The overall objective of this proposal is to understand the mechanisms underlying the cell-type specific and regulated expression of the neurotensin (NT) gene. These studies focus upon the expression of the gene in neural crest-derived cells. Two tissue culture cell lines (PC12 and rMTC 6-23) will be used as model systems. Previous studies have established that NT content and production are regulated in a highly cooperative manner by nerve growth factor (NGF), glucocorticoids, and cAMP levels in PC12 cells with NGF exerting primarily a permissive effect. The combined action of NGF, dexamethasone, and activators of adenylate cyclase results in a hugh increase of NT content up to 600-fold. Using a cDNA for the NT/neuromedin N precursor, we have demonstrated that these induction conditions also result in a striking increase in NT mRNA levels suggesting that the increases in NT content are largely the result of increased NT mRNA levels. These experiments will be extended to examine in detail the effects of different induction conditions on NT mRNA levels, and NT gene transcription rates. The relative rate of synthesis of the NT precursor protein and the translatability of NT mRNA will also be examined. Companion studies will be performed using rMTC 6-23 cells. To verify that these effects are relevant to NT gene expression in vivo, NT mRNA levels will be examined by in situ hybridization both in tissues and in primary cultures of adrenal medullary and superior cervical ganglion cells. We will analyze in detail the cis-regulatory sequences necessary for cell- type specific and regulated expression of the NT gene by DNA transfection and determine whether induction is accompanied by changes in chromatin structure. Specific cis-regulatory sequences will be used as probes to identify specific proteins which bind to these regions. We are particularly interested in the NGF component of the induction. These experiments will set the basis for the purification and cloning of transcription factors which mediate the cell-type specific expression of the NT gene.
| Xing, Y; Johnson, C V; Dobner, P R et al. (1993) Higher level organization of individual gene transcription and RNA splicing. Science 259:1326-30 |
