Functional Evaluation of HbF-Associated Region of BCL11A Locus

Abstract 2148

The severity of the major β-globin disorders, sickle cell disease and β-thalassemia, is modified by the residual level of fetal hemoglobin (HbF, α₂γ₂). Reactivation of HbF by derepression of γ-globin gene expression constitutes a major therapeutic goal, though an incomplete understanding of globin gene regulation has limited such efforts. The postnatal level of HbF is a highly heritable quantitative trait. Genome-wide association (GWA) studies have identified several HbF-associated loci, including the BCL11A gene. BCL11A has been demonstrated to be a bona fide repressor of γ-globin expression in mouse and man. However, the mechanisms by which common genetic variation modulate BCL11A expression remain poorly understood. Numerous GWA studies, conducted in both healthy individuals and those with hemoglobinopathies, and including populations of European, Asian, and African ancestries, have consistently found that HbF-associated variants cluster within a 14-kb region of the second intron of BCL11A. At least twenty HbF-associated SNPs have been identified within this region, with various of these genetic markers considered as “tag SNPs” most strongly associated with HbF levels depending on the particular GWA study. Based on patterns of linkage disequilibrium among SNPs, several common haplotypes have been identified. The tag SNP-dense intronic region, more than 55 kb downstream of the BCL11A transcription start site, demonstrates evolutionary sequence conservation. Here we show that this region of BCL11A is decorated by epigenetic features in human erythroid precursors indicative of distal regulatory potential. Specifically, this intronic region is marked by the presence of the histone modifications H3K4me1 and H3K27ac and the absence of H3K4me3, indicating an enhancer signature. The erythroid transcription factors GATA1 and SCL/TAL1 co-occupy discrete sequences within this region, with the binding peaks of these master erythroid regulators overlapping those of the histone marks. Furthermore, this tag SNP-dense intronic region of BCL11A displays CpG hypomethylation and exhibits DNase I hypersensitive sites restricted to the erythroid lineage but not other lineages that express BCL11A. Moreover, in in vivo transient transgenic assays, this region displays erythroid-restricted enhancer activity. A minimal 1.8-kb sequence possessing erythroid enhancer function has been mapped. This region includes GATA1 and SCL/TAL1 binding sites. We hypothesize that common polymorphisms are in linkage disequilibrium with functional variants that modulate erythroid enhancer activity. These studies further delineate pathways critical for hemoglobin switching and serve as an example of how common genetic variation may influence traits by affecting non-coding regulatory elements.