Merlin Crossley

Hemoglobinopathies are the most common inherited anemias worldwide. While most carriers are asymptomatic or mildly affected, severe forms of α-thalassemia, including severe hemoglobin H (HbH) disease and surviving individuals with Hb Bart’s hydrops fetalis syndrome (BHFS), require lifelong transfusion support or stem cell transplantation. Reactivating the silenced embryonic α-like globin (ζ-globin) gene is a promising approach for treating these diseases. Here we identify the key cis -regulatory elements responsible for silencing the ζ-globin gene in definitive erythroid cells and subsequently develop gene-editing strategies that reactivate ζ-globin expression to therapeutically relevant levels. In a preclinical mouse model of BHFS, we show that this strategy rescues the otherwise prenatal lethal condition until late in development (E17.5). Using primary erythroid cells derived from patients with HbH disease and BHFS, we show that these approaches reactivate ζ-globin expression to levels exceeding those achieved in the mouse model, producing therapeutic levels of α-like globin.

Krüppel-like factor 3 (KLF3) is a member of the archetypal SP/KLF family of transcription factors that bind GC-rich elements and CACCC boxes in promoters and enhancers via three classical zinc fingers (ZFs) at or near their C-termini. KLF3 can both repress and activate transcription. It represses by recruiting CtBP co-repressors via its N-terminal domain but the mechanism by which it activates was unknown. Here, we show that KLF3 associates with WDR5 and this interaction is associated with gene activation. We also demonstrate that this interaction is required for proper genomic targeting of KLF3. This helps explain previous results indicating that both the C-terminal ZF DNA-binding domain and N-terminal functional domain are required for KLF3 to identify its target genes. This result adds to only a handful of examples that transcriptional co-regulators, in addition to facilitating activation and repression, can also influence target gene selection.

β-hemoglobinopathies caused by mutations in adult-expressed HBB can be treated by re-activating the adjacent paralogous genes HBG1 and HBG2 (HBG), which are normally silenced perinatally. Although HBG expression is induced by global demethylating drugs, their mechanism is poorly understood, and toxicity limits their use. We identify the DNMT1-associated maintenance methylation protein UHRF1 as a mediator of HBG repression through a CRISPR/Cas9 screen. Loss of UHRF1 in the adult-type erythroid cell line HUDEP2 causes global demethylation and HBG activation that is reversed upon localized promoter re-methylation. Conversely, targeted demethylation of the HBG promoters activates their genes in HUDEP2 or primary CD34⁺ cell-derived erythroblasts. Mutation of MBD2, a CpG-methylation reading component of the NuRD co-repressor complex, recapitulates the effects of promoter demethylation. Our findings demonstrate that localized CpGmethylation at the HBG promoters facilitates gene silencing and identify a potential therapeutic approach for β-hemoglobinopathies via epigenomic editing.

Hydroxyurea (also known as hydroxycarbamide) has been the most widely used treatment for children and adults with sickle cell disease since the 1980s. It is thought to act primarily by boosting the expression of fetal globin. The molecular mechanism through which hydroxyurea elevates fetal globin levels has not been understood and we have explored this question here. It has recently been established that expression of the fetal globin gene is directly inhibited by two DNA-binding proteins, the transcriptional repressors ZBTB7A and BCL11A. We therefore tested—in primary human erythroid cells in culture and in cell lines—whether hydroxyurea affects the levels of these proteins. The results were clear. Both ZBTB7A and BCL11A protein levels were reduced by treatment with hydroxyurea. This important result is a first step towards elucidating the molecular mechanism through which hydroxyurea elevates fetal globin expression and an insight into why this small molecule with multiple potential activities can elevate fetal globin gene expression. Hydroxyurea (also known as hydroxycarbamide) is the most widely used treatment for children and adults with sickle cell disease. However, while it is known that treatment with hydroxyurea causes beneficial increases in fetal globin expression, it is not known how hydroxyurea leads to this effect. Two proteins are known to normally keep the expression of fetal globin silent in adult red blood cells. We showed here that hydroxyurea acts to reduce the amounts of these silencing proteins. Lower levels of these proteins could explain how hydroxyurea increases fetal globin expression in people with sickle cell disease and therefore the mechanism by which this drug works.

Inducing fetal hemoglobin (HbF) in red blood cells can alleviate β-thalassemia and sickle cell disease. We compared five strategies in CD34⁺ hematopoietic stem and progenitor cells, using either Cas9 nuclease or adenine base editors. The most potent modification was adenine base editor generation of γ-globin –175A>G. Homozygous –175A>G edited erythroid colonies expressed 81 ± 7% HbF versus 17 ± 11% in unedited controls, whereas HbF levels were lower and more variable for two Cas9 strategies targeting a BCL11A binding motif in the γ-globin promoter or a BCL11A erythroid enhancer. The –175A>G base edit also induced HbF more potently than a Cas9 approach in red blood cells generated after transplantation of CD34⁺ hematopoietic stem and progenitor cells into mice. Our data suggest a strategy for potent, uniform induction of HbF and provide insights into γ-globin gene regulation. More generally, we demonstrate that diverse indels generated by Cas9 can cause unexpected phenotypic variation that can be circumvented by base editing.

The fetal-to-adult switch in hemoglobin production is a model of developmental gene control with relevance to the treatment of hemoglobinopathies. The expression of transcription factor BCL11A, which represses fetal β-type globin (HBG) genes in adult erythroid cells, is predominantly controlled at the transcriptional level but the underlying mechanism is unclear. We identify HIC2 as a repressor of BCL11A transcription. HIC2 and BCL11A are reciprocally expressed during development. Forced expression of HIC2 in adult erythroid cells inhibits BCL11A transcription and induces HBG expression. HIC2 binds to erythroid BCL11A enhancers to reduce chromatin accessibility and binding of transcription factor GATA1, diminishing enhancer activity and enhancer–promoter contacts. DNA-binding and crystallography studies reveal direct steric hindrance as one mechanism by which HIC2 inhibits GATA1 binding at a critical BCL11A enhancer. Conversely, loss of HIC2 in fetal erythroblasts increases enhancer accessibility, GATA1 binding and BCL11A transcription. HIC2 emerges as an evolutionarily conserved regulator of hemoglobin switching via developmental control of BCL11A.

The benign condition hereditary persistence of fetal hemoglobin (HPFH) is known to ameliorate symptoms of co-inherited β-hemoglobinopathies, such as sickle cell disease and β-thalassemia. The condition is sometimes associated with point mutations in the fetal globin promoters that disrupt the binding of the repressors BCL11A or ZBTB7A/LRF, which have been extensively studied. HPFH is also associated with a range of deletions within the β-globin locus that all reside downstream of the fetal HBG2 gene. These deletional forms of HPFH are poorly understood and are the focus of this study. Numerous different mechanisms have been proposed to explain how downstream deletions can boost the expression of the fetal globin genes, including the deletion of silencer elements, of genes encoding noncoding RNA, and bringing downstream enhancer elements into proximity with the fetal globin gene promoters. Here we systematically analyze the deletions associated with both HPFH and a related condition known as δβ-thalassemia and propose a unifying mechanism. In all cases where fetal globin is upregulated, the proximal adult β-globin (HBB) promoter is deleted. We use clustered regularly interspaced short palindromic repeats-mediated gene editing to delete or disrupt elements within the promoter and find that virtually all mutations that reduce ΗΒΒ promoter activity result in elevated fetal globin expression. These results fit with previous models where the fetal and adult globin genes compete for the distal locus control region and suggest that targeting the ΗΒΒ promoter might be explored to elevate fetal globin and reduce sickle globin expression as a treatment of β-hemoglobinopathies.