Identification and characterization of a conserved erythroid-specific enhancer located in intron 8 of the human 5-aminolevulinate synthase 2 gene.
Thirty five kilobases of sequence encompassing the human erythroid 5-aminolevulinate synthase (ALAS2) gene have been determined. Analysis revealed a very low GC content, few repetitive elements, and evidence for the insertion of a reverse-transcribed mRNA sequence and a neighboring gene. We have investigated whether introns 1, 3, and 8, which correspond to DNase I-hypersensitivity sites in the structurally related mouse ALAS2 gene, affect expression of the human ALAS2 promoter in transient expression assays. Whereas intron 3 was marginally inhibitory, introns 1 and 8 of the human gene stimulated promoter activity. Intron 8 harbored a strong erythroid-specific enhancer activity which was orientation-dependent. Deletion analysis of this region localized enhancer activity to a fragment of 239 base pairs. Transcription factor binding sites clustered within this region include GATA motifs and CACCC boxes, critical regulatory sequences of many erythroid cell-expressed genes. These sites were also identified in the corresponding intron of both 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 two functional CACCC boxes each bound Sp1 or a related protein in vitro, but binding of the erythroid Krüppel-like factor and the basic Krüppel-like factor could not be detected. The intron 8 enhancer region was not activated by GATA-1 together with Sp1 in transactivation experiments in COS-1 cells indicating the involvement of a related Sp1 protein or of another unidentified erythroid factor. Overall, these results demonstrate that a GATA-1-binding site and CACCC boxes located within the human ALAS2 intron 8 are critical for the erythroid-specific enhancer activity in transfected erythroid cells, and due to the conserved nature of these binding sites across species, it seems likely that these sites play a functional role in the tissue-restricted expression of the gene in vivo.
Surinya
K H
KH
Department of Biochemistry, University of Adelaide, Adelaide, South Australia, Australia 5005.
Cox
T C
TC
May
B K
BK
D000624:5-Aminolevulinate Synthetase / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D017124:Conserved Sequence; D004247:DNA; D018983:DNA Footprinting; D004285:Dogs; D004742:Enhancer Elements, Genetic; D004912:Erythrocytes / Q000201:enzymology*; D017930:Genes, Reporter; D006801:Humans; D007438:Introns; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010802:Phylogeny; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012045:Regulatory Sequences, Nucleic Acid; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors / Q000378:metabolism
D014157:Transcription Factors; D004247:DNA; D000624:5-Aminolevulinate Synthetase