Graduate School of Biomedical Sciences, Cell Biology
Osteocalcin; Promoter Regions; Rats; Gene Expression; Cells; Academic Dissertations; Dissertations, UMMS
Transcription of the osteocalcin gene, which encodes a bone-specific 10 kDa protein, is controlled by the coordinated utilization of modularly organized basal and hormone-responsive enhancer elements. Activation of these sequences involves the interaction of specific transcription factors to these promoter elements. It is becoming increasingly accepted that nuclear architecture provides a basis for support of tightly regulated modulation of cell growth and tissue-specific transcription which is required for the onset and progression of differentiation. Thus packaging of DNA as chromatin can facilitate the cooperative interaction between activities of independent regulatory elements that contribute to the level of transcription. Here, we show that a specific nucleosomal organization supports the constitutive expression of the osteocalcin gene in ROS 17/2.8 rat osteosarcoma cells and that chromatin remodeling directly correlates with the developmentally regulated transcriptional activation of this gene in normal diploid osteoblasts. By combining DNase I, micrococcal nuclease, and specific restriction endonuclease digestion analysis, we observed that the presence of DNase I hypersensitive sites (proximal: -170 to -70, and distal: -600 to -400) and a selective nucleosome positioning over the osteocalcin gene promoter are directly associated with developmentally stage-specific transcriptional activation in bone-derived cells. In addition, we found that chromatin hyperacetylation prevents a key transition in the chromatin structure which is required for the formation of the distal DNase I hypersensitive site. This transition involves the interaction of specific nuclear factors and is necessary for the subsequent ligand-dependent binding of the vitamin D receptor complex. Finally, we have established a requirement for sequences residing in the proximal region of the osteocalcin gene promoter for both formation of the proximal hypersensitive site and basal transcriptional activity. Our approach was to assay nuclease accessibility in ROS 17/2.8 cell lines stably transfected with promoter deletion constructs driving expression of a CAT reporter gene.
Montecino, MA. Chromatin Structure of the Rat Osteocalcin Gene Promoter in Bone-Derived Cells. (1995). University of Massachusetts Medical School. GSBS Dissertations and Theses. Paper 33. http://escholarship.umassmed.edu/gsbs_diss/33
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