BRAIN-SPECIFIC ENHANCEMENT OF THE MOUSE NEUROFILAMENT HEAVY GENE PROMOTER IN-VITRO

We have investigated the DNA elements responsible for transcription fr om the proximal portion of the mouse neurofilament heavy gene (NF-H) p romoter by in vitro transcription using extracts from expressing (brai n) and non-expressing (liver) tissues. We have found that constructs c ontaining 5' region from -1314 to -115 exhibit a 3-5-fold higher level of NF-H promoter activity, relative to the adenovirus major late prom oter (pML), in brain versus liver extracts. Deletion to -85 lowers the level of brain transcription by 2-fold, while deletion from -65 throu gh -31 reduces transcription by 5-fold to a relatively strong (10% of pML) basal level. Basal level expression is observed in all deletions transcribed with liver extract. Deletion to -24 (TATA-less) abolishes promoter activity with both extracts. Deletion of the -115 to -65 regi on from a larger construct reduces transcription in brain extracts to basal levels, suggesting that this region contains the elements necess ary for the brain specific enhancement of promoter function. Mutation of a palindromic sequence within this region abolishes brain specific enhanced promoter activity. This loss of enhanced transcriptional acti vity is correlated with the loss of a shifted band in gel shift assays . Our studies suggest that the sequence (-106)GGGGAG-GAGG-(15 bp)-CCTC CTCCCC(-72) (where bp = base pairs) is important in brain-specific enh ancement of transcription from the mouse NF-H promoter.