Synthesis of Osteocalcin in Bone

描述(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)成熟的诱导和进展的因素。在当前的计划阶段，我们发现了一个同源结构域(HD)蛋白的调控网络，它为介导OC的瞬时表达提供了分子开关，在OC转录开始时对DLX3起着关键的激活作用。发现了一种新的HOX转录因子调控元件，它与HOXA10结合以激活OC转录。Hoxa10和DLX3在Runx2之前与OC基因结合，与Runx2结合后再次增加。此外，这些因子可以激活Runx2缺失细胞中的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。这些发现为进一步研究它们在体内和体外成骨细胞中的功能作用提供了依据。我们推测，这两个调节因子在OB分化过程中对基因表达起着关键作用，这一功能不同于它们在骨骼胚胎模式形成中的活动。我们将通过确定它们调节OC发育表达的机制来识别这些与OB相关的功能，包括促进染色质重塑，通过与共同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和与Runx2协同作用来建立OB表型。AIM1验证了这一假说，即这些因子与骨钙素染色质的动态结合/解离发生在表型发育的特定阶段，原因是转录因子复合体的形成改变了OC的染色质结构。目的2提出Hoxa10在Runx2上游发挥作用的假说，促进OC基因的表观遗传标记，以实现组织特异性激活，并调节OBS中靶基因的表达。在体外和体内，它从OB中缺失被认为改变了OB的成熟度，并有助于成年小鼠的轻度骨表型。目的3推测DLX3在基因调控中具有短暂的作用，负责OC基因转录的时间和Runx2的协调占据，这将在DLX3缺失和条件性基因敲除小鼠的细胞中得到解决。我们进一步假设，人类DLX3三齿骨(TDO)突变改变了成骨细胞中DLX3的瞬时功能，这是相互作用的共调节蛋白功能丧失的结果。这些研究将确定在矿化基质中诱导和调节OC以及调节OB成熟到分化表型所需的新机制和调控途径。

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