ESET histone methyltransferase is essential to the control of chondrocyte
hypertrophy and epiphyseal plate
1, 2
Yang, L; 1, 2Hacquebord, J; 1, 2Zou, J; 1, 2Patterson, DP; 1, 2Ghatan, AC; 1, 2Mei, Q; 1,
2
Kiatkowska, AZ; 1, 2Lawson, KA; 1, 2Chansky, HA
1
University of Washington, Seattle, WA, USA, 2Seattle VA Medical Center, Seattle,
WA, USA
lyang@u.washington.edu
Introduction: ESET (an ERG-interacting protein with a SET domain) is a histone
methyltransferase that methylates histone H3 at lysine 9 (H3-K9). We have found that
expression of ESET protein is transiently upregulated in prehypertrophic
chondrocytes during development. To investigate how histone modification enzymes
affect chondrocyte differentiation in vivo. we generated mice with conditional
deletion of the ESET gene from mesenchymal cells that give rise to chondrocytes
during limb development. Phenotype analysis of ESET knockout mice reveals that
ESET protein plays an essential role during hypertrophic differentiation of
chondrocytes and development of the epiphyseal plate. Though H3-K9 methylation is
increasingly recognized as a major marker for gene silencing during mammalian
development, disruptions of other bona fide H3-K9 methyltransferases such Suv39h1,
G9a and GLP have failed to show specific effects on an organ system in mice. our
study therefore provides clear in vivo evidence that proper differentiation of certain
tissue (cartilage) requires the H3-K9 mehtyltransferase activity of a specific H3-K9
methyltransferase.
Methods: Generation and analysis of ESET knockout mice --- Mice harboring the
floxed ESET allele were mated with Prx1-Cre mice on a C57BL/6 background. The
skeletal preparations were stained using Alcian blue and Alizarin red. Newborn pups
were kept at five or fewer per litter to ensure survival of the mutant animals. Weaned
mutant mice were fed with DietGel soft maintenance diet to ensure adequate intake of
food and water. Mouse embryos or postnatal tissues were fixed in 4%
paraformaldehyde overnight, decalcified in 14% EDTA for at least 24 hrs before
further treatments for embedding. Staining of distal femurs was carried out with 18
μm tissue sections using the NovaUltra Safranin O stain kit. Immunohistostaining was
carried out with a rabbit polyclonal anti-ESET antibody (against residues 1-167 of
mouse ESET) from Millipore (catalog # 07-378), followed by a Cy3-conjugated goat
anti-rabbit IgG from Jackson ImmunoResearch Laboratories.
Results: We chose Prx1-Cre as the deleter strain to achieve conditional knockout of
the ESET gene since the Cre activity first appears in the forelimb mesenchyme at
embryonic stage E9.5, followed by appearance in the hind limb bud within one day.
By E16.5, Cre is uniformly active in the limb buds while sparing the vertebrae and
ribs. In mice that are positive for Prx1-Cre and homozygous for the floxed ESET
allele, the bifurcated SET domain (and the H3-K9 methyltransferase activity) is
eliminated from ESET due to a frame-shift mutation in mesenchymal cells.
Genotyping of more than 80 embryos revealed that distribution of the knockout
mutants largely followed the Mendelian ratio. ESET knockout mice are viable and
characterized by shortened forelimbs but no missing bones (Fig. 1). Mice with
mesenchymal deletion of the ESET gene exhibit deformed scapulae, shortened digits,
and a lack of deltoid tuberosity.
While hindlimbs of the knockout mice did not show overt signs of gross defects at
birth, further examination revealed abnormal hypertrophic differentiation in the
hindlimbs as well as in all developing cartilage anlagen. In contrast to an orderly
appearance of distinct differentiation zones of chondrocytes within the growth plates
in wild-type animals, growth plates in ESET knockout mice were disorganized and
exhibited obvious signs of accelerated chondrocyte hypertrophy. By postnatal day 10
when secondary ossification centers start to develop in wild-type animals, there were
barely enough chondrocytes left behind within the growth plates of ESET knockout
mice for such developments. In 14-day old wild-type animals, epiphyseal plates
(physis) clearly separate primary ossification centers from secondary ossification
centers, but there is a total absence of such epiphyseal plates at the ends of long bones
in ESET knockout mice.
ESET protein is transiently upregulated in prehypertrophic chondrocytes and partially
overlaps with expression of Runx2, a transcription factor known to accelerate
chondrocyte hypertrophy. To investigate whether ESET protein physically interacts
with Runx2, we lysed 293T cells that express Flag-epitope tagged ESET and HAepitope tagged Runx2 for co-immunoprecipitation experiments. In anti-Flag-ESET
immunoprecipate, we detected the presence of HA-Runx2. In reciprocal
immunoprecipitation with an anti-HA antibody, we detected the presence of FlagESET. In addition, we also detected HDAC4 in these immunoprecipitates,
demonstrating that a subset of the ESET protein forms a multi-protein complex with
both Runx2 and HDAC4.
To functionally assay for ESET-Runx2 interaction, we transfected COS-7 cells with
the murine osteocalcin gene 2 (mOG2)-Luc reporter plus Runx2 and ESET expression
plasmids. The osteocalcin promoter is a well characterized target of Runx proteins and
contains three binding sites for Runx2. While Runx2 activated osteocalcin promoter
by more than 5-fold, ESET inhibited Runx2 activation of this promoter in a dosedependent manner. Interestingly, an ESET mutant (C1243T) that abolishes its
methyltransferase activity failed to inhibit Runx2 activation of osteocalcin promoter,
suggesting that H3-K9 methylation (and subsequent epigenetic silencing) is
responsible for ESET repression of Runx2 target genes.
Discussion: In this study we have demonstrated that the H3-K9 methyltransferase
ESET regulates chondrocyte differentiation during both embryogenesis and postnatal
development, and have provided clear in vivo evidence that formation and
maintenance of the epiphyseal plate requires the H3-K9 methyltransferase activity of
ESET protein. Our findings that ESET interacts with both Runx2 and HDAC4, that
ESET inhibits Runx2 target genes through its H3-K9 methyltransferase activity, that
mesenchymal deletion of ESET in mice generates a phenotype closely resembling the
one observed in HDAC4-null embryos all point to the possibility that an ESETHDAC4-Runx2 complex in the prehypertrophic zone functions as the gate-keeper for
an orderly entry of prehypertrophic chondrocytes into terminal differentiation.
Disruption in any one of these proteins will functionally inactvate such a complex and
causes an abnormal 'histone code', leading to either delayed or accelerated
chondrocyte hypertrophy. As ESET expression and its activity could be influenced by
different extracellular stimuli, it is likely that deregulation of ESET also plays a role
in other pathological processes such as premature hypertrophy and apoptosis of
articular chondrocytes in osteoarthritic patients.
Significance: We have identified a novel regulator of chondrocyte hypertrophy that is
critical to skeletal development and disease.
Fig. 1. Skeletal defects in mice harboring mesenchymal deletion of the ESET gene. a,
diagrams of ESET protein domains, gene structure and exons 15 & 16-floxed allele. b,
photograph of wild-type and mutant newborn pups. c-d, double staining of forelimbs
and hindlimbs with alizarin red and alcian blue. e-j, safranin O staining of cartilage in
distal femurs from mice at different stages of postnatal development.
ORS 2013 Annual Meeting
Paper No: 0277