We propose to analyze the DNA sequences and protein binding factors which are responsible for elevated transcription of the alpha-1(I) collagen gene in bone cells as compared to fibroblast, and also these sequences and factors responsible for the inhibition of alpha-1(I) synthesis by vitamin D in bone cells but not fibroblasts. We will follow-up on our preliminary evidence that there is a bone-specific enhancer between -3521 and -2291, and a vitamin D responsive element between -2291 and -1665 upstream from the RNA transcription start site. We will analyze DNA sequences using deletions and oligonucleotide directed mutagenesis followed by analysis of transcriptional activity and hormone regulation of the mutants in stably transfected osteosarcoma and fibroblast cell lines, and also in transgenic mice. Subfragments of the promoter will be tested for their effect on the transcription of a weak heterologous promoter. In vitro protein/DNA interactions will be analyzed by gel mobility shifts and footprinting using nuclear extracts from hormone treated and control bone cells and fibroblasts. In vivo chromatin structure will be analyzed using DNase sensitivity studies. Appreciating the different regulatory influences on collagen production in bone and fibroblast is essential for understanding genetic acquired disorders of bone.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
5R01AR029983-10
Application #
2078669
Study Section
General Medicine B Study Section (GMB)
Project Start
1981-08-01
Project End
1995-07-31
Budget Start
1993-08-01
Budget End
1995-07-31
Support Year
10
Fiscal Year
1993
Total Cost
Indirect Cost
Name
University of Connecticut
Department
Pediatrics
Type
Schools of Medicine
DUNS #
City
Farmington
State
CT
Country
United States
Zip Code
06030
Erceg, Ivana; Tadic, Tade; Kronenberg, Mark S et al. (2003) Dlx5 regulation of mouse osteoblast differentiation mediated by avian retrovirus vector. Croat Med J 44:407-11
Tadic, Tade; Dodig, Milan; Erceg, Ivana et al. (2002) Overexpression of Dlx5 in chicken calvarial cells accelerates osteoblastic differentiation. J Bone Miner Res 17:1008-14
Tadic, T; Erceg, I; Stover, M L et al. (2001) Dlx5 induces expression of COL1A1 promoter contained in a retrovirus vector. Croat Med J 42:436-9
Dodig, M; Tadic, T; Kronenberg, M S et al. (1999) Ectopic Msx2 overexpression inhibits and Msx2 antisense stimulates calvarial osteoblast differentiation. Dev Biol 209:298-307
Bedalov, A; Salvatori, R; Dodig, M et al. (1998) 1,25-Dihydroxyvitamin D3 inhibition of col1a1 promoter expression in calvariae from neonatal transgenic mice. Biochim Biophys Acta 1398:285-93
Pan, Z; Lichtler, A C; Upholt, W B (1998) DNase I hypersensitive sites in the chromatin of the chicken Msx2 gene differ in anterior and posterior limb mesenchyme, calvarial osteoblasts and embryonic fibroblasts. Biochem Mol Biol Int 46:549-57
Walton, C M; Wu, G Y; Petruff, C A et al. (1996) A collagen enhancer-promoter construct in transgenic mice is markedly stimulated by ethanol administration. Hepatology 23:310-5
Dodig, M; Kronenberg, M S; Bedalov, A et al. (1996) Identification of a TAAT-containing motif required for high level expression of the COL1A1 promoter in differentiated osteoblasts of transgenic mice. J Biol Chem 271:16422-9
Kream, B E; Harrison, J R; Krebsbach, P H et al. (1995) Regulation of type I collagen gene expression in bone. Connect Tissue Res 31:261-4
Bedalov, A; Salvatori, R; Dodig, M et al. (1995) Regulation of COL1A1 expression in type I collagen producing tissues: identification of a 49 base pair region which is required for transgene expression in bone of transgenic mice. J Bone Miner Res 10:1443-51

Showing the most recent 10 out of 20 publications