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 IIA gamma (TFIIA gamma) as a Runx2-interacting factor in a yeast two-hybrid screen. Immunoprecipitation assays confirmed that TFIIA gamma 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 TFIIA gamma 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 TFIIA gamma binding. Although TFIIA gamma interacts with Runx2, it does not activate Runx2. Instead, TFIIA gamma binds to and activates ATF4. Furthermore, TFIIA gamma together with ATF4 and Runx2 stimulates osteocalcin promoter activity and endogenous mRNA expression. Small interfering RNA silencing of TFIIA gamma markedly reduces levels of endogenous ATF4 protein and Ocn mRNA in osteoblastic cells. Overexpression of TFIIA gamma increases levels of ATF4 protein. Finally, TFIIA gamma significantly prevents ATF4 degradation. This study shows that a general transcription factor, TFIIA gamma, facilitates osteoblast-specific gene expression through interactions with two important bone transcription factors ATF4 and Runx2.
Highlights
Transcription factor IIA (TFIIA) was classified as a general transcription factor when it was first identified, more and more evidence shows that this elusive factor may play an important role in the regulation of tissue-specific gene expression via interactions with tissue- or cell type-specific transcription factors (34 –36)
B, nuclear extracts from ROS17/2.8 cells were immunoprecipitated with normal IgG or TFIIA␥ antibody followed by WB using Runx2, ATF4, or Fra-1 antibodies
3, 5 and 7), Runx2 antibody, ATF4 antibody, or Fra-1 antibody followed by WB using TFIIA␥ antibody
Summary
Reagents—Tissue culture media were purchased from Invitrogen and fetal bovine serum from HyClone (Logan, UT). Regular PCR—Regular PCR was performed on a 2720 Thermal Cycler (Applied Biosystem, Foster, CA), using 2.5 l of the cDNA (equivalent to 0.2 g of RNA) and AmpliTaq DNA polymerase (Applied Biosystems, Foster City, CA) in a 25-l reaction according to the manufacturer’s instructions. After 36 h, total RNA was harvested for quantitative real time RT-PCR analysis for TFIIA␥, Ocn, Bsp, Opn (osteopontin), and Atf mRNAs. A second set of mouse TFIIA␥ siRNAs (sense, AUG ACA ACA CUG UGC UAU AUU; antisense, UAU AGC ACA GUG UUG UCA UUU) was designed in the project laboratory and used to confirm the results using the first set of TFIIA␥ siRNA. Differences with a p Ͻ 0.05 was considered as statistically significant
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