General transcription factor IIA-γ increases osteoblast-specific osteocalcin gene expression via activating transcription factor 4 and runt-related transcription factor

Abstract

ATF4 (activating transcription factor 4) is an osteoblast-enriched transcription factor that regulates terminal osteoblast differentiation and bone formation. ATF4 knock-out mice have reduced bone mass (severe osteoporosis) throughout life. Runx2 (runt-related transcription factor 2) is a runt domain-containing transcription factor that is essential for bone formation during embryogenesis and postnatal life. In this study, we identified general transcription factor IIAγ (TFIIAγ) as a Runx2-interacting factor in a yeast two-hybrid screen. Immunoprecipitation assays confirmed that TFIIAγ interacts with Runx2 in osteoblasts and when coexpressed in COS-7 cells or using purified glutathione S-transferase fusion proteins. Chromatin immunoprecipitation assay of MC3T3-E1 (clone MC-4) preosteoblast cells showed that in intact cells TFIIAγ is recruited to the region of the osteocalcin promoter previously shown to bind Runx2 and ATF4. A small region of Runx2 (amino acids 258-286) was found to be required for TFIIAγ binding. Although TFIIAγ interacts with Runx2, it does not activate Runx2. Instead, TFIIAγ binds to and activates ATF4. Furthermore, TFIIAγ together with ATF4 and Runx2 stimulates osteocalcin promoter activity and endogenous mRNA expression. Small interfering RNA silencing of TFIIAγ markedly reduces levels of endogenous ATF4 protein and Ocn mRNA in osteoblastic cells. Overexpression of TFIIAγ increases levels of ATF4 protein. Finally, TFIIAγ significantly prevents ATF4 degradation. This study shows that a general transcription factor, TFIIAγ, facilitates osteoblast-specific gene expression through interactions with two important bone transcription factors ATF4 and Runx2. © 2008 by The American Society for Biochemistry and Molecular Biology, Inc.