Fine-mapping GWAS followed by genome editing identifies an essential erythroid enhancer at the HbF-associated BCL11A locus. D. E. Bauer1, S. Lessard3, S. C. Kamran1,2, J. Xu1, Y. Fujiwara1, C. Lin1, Z. Shao1, M. C. Canver1, E. C. Smith1, L. Pinello6, P. J. Sabo4, J. Vierstra4, R. A. Voit5, G. C. Yuan6, M. H. Porteus5, J. A. Stamatoyannopoulos4, G. Lettre3, S. H. Orkin1,2 1) Pediatric Hematology/Oncology, Boston Children's Hospital, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA., United States; 2) Howard Hughes Medical Institute, Boston, MA; 3) Montreal Heart Institute and Université Montréal, Montreal, Quebec, Canada; 4) Department of Genome Sciences, University of Washington, Seattle, WA; 5) Department of Pediatrics, Stanford University School of Medicine, Palo Alto, CA; 6) Harvard School of Public Health, Boston, MA.

   Introduction: Genome-wide association studies (GWAS) have ascertained numerous trait-associated common genetic variants localized to regulatory DNA. The hypothesis that regulatory variation accounts for substantial heritability has undergone scarce experimental evaluation. Common variation at BCL11A is estimated to explain ~15% of the trait variance in fetal hemoglobin (HbF) level but the functional variants remain unknown. Materials and Methods: We use chromatin immunoprecipitation (ChIP), DNaseI sensitivity and chromosome conformation capture to evaluate the BCL11A locus in primary erythroblasts. We extensively genotype 1263 samples from the Collaborative Study of Sickle Cell Disease within three HbF-associated erythroid DNase I hypersensitive sites (DHSs) at BCL11A. We pyrosequence heterozygous erythroblasts to assess allele-specific transcription factor binding and gene expression. We conduct transgenic analysis by mouse zygotic microinjection and genome editing with transcription activator-like effector nucleases (TALENs). Results: Common genetic variation at BCL11A associated with HbF level lies in noncoding sequences decorated by an erythroid enhancer chromatin signature. Fine-mapping this putative regulatory DNA uncovers a motif-disrupting common variant associated with reduced transcription factor binding, modestly diminished BCL11A expression and elevated HbF. The surrounding sequences function in vivo as a developmental stage-specific lineage-restricted enhancer. Genome editing reveals that the enhancer is required in erythroid but dispensable in B-lymphoid cells for expression of BCL11A. Conclusions: We describe a comprehensive and widely applicable approach, including chromatin mapping followed by fine-mapping, allele-specific ChIP and gene expression studies, and functional analyses, to reveal causal variants and critical elements. We assert that functional validation of regulatory DNA ought to include perturbation of the endogenous genomic context by genome editing and not solely rely on in vitro or ectopic surrogate assays. These results validate the hypothesis that common variation modulates cell type-specific regulatory elements, and reveal that although functional variants themselves may be of modest impact, their harboring elements may be critical for appropriate gene expression. We speculate that the GWAS-marked BCL11A enhancer represents a highly attractive target for therapeutic genome editing for the major -hemoglobin disorders.

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