Identification of the Novel Erythroid-Specific Enhancer in the First Intron of Human ALAS2 Gene; Tthe Mutation Disrupting GATA Transcription Factor-Binding Site in the Enhancer Causes X-Linked Sideroblastic Anemia

Abstract 3185

Human ALAS2 gene encodes erythroid-specific 5-aminolevulinate synthase, the rate-limiting enzyme of heme biosynthetic pathway in erythroid cells. The mutation of ALAS2 gene causes X-linked sideroblastic anemia (XLSA), and approximate 50 different mutations have been reported in the coding region of ALAS2 gene as disease causative mutations. Here, we report two novel mutations of ALAS2 gene identified in a pedigree with XLSA and one male patient with congenital sideroblastic anemia (CSA). Incidentally, both mutations were predicted to impair or abolish the function of the specific GATA transcription factor-binding motif (GATA element) located at the middle of the first intron of ALAS2 gene (referred as ALAS2int1GATA). In a proband of the pedigree of XLSA, the “GATA” sequence in ALAS2int1GATA, which is the core sequence of consensus for GATA element (WGATAR), was changed to “GGTA”, and the same mutation was identified in two male relatives, his mother's cousins, both of whom were diagnosed as sideroblastic anemia. Furthermore, a patient with CSA carries a deletion of 35 bps in the first intron of ALAS2 gene, the deleted region of which contains ALAS2int1GATA, although this deletion was not detected in his parents' ALAS2 gene. No other mutation was detected in the proximal promoter region, the known enhancer region present in eighth intron, and the coding region and exon-intron boundaries of ALAS2 gene. Moreover, no mutation was detected in the coding region and exon-intron boundaries of SLC25A38, ABCB7, GLRX5, PUS1 or SLC19A2 gene, each of which was reported as a responsible gene for CSA. It is of interest that ALAS2int1GATA is present within one of “GATA transcription factor-occupying regions in K562 erythroleukemia cells” identified by genome-wide analysis using chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq) (Fujiwara et al., Mol Cell, vol. 36: p667–681, 2009), suggesting that ALAS2int1GATA may act as a cis-regulatory element for ALAS2 transcription in vivo. In fact, our ChIP-PCR analysis in K562 cells, which focused on the proximal promoter (−267 to +29) and the first intron of ALAS2 gene, confirmed that GATA-1 protein selectively bound to the GATA element in the proximal promoter region as well as ALAS2int1GATA out of 17 consensus GATA elements present in the first intron. Then, we examined whether the 467-bp region in the first intron of ALAS2 gene, defined by ChIP-seq analysis (referred as “ALAS2 ChIP-peak”), acts as an enhancer for ALAS2 expression using transient reporter assay, because ALAS2 ChIP-peak contains ALAS2int1GATA. For this assay, we used pGL3-AEpro, which contains the firefly luciferase gene under the control of the ALAS2 proximal promoter, as a parent reporter vector. The presence of ALAS2 ChIP-peak in the downstream region of the luciferase gene increased ALAS2 proximal promoter activity about ten-folds in K562 cells. However, this enhancing activity of ALAS2 ChIP-peak was not observed in non-erythroid HEK293 embryonic kidney cells. Importantly, each newly identified mutation at ALAS2int1GATA diminished the enhancer activity of ALAS2 ChIP-peak on ALAS2 proximal promoter in K562 cells. Moreover, electrophoretic mobility shift assay (EMSA) revealed that GATA-1 protein in nuclear extracts of K562 cells or HEK293 cells overexpressing GATA-1 could bind to a wild-type probe containing ALAS2int1GATA, but GATA-1 failed to bind to a mutant probe, which carries a single base change in ALAS2int1GATA or a deletion of ALAS2int1GATA that was identified in patients with XLSA or CSA, respectively. These results suggest that ALAS2int1GATA plays an essential role for enhancing ALAS2 expression as a core of the ALAS2 ChIP-peak, the function of which may depend on the erythroid-specific transcription factor, GATA-1. Thus, the mutation at or the deletion of ALAS2int1GATA impairs the recruitment of GATA transcription factor(s) to the ALAS2 ChIP-peak, which in turn decreases the transcription of ALAS2 gene, thereby causing XLSA or CSA. In conclusion, we provide the evidence for the existence of an enhancer element in the first intron of ALAS2 gene. Moreover, the loss-of-function mutation at the newly identified enhancer element of ALAS2 gene is associated with XLSA or CSA.