Experiments will be carried out to characterize the molecular mechanisms that regulate and coordinate the expression of the neurofilament (NF) triplet proteins. Cis-acting elements in the 5' flanking (and other) regions of the three mouse NF genes will be mapped by cell transfection and by in vitro transcription. Binding sites of trans-acting factors that are located in the same regions of the NF genes will be identified by gelshift, methylation interference and DNase I footprinting. The effects of the respective binding factors on NG transcription will be examined by comparing transcriptional activities of NF constructs bearing wild vs mutant (non-binding) binding sites. The presence of binding factors will be compared in cells and in tissues that express varying levels of endogenous NF genes, including neuronal (PC12, and Neuro 2a) vs non- neuronal (HeLa and L cells) cell lines and in high- (DRG), low- (brain) and non- (liver) expressing tissues of the rat (or mouse). Transcriptional factors that are unique to high-expressing tissues and bind to regulatory elements in one or more NF genes will be cloned and further characterized. The extent of transcriptional vs post-transcriptional control of NF expression will be analyzed in primary cultures of rat DRG neurons. The ability of axonal factors (or signals) to regulate NF expression will be studied by further examining the downregulation of NF expression in rat DRG neurons following sciatic nerve transection under conditions in which the microenvironment of the severed axons are controlled and manipulated. The overall intent of the research project is to elucidate the factors that regulate NF expression and to begin to identify and characterize these factors. NF proteins represent the most abundant neuron-specific gene product. In addition, NF expression is highly instrumental in several basic neuronal events, such as neuronal differentiation and development, the maintenance of steady-state neuronal gene expression and the regenerative response following neuronal injury. Insight into the factors that regulate NF gene expression is likely to reflect on the nature of fundamental phenomena that underlie and determine the structure and function of the neuron.
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