HBS1L-MYB intergenic Variants Modulate Fetal Hemoglobin Via Long-Range MYB Enhancers

Abstract

Three quantitative trait loci (QTLs) modifying fetal hemoglobin (HbF) levels have been identified, and these have been shown to have a predictive value of disease severity in β thalassemia and sickle cell disease in diverse ethnic groups. One of the HbF QTLs which consists of a set of common intergenic single nucleotide polymorphisms (SNPs) in the HBS1L-MYB intergenic region on chromosome 6q23, has also been consistently identified as highly associated with clinically important human erythroid traits. Despite extensive genetic evidence, a clear mechanistic basis for the association between the intergenic SNPs and erythroid biology has remained elusive, although the two flanking genes (HBS1L and MYB) are candidate target genes. Here, we set out to characterize the regulatory potential of the human HBS1L-MYB intergenic region in detail and to investigate its functional impact on the erythroid phenotype-associated variants. We profiled chromatin occupancy of the key erythroid LDB1 transcription factor (TF) complex in primary human erythroid progenitors (HEPs) using chromatin immunoprecipitation coupled to high-throughput sequencing (ChIP-Seq) and quantitative PCR (ChIP-qPCR). We detected an intergenic cluster containing 7 binding sites for the LDB1–complex, characterized by strong binding and co-occupancy of core complex proteins LDB1, GATA1, TAL1 and ETO2. One of these sites was co-occupied by the erythroid-specific TF KLF1, a protein which was also found to bind the murine intergenic region. Depletion of LDB1, TAL1 and KLF1 in K562 cells using RNA interference resulted in a specific downregulation of MYB expression while leaving HBS1L levels unaffected, demonstrating that the erythroid TFs occupying the intergenic enhancers are required for MYB expression. Using chromosome conformation capture (3C) coupled to high-throughput sequencing (3C-Seq), we profiled higher order chromatin structure within the locus, and detected several strong chromatin co-associations between the MYB promoter and intergenic sequences, almost all of which correlated with TF binding. The binding activities were distributed over a conserved core region identical to a 24-kb interval containing genetic variants in strong genetic association with erythroid traits in human populations. This block of SNPs is referred to as HBS1L-MYB intergenic polymorphisms block-2 (HMIP-2). The SNPs within HMIP-2 clustered in 2 regions positioned directly under the 2 LDB1-complex ChIP-Seq peaks, at -84 and -71 kb from the MYB transcription start site. Using allele-specific ChIP in K562 cells, we observed diminished (25-50%) binding of LDB1, GATA1, TAL1 and KLF1 to the minor rs66650371 allele, showing that rs66650371 affects local TF binding. An allele-specific 3C analysis also showed reduced interactions between the minor rs66650371 allele at -84 and MYB. We validated and expanded observations made in K562 cells using primary human cells: 1. HEPs from high HbF individuals homozygous for all minor alleles of the phenotype-associated -84kb and -71kb intergenic variants in the conserved core (‘SNP/SNP’) showed consistently lower MYB levels throughout phase II of the culture as compared to wildtype control cells (‘WT/WT’); 37% lower MYB on average; 2. ChIP experiments on SNP/SNP and WT/WT HEPs showed reduced binding of GATA1 and KLF1 to the -84 and -71 regulatory elements (containing the associated variants), the results were further confirmed by allele-specific ChIP assays in SNP/WT HEPs; 3. 3C-qPCR assays on cultured SNP/SNP and WT/WT cells demonstrated diminished looping between the -84 element and the MYB promoter in SNP/SNP individuals; 4. SNP/WT HEPs also showed allelic imbalance of MYB but not HBS1L transcripts when compared to controls (WT/WT and SNP/SNP HEPs). In conclusion, we show that the HBS1L-MYB gene-free interval contains distal enhancer elements that interact with MYB, a critical regulator of erythroid development and HbF levels. Key variants in the intergenic interval affect MYB expression by reducing TF binding to its regulatory elements and disrupting long-range enhancer-gene interaction. Our study identifies the first causal link between the 6q23 HbF QTL and MYB regulation, provides novel insights into the molecular control of clinically important haematological traits and adds another layer of complexity to the regulation of MYB, suggesting potential targets for therapeutic intervention. Disclosures: Thein: Sangart: Consultancy; Shire: Research Funding; Novartis: Speakers Bureau.

The LDB1 complex co-opts CTCF for erythroid lineage specific long-range enhancer interactions

“…Among these associations are numerous instances where an LDB1 enhancer containing a SNP interacts with a gene relevant to the SNP phenotype whose promoter is occupied by CTCF but not LDB1 (Maurano et al, 2012). In particular, within Myb and Bcl11a enhancers, SNPs cluster at Ldb1 complex binding sites (Bauer et al, 2013;Stadhouders et al, 2014). Overall, SNPs are known to cluster at looped gene regulatory sites on a genome wide scale (Rao et al, 2014).…”

Advances in understanding erythropoiesis: evolving perspectives

“…BCL11A appears to be a promising target for therapeutic HbF induction and recent work has shown that humans with haploinsufficiency of this gene have substantial persistence of HbF, although there appear to be adverse neurodevelopmental phenotypes found in such patients, which emphasizes the need to develop targeted approaches to suppress BCL11A (Basak et al, 2015). GWAS studies have also implicated the transcription factor, MYB, as a regulator of HbF levels and reduction of this factor does appear to robustly induce HbF production, although its mechanism of action remains unknown Stadhouders et al, 2014). Additionally, rare mutations in KLF1 have been associated with increased HbF levels in some patients, but the effects are variable and certain mutations in this factor have been associated with both increased HbF and congenital anaemias (Sankaran & Orkin, 2013).…”

“…Mouse erythroleukaemia cells have been particularly valuable in broadening our understanding of certain aspects of gene regulation, particularly given their adult globin gene expression pattern (Bauer et al, 2013). Human erythroleukaemia cell lines, including TF-1, which is cytokine responsive, and K562 cells, which are cytokine independent, have also proven to be valuable models (Sankaran et al, 2012a;Stadhouders et al, 2014;Ulirsch et al, 2014). More recently, immortalized human erythroid cell lines have been created through a variety of methods, and some of these lines may be valuable in future studies (Kurita et al, 2013).…”

The association of HBG2, BCL11A, and HMIP polymorphisms with fetal hemoglobin and clinical phenotype in Iraqi Kurds with sickle cell disease

“…This SNP is known to be in linkage disequilibrium with rs66650371, a 3 bp deletion at HMIP, which has been reported as the most significant motif accounting for HMIP modulation of HbF. [29][30][31][32] It is located near the erythroid-specific DNase I hypersensitivity site 2 within HMIP block 2, in close proximity to the binding sites for erythropoiesisrelated transcription factors TAL1, E47, GATA2, and RUNX1. 13 In the present study, MAF of rs9399137 is 0.137, which is among the highest frequencies observed in SCD populations (Table 3).…”