Identification and characterization of a conserved erythroid-specific enhancer located in intron 8 of the human 5-aminolevulinate synthase 2 gene
| Autor: | Brian K. May, Timothy C. Cox, Katharina H. Surinya |
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| Rok vydání: | 1998 |
| Předmět: |
Erythrocytes
Molecular Sequence Data DNA Footprinting Biology Regulatory Sequences Nucleic Acid Biochemistry Transactivation Mice Dogs Genes Reporter Animals Humans Enhancer Promoter Regions Genetic Molecular Biology Gene Conserved Sequence Phylogeny Sp1 transcription factor Base Sequence Sequence Homology Amino Acid GATA2 Intron Cell Biology DNA Molecular biology Introns DNA binding site Enhancer Elements Genetic Regulatory sequence Mutagenesis Site-Directed 5-Aminolevulinate Synthetase Protein Binding Transcription Factors |
| Zdroj: | The Journal of biological chemistry. 273(27) |
| ISSN: | 0021-9258 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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