The ESET histone methyltransferase is critical to osteoblast differentiation and
trabecular bone formation
Lawson, KA; Patterson, DP; Bain, SD; Ghatan, AC; Kwiatkowska, AZ; Zou, J; Qi,
M; Gao, J; Chansky, HA; Yang, L
University of Washington, Seattle, WA, USA
Introduction: The ESET protein contains three major functional domains: the Nterminal tudor domain and the internal methyl CpG binding domain are responsible
for interaction with other chromatin modification enzymes, while the C-terminal
bifurcated SET domain catalyzes histone methylation. Since ubiquitous deletion of the
ESET gene results in lethality during early embryonic development, we have
generated viable mice with conditional deletion of the ESET gene in mesenchymal
cells. The availability of these mutant mice makes it possible to examine the effects of
ESET histone methyltransferase on post-natal bone development. In this study we
have carried out comparison of bone properties between wild-type and ESETknockout mice, and isolated mesenchymal stem cells from bone marrow of wild-type
and mutant animals for osteobalst differentiation.
Methods: 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.
Microcomputed tomography (μCT) scans of the whole body and tibia were performed
at 76 μm and 10.5 μm resolution, respectively (Scanco vivaCT 40; 10.5 μm voxel).
Serial scans of whole body and tibia were reconstructed using machine software.
Histological characterization of osteoblasts in wild-type and ESET-deficient mice was
carried out with tissue sections after PLP (2% paraformaldehyde, 0.075 M Lysine, and
0.01 M sodium periodate) fixation. Tissue sections were then renatured in 1% MgCl2
and stained using the Sigma alkaline phosphatase staining kit. For differentiation of
mesenchymal stem cells into osteoblasts, mesenchymal stem cells were isolated from
the bone marrow of 6-8 week old mice and enriched, then cultured in standard
osteogenic medium for 10 days before staining for alkaline phosphatase-positive
osteoblasts.
Results: To achieve specific deletion of the ESET gene in mesenchymal cells, we
used the Prx1-Cre mouse strain for breeding with mice harboring the floxed ESET
allele. Deletion of the ESET gene starts in the forelimb mesenchyme at embryonic
stage E9.5 and in the hindlimb bud at E10.5. Out of more than 80 embryos,
distribution of the knockout mutants largely followed the Mendelian ratio. At birth,
pups that are homozygous for the floxed ESET allele and positive for the Prx1-Cre
transgene are viable but easily recognizable by their significantly shortened forelimbs.
Due to acceleration of hypertrophy among growth plate chondrocytes in the knockout
animals, there were no growth plate chondrocytes left behind at postnatal day 14 to
form physis(epiphyseal plate) at the end of any long bone. Since physis is the skeletal
element responsible for long bone growth, all four limbs of ESET knockout mice are
therefore very short and these animals had difficulty with locomotion. As a result,
ESET knockout mice had to be fed with DietGel soft maintenance diet to ensure
adequate intake of food and water.
To investigate the bone properties of ESET knockout mice, age- and sex-matched
wild-type and ESET knockout mice were sacrificed for microcomputed tomography
(μCT) imaging 6-weeks after birth. As shown in Fig. 1, the tibia of wild-type animal
is significantly longer than that of the knockout mutant, and that the physis is clearly
absent from the knockout animal. Another prominant feature of the knockout mice is
the lack of trabecular bone. To investigate what caused such a decrease in trabecular
bone volume, we prepared 10 μm sections of proximal tibia from wild-type and
knockout mice. Histological staining for alkaline phosphatase-positive osteoblasts
again revealed the presence of physis in the wild-type animal and an absence of physis
in the mutant. In addition, alkaline phosphatase activity was significantly stronger in
the wild-type tibia and more cells exhibited positive staining than in the knockout
counterpart.
Since osteoblasts are derived from mesenchymal stem cells, we speculate that ESET
knockout in mesenchymal stem cells may have impaired the ability of these cells to
differentiate into osteoblasts. To test this hypothesis, we isolated mesenchymal stem
cells from the bone marrow of either wild-type or knockout animals, then cultured
equal number of enriched mesenchymal cells in osteogenic medium to force
differentiation into osteoblasts for 10 days. When stained for alkaline phosphatase
activity, mesenchymal stem cells from wild-type bone marrow efficiently grew to
become osteoblasts, whereas very few mesenchymal stem cells from the ESET
knockout bone marrow were able to differentiate along the osteoblast lineage.
Discussion: In this study, we have demonstrated that the ESET histone
methyltransferase is critical to trabecular bone formation while mesenchymal deletion
of the ESET gene led to near complete absence of trabeculae in adult animals. Since
osteoclastogenesis is not affected by ESET knockout in mesenchymal cells,
impairment of trabecular bone appears to be mainly caused by significantly fewer and
less active osteoblasts in the bone marrow of ESET knockout mice, and this trabecular
bone defect in turn has been traced back to the inability of ESET-null mesenchymal
stem cells to differentiate into osteoblasts.
How could ESET protein exert such a drastic effect on the differentiation of
mesenchymal stem cells along the osteogenic lineage? We have found evidence that
ESET interacts with Runx2 and represses Runx2 target genes. Since Runx2 can
function both as a transcriptional activator and repressor, it could be explained that
recruitment of ESET to Runx2 target genes may be necessary for their repression in
mesenchymal cells in order for osteogenic differentiation to take place. In fact,
mammalian development is an intrinsically epigenetic process in which a zygote
generates hundreds of different cell types with unique epigenetic landscape. ESET
histone methyltransferase, with its in vivo functions described here, may indeed
represent an irreplaceable epigeneic enzyme that writes methylation of H3-K9 as the
critical 'histone code' for osteogenic differnetiation.
Significance: We have identified a histone methyltransferase as a critical regulator of
osteoblast differentiaiton and trabecular bone formation, thus gaining new insight into
skeletal development and disease process.
Requirement for ESET protein in trabecular bone formation and osteoblast
differentiation from mesenchymal stem cells. a, ESET protein domains and genomic
structure. Note that exons 15 & 16 are flanked by two loxP sites. b-c, proximal tibia
from 6-week old wild-type and knockout mice were analyzed by micro-CT scanning
to show differences in trabecular bones. d-e, sagittal sections of proximal tibia from
wild-type and knockout mice were stained for alkaline phosphatase activity to identify
osteoblasts. f-g, mesenchymal stem cells were isolated from the bone marrows of 8week old wild-type and knockout mice. The cells were cultured for 14 days in
osteogenic medium, then stained for alkaline phosphatase activity to measure
osteoblasts.
ORS 2013 Annual Meeting
Poster No: 0636