We propose a systematic investigation of the detailed structure of extended stretches of chromatin encompassing the rat osteocalcin and H4 histone genes as a function of changes in their expression during osteoblast differentiation. A major goal will be to interrelate information at the molecular and cytological levels to understand the relationship of sequence context with higher level chromatin packaging. The short and long-range sequence context of genes involved in osteoblast differentiation will be investigated for specific sequence landmarks, including immediate regulatory regions, enhancers and activators, CpG islands, and the distribution of different classes of repetitive elements. At another level, the chromatin packaging of these DNA sequences will be examined using assays for DNase I sensitivity and hypersensitivity, as well as assays for matrix attachment regions (MARs). While these studies will make a significant contribution to understanding the molecular elements involved in osteoblast gene regulation, we hop to extend this work to address fundamental questions concerning chromatin packaging that have not been addressed for any gene. A recently developed cytological approach will be used to visualize specific gene packaging with respect to the loop/scaffold structure, as a function of osteoblast differentiation or developmental commitment. By coupling defined chromatin distension protocols to reveal loop domain organization with high resolution in situ hybridization to specific sequences, we will examine changes in overall packaging of individual genes. In addition, the relative positions of specific molecular using both fluorescence and electron microscopy. The studies proposed here will provided basic information as to the regulatory elements involved in osteoblast gene regulation, but also will have the potential to advance significantly our understanding of how specific molecular landmarks are packaged relative to the loop/scaffold structure and how changes in gene expression correlate with and may be mediated by, chromatin packaging.
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