Chromatin Organization and Nuclear compartmentalization in Osteoblast Gene Exp...

骨细胞生物学中的一个基本问题是，骨骼特异基因是如何被赋予能力的 成骨细胞发育过程中适当阶段的表达和类固醇激素反应 序列。该程序确定了骨形成所需的关键转录因子Runx2， 在表型过程中，在骨钙素(OC)启动子上活性染色质的组装中起着关键作用 进展，是类固醇激素反应所必需的。这个项目的长期目标是 了解核下局灶骨特异性基因染色质的动态重组 有助于激活成骨细胞发育转录程序以响应生理学 监管方面的暗示。重要的是，在当前的颁奖期间，我们已经展示了 SWI/SNF染色质重塑复合体也是表达和维生素D增强所必需的 OC基因在成骨细胞和成骨细胞分化中的作用。我们现在提议将我们激动人心的 关于成骨细胞表型诱导必须涉及组蛋白这一假说的发现 以成骨细胞特异性的方式对染色质进行修饰和有序重塑，以响应组织- 特定的因子和多组分的酶复合体顺序结合以激活和调节 转录和调节维生素D的反应。为了更广泛地了解基因表达是如何 在骨骼发育过程中，我们将采取高度合作的方式来描述：1. 结合事件和染色质重塑的Runx2依赖和维生素D反应程序 骨钙素启动子；2.骨表型基因的表观遗传标记和染色质重塑 成骨细胞分化过程中对Runx2和维生素D的反应；3.Runx2和维生素D 建立和维持成熟骨细胞所需的反应性基因表达程序 表型。这些研究将揭示Runx2和发育表达的关键辅助因素的作用。 维生素D对成骨细胞分化所需基因的调节。与以下公司合作 项目1和2将为Runx2在支持当地建筑方面的功能提供新的见解 骨特异性转录调控的基因启动子和共同调节因子的组织。 临床相关性：确定控制染色质组织的机制是令人难以理解的 靶向染色质的抑制剂在治疗影响骨骼的疾病中的作用和后果 成人骨骼的周转。 马萨诸塞大学医学院 湖畔大道北55号 马萨诸塞州伍斯特，邮编01655 小灵通398(04/06版)第240页表格第2页 项目3 首席研究员/项目主任(最后、第一、中间)：Stein，Gary S. 关键人员。请参阅说明。根据需要使用续页，以如下所示的格式提供所需信息。 从首席调查员(S)开始。按字母顺序列出所有其他关键人员，姓氏在前。 名称时代共享用户名项目上的组织角色 首页--期刊主要分类--期刊细介绍--期刊题录与文摘内容首席调查员 蒙特西诺，马丁·姆蒙蒂奇大学 智利康塞普西翁 书名/作者The Imbalzano Uma Medical Sch.Co-PI 其他重要贡献者 命名项目中的组织角色 首页--期刊主要分类--期刊细介绍--期刊题录与文摘内容协作者 题名/责任者：MASS MEDICAL Sch.协作者 题名/责任者：The Healthing of the MASH.协作者 作者声明：David L.协作者 人类胚胎干细胞[X]否D是 如果建议的项目涉及人类胚胎干细胞，请从以下列表中列出特定细胞(S)的注册号： Http://stemcells.nih.qov/registrv/index.asp.根据需要使用续页。 如果此时不能引用某一特定行，请附上一条声明，说明将使用注册处的一行。 细胞系 小灵通398(04/06版)第241页表格第2页-续 下面的页码从头到尾都要连续编号 应用程序。不要使用诸如4a、4b之类的后缀。 项目3首席调查员/项目主任(最后、第一、中间)：Stein，Gary S. 初始预算期间的详细预算，从 直接成本仅7/1/07 6/30/08 人员(仅限申请组织)用于项目的月份 在Cal上的角色。阿卡德。总和。INST.BASE 命名项目月薪 本金 珍妮特·L·斯坦1.80 166,683 调查员 共同校长 马丁·蒙特西诺1.20 0 调查员 共同校长 安东尼·因巴尔扎诺1.20 126,764 调查员 研究 Thomas Barthel Associate 12.00 38,000 博士后 Sharanjot Saini 12.00 36,000 联想 毕业生 卡拉·魏斯曼12.00 25,740 学生 小计 顾问费 设备(分项) 请求的美元金额(省略美分) 工资福利 申请的福利总额 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 用品(按类别分项列出) 放射性同位素和放射自显影5,400通用化学品7,000 分子生物学酶和试剂盒13,000抗体/免疫学9,300 电泳剂/用品5,100 实验室用品和塑料10,437 50,237 旅行 ASBMR年会(3人)，4800人；M.Montecino(每年4次)，8000人，12800人 住院病人护理费用 门诊病人 改建和翻新(按类别分列) 其他费用(按类别分项列出) 同位素费用/人(3x350)1050 设备维护2,500 出版费用3350美元 生物医学成像2,000 8900 联合体/合同成本直接成本 初步预算期的直接费用小计(项目7a，正面页)$251,725 联合体/合同费用、设施和行政费用 初期预算期间的直接费用总额为251,725美元 小灵通398(04/06版)第242页表格第4页

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