Synthesis of Osteocalcin in Bone

描述（由申请人提供）：该项目继续研究骨钙素（OC）基因的组织特异性和发育调节以及促进成骨细胞（OB）成熟诱导和进展的因素。在目前的计划期间，我们发现了一个同源域（HD）蛋白的调节网络，提供分子开关，用于介导OC的时间表达，在OC转录开始时，Dlx3具有关键的激活作用。Hox转录因子的一个新的调控元件被发现，结合Hoxa10激活OC转录。Hoxa10和Dlx3在Runx2之前与OC基因结合，并在Runx2结合时再次增加。此外，这些因子可以激活Runx2无效细胞中的OC基因，从而为促进Runx2上游OC和骨相关基因表达的分子事件提供证据。这些发现证明了进一步研究其在体内和体外成骨细胞中的功能作用。我们推测，这两个调节因子在调节整个OB分化的基因表达中具有关键作用，这一功能不同于它们在胚胎骨骼模式中的活动。我们将通过确定这些OB相关功能调节OC发育表达的机制来鉴定这些功能，包括促进染色质重塑，通过与共调节因子的相互作用调节基因转录的激活和抑制，以及独立于Runx2并与Runx2协调以建立OB表型。目的1测试的假设，动态协会/解离这些因素与骨钙蛋白染色质发生在特定阶段的表型发展，由于形成转录因子复合物修改染色质结构的OC。目的2地址的假设，Hoxa10的功能上游Runx2促进表观遗传标记的OC基因的组织特异性激活，并在OBs的靶基因表达的调节。其从OB在体外和体内的缺失被假定为改变OB成熟，并有助于一个温和的骨表型在成年小鼠。目的3假设Dlx3在基因调控中具有瞬时作用，负责OC基因的转录时间和Runx2的协调占据，这将在来自Dlx3缺失和条件性敲除小鼠的细胞中离体解决。我们进一步假设，人类Dlx 3牙-牙-骨（TDO）突变改变了Dlx 3在成骨细胞中的瞬时功能，这是相互作用的共调节蛋白功能丧失的结果。这些研究将确定新的机制和调控途径所需的诱导和调节OC和调节OB成熟的分化表型在矿化基质。项目叙述

DESCRIPTION (provided by applicant): This program continues to study the tissue specific and developmental regulation of the osteocalcin (OC) gene and the factors that contribute to induction and progression of osteoblast (OB) maturation. In the current program period, we discovered a regulatory network of homeodomain (HD) proteins that provide molecular switches for mediating the temporal expression of OC with a pivotal activating role for Dlx3 at the onset of OC transcription. A novel regulatory element for the Hox transcription factors was discovered that binds Hoxa10 for activation of OC transcription. Hoxa10 and Dlx3 are bound to the OC gene prior to Runx2 and increase again with Runx2 binding. In addition, these factors can activate the OC gene in Runx2 null cells, thus providing evidence for molecular events contributing to expression of the OC and bone related genes upstream of Runx2. These findings justify further investigation of their functional roles in osteoblasts in vivo and in vitro. We postulate that these two regulatory factors have key roles in regulating gene expression throughout OB differentiation, a function distinct from their activities in embryonic patterning of the skeleton. We will identify these OB- related functions by determining the mechanism by which they regulate developmental expression of OC which include contributing to chromatin remodeling, regulating activation and repression of gene transcription through interactions with co-regulatory factors, and functioning both independent of Runx2 and coordinated with Runx2 for establishment of the OB phenotype. Aim1 tests the hypothesis that dynamic association/dissociation of these factors with osteocalcin chromatin occurs at specific stages of phenotype development due to formation of transcription factor complexes modifying chromatin architecture of OC. Aim 2 addresses the hypothesis that Hoxa10 functions upstream of Runx2 to promote epigenetic marking of the OC gene for tissue specific activation, and regulation of target gene expression in OBs. Its deletion from OB in vitro and in vivo is postulated to alter OB maturation and contribute to a mild bone phenotype in adult mice. Aim 3 postulates that Dlx3 has a transient role in gene regulation, responsible for the timing of transcription of the OC gene and coordinated occupancy of Runx2 which will be addressed ex vivo in cells from the Dlx3 null and conditional knockout mice. We further postulate that the human Dlx3 tricho-dento-osseous (TDO) mutation alters the transient function of Dlx3 in osteoblasts as a result of loss-of-function of interacting co-regulatory proteins. These studies will identify novel mechanisms and regulatory pathways required for induction and regulation of OC and regulation of OB maturation to the differentiated phenotype in a mineralizing matrix.PROJECT NARRATIVE