A functional element necessary for fetal hemoglobin silencing

Abstract

Background: An improved understanding of the regulation of the fetal hemoglobin genes holds promise for the development of targeted therapeutic approaches for fetal hemoglobin induction in the β-hemoglobinopathies. Although recent studies have uncovered trans-acting factors necessary for this regulation, limited insight has been gained into the cis-regulatory elements involved.

Methods: We identified three families with unusual patterns of hemoglobin expression, suggestive of deletions in the locus of the β-globin gene (β-globin locus). We performed array comparative genomic hybridization to map these deletions and confirmed breakpoints by means of polymerase-chain-reaction assays and DNA sequencing. We compared these deletions, along with previously mapped deletions, and studied the trans-acting factors binding to these sites in the β-globin locus by using chromatin immunoprecipitation.

Results: We found a new (δβ)(0)-thalassemia deletion and a rare hereditary persistence of fetal hemoglobin deletion with identical downstream breakpoints. Comparison of the two deletions resulted in the identification of a small intergenic region required for γ-globin (fetal hemoglobin) gene silencing. We mapped a Kurdish β(0)-thalassemia deletion, which retains the required intergenic region, deletes other surrounding sequences, and maintains fetal hemoglobin silencing. By comparing these deletions and other previously mapped deletions, we elucidated a 3.5-kb intergenic region near the 5' end of the δ-globin gene that is necessary for γ-globin silencing. We found that a critical fetal hemoglobin silencing factor, BCL11A, and its partners bind within this region in the chromatin of adult erythroid cells.

Conclusions: By studying three families with unusual deletions in the β-globin locus, we identified an intergenic region near the δ-globin gene that is necessary for fetal hemoglobin silencing. (Funded by the National Institutes of Health and others.).

Figure 1. Pedigree and Mapping of New β-Globin Locus Deletions

Panel A shows pedigrees for Families A, B, and C, with mutations at the β-globin locus and fetal hemoglobin (HbF) levels indicated for each family member. Squares denote male family members, and circles female family members; arrows indicate the proband in each family. Panel B shows the deletions that were mapped by means of array comparative genomic hybridization and confirmed by means of sequencing, along with corresponding base-pair locations on chromosome 11: the Sri Lankan hereditary persistence of the fetal hemoglobin deletion (HPFH), the Sri Lankan (δβ)⁰-thalassemia deletion, and the Kurdish β⁰-thalassemia allele (with two deletions in cis). The corresponding genes, according to the Reference Sequence (RefSeq) database, are shown below the deletions: HBE1 (ε-globin), HBG2 (Gγ-globin), HBG1 (Aγ-globin), HBBP1 (the globin pseudogene), HBD (δ-globin), and HBB (β-globin). OR51V1 denotes olfactory receptor, family 51, subfamily V, member 1.

Figure 2. Results of Comparative Mapping ofβ-Globin Locus Deletions

Panel A shows the deletions that were mapped in this and prior studies, along with corresponding base-pair locations on chromosome 11: various hereditary persistence of fetal hemoglobin (HPFH) deletions, the (δβ)⁰-thalassemia deletions, and the Kurdish β⁰-thalassemia allele (with two deletions in cis). The corresponding genes, according to the Reference Sequence (RefSeq) database, are shown below the β-globin genes: HBG2 (Gγ-globin), HBG1 (Aγ-globin), HBBP1 (the globin pseudogene), HBD (δ-globin), and HBB (β-globin). A 3.5-kb region was delineated (indicated within the dashed vertical lines) by the upstream breakpoint of HPFH1 at one end and the 5′-untranslated region of the δ-globin at the other end. The HPFH1 breakpoint represents one of the best-documented and most precisely mapped HPFH deletions with one of the most 3′ ends of the upstream deletions (with the exception of the French HPFH breakpoint). The upstream breakpoint of the Macedonian (Macedonian–Turkish) (δβ)⁰-thalassemia deletion^, has been mapped and was used to define the downstream breakpoint, though the downstream end of the deletion remains imprecisely mapped (as indicated with hatch marks). Panel B shows the results of chromatin immunoprecipitation experiments performed on a microarray for several transcription factors and histone modifications. These data were analyzed in a manner similar to work described previously. The y axis represents the relative enrichment at any particular chromosomal coordinate.