A PEBP2/AML-1-related Factor Increases Osteocalcin Promoter Activity through Its Binding to an Osteoblast-specific cis-Acting Element

doi:10.1074/jbc.270.52.30973

A PEBP2/AML-1-related Factor Increases Osteocalcin Promoter Activity through Its Binding to an Osteoblast-specific cis-Acting Element (*)

From the Department of Molecular Genetics, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030

** To whom correspondence should be addressed:
The University of Texas M. D. Anderson Cancer Center, Dept. of Molecular Genetics, Box 45, 1515 Holcombe Blvd., Houston, TX 77030
. Tel.: 713-792-8934; Fax: 713-794-4394; gkarsenty{at}molgen.mda.uth.tmc.edu.

Abstract

To identify osteoblast-specific cis-acting elements and trans-acting factors, we initiated an analysis of the promoter of a mouse osteocalcin gene, an osteoblast-specific gene. In this promoter, we identified two osteoblast-specific cis-acting elements (Ducy, P. and Karsenty, G.(1995) Mol. Cell. Biol. 15, 1858-1869). The sequence of one of these elements, OSE2, is identical to the DNA-binding site of the PEBP2α/AML-1 transcription factors, the mammalian homologues of the Drosophila Runt protein. Here we show, using nuclear extracts, recombinant protein, and a specific antiserum against AML-1 proteins in DNA-binding assays, that one member of this family, AML-1B, binds specifically to OSE2 and is immunologically related to OSF2, the factor present in osteoblast nuclear extracts that binds to OSE2. By DNA cotransfection experiments, we also demonstrate that AML-1B can increase the activity of a short osteocalcin promoter through its binding to OSE2. Lastly, the different mobilities of osteoblast nuclear extract-DNA complexes compared with T-cell nuclear extract-DNA complexes, along with the inability of OSF2 to be up-regulated by retinoic acid, unlike the other PEBP2α factors, suggest that OSF2 is a new member of this family of transcription factors. Thus, this study demonstrates that AML-1B can increase gene expression of an osteoblast-specific gene through its binding to an osteoblast-specific cis-acting element and presents evidence that OSF2 is a member of the PEBP2α/AML-1 family of transcription factors.

Footnotes

↵^§ The first two authors contributed equally to this work and should be both considered first author.
↵^¶ Supported by a Fellowship from Institut Scientifique Roussel.
↵* This work was supported by National Institute Health Grants AR-41059 and DE/AR-11290 and Basic Research Award IFY 92-0871 from the March of Dimes Foundation (to G. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵⁽¹⁾ The abbreviations used are:

bp

base pair(s)

CMV

cytomegalovirus

DHFR

dihydrofolate reductase

RA

retinoic acid

ivt

in vitro transcribed and translated

GRA

gel retardation assays

luc

luciferase.
- Received September 7, 1995.
- Revision received October 24, 1995.