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成骨细胞分化过程中必不可少的转录因子。在胚胎的骨形成过程中，OSX承诺前体细胞成为功能齐全的成骨细胞，成骨细胞合成和分泌骨基质的成分。根据我们的初步结果，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，在生理学上控制出生后的骨内稳态。为了了解OSX在出生后的这种作用，已经产生了OSX在出生后失活的小鼠。这些小鼠的骨骼将通过显微CT、组织形态计量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的定量来广泛地表征。突变的小鼠还将测试Ox是成骨细胞中典型的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控因子。No66与OSX结合，具有去甲基酶活性，靶向转录活性染色质的组蛋白H3K4和H3K36标记的甲基化形式。这种特异性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。基于成骨细胞的基因敲除实验，我们推测染色质蛋白NO66在成骨细胞中具有重要的生理功能。在表达OSX的细胞中，No66条件失活或条件过表达的小鼠突变体的特征，对于了解No66在骨骼中的生理作用是必不可少的。为了从机制上了解OSX和NO66之间的关系，我们还建议在成骨细胞分化过程中检测OSX靶基因染色质中OSX和NO66的动态占据，并研究在NO66有条件失活或在这些细胞中过度表达的小鼠成骨细胞中，OSX靶基因染色质中OSX占据的动态是否被破坏。总体而言，拟议的实验将为OSX在骨重建的动态平衡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：了解骨形成被控制的机制，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，是胚胎发育过程中骨形成所必需的。我们的假设是，Osterix在出生后和一生中也是骨基质成分合成所必需的，也是骨细胞正常功能所必需的，骨细胞是嵌入骨基质中的细胞。我们还假设，除了Osterix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中，我们将测试一种假设，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.