OSTEOBLAST CHROMATIN STRUCTURE AND NUCLEAR DOMAINS

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The goal of this project is to understand how chromatin organization and nuclear compartmentalization contribute to and support regulation of osteoblast-related gene expression. Transcription factors and protein- DNA interactions clearly play an important role in cell and tissue- specific gene regulation. However, such gene-regulatory interactions occur within DNA that is packaged as chromatin and further organized within the nucleus. We are interested in understanding how chromatin packaging affects the ability of a gene to be expressed and how sequence elements and binding factors mediate bone tissue-specific chromatin structure/organization. We will also address the role of nuclear location and spatial organization of osteoblast-expressed genes in osseous and non-osseous cells. We hypothesize that alterations in chromatin structure and nuclear organization accompany and mediate changes in gene expression. Our specific aims are: (1) To determine regulatory parameters that mediate chromatin structure of a bone tissue- specific gene in osteoblastic cells. Sequence-specific gene regulatory elements, nucleosome positioning, and histone acetylation will be manipulated and effects on chromatin structure and expression will be evaluated in stable cell lines. (2) To analyze biochemical and molecular mechanisms that alter the chromatin structure of the osteocalcin gene promoter. We will address these mechanisms by using an in vitro, cell-free system to monitor alterations in chromatin structure that result from interactions of tissue-specific transcription factors, hormone receptors, and the SWI/SNF complex with DNA in a nucleosomal context. (3) To examine the spatial relationship of osteoblast expressed genes to intra-nuclear compartmentalization in osteogenic cells, contrasted with non-osseous cells. We will analyze the distribution of bone and muscle chromatin, relative to Sc-35 domains in cells induced to switch from a myogenic to an osteogenic developmental program. These studies will contribute to understanding regulation of a key component of nuclear architecture that supports transcription during osteoblast differentiation.