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 the 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 extracts. Deletion to -49 resulted in a 10-fold loss of promoter activity in brain extracts and 6-fold drop in liver. Transcription in both extracts was TATA box-dependent. The region between -65 and -40 was shown to contain sequences responsible for high-level NF-M promoter activity in brain and liver extracts. Within this region are Sp1 and NF-1-like binding sites. Mutation of the NF-1-like site (-53/-39) caused a large drop in the activity of the NF-M promoter while mutation of the Sp1 site (-64/-57) possibly slightly diminished promoter activity in brain and liver extracts. Both the Sp1 and NF-1-like sites were shown by gel shift competition and supershift assays to be able to bind their respective factors. We conclude that the basic mouse NF-M promoter is a promiscuous promoter whose activity is modulated by a NF-1-like transcription factor. The lack of tissue specificity in an in vitro system strongly suggests an important role for chromatin structure in the regulation of the mouse NF-M promoter.