REGULATION OF CHROMATINIZED GENES IN OSTEOBLASTS

描述（改编自研究者摘要）：糖皮质激素（GC）

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.