HbF-Associated Genetic Variation Marks an Erythroid Regulatory Element Essential for BCL11A Transcription and Subsequent Stage-Specific Globin Expression

Abstract 828

Genome-wide association studies (GWAS) have identified common genetic variation within a 14-kb segment of intron-2 of the BCL11A gene that is associated with the level of fetal hemoglobin (HbF) and the clinical severity of the β-globin disorders sickle cell disease and β-thalassemia. Functional studies have validated BCL11A as a major repressor of HbF. Here, we explore the consequences of genetic variation on BCL11A expression. The HbF-associated variants overlap BCL11A sequences that possess an evolutionarily conserved enhancer chromatin signature, including the presence of H3K4me1 and H3K27ac and the absence of H3K4me3 histone modifications. Moreover, these sequences show a unique pattern of DNase I hypersensitivity in erythroid precursors compared to other BCL11A-expressing lineages including fetal brain and B-lymphocytes. Occupancy of the major erythroid transcription factors GATA1 and TAL1 is observed at these DNase I hypersensitive sites (DHS). Chromosome conformation capture studies demonstrate that these DHSs form a long-range interaction with the BCL11A promoter.

To address the function of the putative regulatory element, we generated transgenic mice with the 12-kb intronic region attached to a minimal promoter and reporter gene. The element enhanced reporter gene expression in an erythroid-lineage restricted, definitive erythroid stage-specific manner. The minimal enhancer activity was mapped to the +58-kb DHS. Similar enhancer activity was observed in primary human erythroid precursors. To address the possible requirement of this element for BCL11A expression, we deleted the corresponding region in adult-stage murine erythroleukemia cells with TALEN-mediated gene targeting. Expression of BCL11A was dramatically reduced and embryonic globins Hbby and Hbb-bh1 were markedly derepressed with concomitant reduction of adult globin Hbb-b1. Analysis of heterozygous primary human erythroid precursors revealed that low-HbF haplotype alleles are preferentially occupied by GATA1 and TAL1 at sequences surrounding rs1427407. A composite half-E-box–GATA motif is disrupted at this SNP in the high-HbF allele. Furthermore, the low-HbF haplotype allele of BCL11A is expressed at a 1.7-fold higher level relative to the high-HbF allele. These data demonstrate that HbF-associated genetic variation in BCL11A modulates the function of a critical erythroid enhancer of BCL11A, and consequently expression of BCL11A itself. The quantitative level of BCL11A in turn regulates the relative expression of the stage-specific globin genes. The essential erythroid enhancer of the BCL11A gene might itself serve as a target for genetic manipulation as a novel approach to the human β-globin disorders.