Disruption of the Hbs1l-Myb Locus Causes Hereditary Persistence of Fetal Hemoglobin in a Mouse Model

Abstract

The human ␤-globin locus is comprised of embryonic, fetal, and adult globin genes, each of which is expressed at distinct stages of pre-and postnatal development. Functional defects in globin proteins or expression results in mild to severe anemia, such as in sickle-cell disease or ␤-thalassemia, but the clinical symptoms of both disorders are ameliorated by persistent expression of the fetal globin genes. Recent genome-wide association studies (GWAS) identified the intergenic region between the HBS1L and MYB loci as a candidate modifier of fetal hemoglobin expression in adults. However, it remains to be clarified whether the enhancer activity within the HBS1L-MYB regulatory domain contributes to the production of fetal hemoglobin in adults. Here we report a new mouse model of hereditary persistence of fetal hemoglobin (HPFH) in which a transgene was randomly inserted into the orthologous murine Hbs1l-Myb locus. This mutant mouse exhibited typically elevated expression of embryonic globins and hematopoietic parameters similar to those observed in human HPFH. These results support the contention that mutation of the HBS1L-MYB genomic domain is responsible for elevated expression of the fetal globin genes, and this model serves as an important means for the analysis of networks that regulate fetal globin gene expression. The human ␤-type globin locus consists of embryonic, fetal, and adult globin genes. The embryonic and fetal globin genes are typically expressed in the yolk sac and fetal liver, respectively (1). In the adult spleen and bone marrow, the adult globin gene is activated, while the embryonic and fetal globin genes are silenced (1). Two mechanisms have been proposed to regulate silencing of the embryonic and fetal globin genes in the adult: autonomous active silencing of the embryonic and fetal globin genes by gene autonomous recruitment of repressors and passive silencing due to competitive sequestration of locus control region (LCR) enhancer activity by the adult ␤-globin gene (1). Reactivation of the embryonic and/or fetal globin genes could be a critically useful ploy for establishing therapies for hereditary anemias and the associated pathophysiology caused by adult globin gene mutations, such as sickle-cell disease and ␤-thalassemia (2-5).A couple of genome-wide association study (GWAS) experiments independently identified two candidate regions that might influence the expression of fetal hemoglobin in adults (6, 7). One is the BCL11A locus. BCL11A encodes a well-characterized hematopoietic transcription factor (8), and persistent expression of ␥-globin was observed in BCL11A-deficient human and murine erythroid cells, demonstrating a contribution of BCL11A to hereditary persistence of fetal hemoglobin (HPFH) (9, 10). The other is the intergenic region between the human HBS1L and MYB genes. A 24-kbp linkage disequilibrium (LD) block located 33 kbp 5= to HBS1L and 65 kbp 5= to MYB was found to exhibit strong association with elevated levels of fetal hemoglobin (7), and the LD block was designated ...

HBS1L-MYB intergenic variants modulate fetal hemoglobin via long-range MYB enhancers

“…In accordance with these results, ChIP-Seq experiments (29) detected c-Myb binding to key cell-cycle regulators (i.e., Bcl2, Cdk6, Myc) in murine erythroid progenitors (Supplemental Figure 7B). Furthermore, several of these genes were found to be misregulated in an analysis of published MYB loss-of-function studies (20,21,61) in HEPs (Supplemental Figure 7C). Accelerated differentiation in an environment of lower MYB levels could favor premature cell-cycle termination during the proliferation cycles of adult erythropoiesis, producing more erythroid cells that synthesize predominantly HbF ("F-cells") before the switch to adult Hb synthesis occurs (Supplemental Figure 7D) (62).…”

“…Remarkably, we noticed that c-Myb in murine erythroid progenitors occupied the β-globin locus and many of the established γ-globin repressor genes, including Bcl11a and Klf1 (Supplemental Figure 7A). Analysis of previous c-MYB loss-of-function studies (20,21,61) indeed showed that several of the c-Myb-bound γ-globin repressor genes (i.e., Bcl11a, Klf1) are downregulated upon MYB depletion (Supplemental Figure 7C). These observations suggest that c-MYB directly activates key γ-globin repressor genes and thus fulfills an important role within the established molecular HbF repression mechanisms (Supplemental Figure 7D).…”

“…Association of these HMIP-2 SNPs with the erythroid traits has been replicated and validated in populations from diverse ethnic backgrounds (6)(7)(8)10). Despite extensive genetic evidence, a clear mechanistic basis for the association between the intergenic SNPs and erythroid biology has remained elusive, although the 2 flanking genes (HBS1L and MYB) are candidate target genes (4,(19)(20)(21)(22).…”

Fetal Globin Gene Repressors as Drug Targets for Molecular Therapies To Treat the β-Globinopathies

“…While the Myb gene is highly expressed in hematopoietic stem cells and progenitors, its expression progressively diminishes as erythroid cells mature (72). Since the developmental stages at which the MYB and ␤-type globins are expressed differ markedly (67), the data can be interpreted to suggest that MYB probably induces fetal globin transcription through an indirect mechanism. In support of this contention, Bianchi et al and we have found that MYB activates KLF1 and TR2/TR4 in human erythroid cells (67,73).…”

Cell signaling pathways involved in drug-mediated fetal hemoglobin induction: Strategies to treat sickle cell disease

“…Later studies in newborns with trisomy 13 and high HbF levels led to the discovery of two microRNAs, miR-15 a and miR-16-1 mediating repression of MYB gene expression in adult CD34þ stem cell-derived erythroid progenitors. 16 Disruption of MYB in mice produced increased e-globin and g-globin expression 17 supporting a role of MYB in g-gene silencing during development. Additional studies demonstrated the ability of MYB to activate KLF1 and TR2/TR4 in human erythroid cells 18 as an indirect mechanism of g-globin silencing.…”