IN-VITRO ACTIVATION OF THE MOUSE MID-SIZED NEUROFILAMENT GENE BY AN NF-1-LIKE TRANSCRIPTION FACTOR

In vitro transcription using nuclear extracts from rat brain and Liver were used to assess the tissue-specific and functional elements of th e mouse neurofilament mid-sized gene promoter (pNF-M). Deletion from - 2.7 to -103 (relative to the start site of transcription) resulted in a small increase (2-fold) in the activity of the NF-M promoter in both extracts. Promoter strength was slightly higher in brain vs. liver ex tracts. Deletion to -49 resulted in a 10-fold loss of promoter activit y in brain extracts and 6-fold drop in liver. Transcription in both ex tracts was TATA box-dependent. The region between -65 and -40 was show n to contain sequences responsible for high-level NF-M promoter activi ty in brain and liver extracts. Within this region are Spl and NF-1-li ke binding sites. Mutation of the NF-1-Like site (-53/-39) caused a la rge drop in the activity of the NF-M promoter while mutation of the Sp l site (-64/-57) possibly slightly diminished promoter activity in bra in and liver extracts. Both the Spl and NF-l-like sites were shown by gel shift competition and supershift assays to be able to bind their r espective factors. We conclude that the basic mouse NF-M promoter is a promiscuous promoter whose activity is modulated by a NF-l-like trans cription factor. The lack of tissue specificity in an in vitro system strongly suggests an important role for chromatin structure in the reg ulation of the mouse NF-M promoter.