Characterization of the rat type III hexokinase gene promoter - A functional Octamer 1 motif is critical for basal promoter activity

A 1532-base pair 5'-flanking region of the gene encoding rat type Ill hexok inase has been cloned and sequenced. The total sequence includes positions -1548 to -17 (A of the translational start ATG; as position +1). Using luci ferase reporter constructs transfected into PC12 (rat pheochromocytoma) and L2 (rat lung) cells, basal promoter activity has been associated with sequ ence between -182 and -89. This includes a single transcriptional start sit e, an adenine at position -134 identified by primer extension. Together wit h previously cloned cDNA sequence, this accounts for an mRNA of approximate ly 3.9 kilobases, found by Northern blotting of RNA from rat lung and kidne y. Sequence upstream of the transcriptional start site was devoid of canoni cal TATA and CAAT elements, An octamer 1 (Oct-1) binding site, located betw een positions -166 and -159 was shown by deletion analysis and site-directe d mutation to be critical for promoter activity. Nuclear extracts from PC12 cells contained a protein (or proteins) specifically binding the octamer s equence, and supershift experiments with anti-Oct-1 indicated involvement o f this ubiquitously expressed transcription factor in the complex. Sequence including the Oct-1 site and immediately adjacent regions was protected fr om DNase I digestion in footprinting experiments with nuclear extracts from PC12 cells. Reverse transcription polymerase chain reaction indicated that levels of type IPI hexokinase mRNA in rat tissues increased in the order b rain < liver < lung approximate to kidney; immunoblotting indicated that ty pe III hexokinase protein in these tissues increased in a similar manner.