BCL11A Enhancer Research Articles

BCL11A enhancer dissection by Cas9-mediated in situ saturating mutagenesis.

SummaryEnhancers, critical determinants of cellular identity, are commonly identified by correlative chromatin marks and gain-of-function potential, though only loss-of-function studies can demonstrate their requirement in the native genomic context. Previously we identified an erythroid enhancer of BCL11A, subject to common genetic variation associated with fetal hemoglobin (HbF) level, whose mouse ortholog is necessary for erythroid BCL11A expression. Here we develop pooled CRISPR-Cas9 guide RNA libraries to perform in situ saturating mutagenesis of the human and mouse enhancers. This approach reveals critical minimal features and discrete vulnerabilities of these enhancers. Despite conserved function of the composite enhancers, their architecture diverges. The crucial human sequences appear primate-specific. Through editing of primary human progenitors and mouse transgenesis, we validate the BCL11A erythroid enhancer as a target for HbF reinduction. The detailed enhancer map will inform therapeutic genome editing. The screening approach described here is generally applicable to functional interrogation of noncoding genomic elements.

53. From GWAS To the Clinic: Genome-Editing the Human BCL11A Erythroid Enhancer for Fetal Globin Elevation in the Hemoglobinopathies

We describe here a fundamentally novel way to approach the development of a therapeutic: use of data from genome-wide association studies (GWAS) as a guide to create, in a targeted fashion, a disease-ameliorating genotype in the patient's own cells. We exemplify this by focusing on the hemoglobinopathies, β-thalassemia and sickle cell disease (SCD): in both cases elevated levels of fetal hemoglobin (HbF) have been shown to lessen or eliminate disease symptoms. Thus, reversing HbF silencing in patients is an attractive strategy for the development of therapies. To this end, GWAS data pointed to loss-of-function variants in the erythroid-specific enhancer of the fetal globin repressor, BCL11A, as causative for HbF elevation. While such regulatory elements are challenging to affect in a clinical setting via a conventional small molecule approach, the development of genome editing with engineered nucleases allows, in principle, a targeted intervention at the DNA level to disable enhancer function. Here, we reduce this notion to practice.The BCL11A enhancer consists of three separate cis-regulatory elements spanning a total of~1,500 bp that together drive erythroid-specific expression of BCL11A and lead to a silencing of fetal hemoglobin post-birth. Guided in part by fine-scale in vivo protein-DNA interaction data, we performed a reverse-genetic analysis of the enhancer at its endogenous location in the physiologically relevant cell type, primary human erythroid cells, using zinc finger nucleases (ZFNs). We developed highly optimized ZFNs that yield 60-80% target locus editing in human mobilized peripheral blood CD34 cells. Remarkably, we find that the ZFN-driven ablation of a single 5 bp element, GATAA, resident in the enhancer reproducibly elevates fetal globin in erythroid progeny of these CD34 cells to levels indistinguishable from those resulting from an essentially complete ZFN-driven coding knockout of BCL11A itself. We demonstrate high-efficiency ZFN-driven marking at the enhancer in peripheral blood mobilized CD34 cells at clinical scale production in a GMP-compliant setting, observe the successful engraftment and differentiation of genome-edited cells in an immunodeficient mouse model, and use an unbiased deep-sequencing based assay to comprehensively characterize the nucleus-wide specificity profile of ZFN action.Together these data illustrate the feasibility of using findings from genome-wide association studies to pursue novel therapeutic approaches in monogenic disease. In particular, our work supports the further development of fetal globin elevation via the targeted genome editing of the BCL11A erythroid-specific enhancer in both peripheral blood- and bone marrow-derived CD34 cells as a potential treatment for the hemoglobinopathies.

BCL11A enhancer haplotypes and fetal hemoglobin in sickle cell anemia.

Fetal hemoglobin (HbF) levels in sickle cell anemia patients vary. We genotyped polymorphisms in the erythroid-specific enhancer of BCL11A to see if they might account for the very high HbF associated with the Arab-Indian (AI) haplotype and Benin haplotype of sickle cell anemia. Six BCL112A enhancer SNPs and their haplotypes were studied in Saudi Arabs from the Eastern Province and Indian patients with AI haplotype (HbF ~20%), African Americans (HbF ~7%), and Saudi Arabs from the Southwestern Province (HbF ~12%). Four SNPs (rs1427407, rs6706648, rs6738440, and rs7606173) and their haplotypes were consistently associated with HbF levels. The distributions of haplotypes differ in the 3 cohorts but not their genetic effects: the haplotype TCAG was associated with the lowest HbF level and the haplotype GTAC was associated with the highest HbF level and differences in HbF levels between carriers of these haplotypes in all cohorts were approximately 6%. Common HbF BCL11A enhancer haplotypes in patients with African origin and AI sickle cell anemia have similar effects on HbF but they do not explain their differences in HbF.

BCL11A enhancer Haplotypes Are Associated with the Distribution of HbF in Arab-Indian and African Haplotype Sickle Cell Anemia but Not the Different Population Levels of HbF

Fetal hemoglobin (HbF) modulates the phenotype of sickle cell anemia. In the Middle East and India the HbS gene is often on an Arab-Indian HBB haplotype that is associated with high HbF levels. HbF is “normally” distributed in this population with a mean ~20%. In African HbS haplotypes, HbF levels are much lower (mean value ~6%) with a highly skewed distribution. BCL11A is an important modulator of γ-globin gene (HBG2 and HBG1) expression and BCL11A is regulated by erythroid specific enhancers in its 2nd intron. The enhancers consist of 3 DNase hypersensitive sites (HS) +62, +58 and +55 kb from the transcription initiation site of this gene. Polymorphisms (SNPs) in these enhancers are associated with HbF. The strongest association with HbF levels in African Americans with sickle cell anemia was with rs1427407 in HS +62 and to a lesser extent, rs7606173 in HS+55. Using the results of whole genome sequencing of 14 AI haplotype patients—half with HbF <10%, half with HbF >20%—6 SNPs in the BCL11A enhancer region, rs1427407, rs7599488, rs6706648, rs6738440, rs7565301, rs7606173 and 2 indels rs3028027 and rs142027584 (CCT, CCTCT and AAAAC respectively), were detected as possibly associated with HbF level. There were no novel polymorphisms detected. We genotyped the 6 SNPs and studied their associated haplotypes in 137 Saudi (HbF18.0±7.0%) and 44 Indian patients (HbF23.0±4.8%) with the Arab-Indian HBB haplotype; 50 African Americans with diverse African haplotypes, including 4 Senegal haplotype heterozygotes, (20 with HbF 17.2±4.6% and 30 with HbF 5.0±2.5%) and imputed genotypes for these SNPs in 847 African Americans with sickle cell anemia and diverse haplotypes (HbF 6.6±5.5%). Four SNPs (rs1427407, rs6706648, rs6738440, and rs7606173) in the HS sites showed consistent associations with HbF levels in all 4 cohorts. Haplotype analysis of these 4 SNPs showed that there were 4 common and 10 rare haplotypes. The most common, GCAG, was found in ~54% of Arab-Indian haplotype carriers (HbF, ~20%) and in ~33% of African origin haplotype carriers (HbF, ~5.5%). Two haplotypes, GTAC and GTGC, were carried by ~40% of African American patients and were associated with lower levels of HbF (3.6%-4%). These same haplotypes were carried by 18% of Arab-Indian haplotype carriers and their average HbF level was 17%. These differences were significant. Haplotype TCAG was present in 20% of Arab-Indian and 25% of African haplotype cases, and carriers had on average higher HbF levels (~22% in the Arab-Indian haplotype, ~8% in African Americans). The analysis shows that: BCL11A enhancer haplotypes are differentially distributed among patients with the HbS gene on Arab-Indian or African origin haplotypes; haplotype pairs TCAG/TCAG and GTAC/GTGC are associated with the highest and lowest HbF levels in all the studied groups; the population-specific prevalence of HbF BCL11A enhancer haplotypes are likely to explain the different distributions of HbF in African origin and Arab-Indian haplotypes but do not account for the differences in average population levels of HbF or the high HbF of the Arab-Indian haplotype. Novel SNPs in BCL11A do not explain the high HbF of the Arab-Indian haplotype. Other important loci must have a predominant role in the differential expression of HbF among HbS Arab-Indian haplotype carriers. DisclosuresNo relevant conflicts of interest to declare.

Reactivation of Fetal Hemoglobin in Thalassemia and Sickle Cell Disease

Considerable attention has been recently devoted to mechanisms involved in the perinatal hemoglobin switch, as it was long ago established that the survival of fetal hemoglobin (HbF) production in significant amount can reduce the severity of the clinical course in severe disorders like β-thalassemia and sickle cell disease (SCD). For instance, when β-thalassemia is associated with hereditary persistence of fetal hemoglobin (HPFH) the disease takes a mild course, labeled as thalassemia intermedia. The same clinical amelioration occurs for the association between HPFH and SCD. As for the mechanism of this effect, some information has been obtained from the study of natural mutations at the human β-globin locus in patients with increased HbF, like the Corfu thalassemia mutations. Important evidence came from the discovery that drugs capable of improving the clinical picture of SCD, like decitabine ad hydroxycarbamide, are acting through the reactivation, to some extent, of HbF synthesis. The study of the mechanism of action of these compounds was followed by the identification of some genetic determinants, which promote this event. In particular, among a few genetic factors involved in this process, the most relevant appears the BCL11A gene, which is now credited to be able to silence γ-globin genes in the perinatal period by interaction with several erythroid-specific transcription factors and is actually considered as a barrier to HbF reactivation by known HbF inducing agents. Epigenetics is also a player in the process, mainly through DNA demethylation. This is certified by the recent demonstration that hypomethylating agents such as 5-azacytidine and decitabine, the first compounds used for HbF induction by pharmacology, act as irreversible inhibitors of demethyltransferase enzymes. Great interest has also been raised by the finding that several micro-RNAs, which act as negative regulators of gene expression, have been implicated in the progression of globin gene expression and, particularly, in the reactivation of γ-globin gene expression associated with increased HbF synthesis. Probably, this reactivation is achieved by post-transcriptional inhibition of BCL11A expression. Finally, attention is presently focused on a recently discovered BCL11A enhancer, essential for erythroid expression of BCL11A, which might become a therapeutic target for genome engineering in the β-hemoglobinopathies as its disruption affects only the erythropoietic lineage, without hurting other cell or tissue compartments.

Identification Of BCL11A Structure-Function Domains For Fetal Hemoglobin Silencing

Reactivation of fetal hemoglobin (HbF, α2γ2) expression in adults ameliorates the clinical symptoms in patients with the major β-hemoglobin disorders, sickle cell disease (SCD) and β-thalassemias. The zinc-finger protein BCL11A is a major modulator of hemoglobin switching and HbF silencing. BCL11A was initially identified by genome-wide association studies (GWAS) as a new HbF-associated gene. Down-regulation of BCL11A in primary human erythroid cells induces HbF expression. Knockout of BCL11A in mice impairs HbF silencing in adult erythroid cells. Most importantly, inactivation of BCL11A alone in humanized SCD mice corrects the hematologic and pathologic defects through high-level HbF induction. These studies established BCL11A as a genetically and functionally validated transcriptional regulator of HbF switching and silencing.In human and mouse erythroid cells, BCL11A is expressed as several isoforms, yet their individual roles in globin gene expression remain unexplored. Furthermore, the functional domains within the BCL11A protein responsible for its activity in HbF repression are largely unknown. To further understand the mechanistic roles of BCL11A in globin expression, we established a functional assay based on a BCL11A-null erythroid cell line generated by transcription activator-like effector nucleases (TALENs)-mediated deletion of an obligate erythroid-specific enhancer of BCL11A in murine erythroleukemia (MEL) cells. In the BCL11A-null cells, the expression of β-like embryonic globin genes is markedly induced (>200-fold), consistent with the role of BCL11A in repression of murine embryonic globin genes. To examine the activity of known BCL11A isoforms in HbF silencing, we expressed various BCL11A isoforms in these engineered BCL11A-null cells. Ectopic expression of full-length BCL11A-XL isoform, but not the alternatively spliced, C-terminally truncated L isoform, restored the full repression of β-like embryonic globins in BCL11A-null cells. Since XL and L differ only by 91 amino acids containing three tandem C2H2-type zinc finger motifs, these results indicate that the C-terminal zinc finger motifs are indispensable for BCL11A-mediated transcriptional repression.To systemically define BCL11A functional domains for globin gene repression, we next generated a panel of BCL11A mutant cDNAs, including deletion of the N-terminal NuRD-interacting motif and one or more C2H2-type zinc finger domains. Analysis of various BCL11A mutants in the functional rescue assay identified several functional domains, including the N-terminal NuRD-interacting motif and five out of the six C2H2 zinc fingers, that are required for BCL11A-mediated repression. These findings provide the foundation for further molecular analysis of BCL11A functional domains in globin gene repression. BCL11A is known to interact with several transcriptional co-repressor complexes including Mi-2β/NuRD/HDAC1/HDAC2, LSD1/CoREST and SWI/SNF complexes, occupy discrete regions within the human β-globin cluster, and promote long-range chromosomal interactions. Our results suggest that BCL11A functional domains may be involved in protein-protein interactions, protein homo-/heterodimerization, and/or chromatin/DNA association that are required for its activity in HbF silencing.In summary, we demonstrate that several functional domains on BCL11A protein are indispensable for its transcriptional activity in HbF silencing. Further focused studies of BCL11A structure-function domains in HbF silencing not only will advance our understanding of the molecular mechanisms by which BCL11A controls the clinically important fetal-to-adult globin switch, but may identify novel cellular targets for therapeutic HbF induction in β-hemoglobinopathies. Disclosures:No relevant conflicts of interest to declare.

Fine-Mapping and Genome Editing Reveal An Essential Erythroid Enhancer At The HbF-Associated BCL11A Locus

IntroductionGenome-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 MethodsWe use chromatin immunoprecipitation (ChIP), DNase I sensitivity and chromosome conformation capture to evaluate the BCL11A locus in mouse and human primary erythroblasts. We extensively genotype 1,263 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) and clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9) RNA-guided nucleases. ResultsCommon 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 GATA1 and TAL1 transcription factor binding, modestly diminished BCL11A expression and elevated HbF. This variant, rs1427407, accounts for the HbF association of the previously reported sentinel SNPs. The composite element functions 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. We demonstrate species-specific functional components of the composite enhancer in mouse as compared to human erythroid precursor cells. The mouse sequences homologous to the human DHS sufficient to drive reporter activity are dispensable from the mouse composite element, whereas the adjacent DHS, whose human homolog does not direct reporter activity, is absolutely required for BCL11A expression. ConclusionsWe 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 species-level functional differences in components of the composite enhancer might partially account for differences in timing of globin gene expression among animals. We suggest that the GWAS-marked BCL11A enhancer represents a highly attractive target for therapeutic genome editing for the major b-hemoglobin disorders. Disclosures:No relevant conflicts of interest to declare.

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

Abstract 828Genome-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. Disclosures:Porteus:Alex's Lemonade Stand Foundation: Research Funding.