CHARACTERIZATION OF THE RAT LIGHT NEUROFILAMENT (NF-L) GENE PROMOTER AND IDENTIFICATION OF NGF AND CAMP-RESPONSIVE REGIONS

We have isolated a genomic DNA clone covering the coding and 14 kb ups tream region of the rat light neurofilament (NF-L) gene and sequenced 2.3 kb of its promoter. DNase I hypersensitive sites have been mapped in PC12 cells. For functional analysis of the NF-L promoter, construct s carrying 38, 97, 407, 564, 650, 1,099, 1,660, 2,003 base pairs (bp) upstream region in front of the chloramphenicol acetyltransferase (CAT ) reporter gene were tested for their capability to direct CAT express ion after transient transfection into various cell lines. Similar CAT activities were recorded both in rat pheochromocytoma (PC12) and mouse neuroblastoma N115 cells and also in several nonneural cell lines (He La, C127, NIH 3T3). Regions responsible for the basic promoter activit y were located between -407 and +75 bp from the transcription initiati on site. The NGF-responsive element was located between -38 and +75 bp , and sequence -97 to -38 was found to contain a functional cAMP-respo nsive element. In PC12 cells in which nerve growth factor (NGF) induce s neurite outgrowth and NF-L transcription, NF-L promoter-driven CAT e xpression was stimulated up to 12-fold within three days of NGF treatm ent, whereas epidermal growth factor (EGF) had no effect. Rat NF-L pro moter contained Sp1, AP-2 and CGCCCCCGC elements. In PC12 cells, NGF t ransiently induced the binding of transcription factors to the deoxyol igonucleotide probes containing the binding sites of these elements. T he role of these factors in NF-L gene transcriptional induction by NGF in PC12 cells is discussed. (C) 1995 Wiley-Liss, Inc.