CCAAT/enhancer-binding proteins (C/EBP) beta and delta activate osteocalcin gene transcription and synergize with Runx2 at the C/EBP element to regulate bone-specific expression.
CCAAT/enhancer-binding proteins (C/EBP) are critical determinants for cellular differentiation and cell type-specific gene expression. Their functional roles in osteoblast development have not been determined. We addressed a key component of the mechanisms by which C/EBP factors regulate transcription of a tissue-specific gene during osteoblast differentiation. Expression of both C/EBPbeta and C/EBPdelta increases from the growth to maturation developmental stages and, like the bone-specific osteocalcin (OC) gene, is also stimulated 3-6-fold by vitamin D(3), a regulator of osteoblast differentiation. We characterized a C/EBP enhancer element in the proximal promoter of the rat osteocalcin gene, which resides in close proximity to a Runx2 (Cbfa1) element, essential for tissue-specific activation. We find that C/EBP and Runx2 factors interact together in a synergistic manner to enhance OC transcription (35-40-fold) in cell culture systems. We show by mutational analysis that this synergism is mediated through the C/EBP-responsive element in the OC promoter and by a direct interaction between Runx2 and C/EBPbeta. Furthermore, we have mapped a domain in Runx2 necessary for this interaction by immunoprecipitation. A Runx2 mutant lacking this interaction domain does not exhibit functional synergism. We conclude that, in addition to Runx2 DNA binding functions, Runx2 can also form a protein complex at C/EBP sites to regulate transcription. Taken together, our findings indicate that C/EBP is a principal transactivator of the OC gene and the synergism with Runx2 suggests that a combinatorial interaction of these factors is a principal mechanism for regulating tissue-specific expression during osteoblast differentiation.
Gutierrez
Soraya
S
Department of Cell Biology, University of Massachusetts Medical School, Worcester, Massachusetts 01655-0106, USA.
Javed
Amjad
A
Tennant
Daniel K
DK
van Rees
Monique
M
Montecino
Martin
M
Stein
Gary S
GS
Stein
Janet L
JL
Lian
Jane B
JB
D000818:Animals; D022763:CCAAT-Enhancer-Binding Protein-alpha / Q000378:metabolism*; D051743:CCAAT-Enhancer-Binding Protein-delta; D022762:CCAAT-Enhancer-Binding Proteins / Q000378:metabolism*; D002454:Cell Differentiation / Q000502:physiology; D002478:Cells, Cultured; D002762:Cholecalciferol / Q000494:pharmacology; D050659:Core Binding Factor Alpha 1 Subunit; D018507:Gene Expression Regulation, Developmental; D017930:Genes, Reporter; D016297:Mutagenesis, Site-Directed; D009363:Neoplasm Proteins; D010006:Osteoblasts / Q000187:drug effects / Q000502:physiology*; D015675:Osteocalcin / Q000235:genetics* / Q000378:metabolism; D051381:Rats; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D014157:Transcription Factors / Q000378:metabolism*
D022763:CCAAT-Enhancer-Binding Protein-alpha; D022762:CCAAT-Enhancer-Binding Proteins; C497254:Cebpd protein, rat; D050659:Core Binding Factor Alpha 1 Subunit; D009363:Neoplasm Proteins; D014157:Transcription Factors; D015675:Osteocalcin; D051743:CCAAT-Enhancer-Binding Protein-delta; D002762:Cholecalciferol