Activation of γ-globin gene expression by GATA1 and NF-Y in hereditary persistence of fetal hemoglobin

Abstract

Hereditary persistence of fetal hemoglobin (HPFH) ameliorates β-hemoglobinopathies by inhibiting the developmental switch from γ-globin (HBG1/HBG2) to β-globin (HBB) gene expression. Some forms of HPFH are associated with γ-globin promoter variants that either disrupt binding motifs for transcriptional repressors or create new motifs for transcriptional activators. How these variants sustain γ-globin gene expression postnatally remains undefined. We mapped γ-globin promoter sequences functionally in erythroid cells harboring different HPFH variants. Those that disrupt a BCL11A repressor binding element induce γ-globin expression by facilitating the recruitment of nuclear transcription factor Y (NF-Y) to a nearby proximal CCAAT box and GATA1 to an upstream motif. The proximal CCAAT element becomes dispensable for HPFH variants that generate new binding motifs for activators NF-Y or KLF1, but GATA1 recruitment remains essential. Our findings define distinct mechanisms through which transcription factors and their cis-regulatory elements activate γ-globin expression in different forms of HPFH, some of which are being recreated by therapeutic genome editing.

Extended Data Fig. 1. Derivation of HUDEP-2 cells containing a single γ-globin gene.

Genome browser view of deletions introduced into HUDEP-2 cells to generate the HUDEP-2^{Δεγδβ/GγAγ} line, which contains a single γ-globin gene. The region of the β-like globin gene cluster that was deleted on one chromosome is shown in blue. Chromatin immunoprecipitation (ChIP-seq) analysis for CTCF occupancy and ATAC-seq analysis of HUDEP-2 cells were derived from publicly available data. b, Generation of a single HBG2-HBG1 fusion gene on the remaining β-like globin gene locus. Positions of the gRNAs targeting intron 2 of HBG1 and HBG2 are in shown with arrows. c, Fluorescence in situ hybridization analysis of wild-type (WT) HUDEP-2, HUDEP-2^Δεγδβ, and HUDEP-2^{Δεγδβ/GγAγ} score correlation comparing the frequency of the union (top) and intersection (bottom) between HUDEP-2 and HUDEP-2^{Δεγδβ/GγAγ} cells. The TAD scores identify the degree of separation between the left and right boundaries based on the Hi-C interaction matrix. A TAD will be called at local minima. The union indicates the left and right boundaries across HUDEP-2 and HUDEP-2^{Δεγδβ/GγAγ} cells. The intersection represents the fraction of shared boundaries between the HUDEP-2 and HUDEP-2^{Δεγδβ/GγAγ} TAD sets. The Spearman correlation coefficients (ρ) are shown. Results were generated from merged reads derived from two independent experiments.

Extended Data Fig. 2. Characterization of the HUDEP-2^{Δεγδβ/GγAγ} cell line

a, Next generation sequencing (NGS) analysis of the indicated HUDEP-2 lines showing percentages of reads corresponding to HBG1 or HBG2 exon 3. (mean ± SD; n = 6 independent clones for each genotype). b, %HbF in WT and HUDEP-2^{Δεγδβ/GγAγ} clones, measured by ion-exchange high-performance liquid chromatography (IE-HPLC) after 7 days of erythroid differentiation. Box and whisker plots show minimum, maximum, median, and interquartile ranges. n = 6 independent clones for each genotype. *p = 0.0156 uncorrected two-tailed unpaired t-test. c, Kinetics of erythroid maturation of WT and HUDEP-2^{Δεγδβ/GγAγ} cells determined by flow cytometry for CD49d and Band3 in the CD235a⁺ population at the indicated timepoints after culture in erythroid differentiation medium. Mean ± SD is shown in each quadrant. n = 6 independent clones analyzed for each genotype.

Extended Data Fig. 3. Hi-C analysis showing chromatin structure of the extended β-globin locus in HUDEP-2 cells containing a single γ-globin gene

a, Heat maps comparing chromatin interactions of the extended β-like globin locus in WT HUDEP-2 cells (red) and in HUDEP-2^{Δεγδβ/GγAγ} cells, which contain a single, modified β-like globin locus (blue). Tracks below show transcriptionally open or closed compartments as positive (blue) or negative (magenta) according to Hi-C analysis. CTCF ChIP-seq analysis and ATAC seq analysis are shown for WT HUDEP-2 cells. The 91.5 kb deletion of the extended β-globin locus (Δεγδβ) is shown as a blue rectangle; the 5.4 kb deletion generating a single HBG2-HBG1 fusion gene (GγAγ) is designated in grey. Genes are designated as black vertical bars in the bottom track. b, The topologically associated domain (TAD) separation score correlation comparing the frequency of the union (top) and intersection (bottom) between HUDEP-2 and HUDEP-2^{Δεγδβ/GγAγ} cells. The TAD scores identify the degree of separation between the left and right boundaries based on the Hi-C interaction matrix. A TAD will be called at local minima. The union indicates the left and right boundaries across HUDEP-2 and HUDEP-2^{Δεγδβ/GγAγ} cells. The intersection represents the fraction of shared boundaries between the HUDEP-2 and HUDEP-2^{Δεγδβ/GγAγ} TAD sets. The Spearman correlation coefficients (ρ) are shown. Results were generated from merged reads derived from two independent experiments.

Extended Data Fig. 4. Effects of HPFH variants on HbF expression in HUDEP-2 cells.

a, HPLC tracings showing hemoglobin analysis of WT HUDEP-2, HUDEP-2^{Δεγδβ/GγAγ} and HUDEP-2^{Δεγδβ/GγAγ} cells with the −110 A>C HPFH variant after 7 days of erythroid differentiation. b, ATAC-seq tracks showing open chromatin at the β-like globin gene cluster in single clones with distal CCAAT box HPFH variants −117 G>A and −114 C>A and control mutations −110 A>G and −110 A>T. The shaded area highlighting the HBG promoter is shown in higher resolution on the right. The reference genes are shown at the bottom and the dotted lines indicate the region deleted to create the single in-frame HBG fusion gene.

Extended Data Fig. 5. GATA1 occupancy at the γ-globin promoters is associated with HbF expression.

a, ChIP-seq analysis showing GATA1 occupancy at the β-like globin gene cluster in primary fetal and adult proerythroblasts. The shaded areas highlighting the HBG1 and HBG2 promoters are shown in higher resolution below. b, ChIP seq analysis for GATA1 in HUDEP-1 and HUDEP-2 cells, which express predominantly γ-globin and β-globin respectively, shown as described for panel a. GATA1 occupancy in HUDEP-2 cells was derived from publicly available data.

Extended Data Fig. 6. Disruption of the bipartite GATA motif via −186 C>T impairs HbF expression associated with HPFH variants

a, Representative ion-exchange high-performance liquid chromatography (HPLC) traces showing reduced fetal hemoglobin (HbF) peak intensity in HPFH clones without (top) and with −186 C>T (bottom). Cells were grown in culture for 7 days under erythroid differentiation conditions. b, Representative F-cell staining flow cytometry plots in undifferentiated HPFH clones without (top) and with −186 C>T (bottom). c, Replicate ChIP-seq analysis showing GATA1 occupancy at the β-like globin gene cluster in clones harboring distal CCAAT box HPFH variants ± the −186 C>T GATA motif mutation (related to Fig. 2d). The shaded area highlighting the HBG promoter is shown in higher resolution on the right. The reference genes are shown at the bottom and the dotted lines indicate the region deleted to create the single in-frame HBG fusion gene.

Extended Data Fig. 7. Disruption of the - 189 GATA motif in HUDEP-2^{Δεγδβ/GγAγ} cells does not induce γ-globin expression

a, Sequence of the HBG promoter showing the bipartite GATA motif (blue), BCL11A binding motif (grey) and the distal CCAAT box (dotted rectangle; hg19 – chr11:5,276,112–5,276,201). The −186 C>T mutation (lower case bold), disrupts GATA1 binding. b, %HbF (left) and %F-cells (right) after 7 days of erythroid differentiation in HUDEP-2^{Δεγδβ/GγAγ} cells ± the −186 C>T mutation (lower case bold). Each dot represents an individual clone (n = 12 per genotype). Box and whisker plots show minimum, maximum, median, and interquartile ranges. **p = 0.0017, uncorrected two-tailed unpaired t-test. An uncorrected two-tailed unpaired t-test indicated no significant effect of the −186T mutation on %HbF in WT CCAAT box clones, p > 0.9999.

Extended Data Fig. 8. −110 A>C at the distal CCAAT box enhances NF-Y binding

a, Electrophoretic mobility shift assay (EMSA) for NF-Y binding to WT or mutant γ-globin promoter distal CCAAT box oligonucleotides in K562 cell nuclear extracts. Mutations are indicated in lower case bold. Bound probe is indicated by the closed triangle and supershift product of the NF-Y:probe complex is indicated by the open triangle. Graph on the right shows densitometry analysis of NF-Y band intensity relative to WT signal. b, Motif analysis showing the predicted effects of single nucleotide alterations on BCL11A binding to the −115 distal CCAAT box. The −110 A>C HPFH variant (asterisk) is predicted to decrease BCL11A affinity for the motif. c, Competitive EMSA assay for BCL11A binding to distal CCAAT box probes. The autoradiogram shows competition of cold WT or −110 A>C probes (1X, 5X, 10X, 25X, and 50X molar excess) with radiolabeled WT probe for binding to BCL11A zinc fingers 4–6 expressed in COS-7 cells. Bound probe is indicated by a closed triangle. The graph shows densitometry analysis of this band after incubation with cold competitor oligonucleotides, normalized to intensity with no competitor.

Extended Data Fig. 9. Flow cytometry gating strategies

a, Gating strategy for monitoring the differentiation of HUDEP-2 cells (see Extended Data Fig. 2c). b, Gating strategy for F-cell determination in undifferentiated HUDEP-2 cells (see Extended Data Fig. 6b). Antibodies used are listed in the methods section.

Figure 1.. HPFH variants in the γ-globin promoter distal CCAAT box facilitate recruitment of GATA1 to an upstream motif.

a, The HBG1/2 promoter sequence with transcriptional start at position +1 (hg19 – chr11:5,276,105–5,276,215). HPFH variants examined in this study and their associated %HbF range in RBCs of heterozygous individuals are shown^,,. Transcription factor binding motifs for ZBTB7A (red), BCL11A (grey) and GATA1 (blue), are shown. The −115 distal CCAAT box is indicated by a dashed rectangle. b, Distal CCAAT box HPFH variants were generated in HUDEP-2^{Δεγδβ/GγAγ} cells harboring a single wild-type γ-globin (HBG) gene. Nucleotide substitutions are shown in bold lower case; the 13Δ HPFH deletion is dashed. −198 T>C generates a de novo KLF1 binding motif; 13Δ, −117 G>A and −114 C>A disrupt the BCL11A binding motif; −113 A>G generates a de novo GATA1 binding motif; −110 A>C generates a de novo NF-Y motif (this study); −110 A>G and −110 A>T represent inert controls. c, Fetal hemoglobin (HbF) levels measured by ion-exchange high performance liquid chromatography (IE-HPLC) in WT and mutant clones grown after erythroid differentiation for 7 days. Each dot represents an individual clone (n = 12 per genotype). Box and whisker plots show minimum, maximum, median, and interquartile ranges of independent clones. Multiplicity adjusted p-values of each variant versus WT by ordinary one-way ANOVA with Dunnett’s multiple comparisons test: 13Δ, −117A, −114A, −110C (p < 0.0001); −113G (p = 0.005). d, ATAC-seq analysis at the β-like globin gene cluster in WT HUDEP-2^{Δεγδβ/GγAγ} cells and selected mutant clones. Vertical dotted lines indicate the region deleted to generate an in-frame HBG fusion gene. The shaded area highlighting the single HBG promoter is shown in higher resolution on the right. Reference genes are shown at the bottom.

Figure 2.. The −189 GATA motif facilitates γ-globin gene activation in HPFH.

a, The HBG promoters showing bipartite GATA motifs (blue), the −115 distal CCAAT box (dotted rectangle), and the BCL11A binding motif (grey; hg19 – chr11:5,276,112–5,276,201). b, GATA1 ChIP-seq analysis at the β-like globin gene cluster in WT HUDEP-2^{Δεγδβ/GγAγ} cells and selected mutant clones. c, Graph on the left shows %HbF in HUDEP-2^{Δεγδβ/GγAγ} clones harboring HPFH ± −186 C>T GATA motif mutations after 7 days of erythroid differentiation. Graph on the right shows, %HbF-immunostaining “F-cells”, measured prior to differentiation. Each dot represents an individual clone (n = 12 per genotype). Box and whisker plots show minimum, maximum, median, and interquartile ranges of independent clones. ****p < 0.0001, uncorrected two-tailed unpaired t-test. d, ChIP-seq analysis of GATA1 occupancy at the β-like globin gene cluster in clones harboring distal CCAAT box HPFH variants ± −186 C>T GATA motif mutations. e, GATA1 ChIP-seq signals at the HBG promoter between clones harboring HPFH variants ± GATA motif −186 C>T mutations in two biological replicate experiments using S3norm to adjust for differences in sequencing depth and signal-to-noise ratios.

Figure 3.. Disruption of the −189 GATA motif inhibits HbF expression in primary erythroblasts.

Normal donor umbilical cord blood (UCB) CD34⁺ cells were electroporated with ribonucleoprotein (RNP) containing the adenosine base editor ABE7.10 and gRNA targeting the −189 GATA motif. Edited cells were maintained in liquid culture or seeded into methylcellulose medium with erythroid cytokines after 48 hours. Control cells were not electroporated. a, Sequence of the gRNA target recognition sequence with the protospacer adjacent motif (PAM) in red. The −189 GATA motif is shaded grey (hg19 – chr11:5,276,192–5,276,214). Potential edits within or outside of the WGATAR motif are shown in blue or purple, respectively. Editing frequencies of individual adenosines, measured by next generation sequencing (NGS) after 96 hours, are shown for each position and color-coded according to the heat map (mean ± SD; n = 4 biological replicates across two independent experiments). b, Frequencies of mutant genotypes after base editing (mean ± SD; n = 4 biological replicates across two independent experiments). c, %HbF in bulk-edited or unedited control populations after 10 days of in vitro erythroid differentiation (mean ± SD; n = 3 biological replicates). *p = 0.0255, uncorrected two-tailed unpaired t-test d, %HbF in 14-day-old burst forming unit erythroid (BFU-E) colonies versus number of HBG1 and HBG2 alleles with mutations in the core −189 GATA motif (positions A₇ and/or A₉). Each dot represents a BFU-E colony from the same UCB cells analyzed in panel c. Linear regression analysis and two-tailed Pearson’s correlation coefficient are shown. No adjustments for multiple corrections were made. e, BFU-E colony analysis performed as shown in panel d using UCB cells from a different donor. The mean is indicated by the blue line with the 95% confidence interval shaded between the black curves for d and e. f, HbF expression in BFU-E colonies with ≤10% or ≥90% editing of the −189 GATA motif, indicated by the shaded regions in panels d (Rep. 1) and e (Rep. 2). n = (14) ≤10% edited Rep. 1 colonies, (27) ≥90% edited Rep. 1 colonies, (23) ≤10% edited Rep. 2 colonies, and (11) ≥90% edited Rep. 2 colonies. ***p = 0.0001, ****p < 0.0001, uncorrected two-tailed unpaired t-test.

Figure 4.. Distal CCAAT box HPFH variants recruit NF-Y to the γ-globin promoter.

a, Sequence of the HBG promoter showing the −85 proximal and −115 distal CCAAT boxes (hg19 – chr11:5,276,090–5,276,131). HPFH nucleotide substitutions are indicated by filled triangles. The 13Δ HPFH deletion is shown as a black line. b, ChIP-seq analysis showing NF-YB occupancy in HUDEP-2^{Δεγδβ/GγAγ} clones harboring distal CCAAT box HPFH variants. c, Motif analysis showing the predicted effects of single nucleotide alterations on NF-Y binding to the −115 distal CCAAT box. The −110 A>C HPFH variant (asterisk) is predicted to increase NF-Y affinity for the motif. d, Electrophoretic mobility shift assay (EMSA) for NF-Y binding to WT or mutant oligonucleotides representing the γ-globin promoter distal CCAAT box using K562 cell nuclear extracts. Mutations are indicated in lower case bold. Bound probe is indicated by the closed triangle and supershift product of the NF-Y:probe complex by the open triangle. Graph on the right shows densitometry analysis of NF-Y band intensity for the −110 A>C probe relative to WT. e, Competitive EMSA assay for NF-Y binding to distal CCAAT box probes. The autoradiogram shows competition of cold WT or −110 A>C probes (1X, 5X, 10X, 25X, and 50X molar excess) with radiolabeled WT probe for binding to NF-Y in K562 nuclear extracts. Bound probe is indicated by a closed triangle. Graph on the right shows densitometry analysis of band intensities normalized to intensity of the band with no added competitor.

Figure 5.. GATA1 and NF-Y cooperate to activate γ-globin gene expression.

a, The γ-globin promoter showing transcription factor binding motifs and mutations analyzed according to designations described for Figure 2a (hg19 – chr11:5,276,085–5,276,201). b, %HbF in clones with the indicated mutations, measured after 7 days of erythroid differentiation. Box and whisker plots show minimum, maximum, median, and interquartile ranges. Each dot represents an individual clone (n = 12 per genotype). Ordinary one-way ANOVA with Tukey’s multiple comparisons test shows the multiplicity adjusted p-values between genotypes. ****p < 0.0001. c, ChIP-seq analysis showing GATA-1 and NFY occupancy at the β-like globin gene cluster in HUDEP-2^{Δεγδβ/GγAγ} clones with the indicated mutations.

Figure 6.. NF-Y binding to the −85 proximal CCAAT box is dispensable for HPFH mutations that create de novo transcription factor binding sites.

a, The γ-globin promoter showing transcription factor binding motifs and mutations analyzed according to designations described for Figure 1a (hg19 – chr11:5,276,085–5,276,215. HPFH variants that create de novo transcription factor binding sites include −198 T>C (KLF1), −113 A>G (GATA1), and −110 A>C (NF-Y, this report). b, %HbF in clones with the indicated mutations, measured after 7 days of erythroid differentiation. Box and whisker plots show minimum, maximum, median, and interquartile ranges. Each dot represents an individual clone (n = 12 per genotype). A two-tailed unpaired t-test indicated no significant effect of the 85Δ mutation on either HPFH variant. c, %HbF in clones with the indicated mutations, analyzed as described for panel b. n = (27) −198C clones, (17) −198C + −186T clones, and (12) −198C + −85Δ clones). Ordinary one-way ANOVA with Tukey’s multiple comparisons test shows the multiplicity adjusted p-values between genotypes. *p = 0.0351; ****p < 0.0001.

Figure 7.. Competition between transcriptional repressors and activators for the γ-globin promoter in HPFH.

a, In adult-stage erythroid cells, ZBTB7A, BCL11A and their associated NuRD repressor complex (not shown) bind their indicated motifs and inhibit the recruitment of transcriptional activators GATA1 and NF-Y via steric effects and/or by establishing epigenetic modifications that inhibit chromatin occupancy (yellow and orange arrows). b, Numerous HPFH mutations disrupt the BCL11A binding motif, leading to GATA1 and NF-Y chromatin occupancy and transcriptional activation. Double arrows indicate that ZBTB7A may still bind its motif to exert a partial inhibitory effect. c, The HPFH variant −110 A>C stabilizes ectopic binding of NF-Y to the distal CCAAT box, which activates transcription by displacing BCL11A and promoting GATA1 occupancy. d, The −113 A>G HPFH variant creates a new GATA1 binding site at the distal CCAAT box. GATA-1 displaces BCL11A to activate transcription, in part by facilitating GATA1 binding to the upstream −189 motif. e, The HPFH variant −198 T>C creates a new binding motif for KLF1, which displaces ZBTB7A and activates GATA1-dependent transcription. BCL11A may still bind its motif, resulting in partial gene silencing, as indicated by the double arrow. Binding of GATA1 to the −189 motif is required for normal fetal γ-globin expression and for all HPFH variants tested. In contrast, NF-Y binding to the proximal CCAAT box is dispensable for transcriptional activation by the −110 A>C, −113 A>G, and −198 T>C HPFH variants (panels c-e).