Genetic Interaction Between SOX9 and RUNX2/CBFA1during chondrogenesis

描述（由申请人提供）：广州博士的NICDR-K22职业发展奖申请提出了一年的学者发展阶段和四年的教师过渡阶段。他的学者发展阶段培训将在贝勒医学院进行，由Brendan Lee博士和Gerard Karsenty博士指导。在此期间，拟议的职业发展活动包括德克萨斯州儿童医院的骨骼发育不良临床，UT牙科分支的颅面和牙齿遗传学特别课程，M.D.安德森癌症中心，原位杂交和ES细胞技术在李博士的实验室，和骨组织学培训在卡森蒂博士的实验室。Zhou博士的近期职业目标是完成博士后培训，并在学术/医疗中心环境中获得独立研究员职位。在他的长期职业生涯中，周博士将致力于骨和软骨病理生理学的生物医学研究。 骨骼发生涉及模式化、细胞命运定型、分化、生长和重塑的协调过程。RUNX 2/CBFA 1是成骨细胞分化、软骨细胞肥大和牙齿形成所必需的转录因子。然而，对RUNX 2表达的调控知之甚少，特别是在软骨形成过程中。我们最近发现SOX 9，软骨形成，包括颅神经嵴细胞分化所必需的转录因子，作为一个有效的转录抑制RUNX 2反式激活。我们建议利用体内和体外的工具，以进一步了解SOX 9和RUNX 2之间的相互作用，在软骨细胞肥大，特别是。我们将利用原位杂交技术平行研究Sox 9和Runx 2在软骨形成过程中的表达模式。将进行GST下拉实验以检查SOX 9是否与RUNX 2发生物理相互作用。在组织培养系统中，我们将在软骨形成细胞系ATDC 5和多能间充质前体细胞系C2 C12中过表达SOX 9或其抑制结构域，以确定它们是否可以在体外破坏软骨细胞肥大和成骨。将通过北方分析和酶测定来分析SOX 9对Runx 2表达及其下游基因的影响。我们还将产生在肥大软骨细胞中错误表达SOX 9的转基因小鼠，以确定体内长骨生长的后果。这项研究将帮助我们了解如何实现正确的骨骼模式和细胞命运的承诺。

DESCRIPTION (provided by applicant): This NICDR-K22 career development award application for Dr. Guang Zhou proposes a one-year Scholar Development Phase and a four-year Faculty Transition Phase. His Scholar Development Phase training will be performed at Baylor College of Medicine under the mentoring of Dr. Brendan Lee and Dr. Gerard Karsenty. During this period the proposed career development activities include Skeletal Dysplasia Clinical at Texas Children Hospital, special courses on craniofacial and dental genetics at UT-Dental Branch, mouse genetic seminar at M.D. Anderson Cancer Center, in situ hybridization and ES cell technique at Dr. Lee's laboratory, and bone histology training at Dr. Karsenty's laboratory. Dr. Zhou's immediate career goal is to complete his postdoctoral training and obtain an independent investigator position in an academic/medical center environment. For his long-term career Dr. Zhou will be committed to the biomedical research on bone and cartilage pathophysiology. Skeletogenesis involves the coordinated process of patterning, cell fate commitment, differentiation, growth, and remodeling. RUNX2/CBFA1 is a transcription factor essential for osteoblast differentiation, chondrocyte hypertrophy, and tooth formation. However little is known about the regulation of RUNX2 expression, especially during chondrogenesis. We recently identified SOX9, a transcription factor essential for cartilage formation including cranial neural crest cell differentiation, as a potent transcription repressor for RUNX2 transactivation. We propose to utilize both in vivo and in vitro tools to further understand the interaction between SOX9 and RUNX2 during skeletongenesis, especially chondrocyte hypertrophy. We will use in situ hybridization technique to study in parallel the expression patterns of Sox9 and Runx2 during chondrogenesis. GST-pulldown experiments will be performed to examine whether SOX9 physically interacts with RUNX2. In tissue culture systems, we will over-express SOX9 or its repression domain in chondrogenic cell line ATDC5 and pluripotent mesenchymal precursor cell line C2C12 to determine whether they can disrupt chondrocyte hypertrophy and osteogenesis in vitro. SOX9 effects on Runx2 expression and its downstream genes will be analyzed by Northern analysis and enzymatic assays. We will also generate transgenic mice mis-expressing SOX9 in hypertrophic chondrocytes to determine the consequences in vivo on long bone growth. This study will help us understand how proper skeletal patterning and cell fate commitment are achieved.