REGULATION OF CHROMATINIZED GENES IN OSTEOBLASTS

描述（改编自研究者摘要）：糖皮质激素（GC） 通常用作抗炎和免疫抑制药物。主要 长期治疗的副作用是骨质疏松。虽然机制 GC对成骨细胞的影响被认为是一个关键， 成分骨钙素（OC）基因是一个建立良好的有用的模型， 研究成骨细胞特异性基因表达和GC已知下调 骨钙素转录。研究者开发了成骨细胞系 其稳定表达与报道基因连接的骨钙素启动子区。的 稳定的细胞系下调报告基因的表达，以响应 GC处理。重要的是，在这些细胞中瞬时表达的相同构建体， 行不响应GC。因此，研究者建议， 骨钙素和其他成骨细胞表型基因可能最初处于一种 染色质抑制状态。这些基因在特定的时间被去抑制， 发育和它们的表达导致细胞向细胞的承诺。 成骨细胞分化途径该假说认为GC的作用是 防止去抑制并维持成骨细胞基因（如骨钙素）在一个 压抑的状态稳定整合的骨钙蛋白启动子/报告基因将 作为模型系统来检验这一假设。染色质修饰 以及GC对这些修饰的影响 将通过ChIP测定和核酸内切酶可及性测定进行检查。的 稳定整合的启动子构建体将用于鉴定DNA元件， 转录因子介导GC的抑制作用， 成骨细胞系中的天然染色质背景。特定组蛋白 将检查乙酰转移酶活性（CBP和P300），以确定 GC对特定HAT蛋白水平或活性的影响。

DESCRIPTION (Adapted from the Investigator's Abstract): Glucocorticoids (GC) are commonly used as anti-inflammatory and immunosuppressive drugs. The major adverse effect of long-term therapy is osteoporosis. Although the mechanisms are not well known, effects of GC on osteoblasts are thought to be a key component. The osteocalcin (OC) gene is a well-established useful model to study osteoblast-specific gene expression and GC are known to down-regulate osteocalcin transcription. The investigator has developed osteoblast cell lines that stably express osteocalcin promoter regions linked to a reporter gene. The stable cell lines down-regulate expression of the reporter gene in response to GC treatment. Importantly, identical constructs transiently expressed in these lines do not respond to GC. Therefore, the investigator proposes that osteocalcin and perhaps other osteoblast phenotypic genes may be initially in a chromatin-repressed state. These genes are de-repressed at specific times in development and their expression results in the commitment of the cell to the osteoblast differentiation pathway. The hypothesis asserts that GC act to prevent the de-repression and maintain osteoblast genes (eg osteocalcin) in a repressed state. The stably integrated osteocalcin promoter/reporter genes will serve as the model system to test this hypothesis. Chromatin modifications within the osteocalcin promoter and the effect of GC on these modifications will be examined by ChIP assays and endonuclease accessibility assays. The stably integrated promoter constructs will be used to identify DNA elements and transcription factors that mediate the suppressive effects of GC within the native chromatin context in osteoblast cell lines. Specific histone acetyltransferase activities (CBP and P300) will be examined to determine the effect of GC on the level or activity of specific HAT proteins.