Identification and Characterization of a Conserved Erythroid-specific Enhancer Located in Intron 8 of the Human 5-Aminolevulinate Synthase 2 Gene

Abstract

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 Krü ppel-like factor and the basic Krü ppel-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 tissuerestricted expression of the gene in vivo.

Insight into GATA1 transcriptional activity through interrogation ofciselements disrupted in human erythroid disorders

“…5E). Exogenous reporter assays have shown that, depending on the exact "chunks" of DNA tested, this region can vary in activation strength and even repress (51). Intriguingly, when we separately targeted two GATA1 motifs in ALAS2 intron 8 with CRISPR/Cas9, we observed an increase in ALAS2 mRNA expression, similar to the results obtained by targeting two CREs near SLC4A1, suggesting that these regions may have a particularly complex regulatory architecture (Fig.…”

“…Intriguingly, when we separately targeted two GATA1 motifs in ALAS2 intron 8 with CRISPR/Cas9, we observed an increase in ALAS2 mRNA expression, similar to the results obtained by targeting two CREs near SLC4A1, suggesting that these regions may have a particularly complex regulatory architecture (Fig. S9) (51). Taken together, our analyses provide additional evidence that erythroid CREs are dependent on the binding of multiple TFs for proper transcriptional regulation and RBC development.…”

Differential sensitivities of transcription factor target genes underlie cell type-specific gene expression profiles

“…To establish whether differential chromatin access underlies the distinct sensitivities, the ER-GATA-1 level͞activity was titrated with 5, 10, or 40 nM ␤-estradiol for 16 h, and chromatin occupancy was measured at sites defined previously (13,14,20,22,28,(43)(44)(45). ER-GATA-1 occupied ␤-globin HSs1-4 maximally and the ␤major promoter nearly maximally in 10 nM ␤-estradiol-treated cells ( Fig.…”

A nonsense mutation in zebrafish gata1 causes the bloodless phenotype in vlad tepes

“…In mammals, sptb (39) and alas2 (40)(41)(42) genes have been shown to have GATA binding sites within their promoters that are required for transactivation in tissue culture. In the zebrafish, embryonic globin gene expression was observed earlier (at 3-6 somites) than sptb, alas2, and band3 (at 16 somites) (S.E.L., unpublished results).…”