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）是本实验室发现的一种在成骨细胞分化过程中起重要作用的转录因子。在胚胎骨形成过程中，Osx将前体细胞转化为功能齐全的成骨细胞，合成并分泌骨基质的成分。根据我们的初步结果，我们假设Osx继续在成骨细胞和骨细胞功能中发挥关键作用，超越胚胎发育，在出生后骨稳态的生理控制中。为了理解Osx在出生后的作用，已经产生了Osx在出生后失活的小鼠。这些小鼠的骨将通过microCT、组织形态计量学和电子显微镜方法以及通过成骨细胞、骨细胞和破骨细胞的分子标志物的定量来广泛表征。突变小鼠还将测试Ox是成骨细胞中典型Wnt信号传导的负调节剂的假设。我们最近已经确定了染色质蛋白NO66作为Osx转录活性的第一个负调节因子。NO66与Osx结合，具有脱甲基酶活性，其靶向转录活性染色质的组蛋白H3K4和H3K36标记物的甲基化形式。这种特异性的组蛋白去甲基化酶活性是NO66抑制Osx转录激活功能所必需的。基于成骨细胞的敲除实验，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。表征小鼠突变体，其中NO66在Osx表达细胞中条件性失活或条件性过表达，对于理解NO66在骨中的生理作用至关重要。为了获得一个机械的理解Osx和NO66之间的关系，我们还建议检查Osx和NO66占用在成骨细胞分化过程中的Osx靶基因的染色质中的动态，并调查是否Osx占用在Osx靶基因的染色质中的动态被破坏的小鼠成骨细胞中，NO66是有条件地失活或过表达在这些细胞。总体而言，拟议的实验应该提供相当多的新的见解骨形成的机制Osx在骨重建的稳态控制。公共卫生相关性：了解控制骨形成的机制对于确定骨疾病适当治疗的靶点至关重要。Osterix是一种转录因子，在胚胎发育期间骨形成完全需要。我们的假设是，出生后和整个生命过程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还控制骨中细胞增殖的特定信号的活性。在这个项目的另一部分，我们将测试的假设，一个特定的染色质蛋白质，我们最近发现，控制骨中的Osterix的活动。

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.