An erythroid enhancer of BCL11A subject to genetic variation determines fetal hemoglobin level

Abstract

Genome-wide association studies (GWASs) have ascertained numerous trait-associated common genetic variants, frequently localized to regulatory DNA. We found that common genetic variation at BCL11A associated with fetal hemoglobin (HbF) level lies in noncoding sequences decorated by an erythroid enhancer chromatin signature. Fine-mapping uncovers a motif-disrupting common variant associated with reduced transcription factor (TF) binding, modestly diminished BCL11A expression, and elevated HbF. The surrounding sequences function in vivo as a developmental stage-specific, lineage-restricted enhancer. Genome engineering reveals the enhancer is required in erythroid but not B-lymphoid cells for BCL11A expression. These findings illustrate how GWASs may expose functional variants of modest impact within causal elements essential for appropriate gene expression. We propose the GWAS-marked BCL11A enhancer represents an attractive target for therapeutic genome engineering for the β-hemoglobinopathies.

Fig. 1. Chromatin state and TF occupancy at BCL11A

(A) ChIP-seq from human erythroblasts with indicated antibodies. DNase I cleavage density from indicated human tissues. Three erythroid DHSs termed +62, +58 and +55 based on distance in kb from BCL11A TSS. BCL11A transcription from right to left. (B) ChIP-qPCR from human erythroblasts at BCL11A intron-2. DHSs +62, +58 and +55 boxed. Enrichment at negative (GAPDH, OCT4) and positive control (β-globin LCR HS3 and α-globin HS-40) loci displayed. (C) Chromosome conformation capture in human erythroblasts using BCL11A promoter as anchor. Error bars indicate s.d.

Fig. 2. Regulatory variants at BCL11A

(A) Genotype data obtained in 1,178 individuals from CSSCD for 38 variants within BCL11A +62, +58 or +55 DHSs. Most highly significant associations to HbF level among common (MAF > 1%) SNPs (n = 10) prior to (rs1427407) or following (rs7606173) conditional analysis on rs1427407. SNP coordinates chromosome 2, build hg19. (B) Chromatin from erythroblasts of individuals heterozygous for rs1427407, immunoprecipitated by GATA1 or TAL1 and pyrosequenced to quantify the relative abundance of the rs1427407-G allele. Composite half E-box–GATA motif previously identified (23). (C) gDNA and cDNA from erythroblasts of individuals heterozygous for rs1427407, rs7606173 and rs7569946. Haplotyping demonstrated rs7569946-G, rs1427407-G and rs7606173-C on the same chromosome in each. Pyrosequencing to quantify the relative abundance of the rs7569946-G allele.

Fig. 3. The GWAS-marked BCL11A enhancer is sufficient for adult-stage erythroid expression

(A) A 12.4-kb fragment of BCL11A intron-2 (+52.0–64.4 kb from TSS) was cloned to a lacZ reporter construct. Transient transgenic mouse embryo from 12.5 dpc X-gal stained. Arrowhead indicates liver. (B) Cell suspensions isolated from peripheral blood (PB) and fetal liver (FL) of stable transgenic embryos at 12.5 dpc X-gal stained. (C) Sorted erythroblasts and B-lymphocytes from young adult stable transgenic mice subject to X-gal staining or RNA isolation followed by RT-qPCR. Gene expression normalized to GAPDH and expressed relative to T-lymphocytes. Error bars indicate s.d.

Fig. 4. The GWAS-marked BCL11A enhancer is necessary for erythroid but dispensable for non-erythroid expression

(A) Three mouse erythroleukemia (MEL) and two pre-B lymphocyte clones with biallelic deletion of the orthologous Bcl11a erythroid enhancer (Δ50.4–60.4) subject to RT-qPCR. (B) Immunoblot of Δ50.4–60.4 MEL and pre-B lymphocyte clones.