Genomic deletion of a long-range bone enhancer misregulates sclerostin in Van Buchem disease

Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA.
Genome Research (Impact Factor: 13.85). 07/2005; 15(7):928-35. DOI: 10.1101/gr.3437105
Source: PubMed

ABSTRACT Mutations in distant regulatory elements can have a negative impact on human development and health, yet because of the difficulty of detecting these critical sequences, we predominantly focus on coding sequences for diagnostic purposes. We have undertaken a comparative sequence-based approach to characterize a large noncoding region deleted in patients affected by Van Buchem (VB) disease, a severe sclerosing bone dysplasia. Using BAC recombination and transgenesis, we characterized the expression of human sclerostin (SOST) from normal (SOST(wt)) or Van Buchem (SOST(vbDelta) alleles. Only the SOST(wt) allele faithfully expressed high levels of human SOST in the adult bone and had an impact on bone metabolism, consistent with the model that the VB noncoding deletion removes a SOST-specific regulatory element. By exploiting cross-species sequence comparisons with in vitro and in vivo enhancer assays, we were able to identify a candidate enhancer element that drives human SOST expression in osteoblast-like cell lines in vitro and in the skeletal anlage of the embryonic day 14.5 (E14.5) mouse embryo, and discovered a novel function for sclerostin during limb development. Our approach represents a framework for characterizing distant regulatory elements associated with abnormal human phenotypes.

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Available from: Hansjoerg Keller, Jul 30, 2015
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    • "Female Sost −/− mice, previously described [14] [35] were backcrossed to C57BL/6J genetic background using founders with above 99.09% isogenicity/identity to the C57BL/6J strain. "
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    ABSTRACT: Sclerostin deficiency, via genetic knockout or anti-Sclerostin antibody treatment, has been shown to cause increased bone volume, density and strength of calluses following endochondral bone healing. However, there is limited data on the effect of Sclerostin deficiency on the formative early stage of fibrocartilage (non-bony tissue) formation and removal. In this study we extensively investigate the early fibrocartilage callus. Closed tibial fractures were performed on Sost(-/-) mice and age-matched wild type (C57Bl/6J) controls and assessed at multiple early time points (7, 10 and 14 days), as well as at 28 days post-fracture after bony union. External fixation was utilized, avoiding internal pinning and minimizing differences in stability stiffness, a variable that has confounded previous research in this area. Normal endochondral ossification progressed in wild type and Sost(-/-) mice with equivalent volumes of fibrocartilage formed at early day 7 and day 10 time points, and bony union in both genotypes by day 28. There were no significant differences in rate of bony union; however there were significant increases in fibrocartilage removal from the Sost(-/-) fracture calluses at day 14 suggesting earlier progression of endochondral healing. Earlier bone formation was seen in Sost(-/-) calluses over wild type with greater bone volume at day 10 (221%, p < 0.01). The resultant Sost(-/-) united bony calluses at day 28 had increased bone volume fraction compared to wild type calluses (24%, p < 0.05), and the strength of the fractured Sost(-/-) tibiae was greater than that that of wild type fractured tibiae. In summary, bony union. was not altered by Sclerostin deficiency in externally-fixed closed tibial fractures, but fibrocartilage removal was enhanced and the resultant united bony calluses had increased bone fraction and increased strength. Crown Copyright
    Bone 02/2015; 71. DOI:10.1016/j.bone.2014.10.018 · 4.46 Impact Factor
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    • "Inhibition of the Wnt antagonist SOST to stimulate anabolic osteoblastic Wnt signaling is being developed for the treatment of the low bone mass of osteoporosis [16]. Of note, humans with sclerosteosis or the related van Buchem disease, both of which result from mutations reducing SOST expression or function, do not appear to be at increased risk of developing OS [17] [18] [19] [20] [21] [22]. The report that WIF1 was epigenetically silenced in OS, and that its absence could augment OS in murine models, has potentially significant clinical implications for the treatment of osteoporosis with agents that activate or enhance Wnt signaling in the bone [13]. "
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    ABSTRACT: Wnt pathway targeting is of high clinical interest for treating bone loss disorders such as osteoporosis. These therapies inhibit the action of negative regulators of osteoblastic Wnt signaling. The report that Wnt inhibitory factor 1 (WIF1) was epigenetically silenced via promoter DNA methylation in osteosarcoma (OS) raised potential concerns for such treatment approaches. Here we confirm that Wif1 expression is frequently reduced in OS. However, we demonstrate that silencing is not driven by DNA methylation. Treatment of mouse and human OS cells showed that Wif1 expression was robustly induced by HDAC inhibition but not by methylation inhibition. Consistent with HDAC dependent silencing, the Wif1 locus in OS was characterized by low acetylation levels and a bivalent H3K4/H3K27-trimethylation state. Wif1 expression marked late stages of normal osteoblast maturation and stratified OS tumors based on differentiation stage across species. Culture of OS cells under differentiation inductive conditions increased expression of Wif1. Together these results demonstrate that Wif1 is not targeted for silencing by DNA methylation in OS. Instead, the reduced expression of Wif1 in OS cells is in context with their stage in differentiation. Copyright © 2014. Published by Elsevier Inc.
    Bone 01/2015; 73. DOI:10.1016/j.bone.2014.12.063 · 4.46 Impact Factor
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    • "This is consistent with prior reports of low levels of sclerostin mRNA and protein in osteoclasts and endothelial cells during bone development [58] and in conditioned medium from isolated osteoclasts from aged mice [29]. Although sclerostin is generally considered to be osteocytespecific in adult bone, it is expressed in multiple tissues in the developing embryo, including the kidney, liver and heart [59], and this may explain the detection of sclerostin in non-mesenchymal GFP + cells in the present study of neonatal mice. Similarly, DMP1 and MEPE protein have been identified in the kidney and pancreas [60,61], but expression in haematopoietic lineage cells has not been shown previously. "
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    ABSTRACT: To define their gene expression and function, osteocytes are commonly isolated and purified by fluorescence-activated cell sorting (FACS) from mice expressing GFP directed by the dentin matrix protein 1 (Dmp1) promoter (DMP1-GFP). These cells express mRNA for osteocyte genes, including sclerostin (Sost) and Dmp1, and genes associated with the osteoclast phenotype: Dcstamp, Oscar, Cathepsin K (Ctsk), tartrate resistant acid phosphatase (TRAP/Acp5) and calcitonin receptor (Calcr). This suggests either that osteoclasts and osteocytes share genes and functions or that DMP1-GFP(+) preparations contain haematopoietic osteoclasts. To resolve this we stained DMP1-GFP cells for haematopoietic lineage (Lin) surface markers (CD2, CD3e, CD4, CD45, CD5, CD8, CD11b, B220, Gr1, Ter119) and CD31. Lin(-)CD31(-) (Lin(-)) and Lin(+)CD31(+) (Lin(+)) populations were analysed for GFP, and the four resulting populations assessed by quantitative real-time PCR. Lin(-)GFP(+) cells expressed mRNAs for Sost, Dmp1, and Mepe, confirming their osteocyte identity. Dcstamp and Oscar mRNAs were restricted to haematopoietic (Lin(+)) cells, but Calcr, Ctsk and Acp5 were readily detected in purified osteocytes (Lin(-)GFP(+)). The capacity of these purified osteocytes to support osteoclastogenesis was assessed: no TRAP+ cells with >2 nuclei were formed when purified osteocytes were cultured with bone marrow macrophages and stimulated with 1,25-dihydroxyvitamin-D3/prostaglandin E2. Lin(-)GFP(+) osteocytes also expressed lower levels of Tnfsf11 (RANKL) mRNA than the osteoblast-enriched population (Lin(-)GFP(-)). This demonstrates the importance of haematopoietic depletion in generating highly purified osteocytes and shows that osteocytes express Acp5, Ctsk and Calcr, but not other osteoclast markers, and do not fully support osteoclast formation in vitro. Copyright © 2014 Elsevier Inc. All rights reserved.
    Bone 11/2014; 72C:34-42. DOI:10.1016/j.bone.2014.11.005 · 4.46 Impact Factor
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