MOLECULAR BASIS OF NERVE GROWTH FACTOR
Principal Investigator: Jeffrey Milbrandt
Abstract: Nerve growth factor (NGF) is an important modulator of neuronal development and survival. To understand how NGF alters the pattern of gene expression in responsive cells, we have identified several genes that are rapidly induced by NGF in PC12 cells. Each of these three genes, c-fos, NGFI-A and NGFI-B, are rapidly transcribed in response to NGF, phorbol esters (TPA), and calcium ionophores (A23187), and the induction process does not require de novo protein synthesis. The c-fos protein is also a transcriptional activating factor, and NGFI-A and -B are homologous to transcriptional activating factors; therefore, each of them could play a role in controlling the differentiation process. To determine the sequence elements, or enhancers, that control the NGF-mediated induction of these genes, we plan to isolate the 5'flanking regions of each of these genes. The transcription start site will be established, and the nucleotide sequence will be determined from the start site upstream 1 to 2 kb. DNA fragments from each of these regions will be cloned upstream of the chloramphenicol acetyltransferase (CAT) gene, and each construct will be transfected into PC12 cells. In each instance, expression of the CAT gene will be monitored in the presence and absence of NGF to determine whether an NGF responsive element (NRE) resides in the fragment. Deletion mutants constructed from fragments that contain the NREs will be used to further delimit their location. "Trans-acting" factors that bind to the NREs and presumably promote the transcription of these genes will be detected by gel retardation and DNAse I protection experiments. To explore whether these genes are activated by a common mechanism, we will perform a comparison of the NRE sequences from each gene, and carry out competition experiments aimed at determining whether the same proteins bind to NREs located in different genes. Once the nucleotide sequence of the NREs is determined, oligonucleotide affinity chromatography will be exploited to purify these "trans-acting" factors. Elucidating the molecular mechanism by which NGF regulates gene expression will help us understand how NGF influences the survival and differentiation of neural-crest derived cell types.
Funding Period: 1988-07-01 - 1994-08-31
more information: NIH RePORT