The goal of this project is to understand how chromatin organization and nuclear compartmentalization contribute to and support regulation of osteoblast-related gene expression. Transcription factors and protein- DNA interactions clearly play an important role in cell and tissue- specific gene regulation. However, such gene-regulatory interactions occur within DNA that is packaged as chromatin and further organized within the nucleus. We are interested in understanding how chromatin packaging affects the ability of a gene to be expressed and how sequence elements and binding factors mediate bone tissue-specific chromatin structure/organization. We will also address the role of nuclear location and spatial organization of osteoblast-expressed genes in osseous and non-osseous cells. We hypothesize that alterations in chromatin structure and nuclear organization accompany and mediate changes in gene expression.
Our specific aims are: (1) To determine regulatory parameters that mediate chromatin structure of a bone tissue- specific gene in osteoblastic cells. Sequence-specific gene regulatory elements, nucleosome positioning, and histone acetylation will be manipulated and effects on chromatin structure and expression will be evaluated in stable cell lines. (2) To analyze biochemical and molecular mechanisms that alter the chromatin structure of the osteocalcin gene promoter. We will address these mechanisms by using an in vitro, cell-free system to monitor alterations in chromatin structure that result from interactions of tissue-specific transcription factors, hormone receptors, and the SWI/SNF complex with DNA in a nucleosomal context. (3) To examine the spatial relationship of osteoblast expressed genes to intra-nuclear compartmentalization in osteogenic cells, contrasted with non-osseous cells. We will analyze the distribution of bone and muscle chromatin, relative to Sc-35 domains in cells induced to switch from a myogenic to an osteogenic developmental program. These studies will contribute to understanding regulation of a key component of nuclear architecture that supports transcription during osteoblast differentiation.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
5R01AR045689-04
Application #
6375182
Study Section
Special Emphasis Panel (ZAR1-TLB-A (J1))
Program Officer
Sharrock, William J
Project Start
1998-05-01
Project End
2003-04-30
Budget Start
2001-05-01
Budget End
2003-04-30
Support Year
4
Fiscal Year
2001
Total Cost
$365,812
Indirect Cost
Name
University of Massachusetts Medical School Worcester
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
660735098
City
Worcester
State
MA
Country
United States
Zip Code
01655
Stein, G S; Stein, J L; Lian, J B et al. (2004) Nuclear microenvironments: an architectural platform for the convergence and integration of transcriptional regulatory signals. Eur J Histochem 48:65-76
Stein, Gary S; Lian, Jane B; van Wijnen, Andre J et al. (2004) Runx2 control of organization, assembly and activity of the regulatory machinery for skeletal gene expression. Oncogene 23:4315-29
Shen, Jiali; Hovhannisyan, Hayk; Lian, Jane B et al. (2003) Transcriptional induction of the osteocalcin gene during osteoblast differentiation involves acetylation of histones h3 and h4. Mol Endocrinol 17:743-56
Stein, Gary S; Lian, Jane B; Montecino, Martin et al. (2003) Nuclear microenvironments support physiological control of gene expression. Chromosome Res 11:527-36
Lian, Jane B; Stein, Janet L; Stein, Gary S et al. (2003) Runx2/Cbfa1 functions: diverse regulation of gene transcription by chromatin remodeling and co-regulatory protein interactions. Connect Tissue Res 44 Suppl 1:141-8
Stein, Gary S; Lian, Jane B; Stein, Janet L et al. (2003) Intranuclear organization of RUNX transcriptional regulatory machinery in biological control of skeletogenesis and cancer. Blood Cells Mol Dis 30:170-6
Stein, Gary S; Lian, Jane B; Stein, Janet L et al. (2003) Intranuclear trafficking of transcription factors: Requirements for vitamin D-mediated biological control of gene expression. J Cell Biochem 88:340-55
Gutierrez, Soraya; Javed, Amjad; Tennant, Daniel K et al. (2002) 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. J Biol Chem 277:1316-23
Paredes, Roberto; Gutierrez, Jose; Gutierrez, Soraya et al. (2002) Interaction of the 1alpha,25-dihydroxyvitamin D3 receptor at the distal promoter region of the bone-specific osteocalcin gene requires nucleosomal remodelling. Biochem J 363:667-76
Villagra, Alejandro; Gutierrez, Jose; Paredes, Roberto et al. (2002) Reduced CpG methylation is associated with transcriptional activation of the bone-specific rat osteocalcin gene in osteoblasts. J Cell Biochem 85:112-22

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