Bmp2 Transcription in Osteoblast Progenitors Is Regulated by a Distant 3′ Enhancer Located 156.3 Kilobases from the Promoter

Abstract

Bone morphogenetic protein 2 (encoded by Bmp2) has been implicated as an important signaling ligand for osteoblast differentiation and bone formation and as a genetic risk factor for osteoporosis. To initially survey a large genomic region flanking the mouse Bmp2 gene for cis-regulatory function, two bacterial artificial chromosome (BAC) clones that extend far upstream and downstream of the gene were engineered to contain a lacZ reporter cassette and tested in transgenic mice. Each BAC clone directs a distinct subset of normal Bmp2 expression patterns, suggesting a modular arrangement of distant Bmp2 regulatory elements. Strikingly, regulatory sequences required for Bmp2 expression in differentiating osteoblasts, as well as tooth buds, hair placodes, kidney, and other tissues, are located more than 53 kilobases 3′ to the promoter. By testing BACs with engineered deletions across this distant 3′ region, we parsed these regulatory elements into separate locations and more closely refined the location of the osteoblast progenitor element. Finally, a conserved osteoblast progenitor enhancer was identified within a 656-bp sequence located 156.3 kilobases 3′ from the promoter. The identification of this enhancer should permit further investigation of upstream regulatory mechanisms that control Bmp2 transcription during osteoblast differentiation and are relevant to further studies of Bmp2 as a candidate risk factor gene for osteoporosis.

SUPPLEMENTAL MATERIAL

We thank Laura Selenke and Lissett Ramirez for technical assistance and Karen Deal, Maureen Gannon, Anna Means, and Laura Wilding for generously sharing equipment and advice. We thank Kyle Gurley and David Kingsley for assistance with constructing the pIBG-FTet plasmid, Scott Baldwin and Kevin Tompkins for assistance with pronuclear injections, Brigid Hogan for providing the Bmp2 in situ probe, Soren Warming and Neal Copeland for providing BAC recombination reagents, and the Vanderbilt CHGR DNA Resources Core for real-time PCR assistance and TaqMan probe/primer design. We also kindly thank Steve Harris for discussions regarding the previously reported Bmp2 promoter transgene and Nyk Reed and Michelle Southard-Smith for helpful discussions on the manuscript. We thank Michelle Southard-Smith for help with Southern blothing.

Ronald L. Chandler was supported by NIH Developmental Biology Training Grant 5T32HD07502-08. Kelly J. Chandler was supported by NIH Genetics Training Grant 1T32GM62758-03. Douglas P. Mortlock was supported by NIH Grant 1R01HD47880-01. Transgenic mice were generated by the Vanderbilt University Transgenic and ES Cell Shared Resource, which is supported by the Vanderbilt Cancer, Diabetes, Kennedy, and Vision Centers. We acknowledge use of the VUMC CHGR DNA Resources Core Facility.