Human HKR isozyme: Organization of the hexokinase I gene, the erythroid-specific promoter, and transcription initiation site

We previously described a cDNA for the human HKR isozyme, whose sequence is identical to that of the ubiquitous HKI isozyme, except for a unique 5' en d sequence. Screening a human genomic library with a DNA fragment containin g an erythroid-specific sequence we found one clone including 5' ends for b oth HKR and HKI genes. The first HXR exon was located 3 kb 5' of the first HKI exon. These results confirmed that HKR is produced from the HKI gene by alternate promoter and splicing, The HKI gene consisted of 19 exons. All e xon-intron boundaries are conserved among the genes for hexokinase and gluc okinase. The HKI gene length was estimated at over 67 kb. The initiation si te for the HKR was identified by primer extension. Its promoter did not hav e a canonical TATA box, but an inverted GATA at nt -177 (i.e., 36 nt 5' to the transcription initiation site). In the HKR promoter a DNA fragment span ning nt -275 to nt -107 exhibited erythroid-specific activity. However, thi s was absent in shorter promoter fragments (nt -206 to -107 or nt -229 to - 107). The sequence nt -275 to -229, which appeared critical for the erythro id-specific expression of the HKR gene, contained a consensus motif for Sp- 1 and GATA, CCAAT, and GGAA motifs. The electrophoretic mobility shift assa y (EMSA) suggested erythroid-specific cooperative protein-protein interacti on in this region. Deletion of the GATA sequence as well as reaction with a specific antibody identified GATA-1 as one of the interacting proteins. (C ) 1999 Academic Press.