Characterization of the Rat Type III Hexokinase Gene Promoter: A FUNCTIONAL OCTAMER 1 MOTIF IS CRITICAL FOR BASAL PROMOTER ACTIVITY

Abstract

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