Genetic Interaction Between SOX9 and RUNX2/CBFA1during chondrogenesis

描述(申请人提供)：这份面向广州博士的NICDR-K22职业发展奖申请提出了为期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，由布伦丹·李博士和杰拉德·卡森蒂博士指导。在此期间，拟议的职业发展活动包括德克萨斯儿童医院的骨骼发育不良临床课程、德克萨斯大学牙科分校的颅面和牙齿遗传学特别课程、M.D.安德森癌症中心的小鼠遗传学研讨会、Lee博士实验室的原位杂交和ES细胞技术，以及Karsty博士实验室的骨组织学培训。周博士目前的职业目标是完成博士后培训，并在学术/医学中心环境中获得一个独立的研究员职位。在长期的职业生涯中，周博士将致力于骨和软骨病理生理学的生物医学研究。 骨骼发生涉及构型、细胞命运承诺、分化、生长和重塑的协调过程。Runx2/CBFA1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RUNX2的表达调控知之甚少，尤其是在软骨形成过程中。我们最近发现SOX9是一种转录抑制因子，是RUNX2反式激活的有效转录抑制因子，它是包括颅神经脊细胞分化在内的软骨形成所必需的。我们建议利用体内和体外的工具来进一步了解SOX9和RUNX2在骨骼形成，特别是软骨细胞肥大过程中的相互作用。我们将利用原位杂交技术平行研究Sox9和Runx2在软骨形成过程中的表达模式。将进行GST下拉实验，以检查SOX9是否与RUNX2在物理上相互作用。在组织培养系统中，我们将在软骨形成细胞系ATDC5和多能间充质前体细胞系C2C12中过表达SOX9或其抑制域，以确定它们是否能在体外干扰软骨细胞肥大和成骨。Sox9对Runx2表达及其下游基因的影响将通过Northern分析和酶分析来分析。我们还将在肥大的软骨细胞中产生错误表达SOX9的转基因小鼠，以确定体内对长骨生长的影响。这项研究将帮助我们了解适当的骨骼模式和细胞命运承诺是如何实现的。

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.