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 upstream 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, constructs 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 expression 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 (HeLa, C127, NIH 3T3). Regions responsible for the basic promoter activity were located between -407 and +75 bp from the transcription initiation site. The NGF-responsive element was located between -38 and +75 bp, and sequence -97 to -38 was found to contain a functional cAMP-responsive element. In PC12 cells in which nerve growth factor (NGF) induces neurite outgrowth and NF-L transcription, NF-L promoter-driven CAT expression was stimulated up to 12-fold within three days of NGF treatment, whereas epidermal growth factor (EGF) had no effect. Rat NF-L promoter contained Sp1, AP-2 and CGCCCCCGC elements. In PC12 cells, NGF transiently induced the binding of transcription factors to the deoxyoligonucleotide probes containing the binding sites of these elements. The role of these factors in NF-L gene transcriptional induction by NGF in PC12 cells is discussed. (C) 1995 Wiley-Liss, Inc.