Mutation of a GATA1 Binding Site in ALAS2 Intron 1 Arrests Murine Erythroid Development In Vivo

Congenital sideroblastic anemia (CSA) comprises a heterogeneous group of genetic disorders characterized by decreased heme synthesis and the presence of bone marrow (BM) ring sideroblasts. The most common form of CSA is X-linked sideroblastic anemia (XLSA, OMIM#300751), which is caused by mutations in the gene encoding 5-aminolevulinic acid synthase 2 (ALAS2). However, to date the experimentally confirmed ALAS2 mutations mostly occurred at the exons of ALAS2 and are missense mutations.

In our study, we identified a non-coding GATA1 binding site mutation in ALAS2 intron 1 in a large XLSA pedigree. We addressed the functions of the GATA1 binding site in ALAS2 intron 1 in vivoby generating mice lacking this GATA site and its flanking sequence (13bp) and found that this deletion led to an embryonic lethal phenotype due to severe anemia, indicating that this fragment is indispensable during erythroid development. Next we demonstrated that GATA1 activated ALAS2 transcription by forming an enhancer loop with TAL1, LDB1 and Pol II through GATA binding sites in the promoter and in introns 1 and 8. Finally, we discovered that int-1-GATA site deletion completely abolished ALAS2 transcription due to disruption of the enhancer loop and a simultaneous lack of Pol II enrichment at the intron 1 enhancer region and promoter.

Our findings first revealed the essential roles of GATA1 binding site in ALAS2 intron 1 in erythroid development in vivo and uncovered the mechanisms how GATA1 regulated the expression of ALAS2. We believe that these findings will be of wide general interest and will provide valuable information for the clinical diagnosis of XLSA patients with unknown mutations.