Genetic Control of Osteoblast Differentiation

描述（由申请人提供）：了解控制骨形成和骨转换的复杂机制具有重要的医学意义。Osterix （Osx）是我们实验室发现的成骨细胞分化的重要转录因子。在胚胎骨形成过程中，Osx使前体细胞成为功能齐全的成骨细胞，合成和分泌骨基质成分。基于我们的初步结果，我们假设Osx在胚胎发育之外的成骨细胞和骨细胞功能中继续发挥关键作用，在出生后对骨稳态的生理控制中发挥关键作用。为了了解Osx在出生后的作用，我们在出生后培养了Osx失活的小鼠。这些小鼠的骨骼将通过微ct、组织形态学和电子显微镜方法以及成骨细胞、骨细胞和破骨细胞的分子标记物的定量进行广泛的表征。突变小鼠还将验证Ox是成骨细胞中典型Wnt信号的负调节因子的假设。我们最近发现染色质蛋白NO66是Osx转录活性的第一个负调节因子。NO66与Osx结合，具有去甲基化酶活性，靶向转录活性染色质组蛋白H3K4和H3K36标记物的甲基化形式。这种特定的组蛋白去甲基酶活性是NO66抑制Osx转录激活功能所必需的。基于成骨细胞的敲低实验，我们假设染色质蛋白NO66在成骨细胞中具有关键的生理功能。研究小鼠突变体中NO66在Osx表达细胞中条件失活或条件过表达，对于了解NO66在骨骼中的生理作用至关重要。为了从机制上理解Osx和NO66之间的关系，我们还建议研究成骨细胞分化过程中Osx和NO66在Osx靶基因染色质上的占据动力学，并研究在小鼠成骨细胞中，当NO66条件失活或过表达时，Osx在Osx靶基因染色质上的占据动力学是否被破坏。总的来说，所提出的实验应该为Osx在骨重塑的稳态控制中的骨形成机制提供相当多的新见解。公共卫生相关性：了解控制骨形成的机制，对于确定骨疾病的适当治疗靶点至关重要。Osterix是胚胎发育过程中骨形成所完全需要的转录因子。我们的假设是，在出生后和一生中，骨基质成分的合成以及骨细胞（嵌入骨基质中的细胞）的正常功能也需要Osterix。我们还假设，除此之外，Osterix还控制着骨骼中细胞增殖的特定信号的活动。在这个项目的另一部分，我们将测试一个假设，即我们最近发现的一种特定的染色质蛋白，控制着骨骼中Osterix的活性。

DESCRIPTION (provided by applicant): Understanding the complex mechanisms that control bone formation and bone turnover has major medical implications. Osterix (Osx), which was discovered in our laboratory, is an essential transcription factor in osteoblast differentiation. During bone formation in the embryo Osx commits precursor cells to become fully functioning osteoblasts, which synthesize and secrete the components of the bone matrix. Based on our preliminary results, we hypothesize that Osx continues to have a critical role in osteoblast and osteocyte function beyond embryonic development in the physiological control of bone homeostasis postnatally. To understand this role of Osx after birth, mice have been generated in which Osx is inactivated postnatally. Bones of these mice will be extensively characterized by microCT, histomorphometry and electron microscopy methods as well as by quantitation of molecular markers of osteoblasts, osteocytes and osteoclasts. The mutant mice will also test the hypothesis that Ox is a negative regulator of canonical Wnt signaling in osteoblasts. We have recently identified the chromatin protein NO66 as the first negative regulator of the transcriptional activity of Osx. NO66, which binds to Osx, has a demethylase activity that is targeted to the methylated forms of histone H3K4 and H3K36 markers of transcriptionally active chromatin. This specific histone demethylase activity is needed for the inhibition of the transcription activation function of Osx by NO66. Based on knockdown experiments in osteoblasts we hypothesize that the chromatin protein NO66 has a critical physiological function in osteoblasts. Characterization of mouse mutants in which NO66 is either conditionally inactivated or conditionally overexpressed in Osx expressing cells, is essential to understand the physiological role of NO66 in bones. To gain a mechanistic understanding of the relationship between Osx and NO66, we also propose to examine the dynamics of Osx and NO66 occupancy in the chromatin of Osx target genes during osteoblast differentiation and to investigate whether the dynamics of Osx occupancy in the chromatin of Osx target genes are disrupted in osteoblasts of mice in which NO66 is either conditionally inactivated or overexpressed in these cells. Overall, the proposed experiments should provide considerable new insights in the mechanisms of bone formation by Osx in the homeostatic control of bone remodeling. PUBLIC HEALTH RELEVANCE: Understanding the mechanisms by which bone formation is controlled, is of critical importance for identifying targets for appropriate therapies for bone diseases. Osterix is a transcription factor that is completely required for bone formation during embryonic development. Our hypothesis is that Osterix is also required for the synthesis of components of the bone matrix after birth and throughout life and for the normal function of osteocytes, the cells that are embedded in the bone matrix. We also hypothesize, that in addition Osterix controls the activity of specific signals of cell proliferation in bones. In another part of this project we will test the hypothesis that a specific chromatin protein, which we recently identified, controls the activity of Osterix in bones.