.5 and E12.5 forelimb buds, Sox9 protein was observed
in the proximal region (Fig. 3L) and digit cartilages (Fig. 3N),
while HMGN1 protein was expressed in the surface ectoderm
and throughout the distal mesenchyme region but absent from
the prechondrogenic mesenchyme (Fig. 3K and M). In the
more fully differentiated E14.5 forelimb, HMGN1 protein was
abundant in the perichondrium of the phalanges but depleted
from the cartilage, where Sox9 protein was expressed (Fig. 3).
At higher magnification the difference in the expression pat-
terns of HMGN1 and Sox9 in the developing phalanges was
very clear: the expression of Sox9 was confined to the cartilage,
while HMGN1 was expressed in the surrounding perichon-
drium but not in cartilage (Fig. 3Q and 3R; see Fig. S4 in the
Reciprocal expression patterns of Hmgn1 and Sox9 in mi-
cromass cultures. To gain additional insights into the role of
HMGN1 in chondrocyte differentiation, we analyzed the pat-
tern of Hmgn1 and Sox9 expression in a micromass culture
system (Fig. 4A), which has been commonly used as a model to
study in vitro chondrogenesis. When plated at high density,
mouse limb bud mesenchymal cells differentiate into chondro-
cytes and form cartilage nodules (Fig. 4A). The progress of
differentiation can be assessed by Alcian blue staining, which is
most prominent in differentiated nodules (Fig. 4B). Under our
growth conditions, nodules of differentiated chondrocytes can
be seen within 3 days of plating (Fig. 4B, ?) and distinct,
strongly staining nodules containing differentiated chondro-
cytes are clearly visible after 5 days of plating. In situ hybrid-
ization with digoxigenin-labeled RNA probes indicates that the
levels of Hmgn1 transcripts decrease while those of Sox9 in-
crease during differentiation. Thus, 2 days after plating, Hmgn1
was expressed throughout most of the monolayer cells. At day
3, Hmgn1 expression was down-regulated in the regions in
which the cells condensed into the evolving nodules (Fig. 4B,
?), and by day 5, Hmgn1 transcripts were highly depleted and
practically absent in the differentiated cartilage nodule (Fig.
4C, top panels). In contrast, Sox9 expression was first observed
in the small cell aggregates detectable after 2 days of culturing.
In the evolving nodules, the levels of Sox9 RNA gradually
increases and is prominent in the fully differentiated, 5-day-old
nodules (Fig. 4C, bottom panels).
Confocal immunofluorescence analysis of the fully formed
5-day-old nodules revealed reciprocity in HMGN1 and Sox9
protein levels at the single-cell resolution. The fully differen-
tiated nodules present after 5 days of growth in micromass
culture were clearly depleted of HMGN1 protein, while the
undifferentiated cells surrounding the nodules expressed high
levels of HMGN1 protein (Fig. 4D, frame a). In contrast, Sox9
levels were high in the nodules but undetectable in the undif-
ferentiated cells surrounding the nodules. Although higher
magnifications revealed some residual HMGN1 protein in the
nodule, a merge of the DNA and HMGN1 confocal images
clearly demonstrates a reduced level of HMGN1 protein in the
cells at the center of the nodules. In contrast, Sox9 levels are
highest in the cells at the center of the nodule and gradually
decrease toward the periphery of the nodule; Sox9 is absent
from the cells surrounding the nodules. In the DNA-Sox9
image merge, the center of the nodule is green (high protein
levels) and the surrounding cells are red (high DNA, low
protein), while in the DNA-HMGN1 merge the opposite is
visible: the center is red due to low HMGN1 (light green),
while the surrounding cells are green due to relatively high
levels of HMGN1 (Fig. 4D, frame b).
Thus, the expression levels of Hmgn1 and Sox9 in the mi-
cromass cultures faithfully reproduce the Hmgn1 and Sox9
expression patterns observed in mouse embryos. Chondrocyte
differentiation is associated with decreased levels of HMGN1
protein and increased levels of Sox9. Taken together, the data
demonstrate reciprocity in the expression of Hmgn1 and Sox9.
HMGN1 modulates chondrocyte differentiation. The recip-
rocal expression of HMGN1 and Sox9 raises the possibility that
HMGN1 modulates the rate of chondrocyte differentiation. To
test this possibility, we first compared the rate of nodule for-
mation in micromass cultures prepared from cells derived from
the limb buds of wild-type and Hmgn1?/?E10.5 embryos. On
the basis of Alcian blue staining, Hmgn1?/?cultures were
found to be differentiated faster than Hmgn1?/?cultures at all
time points examined (Fig. 5A). Quantification of Alcian blue
staining confirmed that the rate of differentiation of the mi-
cromass prepared from Hmgn1?/?limb bud cells was signifi-
cantly faster than that prepared from Hmgn1?/?limb buds.
After 4 days in culture, the amount of Alcian blue stain recov-
ered from the Hmgn1?/?cultures was almost twice that ob-
tained from Hmgn1?/?cultures, and after 5 days it was still
more than 1.5 times higher (Fig. 5B). Thus, genetic inactiva-
tion of Hmgn1 accelerated differentiation.
To further test this possibility, we examined the effect of
HMGN1 on cartilage nodule formation by reexpressing
HMGN1 protein in Hmgn1?/?cultures. To this end, E10.5
limb bud mesenchymal cells prepared from Hmgn1?/?embryo
were transiently transfected with vectors expressing either YFP
(control) or the HMGN1-YFP fusion protein. Fluorescent
analysis of the cells verified that the expression level of the
VOL. 26, 2006A ROLE FOR HMGN1 IN CHONDROGENESIS 597
FIG. 4. Reciprocal expression patterns of Hmgn1 and Sox9 during chondrogenesis in vitro in micromass cultures. (A) Schematic diagram of
micromass culture method. Mesenchymal limb bud cells obtained from E10.5 embryos are dissociated by trypsin-collagenase treatment, collected
and concentrated by centrifugation, and then resuspended in the culture medium at high density (2.0 ? 107cells/ml). Cells are plated as a 10-?l
spot and flooded with medium after 1 h of incubation and adhesion. (B) Detection of chondrocyte differentiation by Alcian blue staining.
598FURUSAWA ET AL.MOL. CELL. BIOL.
control plasmid transcribing YFP was similar to that of the
plasmid transcribing the HMGN1-YFP fusion protein (Fig.
6A, frames a and b). The number and staining intensity of the
Alcian blue-positive nodule in the cultures expressing the
HMGN1-YFP fusion protein were significantly lower than
those in the cultures expressing the control YFP protein (com-
pare Fig. 6A, frames d and e, and B). Likewise, the levels of
Sox9 in cells expressing HMGN1-YFP were lower than in
those expressing only YFP (Fig. 6A, frames g and h). Thus,
overexpression of HMGN1 inhibited the formation of differ-
entiated nodules and the expression of Sox9, suggesting that
down-regulation of HMGN1 is required for chondrocyte dif-
To test whether the HMGN1-induced inhibition of nodule
formation is related to the ability of the protein to bind to
chromatin, we expressed the double point mutant HMGN1
(S20,24E)-YFP fusion protein, rather than the wild-type fusion
protein, in the Hmgn1?/?
HMGN1(S20,24E) mutant enters the nucleus but does not
bind nucleosomes (26). In contrast to the wild-type HMGN1,
this mutant did not affect Sox9 expression (Fig. 6A, frames f
and i), and the rate of nodule formation was comparable to
that observed in cells transfected with the control plasmid
expressing YFP (Fig. 6A, compare frames d and g to f and i,
and B). Quantification of the effect by measuring the Alcian
blue staining in the cultures (Fig. 6B) revealed that the amount
of stain in the cultures expressing wild-type HMGN1 was 60%
of that in control cells transfected with plasmids expressing
either YFP or the HMGN1(S20,24E) mutant (Fig. 6C). Thus,
reexpression of wild-type but not mutant HMGN1 in
Hmgn1?/?limb bud cells reduced the rate of Sox9 expression
and chondrocyte differentiation. We therefore conclude that
the interaction of HMGN1 with chromatin affects the rate of
chondrocyte differentiation by modulating the expression lev-
els of chondrogenic factors such as Sox9.
Specific binding of HMGN1 to the Sox9 gene. The interre-
lationship between HMGN1 and Sox9 expression raised the
possibility that HMGN1 protein is directly involved in the
regulation of Sox9 gene expression. We therefore tested
whether HMGN1 protein was directly associated with the Sox9
gene by using a ChIP assay with affinity-purified antibodies
against mouse HMGN1. The ChIP analyses were performed
with chromatin isolated either from nonchondrogenic MEFs
or from E10.5 limb bud cells, which contain both prechondro-
genic cells and chondrocytes (Fig. 3A and B). The relative
amounts of Hmgn1 transcripts and protein in the MEFs and
limb bud cells are similar, while those of Sox9 are almost 10
times higher in the limb bud cells (Fig. 7C and D). However,
although Sox9 expression is detected in the E10.5 limb bud,
most of the cells are only poised to express the gene; high-level
E10.5 limb bud cells. The
Sox9 expression occurs at later developmental stages, when
HMGN1 is significantly down-regulated (Fig. 3).
The DNA purified from the immunoprecipitated chromatin
was amplified with 21 primer sets spanning an approximately
10-kb long genomic region encompassing the Sox9 gene and its
4.4-kb 5? and 5.7-kb 3? flanking regions. In limb bud cells, but
not in MEFs, these analyses identified three regions in the
Sox9 chromatin as enriched in HMGN1: a region 2 kb up-
stream of the promoter, exon 2, and exon 3 (Fig. 7A and B).
The DNA sequences of the three regions do not contain any
common motifs (data not shown), supporting previous findings
that the interaction of HMGN1 with chromatin is not regu-
lated by the sequence of the nucleosomal DNA (28).
To test whether the presence of HMGN1 is associated with
changes in chromatin structure, we first focused on the region
spanned by primer set 3 (Fig. 7A), since its location was in the
5? region of the gene and may be involved in the regulation of
Sox9 gene expression. We digested nuclei isolated from E10.5
limb bud cells or MEFs with different amounts of DNase I and
used PCR to determine the amount of undigested DNA in a
748-bp long region amplified by forward primer 3 and reverse
primer 4. The yield of the resulting amplified fragment is a
measure of the relative rate of DNA digestion in chromatin. A
DNase I-hypersensitive site between primer sets 3 and 4 in the
Sox9 promoter in nuclei prepared from E10.5 limb bud cells
was digested faster than in MEF nuclei (Fig. 7E), suggesting
that the chromatin structure of Sox9 promoter in limb bud
cells, which are poised to begin Sox9 expression, is less com-
pact than the corresponding region in MEFs. Similar results
were obtained by analysis of other regions of the Sox9 gene,
suggesting that the HMGN1-containing gene is more suscep-
tible to DNase I digestion. Similar analysis of the DNase I
sensitivity of the beta-globin gene, which is not expressed in
either MEFs or limb bud cells, showed only minor differences
between the two cell types (Fig. 7F). Our finding both in vivo
and in vitro that the expression of Hmgn1 precedes that of Sox9
and that HMGN1 is associated with Sox9 chromatin in limb
bud but not in MEFs argues for a role for HMGN1 in Sox9
gene regulation during chondrocyte development.
Enhanced binding of HMGN2 on the Sox9 gene in
Hmgn1?/?mice. The lack of phenotype in Hmgn1?/?mice
raises the possibility that homeostatic mechanisms, perhaps
involving HMGN2, compensate for loss of HMGN1 protein.
The levels of HMGN2 RNA and protein in Hmgn1?/?mice is
the same as in their Hmgn1?/?littermates (not shown). ChIP
analysis with affinity pure antibodies to HMGN2 revealed the
levels of this protein in the 10.5-day limb bud Sox9 chromatin
are higher than in the Sox9 chromatin of MEFs (Fig. 8). In-
terestingly, while HMGN1 was enriched in distinct regions of
the gene (Fig. 7B), HMGN2 protein was enriched along the
Mesenchymal cells plated at high density differentiate into chondrocytes after 3 to 5 days in culture. (C) Reciprocal expression of Hmgn1 and Sox9
transcripts during mesenchymal differentiation into nodules. Plus symbols indicate developing nodules. After 5 days, Hmgn1 expression is
down-regulated in the fully differentiated region of the chondrogenic nodule, where Sox9 expression is strongly expressed. (D) Confocal
immunofluorescence analysis of HMGN1 and SOX9 expression in 5-day-old differentiated nodules. (a) Low magnification. HMGN1 protein is
absent from the chondrocytic nodule, while SOX9 protein is detected only within the nodule. (b) Higher magnification of the images shown in part
a centered on a differentiating nodule. Note that in the DNA-Sox9 merge, the center of the nodule is mostly green (high Sox protein levels) and
the surrounding cells are red (low Sox protein; DNA stain prevails), while in the DNA-HMGN1 confocal merge the center is red due to low
HMGN1 (light green) and the surrounding cells are green due to relatively high levels of HMGN1. Proteins are shown in green, and DNA
(Hoechst) is shown in red. Phase-contrast images are used to visualize node morphology. Scale bar, 50 ?m.
VOL. 26, 2006A ROLE FOR HMGN1 IN CHONDROGENESIS599
entire length of the gene (Fig. 8A). Significantly, in the Sox9
chromatin derived from the limb bud of Hmgn1?/?mice, the
level of HMGN2 was enriched in several regions, especially
those spanning primer sets 6 to 10 and 17 to 19 (Fig. 8B). The
increased level of HMGN2 in the Sox9 chromatin of the
Hmgn1?/?limb bud suggests functional redundancy among
The widespread occurrence of HMGN proteins in verte-
brate nuclei and their ability to alter the structure of chromatin
and modulate access to nucleosomes raise the possibility that
they play a role in gene expression and cellular differentiation.
To gain insights into the possible role of HMGN in vertebrate
development, we first analyzed the pattern of HMGN1 expres-
sion during mouse embryogenesis and then focused on the
possible role of this protein in modulating the expression of
Sox9, a master regulator of chondrocyte differentiation.
A role for HMGN1 in Sox9 expression. Numerous types of
experiments suggest a role for HMGN1 in transcription from
chromatin templates (4); however, it is still not clear whether
HMGN proteins act indiscriminately as general facilitators or
whether they play a role in specific gene expression. DNA
array analysis of the expression pattern of UV-treated
Hmgn1?/?and Hmgn1?/?fibroblasts (3) and of fibroblasts
expressing increased amounts of HMGN3 (32) linked the
HMGN proteins to changes in the transcript levels of specific
genes. Likewise, studies on Xenopus development suggest that
altered expression levels of HMGNs alter the expression of
specific genes such as Xbra and chordin (15). These findings
and additional studies (17) suggest that HMGN proteins may
affect the transcription of specific genes rather than act only as
general coregulators of transcription from chromatin. Our
present findings that HMGN1 is associated with the Sox9 chro-
matin of limb bud cells but not of nonchondrogenic MEFs
(Fig. 7) provides evidence for the involvement of HMGN1 in
tissue-specific gene regulation. The ChIP analyses with
HMGN2 indicate a moderate enrichment of HMGN2 along
the entire length of the Sox9 gene, suggesting that this protein
is also involved in the transcription of this gene. Our finding
that the Sox9 chromatin in Hmgn1?/?is enriched in HMGN2
is particularly significant. Loss of HMGN1 does not change the
cellular levels of HMGN2, and the expression of HMGN2
during limb development is indistinguishable from that of
HMGN1 (not shown). The results suggest that HMGN2 re-
places the missing HMGN1, the first direct experimental evi-
dence for functional redundancy among members of the
HMGN protein family.
The regulation of Sox9 transcription is not fully understood.
In human SOX9, regulatory elements are scattered through a
FIG. 5. Loss of HMGN1 enhances the rate of differentiation.
(A) Alcian blue staining of micromass cultures derived from E10.5
limb bud mesenchymal cells of Hmgn1?/?or Hmgn1?/?embryos after
different lengths of time in culture. (B) Quantification of Alcian blue-
stained cells. Alcian blue-stained cultures were lysed (12), centrifuged,
and the A595values of the supernatants were determined.
600FURUSAWA ET AL.MOL. CELL. BIOL.
350-kb region upstream of the start of transcription (35). The
enhancer of the human SOX9 is located in the first intron,
while the promoter of the mouse Sox9 gene is located 193 bp
upstream of the transcriptional start site (13, 21). The associ-
ation of HMGNs with the chromatin of E10.5 limb bud cells
may serve to generate a chromatin structure that poises the
Sox9 gene for transcription at the proper developmental stage.
This view is in agreement with the original suggestion that the
binding of HMGNs to chromatin potentiates genes for tran-
scription (31) and with subsequent studies of in vitro reconsti-
tuted chromatin templates (reviewed in reference 4). At later
developmental stages, when cells are committed to a differen-
tiation pathway, HMGNs may still play a positive role in Sox9
expression; however, high levels of HMGNs may interfere with
proper development and Sox9 expression. This view is in agree-
ment with our present observations that ectopic expression of
HMGN1 in Hmgn1?/?micromass cultures inhibits the rate of
differentiation and Sox9 expression and with previous studies
in Xenopus (15) and mice (20) indicating that either depletion
or overexpression of HMGN protein interferes with proper
Developmental role for HMGN proteins. Our studies link
the expression of HMGN1 protein to cellular differentiation.
We observe a widespread, progressive down-regulation of
Hmgn1 expression during embryonic development in every tis-
sue examined, only a few types of which are shown in Fig. 2.
We also found that these expression patterns were not identi-
cal to the PCNA expression pattern, suggesting that the ex-
pression level of HMGN1 does not simply reflect the cellular
proliferation state. HMGN1 expression is significantly reduced
in differentiated tissues but remains robust in undifferentiated
cells or cells with regenerative-renewal capacity, such as the
basal layer of skin, the inner lumen of stomach, the branching
distal epithelium of lung, or the peripheral mesenchymal cells
layer of the kidney (Fig. 2 and T. Furusawa, unpublished data).
These results are consistent with tissue culture studies that
demonstrated down-regulation of Hmgn1 expression during
erythropoiesis, myogenesis, and osteoblast differentiation (8,
23, 27) and with biochemical studies that linked this down-
regulation specifically to differentiation rather than a de-
creased rate of cellular replication (4). Likewise, during kidney
organogenesis, the closely related protein HMGN2 is ex-
pressed mainly in cells undergoing inductive interactions and
differentiation (16) and is down-regulated in terminally differ-
entiated cells. In the developing limb bud, the expression of
Hmgn1 was restricted to the undifferentiated mesenchymal
regions. The prechondrogenic regions and the differentiated
digits contained very little, if any, HMGN1 (Fig. 2). These
observations raise the possibility that down-regulation of
HMGN1 expression is a prerequisite of proper differentiation
and, conversely, that elevated expression of Hmgn1 may retard
cellular differentiation. Indeed, overexpression of Hmgn1 re-
duced the number and size of the differentiated nodules (Fig.
4). Likewise, previous studies with differentiation of C2C12
myoblasts demonstrated that ectopic expression of HMGN1
inhibits myotube formation (23). Thus, down-regulation of
Hmgn1 is required for both chondrogenesis and myogenesis in
vitro. These findings and previous studies with microinjected
one-cell mouse embryos (20) and with Xenopus laevis embryos
FIG. 6. HMGN1 reduces the rate of chondrocyte differentiation.
(A) E10.5 limb bud mesenchyme cells derived from Hmgn1?/?em-
bryos were transfected with plasmids expressing either YFP (Control),
HMGN1-YFP (HMGN1) fusion protein or HMGN1(S20,24E)-YFP
(HMGN1 S20,24E), a mutant that cannot bind to nucleosomes. Trans-
fection efficiency was assessed by YFP after 2 days of culture, and
chondrocyte differentiation was assessed by Alcian blue staining or
immunostaining for Sox9, as indicated, after 4 days of culture. Note
that the numbers and the sizes of Alcian blue- or Sox9-positive nodules
(arrows point to representative nodules) were lower in the HMGN-
transfected micromass (e and h), whereas the mutant HMGN1(S20,24E)
had no effect (f and i). (B) Representative high-magnification micro-
graphs of fields taken from different plates of Alcian blue-stained 8-day-
old micromass cultures transfected with vectors expressing the protein
indicated at the top of the fields. (C) Quantification of Alcian blue stain-
ing by measuring absorbance at 595 nm.
VOL. 26, 2006A ROLE FOR HMGN1 IN CHONDROGENESIS 601
FIG. 7. HMGN1 binds to Sox9 chromatin. (A and B) ChIP analysis (with anti HMGN1) of mouse MEFs and E10.5 limb bud cells.
(A) Schematic diagram of Sox9 gene, with the positions of primer sets used to amplify the immunoprecipitated DNAs are indicated at the bottom.
The arrow indicates transcription start site. (B) ChIP assay for HMGN1 in MEFs and limb bud cells. Enrichment of each DNA sequence in the
HMGN1 immunoprecipitate relative to the input DNA was normalized and plotted as the position of the PCR primer pair within the Sox9 gene
locus. Note that HMGN1 is associated with Sox9 chromatin in limb bud cells but not in MEFs. (C) Expression levels of Hmgn1 and Sox9 mRNA
in MEFs and limb bud cells. Note that while HMGN1 is expressed in both cell types, Sox9 is expressed only in limb bud cells. (D) Western analysis
of HMGN1 and Sox9 proteins in limb bud cells and MEFs. Coomassie blue staining of histones indicates equal loading of extracts from MEFs and
limb bud cells. (E) Enhanced DNase I sensitivity of Sox9 chromatin in limb bud cells. Nuclei isolated from MEF or E10.5 limb bud cells were
digested with the indicated concentrations of DNase I, the DNA from the digested nuclei purified and amplified with forward primer of primer
pair 3 and reverse primer of primer pair 4. The level of amplification is inversely related to degree of digestion. Each point is an average from three
experiments, with a new preparation of cells each time. (F) Similar rate of DNase I sensitivity of the beta-globin gene in chromatin of limb bud
cells and MEFs. Primers used for amplification are described in Materials and Methods. LB, limb bud cells.
602 FURUSAWA ET AL.MOL. CELL. BIOL.
(15) argue for a role of HMGNs in vertebrate embryonic
In X. laevis, misexpression of either of the two major mem-
bers of the HMGN protein family, HMGN1 or HMGN2, leads
to specific, developmentally timed abnormalities in the grow-
ing embryos (15). Either overexpression or depletion of
HMGNs in Xenopus zygotes caused severe malformations in
postblastula development without affecting preblastula devel-
opment. Significantly, HMGN proteins are expressed only af-
ter midblastula transition. The data suggest that once the pro-
teins are synthesized and functional, their levels need to be
monitored, and either too much or too little protein leads to
In considering molecular mechanisms whereby HMGN pro-
teins affect differentiation processes, we note that in the in
vitro micromass cultures, loss of Hmgn1 resulted in faster
chondrogenesis. In this system, reexpression of HMGN1 pro-
tein in Hmgn1?/?cells restores the wild-type phenotype and
retards the rate of differentiation, whereas reexpression of
HMGN1(S20,24E), the double point mutant form of HMGN1
which does not bind to nucleosomes, did not restore the wild-
type phenotype (Fig. 5 and 6). Likewise, this mutant protein
did not lead to developmental abnormalities in X. laevis (15).
In one-cell stage mouse embryos, disruption of the interaction
of HMGN with chromatin led to an abnormal rate of devel-
opment (20). Taken together, these results argue that HMGNs
modulate a differentiation-related process by interacting with
Since Hmgn1?/?mice seem to develop normally (2), it is
likelythat homeostatic mechanisms,
HMGN2, compensate for the loss of the HMGN1 protein.
Indeed, our ChIP analyses indicate an increase of HMGN2 in
the Sox9 chromatin obtained from Hmgn1?/?mice, suggesting
functional redundancy among these proteins. We also ob-
served that during embryogenesis and during limb bud devel-
opment, Hmgn2 is expressed in a pattern that is indistinguish-
able from that of Hmgn1 (T. Furusawa, unpublished data).
Furthermore, we already demonstrated that transient deple-
tion of both Hmgn1 and Hmgn2 transcripts in 1-day-old mouse
embryos was necessary to retard the progression of preimplan-
tation development. Depletion of only one of the Hmgn tran-
scripts had no significant effect on the rate of development and
FIG. 8. Enhanced binding of HMGN2 on the Sox9 gene in Hmgn1?/?mice. (A) ChIP assay for HMGN2 on Sox9 in MEF and limb bud cells.
Enrichment of each DNA sequence in the HMGN2 immunoprecipitate relative to the input DNA was normalized to the expression of the
beta-globin gene and plotted as the position of the PCR primer pair within the Sox9 gene locus (as described in the legend of Fig. 7). (B) ChIP
assay for HMGN2 on Sox9 chromatin in limb bud cells obtained from Hmgn1?/?mice (KO-LB). The results are shown together with the plots
of HMGN2 and HMGN1 in Hmgn1?/?mice, superimposed for comparison. Each point is an average from three experiments, with a new
preparation of each time limb bud cells. LB, limb bud cells.
VOL. 26, 2006A ROLE FOR HMGN1 IN CHONDROGENESIS603
did not cause an embryonic developmental delay (20), suggest-
ing a functional overlap that compensates for loss of one of the
major components of HMGN protein.
In the nucleus, HMGN1 functions as a member of a dynamic
network of chromatin binding proteins, involving other HMG
proteins and histone H1, which compete for nucleosome bind-
ing sites (6, 7). As discussed elsewhere (6), changes in one
component of the network may trigger compensatory adjust-
ments in other components of the network and in chromatin,
all aimed to optimize the cellular requirement at any given
time. Thus, changes in HMGN1 alter the interaction of H1
with chromatin (7) and modify the pattern of posttranslational
modification of histones (17, 18). These changes reflect the
action of homeostatic mechanisms that allow survival and de-
velopment of Hmgn1?/?mice, which mostly appear normal.
Yet their stress response is significantly impaired, and they are
hypersensitive to irradiation by either UV (3) or X ray (2). By
analogy, the Hmgn1?/?limb bud cells grown in micromass
cultures are placed under conditions in which homeostatic
mechanisms operating in the intact embryo are altered, thus
leading to changes in Sox9 expression and in differentiation of
We thank Susan H. Garfield and Stephen M. Wincovitch (Labora-
tory of Experimental Carcinogenesis, Center for Cancer Research,
National Cancer Institute) for help with the confocal microscopy and
the NCI CCR Fellows Editorial Board for constructive criticisms of the
T.F. is a recipient of a fellowship from the Japan Society for Pro-
motion of Sciences. This research was supported by the Center for
Cancer Research, National Cancer Institute, NIH.
1. Begum, N., J. M. Pash, and J. S. Bhorjee. 1990. Expression and synthesis of
high mobility group chromosomal proteins in different rat skeletal cell lines
during myogenesis. J. Biol. Chem. 265:11936–11941.
2. Birger, Y., F. Catez, T. Furusawa, J. H. Lim, M. Prymakowska-Bosak, K. L.
West, Y. V. Postnikov, D. C. Haines, and M. Bustin. 2005. Increased tumor-
igenicity and sensitivity to ionizing radiation upon loss of chromosomal
protein HMGN1. Cancer Res. 65:6711–6718.
3. Birger, Y., K. L. West, Y. V. Postnikov, J. H. Lim, T. Furusawa, J. P. Wagner,
C. S. Laufer, K. H. Kraemer, and M. Bustin. 2003. Chromosomal protein
HMGN1 enhances the rate of DNA repair in chromatin. EMBO J. 22:1665–
4. Bustin, M. 2001. Chromatin unfolding and activation by HMGN(*) chro-
mosomal proteins. Trends Biochem. Sci. 26:431–437.
5. Bustin, M. 1999. Regulation of DNA-dependent activities by the functional
motifs of the high-mobility-group chromosomal proteins. Mol. Cell. Biol.
6. Bustin, M., F. Catez, and J. H. Lim. 2005. The dynamics of histone H1
function in chromatin. Mol. Cell 17:617–620.
7. Catez, F., H. Yang, K. J. Tracey, R. Reeves, T. Misteli, and M. Bustin. 2004.
Network of dynamic interactions between histone H1 and high-mobility-
group proteins in chromatin. Mol. Cell. Biol. 24:4321–4328.
8. Crippa, M. P., J. M. Nickol, and M. Bustin. 1991. Developmental changes in
the expression of high mobility group chromosomal proteins. J. Biol. Chem.
9. Crippa, M. P., J. M. Nickol, and M. Bustin. 1991. Differentiation-dependent
alteration in the chromatin structure of chromosomal protein HMG-17 gene
during erythropoiesis. J. Mol. Biol. 217:75–84.
10. de Crombrugghe, B., V. Lefebvre, and K. Nakashima. 2001. Regulatory
mechanisms in the pathways of cartilage and bone formation. Curr. Opin.
Cell Biol. 13:721–727.
11. Ding, H. F., S. Rimsky, S. C. Batson, M. Bustin, and U. Hansen. 1994.
Stimulation of RNA polymerase II elongation by chromosmal protein
HMG-14. Science 265:796–799.
12. Hatakeyama, Y., R. S. Tuan, and L. Shum. 2004. Distinct functions of BMP4
and GDF5 in the regulation of chondrogenesis. J. Cell Biochem. 91:1204–
13. Kanai, Y., and P. Koopman. 1999. Structural and functional characterization
of the mouse Sox9 promoter: implications for campomelic dysplasia. Hum.
Mol. Genet. 8:691–696.
14. Kelly, R. G., and M. E. Buckingham. 2002. The anterior heart-forming field:
voyage to the arterial pole of the heart. Trends Genet. 18:210–216.
15. Korner, U., M. Bustin, U. Scheer, and R. Hock. 2003. Developmental role of
HMGN proteins in Xenopus laevis. Mech. Dev. 120:1177–1192.
16. Lehtonen, S., and E. Lehtonen. 2001. HMG-17 is an early marker of induc-
tive interactions in the developing mouse kidney. Differentiation 67:154–163.
17. Lim, J. H., F. Catez, Y. Birger, K. L. West, M. Prymakowska-Bosak, Y. V.
Postnikov, and M. Bustin. 2004. Chromosomal protein HMGN1 modulates
histone H3 phosphorylation. Mol. Cell 15:573–584.
18. Lim, J. H., K. L. West, Y. Rubinstein, M. Bergel, Y. V. Postnikov, and M.
Bustin. 2005. Chromosomal protein HMGN1 enhances the acetylation of
lysine 14 in histone H3. EMBO J.
19. Megason, S. G., and A. P. McMahon. 2002. A mitogen gradient of dorsal
midline Wnts organizes growth in the CNS. Development 129:2087–2098.
20. Mohamed, O. A., M. Bustin, and H. J. Clarke. 2001. High-mobility group
proteins 14 and 17 maintain the timing of early embryonic development in
the mouse. Dev. Biol. 229:237–249.
21. Morishita, M., T. Kishino, K. Furukawa, A. Yonekura, Y. Miyazaki, T.
Kanematsu, S. Yamashita, and T. Tsukazaki. 2001. A 30-base-pair element
in the first intron of SOX9 acts as an enhancer in ATDC5. Biochem. Bio-
phys. Res. Commun. 288:347–355.
22. Paranjape, S. M., A. Krumm, and J. T. Kadonaga. 1995. HMG17 is a
chromatin-specific transcriptional coactivator that increases the efficiency of
transcription initiation. Genes Dev. 9:1978–1991.
23. Pash, J. M., P. J. Alfonso, and M. Bustin. 1993. Aberrant expression of high
mobility group chromosomal protein 14 affects cellular differentiation.
J. Biol. Chem. 268:13632–13638.
24. Pash, J. M., J. S. Bhorjee, B. M. Patterson, and M. Bustin. 1990. Persistence
of chromosomal proteins HMG-14/-17 in myotubes following differentia-
tion-dependent reduction of HMG mRNA. J. Biol. Chem. 265:4197–4199.
25. Phair, R. D., and T. Misteli. 2000. High mobility of proteins in the mam-
malian cell nucleus. Nature 404:604–609.
26. Prymakowska-Bosak, M., T. Misteli, J. E. Herrera, H. Shirakawa, Y. Birger,
S. Garfield, and M. Bustin. 2001. Mitotic phosphorylation prevents the
binding of HMGN proteins to chromatin. Mol. Cell. Biol. 21:5169–5178.
27. Shakoori, A. R., T. A. Owen, V. Shalhoub, J. L. Stein, M. Bustin, G. S. Stein,
and J. B. Lian. 1993. Differential expression of the chromosomal high mo-
bility group proteins 14 and 17 during the onset of differentiation in mam-
malian osteoblasts and promyelocytic leukemia cells. J. Cell Biochem. 51:
28. Shirakawa, H., J. E. Herrera, M. Bustin, and Y. Postnikov. 2000. Targeting
of high mobility group-14/-17 proteins in chromatin is independent of DNA
sequence. J. Biol. Chem. 275:37937–37944.
29. Shum, L., C. M. Coleman, Y. Hatakeyama, and R. S. Tuan. 2003. Morpho-
genesis and dysmorphogenesis of the appendicular skeleton. Birth Defects
Res. C 69:102–122.
30. Trieschmann, L., P. J. Alfonso, M. P. Crippa, A. P. Wolffe, and M. Bustin.
1995. Incorporation of chromosomal proteins HMG-14/-17 into nascent nu-
cleosomes induces an extended chromatin conformation and enhances the
utilization of active transcription complexes. EMBO J. 14:1478–1489.
31. Weisbrod, S. 1982. Active chromatin. Nature 27:289–295.
32. West, K. L., M. A. Castellini, M. K. Duncan, and M. Bustin. 2004. Chro-
mosomal proteins HMGN3a and HMGN3b regulate the expression of gly-
cine transporter 1. Mol. Cell. Biol. 24:3747–3756.
33. West, K. L., Y. Ito, Y. Birger, Y. Postnikov, H. Shirakawa, and M. Bustin.
2001. HMGN3a and HMGN3b, two protein isoforms with a tissue-specific
expression pattern, expand the cellular repertoire of nucleosome-binding
proteins. J. Biol. Chem. 276:25959–25969.
34. Wilkinson, D. G. 1992. In situ hybridization, p. 75–83. In D. G. Wilkinson
(ed.), In situ hybridization: a practical approach. IRL Press, Oxford Univer-
sity, Oxford, United Kingdom.
35. Wunderle, V. M., R. Critcher, N. Hastie, P. N. Goodfellow, and A. Schedl.
1998. Deletion of long-range regulatory elements upstream of SOX9 causes
campomelic dysplasia. Proc. Natl. Acad. Sci. USA 95:10649–10654.
36. Zechner, D., Y. Fujita, J. Hulsken, T. Muller, I. Walther, M. M. Taketo, E. B.
Crenshaw, 3rd, W. Birchmeier, and C. Birchmeier. 2003. ?-Catenin signals
regulate cell growth and the balance between progenitor cell expansion and
differentiation in the nervous system. Dev. Biol. 258:406–418.
604FURUSAWA ET AL.MOL. CELL. BIOL.