IDENTIFICATION AND CHARACTERIZATION OF A CONSERVED ERYTHROID-SPECIFICENHANCER LOCATED IN INTRON-8 OF THE HUMAN 5-AMINOLEVULINATE SYNTHASE-2 GENE

Thirty five kilobases of sequence encompassing the human erythroid Fi- aminolevulinate synthase (ALAS2) gene have been determined. Analysis r evealed a very low GC content, few repetitive elements, and evidence f or the insertion of a reverse-transcribed mRNA sequence and a neighbor ing gene. We have investigated whether introns 1, 3, and 8, which corr espond to DNase I-hypersensitivity sites in the structurally related m ouse ALAS2 gene, affect expression of the human ALAS2 promoter in tran sient expression assays. Whereas intron 3 was marginally inhibitory, i ntrons 1 and 8 of the human gene stimulated promoter activity. Intron 8 harbored a strong erythroid-specific enhancer activity which was ori entation-dependent. Deletion analysis of this region localized enhance r activity to a fragment of 239 base pairs. Transcription factor bindi ng sites clustered within this region include GATA motifs and CACCC bo xes, critical regulatory sequences of many erythroid cell-expressed ge nes. These sites were also identified in the corresponding intron of b oth the murine and canine ALAS2 genes. Mutagenesis of these conserved sites in the human intron 8 sequence and transient expression analysis in erythroid cells established the functional importance of one GATA motif and two CACCC boxes. The GATA motif bound GATA-1 in vitro, The t wo functional CACCC boxes each bound Spl or a related protein in vitro , but binding of the erythroid Kruppel-like factor and the basic Krupp el-like factor could not be detected. The intron 8 enhancer region was not activated by GATA-1 together with Spl in transactivation experime nts in COS-1 cells indicating the involvement of a related Spl protein or of another unidentified erythroid factor. Overall, these results d emonstrate that a GATA-1-binding site and CACCC boxes located within t he human ALAS2 intron 8 are critical for the erythroid-specific enhanc er activity in transfected erythroid cells, and due to the conserved n ature of these binding sites across species, it seems likely that thes e sites play a functional role in the tissue-restricted expression of the gene in vivo.