Abstract
A major ameliorating factor in sickle cell disease and beta-thalassemia is the inherent ability to produce fetal hemoglobin (HbF, a2g2), a highly heritable trait. We have previously mapped a major quantitative trait locus (QTL) controlling HbF levels to an array of single nucleotide polymorphisms (SNPs) within a 79 kb block between the gene for HBS1L (a G-protein/elongation factor) and the MYB proto-oncogene in Northern European Caucasians. These variants in chromosome 6q23.3, are referred to as HBS1L-MYB intergenic polymorphism (HMIP) and exist in three linkage disequilibrium (LD) blocks, HMIP-1, -2 and -3, of which HMIP-2 shows the strongest association. Association of HMIP with HbF has recently been replicated in two independent cohorts of patients with sickle cell disease, African-American and Brazilian, as well as in a cohort of Chinese patients with beta-thalassemia. Physiologically, MYB and HBS1L expression was simultaneously down-regulated in individuals with high HbF while over-expression of MYB inhibits gamma-globin expression. We also showed that HMIP influences erythrocyte, platelet and monocyte counts in humans. We hypothesised that the genetic variants in the MYB-HBS1L intergenic region are situated in a regulatory region and affect HbF levels by altering the expression of MYB and/or HBS1L.
Initial DNase I hypersensitivity analysis of the HMIP-2 region in K562 cells identified a number of hypersensitive sites which encouraged further functional analysis of the region. To study activity and to map potential regulatory elements in the entire MYB-HBS1L region we used chromatin immunopreciptation on microarray (ChIP–chip) of acetylated histones H3 and 4, RNA polymerase II and GATA-1. ChIP was performed on chromatin from primary human erythroid precursor cells and enrichment for specific sequences was evaluated using a high resolution Nimblegen microarray covering a large part of chromosome 6q.
Our ChIP-chip data show high levels of histone acetylation in the MYB-HBS1L intergenic region with a concentration in the HMIP-2 block, indicating that the region is transcriptionally active in erythroid precursors. Moreover, a number of potential cis-regulatory elements were identified in the same region as strong GATA-1 signals in coincidence with DNase I hypersensitive sites. These results suggest a regulatory region in the MYB-HBS1L intergenic region that could be important in erythroid development by controlling expression in the MYB locus.
Disclosures: No relevant conflicts of interest to declare.
Acknowledgments: Funding: The London University Central Research Fund (SB) and MRC ( G000111, ID51640 to SLT). We thank Jim Hughes, Marco De Gobbi, David Garrick and Doug Higgs (WIMM, Oxford, UK) and Jiannis Ragoussis (WTCHG, Oxford, UK) for help and advice on ChIP-chip.
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