A fundamental problem in bone cell biology, is how bone-specific genes are rendered competent for expression and steroid hormone responsiveness at appropriate stages during the osteoblast developmental sequence. This program established that Runx2, the key transcription factor required for bone formation, plays a critical role in the assembly of active chromatin at the osteocalcin (OC)promoter during phenotype progression and is required for steroid hormone responsiveness. The long term goal of this project is to understand how dynamic reorganization of the chromatin of bone-specific genes at subnuclear foci contributes to activation of the osteoblast developmental transcription program in response to physiologic regulatory cues. Importantly, during the current award period, we have demonstrated that activity of the SWI/SNF chromatin remodeling complex is also essential both for expression and vitamin D enhancement of the OC gene in osteoblastic cells and for osteoblast differentiation. We now propose to extend our exciting findings to address the hypothesis that induction of the osteoblast phenotvpe must involve histone modifications and ordered remodeling of chromatin in an osteoblast-specific manner in response to tissue- specific factors and multi-component enzymatic complexes that bind sequentially to activate and regulate transcription and mediate vitamin D responsiveness. To understand more broadly how gene expression is regulated during skeletal development, we will pursue a highly collaborative approach to characterize: 1. the Runx2-dependent and vitamin D responsive program of binding events and chromatin remodeling at the osteocalcin promoter;2. epigenetic marking and chromatin remodeling of bone phenotypic genes in response to Runx2 and vitamin D during osteoblast differentiation;and 3. the Runx2 and Vitamin D responsive gene expression program required for establishment and maintenance of the mature bone cell phenotype. These studies will reveal the role of Runx2 and key co-factors for developmental expression and vitamin D dependent regulation of genes required for osteoblast differentiation. Collaborations with Projects 1 and 2 will provide novel insights into the function of Runx2 in supporting local architectural organization of gene promoters and co-regulators for bone-specific transcriptional control. Clinical Relevance: Defining mechanisms that control chromatin organization is compelling for understanding actions and consequences of inhibitors that target chromatin for the treatment of diseases that affect bone turnover in the adult skeleton. University of Massachusetts Medical School 55 Lake Avenue North Worcester, MA 01655 PHS 398 (Rev. 04/06) Page 240 Form Page 2 Project 3 Principal Investigator/Program Director (Last, First, Middle): Stein, Gary S. KEY PERSONNEL. See instructions. Use continuation pages as needed to provide the required information in the format shown below. Start with Principal Investigator(s). List all other key personnel in alphabetical order, last name first. Name eRA Commons User Name Organization Role on Project Stein, Janet L. JLSTEIN UMASS Medical Sch. Principal Investigator Montecino, Martin mmonteci Universidad de Co-Pi Concepcion, Chile Imbalzano, Anthony N. AN IMBALZANO UMASS Medical Sch. Co-Pi OTHER SIGNIFICANT CONTRIBUTORS Name Organization Role on Project Lian, Jane B. UMASS Medical Sch. Collaborator Stein, Gary S. UMASS MedicalSch. Collaborator van Wijnen, Andre J. UMASS Medical Sch. Collaborator Lapointe, David UMASS Medical Sch. Collaborator Human Embryonic Stem Cells [X] No D Yes If the proposed project involves human embryonic stem cells, list below the registration number of the specific cell llne(s) from the following list: http://stemcells.nih.qov/registrv/index.asp. Use continuation pages as needed. If a specific line cannot be referenced at this time, include a statement that one from the Registry will be used. Cell Line PHS 398 (Rev. 04/06) Page 241 Form Page 2-continued Number the followingpages consecutively throughout the application. Do not use suffixes such as 4a, 4b. PROJECT 3 Principal Investigator/Program Director (Last, First, Middle): Stein, Gary S. DETAILED BUDGET FOR INITIAL BUDGET PERIOD FROM THROUGH DIRECT COSTS ONLY 7/1/07 6/30/08 PERSONNEL (Applicant organization only) Months Devoted to Project ROLE ON Cal. Acad. Sum. INST.BASE NAME PROJECT Mnths Mnths Mnths SALARY Principal Janet L. Stein 1.80 166,683 Investigator Co-Principal Martin Montecino 1.20 0 Investigator Co-Principal Anthony Imbalzano 1.20 126,764 Investigator Research Thomas Barthel Associate 12.00 38,000 Postdoctoral Sharanjot Saini 12.00 36,000 Associate Graduate Cara Weismann 12.00 25,740 Student SUBTOTALS CONSULTANT COSTS EQUIPMENT (Itemize) DOLLAR AMOUNT REQUESTED (omit cents) SALARY FRINGE REQUESTED BENEFITS TOTAL 25,002 8,686 33,688 0 0 0 12,676 4,404 17,080 38,000 13,201 51,201 36,000 12,506 48,506 25,740 3,573 29,313 137,418 42,370 179,788 SUPPLIES (Itemize by category) Radioisotopes and autoradiography 5,400 General chemicals 7,000 Molecular biology enzymes and kits 13,000 Antibodies/Immunology 9,300 Electrophoresis reagents/supplies 5,100 Laboratory supplies and plastics 10,437 50,237 TRAVEL ASBMR Annual Meeting (3 personnel), 4,800;M. Montecino (4 trips/year), 8,000 12,800 PATIENT CARE COSTS INPATIENT OUTPATIENT ALTERATIONS AND RENOVATIONS (Itemize by category) OTHER EXPENSES (Itemize by category) Isotope fee/person (3 x 350) 1 ,050 Equipment maintenance 2,500 Publication costs 3,350 Biomedical imaging 2,000 8,900 CONSORTIUM/CONTRACTUAL COSTS DIRECT COSTS SUBTOTAL DIRECT COSTS FOR INITIAL BUDGET PERIOD (Item 7a, Face Page) $ 251 ,725 CONSORTIUM/CONTRACTUAL COSTS FACILITIES AND ADMINISTRATIVE COSTS TOTAL DIRECT COSTS FOR INITIAL BUDGET PERIOD $ 251,725 PHS 398 (Rev.04/06) Page 242 Form Page 4