Transcriptional Regulation of the Human Erythroid 5-Aminolevulinate Synthase Gene

Abstract

We have characterized the 5-flanking region of the human erythroid-specific 5-amino levulinate synthase (ALAS) gene (the ALAS2 gene) and shown that the first 300 base pairs of promoter sequence gives maximal expression in erythroid cells. Transcription factor binding sites clustered within this promoter sequence include GATA motifs and CACCC boxes, critical regulatory sequences of many erythroid cell-expressed genes. GATA sites at ؊126/؊121 (on the noncoding strand) and ؊102/ ؊97 were each recognized by GATA-1 protein in vitro using erythroid cell nuclear extracts. Promoter mutagenesis and transient expression assays in erythroid cells established that both GATA-1 binding sites were functional and exogenously expressed GATA-1 increased promoter activity through these sites in transactivation experiments. A noncanonical TATA sequence at the expected TATA box location (؊30/؊23) bound GATA-1-or TATA-binding protein (TBP) in vitro. Conversion of this sequence to a canonical TATA box reduced expression in erythroid cells, suggesting a specific role for GATA-1 at this site. However, expression was also markedly reduced when the ؊30/؊23 sequence was converted to a consensus GATA-1 sequence (that did not bind TBP in vitro), suggesting that a functional interaction of both factors with this sequence is important. A sequence comprising two overlapping CACCC boxes at ؊59/؊48 (on the noncoding strand) was demonstrated by mutagenesis to be functionally important. This CACCC sequence bound Sp1, erythroid Krü ppellike factor, and basic Krü ppel-like factor in vitro, while in transactivation experiments erythroid Krü ppel-like factor activated ALAS2 promoter expression through this sequence. A sequence at ؊49/؊39 with a 9/11 match to the consensus for the erythroid specific factor NF-E2 was not functional. Promoter constructs with 5-flanking sequence from 293 base pairs to 10.3 kilobase pairs expressed efficiently in COS-1 cells as well as in erythroid cells, indicating that an enhancer sequence located elsewhere or native chromatin structure may be required for the tissue-restricted expression of the gene in vivo.

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).…”

Identification of a novel erythroid-specific enhancer for the ALAS2 gene and its loss-of-function mutation which is associated with congenital sideroblastic anemia

“…The human ALAS2 proximal promoter region (g.4820_5115, between -267 and +29 from the transcription start site) 16,24 was cloned into the multiple-cloning site of pGL3basic [referred to as pGL3-AEpro (-267)]. A single DNA fragment (5.2 kbp), carrying the ALAS2 proximal promoter, first exon, first intron and the untranslated region of the second exon, was subcloned into the multiple cloning site of pGL3basic [referred to as pGL3-AEpro(-267)+intron1].…”

“…These results indicate that GATA1 protein bound to the regions amplified with primer sets 1 and 10 in K562 cells; that is, GATA1 protein could bind to the proximal promoter region as well as to ALAS2int1GATA in the first intron of the ALAS2 gene in vivo. Since the GATA element located in the proximal promoter has been well examined in vitro, 16 we further determined the functional features of ALAS2int1GATA. …”

“…21 We also included the proximal promoter region that contains a functional GATA-binding site (g.4961_4966). 16 Overall 13 primer sets were designed to amplify the GATA elements located in the proximal promoter region and the first intron of ALAS2 ( Figure 1A and Online Supplementary Table S1). Among the 12 primer sets targeting the first intron, using primer set 10, we could amplify genomic DNA that was precipitated with anti-GATA1 antibody at a similar level to that of the positive control, but not with other primer sets.…”

A promoter mutation in the erythroid-specific 5-aminolevulinate synthase (ALAS2) gene causes X-linked sideroblastic anemia

“…39 Thus, some as-yet-unidentified transcription factor could be binding to a response element at the Ϫ206C site and activating ALAS2. Further studies of the promoter mutation region will require specific binding assays, such as electrophoretic shift and footprinting in erythroid cells, to uncover the mechanism by which this mutation disrupts the transcription of the ALAS2 gene, perhaps revealing important new functions of the ALAS2 promoter and potentially those of other erythroid-specific genes.…”