The human β-globin locus consists of five globin genes embedded in the olfactory receptor cluster. During early development, these globin genes undergo gene switching from embryonic ε-globin (HBE) to fetal γ-globin (HBG1/2) and finally to adult β-globin (HBB). Inherited mutations in the HBB gene lead to dysfunction of the adult β-globin protein, causing hemoglobinopathies (Bauer et al., 2012). The symptoms of these disorders, including sickle cell disease and β-thalassemia, can be alleviated by persistent expression of fetal hemoglobin (hereditary persistence of fetal hemoglobin [HPFH]) throughout adulthood, which compensates for the mutant adult β-globin (Bank, 2006; Hassell, 2010). As such, multiple genome-editing strategies have been proposed to mimic HPFH as a treatment for hemoglobinopathies (Bauer et al., 2012; Breda et al., 2016; Sankaran et al., 2008; Sankaran et al., 2011; Sankaran et al., 2009; Traxler et al., 2016; Xu et al., 2011; Ye et al., 2016; Zeng et al., 2020). Two types of HPFH have been identified based on patient genetics. First is the non-deletional HPFH caused by point mutations in the BCL11A binding site at the HBG1/2 promoters, and disruption of this transcriptional repressor binding leads to the activation of these genes (Forget, 1998; Liu et al., 2018; Martyn et al., 2018; Traxler et al., 2016). Second is the deletional HPFH that consists of the excision of a large genomic region within the β-globin locus, frequently including HBB and HBD (Forget, 1998; Ye et al., 2016). These deletions can vary in length, and it remains unclear as to how they lead to the expression of fetal globin in adulthood (Ye et al., 2016).

The human β-globin gene locus is flanked by five CTCF binding sites (CBSs), which form the anchors for six chromosomal loops (Huang et al., 2017). Two convergent CBSs, designated as 3′HS1 and HS5, are located at the borders of the globin gene cluster. These two CBSs are nested between a downstream CBS (referred to as 3'-OR52A5-CBS) and two closely spaced upstream CBSs (referred together as 5'-OR51B5-CBSs). The HPFH deletions frequently cover the 3′HS1 CBS (Figure 1A). Therefore, we hypothesized that 3′HS1 may play a role in regulating β-globin cluster gene expression. To explore this, we first deleted the 3′HS1 using CRISPR/Cas9 genome-editing technology in K562 myelogenous leukemia cells, which express high levels of hemoglobin (Figure 1—figure supplement 1A). At the same time, we also deleted HS5 as a control in K562 cells. We observed that deletion of the HS5 CTCF site resulted in the upregulation of the 3′ genes including HBB and HBG1/2. Interestingly, the disruption of 3′HS1 CBS led solely to the upregulation of HBG1/2 (Figure 1—figure supplement 1B and C, Figure 1—figure supplement 1—source data 1). These results show that altering the CTCF binding profile across the locus can significantly change the expression of the β-globin genes.

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As HPFH deletions frequently cover the 3′HS1 CBS, we hypothesized that this site may contribute to the regulation of HBG1/2. To investigate further, we utilized the HUDEP-2 erythroid progenitor cell model, which predominantly express adult β-globin. We performed CRISPR/Cas9 editing to delete and invert the orientation of the 3′HS1 CBS to observe their respective impact on the expression of HBG1/2 (Figure 1B). In bulk edited cells, we found that HS5 deletion did not alter globin gene expression significantly, yet 3′HS1 disruption did increase γ-globin gene expression as observed in K562 cells with low deletion percentage and low editing efficiency (Figure 1—figure supplement 1D and E). Subsequently, we generated two 3′HS1 CBS deletion clones (referred to as B6 and D3) and two 3′HS1 inversion clones (A2 and G3), whose genomic sequences were verified by Sanger sequencing and CTCF binding evaluated by CUT&RUN (Figure 1D and E, Figure 1—figure supplement 1F). To elucidate whether the genetic editing at 3′HS1 caused changes to gene expression, we differentiated the HUDEP-2 clones to activate β-globin expression.

We performed Hi-C and capture Hi-C to examine the changes to 3D chromatin organization at the β-globin locus following alterations to the CBSs (Figure 1—figure supplement 2A, Figure 1C–E). In situ Hi-C data was generated with high resolution at 5 kb. A total of 15,207–16,529 loops could be detected in the HUDEP-2 clones used for in situ Hi-C using Mustache (Figure 1—figure supplement 2A; Roayaei Ardakany et al., 2020). The CTCF bound around the β-globin locus form four chromosomal loops and separate the cluster into three distinct domains (Figure 1A and C, Figure 1—figure supplement 2B). Of notice, we could detect the enhancer to target gene interaction between the LCR and the HBB gene (Figure 1—figure supplement 2B). We also tested the copy number variance (CNV) in the three particular HUDEP-2 clones, we could verify all clones have chromosome number 49–50,XY, which is of normal range in unmodified HUDEP-2 cells (Figure 1—figure supplement 2C; Moir-Meyer et al., 2018; Vinjamur and Bauer, 2018). Next, we tested if the chromosomal loops were altered by the 3′HS1 editing. We applied the HiCCUPS method to call the significant chromosomal loops in the β-globin locus, four loops were identified with q value less than 0.1 (Figure 1C and F). We then use the q value of the called loops by HiCCUPS to quantify the strength of loop interactions between CBSs. Of the convergent CTCF interactions, 3′HS1 to 5′-OR51B5-CBSs was not called as loop with q value over 0.25. One loop was called between the two forward CTCF CBSs – 3′HS1 and 3′-OR52A5 CBS (Figure 1D and F). In the 3′HS1 deletion clone, the loss of CTCF at 3′HS1 resulted in the total loss of loops between 3′HS1 and HS5 as well as loops between 3′HS1 and 5′-OR51B5-CBSs (not called as loop). Concomitantly, a strong increase in the interaction between HS5 and 3′-OR51A5-CBS was observed (Figure 1D and F, Figure 1—figure supplement 2A). This reveals how the loss of a CTCF anchor drastically alters the 3D chromatin organization in the β-globin locus. The inversion of the 3′HS1 CTCF caused a significant increase in the interaction between 3′HS1 and 3′-OR52A5 CBS. Meanwhile, 3′HS1 upstream interactions with HS5 and 5′-OR51B5-CBSs were decreased (Figure 1E and F, Figure 1—figure supplement 2A). This revealed that the inversion of 3′HS1 CTCF drove the formation of chromosomal loops between the convergent CBSs, which may lead to stronger insulation of regulatory elements.

Next, we evaluated the expression of the β-globin genes and found that the HBG1/2 and HBE genes upregulated 2.5- to 8-fold in the Δ3′HS1 clones (Figure 1G and H). In contrast, the inversion of 3′HS1 resulted in a >50% reduction of HBE and near-complete depletion of HBG1/2 (Figure 1H). Most notably, the increase in HBG1/2 upon deletion of the 3′HS1 CTCF site leads to a significant increase in fetal hemoglobin HbF (Figure 1I). Consequently, we evaluated the clones for HbF+ cells by flow cytometry. Consistent with transcription level, we observed an increase in HbF+ cells from 4.3% in the Cas9 control clone to 37.8% and 53.1% in Δ3′HS1 B6 and D3 clones, respectively (Figure 1J). Meanwhile, inversion of 3′HS1 resulted in a decrease of HbF+ cells to below 1%. In contrast, deletion of the upstream HS5 CBS did not induce nor abrogate the quantity of HbF+ cells (Figure 1J). All these clones are well differentiated at the same stage when HbF is measured by flow cytometry (Figure 1—figure supplement 3).

We then performed ATAC-seq and H3K27ac ChIP-seq in the Δ3′HS1 and 3′HS1 inversion clones to examine the regulatory landscape of the β-globin gene cluster. Following 3′HS1 CBS deletion, we observed significant open chromatin at the HBG1/2, BGLT3, and HBBP1 genes (Figure 2—figure supplement 1A). There was also a significant increase in activating H3K27ac in the HBG2 gene body. Interestingly, these epigenetic changes upon deletion of 3′HS1 CBS do not occur at the promoter of HBG2, which suggests that this 3′HS1-dependent regulation is independent from BCL11A transcriptional regulation (Figure 2—figure supplement 1A).

To determine if other regulatory pathways, such as the transcriptional repressor BCL11A, are involved in the upregulation of γ-globin expression in Δ3′HS1 and 3′HS1 inversion clones, we performed RNA-seq to identify differentially expressed genes in these clones. We found 161 upregulated and 153 downregulated genes in the Δ3′HS1 clones with HBG1/2 genes being the most significantly upregulated as well as β-globin cluster genes HBE1, HBBP, and BGLT3 (Figure 2A and C). In the 3′HS1 inversion clones, we identified only 3 upregulated genes and 51 downregulated genes (Figure 2B). In these clones, we observed downregulation of HBG2 and upregulation of the nearby OR52A5 gene; however, we did not observe any change in the expression of known γ-globin regulators (BCL11A, ZBTB7A (LRF), ELF2AK1 (HRI), ATF4, ZNF410, and NFIX) (Figure 2B and D, Supplementary file 1; Grevet et al., 2018; Masuda et al., 2016). We further verified BCL11A protein level by immunoblotting and observed no significant reduction in Δ3′HS1 clones (Figure 2, Figure 2—source data 1). Taken together, we concluded that BCL11A does not contribute to the induction of HbF in the edited HUDEP-2 cells. To further elucidate the role of BCL11A in the regulation of HBG1/2, we performed targeted disruption of a known BCL11A gene enhancer in both WT and Δ3′HS1 HUDEP-2 clones (Figure 2—figure supplement 1B, Figure 2—figure supplement 1—source data 1; Bauer et al., 2013). This mutation resulted in a significantly lower BCL11A expression, and we observed further increase of HBG1/2 expression level and HbF+ cells (Figure 2F, Figure 2—figure supplement 1C and F). Pomalidomide was found to boost the level of HbF in adult erythroblasts by destabilizing the BCL11A protein in cells (Grevet et al., 2018). Therefore, we treated HUDEP-2 3′HS1 deletion clones with pomalidomide and observed a significant reduction in BCL11A protein. This led to further increase in expression of fetal globin (Figure 2—figure supplement 2A and D, Figure 2—figure supplement 2—source data 1). These results show that genetic editing of the regulatory cis-element (3′HS1 CBS) accentuates with depletion of the transcriptional repressor BCL11A. This further indicates that the γ-globin activation in 3′HS1 deletion HUDEP-2 cells was not driven by the BCL11A-associated pathways. The double disruption of BCL11A and 3′HS1 also led to similar level of γ-globin activation as the disruption of BCL11A alone (Figure 2F). This data suggests that 3′HS1-regulated γ-globin repression might be hypostatic to the BCL11A-mediated γ-globin repression.

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The immunoblot data of BCL11A, ZBTB7A, β-actin, β-globin, and γ-globin of clones displayed in Figure 2.

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Deletional HPFH has been proposed to be the result of distal enhancer juxtaposition in the region downstream of 3′HS1, and several HPFH enhancers have been identified (Forget, 1998). The reduction of HbF in the 3′HS1 inversion HUDEP-2 clones suggests that potential enhancers may be located between 3′HS1 and 3′-OR52A5-CBS. These enhancers may be insulated by the loop formed between 3′HS1 to 3′-OR52A5-CBS in the inversion clones. With no significant change in chromatin interaction observed between the LCR and HBG1/2 genes (Figure 1—figure supplement 2C and D), it suggests that the HBG1/2 expression increase is controlled by a cis-element other than the LCR. We then analyzed the ATAC-seq, GATA1 ChIP-seq data together to search for potential cis-regulatory elements. We found the OR52A1 region bound by the GATA1 transcriptional activator in the erythroid lineages (Corces et al., 2016; Feingold and Forget; Liu et al., 2018; Figure 3A). Importantly, the mapping of a previously described HPFH enhancer encompassed both OR52A1 gene and GATA1 binding site (Elder et al., 1990; Feingold and Forget, 1989). We proceeded to delete the GATA1 binding site within the HPFH enhancer site and the entire HPFH enhancer marked by ATAC-seq in Δ3′HS1 clones (Figure 3B). We found that both deletions reduced the HBG1/2 expression and HbF+ cell percentage in these cells, suggesting that the HPFH enhancer contributes to the activation of HBG1/2 (Figure 3C–E). We further evaluated whether there were direct interactions between HBG1/2 and HPFH enhancer after the deletion of 3′HS1 by virtual 4C (v4C) in our Hi-C dataset. We observed mildly increased v4C signal in HBG2 promoter region of Δ3′HS1 cells compared with WT and 3′HS1 inversion clones when HPFH enhancer region acts as viewpoint (Figure 3—figure supplement 1A). Vice versa, we also observed mildly increased v4C signal in HPFH enhancer region of Δ3′HS1 cells compared with WT and 3′HS1 inversion clones when HBG2 promoter region acts as viewpoint (Figure 3—figure supplement 1A). These data suggest that the deletion of 3′HS1 might have induced more frequent interaction between HPFH enhancer and HBG2 promoter regions.

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Previously, mouse and human β-globin locus and its surrounding regions have been shown to be evolutionally conserved (Bulger et al., 2003). More interestingly, previous report showed that the disruption of 3′HS1 site in mouse did not result in any change in the β-like globin gene expression (Bender et al., 2006). We hypothesized that the alteration of HPFH enhancer sequence might contribute to the different outcome of 3′HS1 deletion in mouse and human. Therefore, we evaluated the evolutionary conservation of OR52A1 in mammals and found mouse and rat homolog of human OR52A1–Olfr68 bears two single-nucleotide substitutions right at the core binding site of GATA1 (Figure 3F). When we further checked the GATA1 ChIP-seq in mouse erythroid cells, we also found the absence of binding in the OR52A1’s homolog Olfr68 and its surrounding region (Figure 3G). This data suggests that the mouse olfactory receptor region 3′ to β-globin genes no longer bears GATA1 binding sites and enhancer activity. Overall, the loss of GATA1 binding in mouse clearly explains the difference between mouse and human on the effect of 3′HS1 deletion on globin gene expression.

Previously, it has been proposed that the juxtaposition of HPFH enhancer results in the activation of γ-globin. We wonder if the chromosomal distance between HPFH enhancer and globin genes also contributes to the regulation of globin gene expression. We used paired guide RNA to delete a 48 kb region between HPFH enhancer and 3′HS1, to access the effect of distance between enhancer and target genes in the presence of chromosomal insulators (Figure 3—figure supplement 1B). We obtained a heterozygous clone bearing this 48 kb deletion (Figure 3—figure supplement 1B). We found that moving the HPFH enhancer to the proximity of globin locus mildly increases the HBG1/2 gene expression by threefold with increase of HBE1 gene and reduction of HBB gene (Figure 3—figure supplement 1C and D). Overall, the data suggests that both chromosomal distance and insulator contribute to the low expression of HBG1/2, but chromosomal insulators are dominant to insulate HPFH enhancer to access the globin genes.

To assess the therapeutic potential of 3′HS1 deletion in primary HSPCs, we performed the 3′HS1 and HS5 CBS deletions in adult mobilized peripheral blood CD34+ HSPC from three different donors (Figure 4A). We achieved high deletion percentage of both CBSs in these primary cells (Figure 4B, Figure 4—source data 1). Upon differentiation, we observed a robust increase in HbF+ cells across all three donors with 3′HS1 deletion but not with HS5 deletion (Figure 4C and D). The normal erythroid differentiation was not affected by either CBS deletion (Figure 4—figure supplement 1A and B). While the disruption of the 3'HS1 CBS in primary patient cells did not yield as great of an effect on HbF+ cells as in HUDEP-2 cells, the results do support the involvement of a downstream cis-acting regulatory element on the HBG1/2 genes.

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The gel picture of paired guide deletion for HS5 and 3′HS1 in HSPC.

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Finally, based on our data, we propose a model where the 3′HS1 CBS modulates the HPFH enhancer’s access to the HBG1/2 genes (Figure 4E).

Here, we show that the deletion of a CBS – 3′HS1– in the human β-globin locus can phenocopy HPFH. This condition is driven by alteration of the 3D genomic organization around the β-globin locus, which allows the long-range interaction of a distal enhancer in the OR52A1 gene to drive the expression of HBG1/2. We further show that inversion of 3′HS1 insulates the enhancer element and further suppresses HBG1/2. Previously, induced LCR to HBG1/2 interaction showed the importance of high-order chromatin structure in the regulation of globin gene expression (Breda et al., 2016; Deng et al., 2014). However, the role of CTCF protein bound around the gene cluster was not clear. Our study reveals how CTCF binding at this locus modulates the accessibility of the fetal HBG1/2 genes to a downstream enhancer. In the HPFH enhancer scenario, 3′HS1 limits the HPFH enhancer access to HBG1/2 by forming the sub-TAD with 5′HS (Figure 4E; Oudelaar et al., 2021). When the 3′HS1 CBS is deleted, the HPFH enhancer gains access to HBG1/2 without the hinder of 3′HS1–HS5 loop. When the 3′HS1 CBS motif is inverted, the HPFH enhancer is further restricted by the pairing of 3′-OR52A5-CBS to the inverted 3′HS1 CBS, which results in the strong chromosomal loop formation between the two CBSs. This insulation leads to the reduced HBG1/2 expression and upregulation of OR52A5 (Figures 2B and 4E).

Furthermore, we have elucidated the function of long speculated HPFH enhancer in the induction of HbF with our Δ3′HS1 cells. However, we suspect that other regulatory elements also exist between 3′HS1 and 3′-OR52A5-CBS since we only get a partial decrease of HBG1/2 expression with HPFH enhancer deletion in Δ3′HS1 cells. This may suggest that other previously identified HPFH enhancers also contribute to HbF induction in the Δ3′HS1 cells (Feingold and Forget, 1989; Forget, 1998). Indeed, other GATA1 binding sites can be observed between OR52A1 and OR52A5 genes of erythroid progenitor cells. Overall, these observations suggest that GATA1 binding across the locus works collectively to activate HBG1/2 expression with or without direct promoter-enhancer interactions. Interestingly, despite the evolution conservation of OR52A1 gene in mammals, TF binding disrupting nucleotide substitutions occur at the GATA1 binding site in HPFH enhancer in some mammal species that do not express distinct form of fetal hemoglobin (mouse and rat) (Figure 3F and G). This evolutionary alteration in the TF binding site suggests that HPFH enhancer may also play a role in regulating globin expression in other developmental stages. The consistent binding of GATA1 in high HBE/HBG-expressing K562 cells and high HBB-expressing HUDEP-2 cells suggests that the potential function of HPFH enhancer may still need the modulation of 3D genomic reorganization.

Despite our data suggesting that deletion of 3′HS1 is sufficient to induce the γ-globin activation, yet, the upregulation does not completely phenocopy the HPFH condition, in which the HbF is expressed at pancellular level. The deletion of 3′HS1 only induces a portion of cells to be F+ cells, with an interesting differentiation block phenotype in a large portion of clones we have selected (Figure 1J). Although HUDEP-2 is known to be heterogenous in clonal level, the F+ cell phenotype of 3′HS1-deleted cells suggests that the full HPFH phenotype may require the deletion of HBB and HBD genes to erase the strong enhancer-promoter interaction between LCR and HBB. Furthermore, our results indicate that the distance between HPFH enhancer and the globin locus could also play a role in regulating γ-globin gene expression. This data hints that the deletion of both 3′HS1 and the flanking region between 3′HS1 and HPFH enhancer may activate the fetal hemoglobin to an even higher level. Larger deletions will result in the reorganization of chromosomal loop interactions as well as the decrease of physical distance from HPFH enhancer to globin locus. The enhancers have to locate at certain range of distance from the target gene promoters to exert the maximal activation effect (Jessica Zuin et al., 2021). Therefore, a method that can create large deletion that disrupts 3′HS1 and reduces enhancer-promoter distance simultaneously could be a potential gene therapy to effectively activate the γ-globin expression in hemoglobinopathies.

The differentiation stage of HUDEP-2 cell clones used in Figure 1 was profiled by flow cytometry of CD71 and CD235a (Figure 2—figure supplement 1—source data 1). The immunoblot data of BCL11A, ZBTB7A, β-actin, β-globin, and γ-globin of clones is displayed in Figure 2—figure supplement 2.

Sequencing data have been deposited in GEO under accession codes GSE160425.

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Author details

1. Pamela Himadewi

   Center for Epigenetics, Van Andel Research Institute, Grand Rapids, United States

   Contribution

   Data curation, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing – original draft

   Contributed equally with

   Xue Qing David Wang and Fan Feng

   Competing interests

   No competing interests declared

2. Xue Qing David Wang

   Center for Epigenetics, Van Andel Research Institute, Grand Rapids, United States

   Contribution

   Investigation, Writing – original draft

   Contributed equally with

   Pamela Himadewi and Fan Feng

   Competing interests

   No competing interests declared

3. Fan Feng

   Department of Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, United States

   Contribution

   Data curation, Formal analysis, Software, Writing – original draft, Writing - review and editing

   Contributed equally with

   Pamela Himadewi and Xue Qing David Wang

   Competing interests

   No competing interests declared

4. Haley Gore

   Center for Epigenetics, Van Andel Research Institute, Grand Rapids, United States

   Contribution

   Methodology, Writing – original draft

   Competing interests

   No competing interests declared

5. Yushuai Liu

   Center for Epigenetics, Van Andel Research Institute, Grand Rapids, United States

   Contribution

   Investigation

   Competing interests

   No competing interests declared

6. Lei Yu

   Cell and Development Biology, University of Michigan, Ann Arbor, United States

   Contribution

   Methodology

   Competing interests

   No competing interests declared

7. Ryo Kurita

   Department of Research and Development, Central Blood Institute, Japanese Red Cross Society, Tokyo, Japan

   Contribution

   providing material

   Competing interests

   No competing interests declared

8. Yukio Nakamura

   1. Cell Engineering Division, RIKEN BioResource Research Center, Tsukuba, Japan
   2. Faculty of Medicine, University of Tsukuba, Tsukuba, Japan

   Contribution

   provide research material

   Competing interests

   No competing interests declared

9. Gerd P Pfeifer

   Center for Epigenetics, Van Andel Research Institute, Grand Rapids, United States

   Contribution

   Conceptualization, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-5080-9604

10. Jie Liu

    Department of Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, United States

    Contribution

    Conceptualization, Writing - review and editing

    For correspondence

    drjieliu@umich.edu

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-9504-0587

11. Xiaotian Zhang

    Center for Epigenetics, Van Andel Research Institute, Grand Rapids, United States

    Present address

    Department of Pathology, University of Michigan, Ann Arbor, United States

    Contribution

    Funding acquisition, Data curation, Formal analysis, Project administration, Investigation, Methodology, Supervision, Conceptualization

    For correspondence

    xiaozhan@umich.edu

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-9533-4761

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