Fetal hemoglobin expression is repressed following a transition to adult hemoglobin production at birth. For individuals with β-hemoglobinopathies, which impair the function of adult hemoglobin, reversing this switch is a promising route towards curative therapies. New research by Liu et al. proposes a simple model for hemoglobin switching based on competition between transcription factors BCL11A and NF-Y for binding sites in the γ-globin gene promoter.
Sickle cell disease (SCD) and β-thalassemia, collectively known as β-hemoglobinopathies, are common monogenic disorders in which abnormal hemoglobin production impairs erythrocyte production and function1,2. Approximately 300-400,000 annual global births are afflicted with a β-hemoglobinopathy1,2. Despite a high mortality rate, these diseases have remained extremely prevalent, and easily accessibly therapies are desperately needed1,2,3.
β-hemoglobinopathies are caused by variant alleles in the β-globin gene which alter or disable the β-subunit of hemoglobin. Adult hemoglobin is made up of two α-globin subunits and two β-globin (HbA, α2β2)1. An alternate globin protein, γ-globin, is expressed during fetal development and contributes two subunits of fetal hemoglobin (HbF, α2γ2)1. As expression of β-globin rises during late fetal development, γ-globin production is inhibited; HbF comprises <2% of hemoglobin in adults4. Sustained expression of HbF in adults is occasionally observed and is termed Hereditary Persistence of Fetal Hemoglobin (HPFH)1,4. HPFH has been associated with reduced symptom load in patients with β-hemoglobinopathies, implicating relief of γ-globin repression as a potential treatment avenue2. HPFH is typically caused by mutations in the γ-globin promoter which fall in distinct clusters, suggesting that they might disrupt the binding motif of a regulatory repressor5. A recent study by Liu et al. identified competitive binding between repressive transcription factor BCL11A and activator NF-Y at an HPFH cluster site in the γ-globin promoter as a key mechanism of expression control4.
The β-globin gene cluster on chromosome 16 contains 5 globin genes, including β-globin and γ-globin (Figure 1)1. In addition to the individual gene promoters, the region is regulated by a locus controls region (LCR) containing 5 DNase hypersensitivity sites (HSs) with varying degrees of regional enhancer activity1,2. Transcription of each globin gene is associated with looping between the promoter and the LCR. Developmental specificity is conveyed by the individual promoters while the LCR generally enhances transcription5. Transcription factors BCL11A and LRF have both been shown to repress transcription of γ-globin in adult erythroid cells through promoter binding and recruitment of the NuRD silencing complex6. HPFH mutation sites at -115 and -200 in the γ-globin promoter align with BCL11A and LRF binding sites, respectively4,7. CRISPR-Cas9 mediated disruption of the -115 bp binding motif reduces BCL11A binding and reproduces the HPFH phenotype, but the specific mechanism by which BCL11A silences γ-globin expression has been previously unclear7.
Liu et al.4 performed CRISPR-Cas9 perturbation screens to assess γ-globin expression in adult erythroid cell lines expressing Cas9 variants. Pooled gRNAs targeting the β-globin cluster at 11-bp intervals were introduced, and transfected cells with high HbF expression were isolated for analysis. Enrichment or depletion of gRNAs was quantified to identify sequences at which Cas9 activity was correlated with increased HbF expression. Inactive Cas9 (dCas9) binds to a target region but does not cleave DNA. dCas9 targeted to the LRF repressor binding site at approximately -200bp in the γ-globin promoter was associated with increased HbF expression, consistent with displacement of LRF. However, dCas9 binding at the BCL11A TGACCA binding motif at -115 bp was associated with reduced HbF expression. This effect was replicated by dCas9 targeting to other sites between -150 and -60 bp, suggesting that bound dCas9 was displacing an activating factor with a binding site in this region.
NF-Y is an activating transcription factor with two possible binding sites within this range. It has been previously identified as an activator of globin gene expression, though the specific mechanism remained elusive8. Liu et al.4 found that shRNA knockdown of NF-Y subunit A in HbF-expressing erythroid precursors resulted in reduced LCR looping to the γ-globin promoter. This reduced looping was associated with decreased expression, suggesting a potential role for NF-Y in expression activation through facilitation of chromosomal looping. CUT&RUN located NF-Y binding at a CCAAT motif at -88 to -84 bp in the γ-globin promoter, within the -150 to -60 range identified by dCas9 screening. Mutation of this motif by Cas9 resulted in reduced NF-Y occupancy and decreased HbF levels. Conversely, mutation of the BCL11A motif at -115 bp resulted in increased NF-Y occupancy and γ-globin expression. dCas9 targeting to the -115 bp BCL11A binding site and other adjacent sites within the -60 to -150 bp window partially reduced NF-Y occupancy and decreased γ-globin expression in BLC11A knockout (KO) cells. Based on these results, the authors proposed a simple model of competitive binding between BCL11A and NF-Y as a major regulator of γ-globin expression (Figure 2). BCL11A is dramatically upregulated in adult erythroid cells as compared to fetal progenitors, which likely contributes to out-competition of NF-Y and transition to HbA production4.
This simple model of γ-globin regulation suggests promising molecular targets for treatment of β-hemoglobinopathies via upregulation of HbF. Current clinical trials focus on downregulation of BCL11A via shRNA silencing or Cas9-mediated gene editing9,10. However, BCL11A has a key role in development of B-lymphocytes and hematopoietic stem cells, and downregulation negatively effects red blood cell enucleation1. Treatments targeting the -115 BLC11A site in the promoter may instead allow for highly specific relief of γ-globin repression. Cas9-mediated mutation of the binding site as demonstrated by Liu et al.4 is a potential therapy with long-term effect. Small proteins or ncRNAs may be also engineered to bind the site, but not to inhibit NF-Y binding. This approach lacks the risk of off-target mutation caused by Cas9 and may be translated into an affordable and easily produced therapeutic.
- Cavazzana, M., Antoniani, C. & Miccio, A. Gene Therapy for β-Hemoglobinopathies. Mol Ther 25, 1142–1154 (2017).
- Frati, G. & Miccio, A. Genome Editing for β-Hemoglobinopathies: Advances and Challenges. J Clin Med 10, 482 (2021).
- Piel, F. B. J., Steinberg, M. H. & Rees, D. C. Sickle cell disease. N Engl J Med 376, 1561-1573 (2017).
- Liu, N. et al. Transcription factor competition at the γ-globin promoters controls hemoglobin switching. Nat Genet 53, 511–520 (2021).
- Bender, M. A., Bulger, M., Close, J. & Groudine, M. Beta-globin gene switching and DNase I sensitivity of the endogenous beta-globin locus in mice do not require the locus control region. Mol Cell 5, 387–393 (2000).
- Xu, J. et al. Transcriptional silencing of γ-globin by BCL11A involves long-range interactions and cooperation with SOX6. Genes Dev 24, 783–798 (2010).
- Liu, N. et al. Direct Promoter Repression by BCL11A Controls the Fetal to Adult Hemoglobin Switch. Cell 173, 430-442.e17 (2018).
- Zhu, X. et al. NF-Y recruits both transcription activator and repressor to modulate tissue- and developmental stage-specific expression of human γ-globin gene. PLoS One 7, e47175 (2012).
- Frangoul, H. et al. CRISPR-Cas9 Gene Editing for Sickle Cell Disease and β-Thalassemia. N Engl J Med 384, 252–260 (2021).
- Esrick, E. B. et al. Post-Transcriptional Genetic Silencing of BCL11A to Treat Sickle Cell Disease. N Engl J Med 384, 205–215 (2021).