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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.

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Fig. 1: HPFH variants in the γ-globin promoter distal CCAAT box facilitate recruitment of GATA1 to an upstream motif.
Fig. 2: The −189 GATA motif facilitates γ-globin gene activation in HPFH.
Fig. 3: Disruption of the −189 GATA motif inhibits HbF expression in primary erythroblasts.
Fig. 4: Distal CCAAT box HPFH variants recruit NF-Y to the γ-globin promoter.
Fig. 5: GATA1 and NF-Y cooperate to activate γ-globin gene expression.
Fig. 6: NF-Y binding to the −85 proximal CCAAT box is dispensable for HPFH mutations that create de novo transcription factor binding sites.
Fig. 7: Competition between transcriptional repressors and activators for the γ-globin promoter in HPFH.

Data availability

Datasets used in this study are listed in Supplementary Table 7. Raw and processed sequencing data generated in this study are available from the NCBI Gene Expression Omnibus under accession GSE152338. Source data are provided with this paper.

Code availability

The code used to perform ATAC–seq (HemTools atac_seq), ChIP–seq (HemTools chip_seq_pair) and Hi-C analyses (hicpro_batch.py) is available at https://github.com/YichaoOU/HemTools/ and at https://doi.org/10.5281/zenodo.4783657. Pipeline documentation is available at https://hemtools.readthedocs.io/en/latest/. The code used to perform motif analysis is available at https://github.com/YichaoOU/HPFH_code and at https://doi.org/10.5281/zenodo.4784805.

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Acknowledgements

We are grateful to A. Impagliazzo for illustration assistance as well as R. Hardison, G. Xiang, M. Osborn, G. Newby and D. Liu for valuable discussion and technical expertise. HUDEP-2 cells were a gift from R. Kurita and Y. Nakamura (RIKEN BioResource Center). ABE7.10 was a gift from M. Osborn (University of Minnesota) and D. Liu (Harvard University/HHMI). APC-conjugated anti-Band3 was a gift from X. An (New York Blood Center). This work was supported by National Institutes of Health (NIH) grants P01HL053749 (to S.M.P.-M. and M.J.W.), R01HL156647 (to M.J.W.), R35GM133614 (to Y.C.), R24106766 (to Y.C and M.J.W.) and F32DK118822 (to P.A.D.); The Assisi Foundation of Memphis (to M.J.W.); Doris Duke Charitable Foundation grant 2017093 (to M.J.W.); an Australian Government Research Training Program Scholarship (to H.W.B.); the Australian National Health and Medical Research Council grant APP1164920 (to M.C.); The St. Jude Collaborative Research Consortium for Sickle Cell Disease and St. Jude/ALSAC. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. We thank the members of the St. Jude Children’s Research Hospital Cytogenetics, Hartwell Center, Center for Advanced Genome Engineering and Flow Cytometry core facilities. The St. Jude Cytogenetics, Hartwell Center, Center for Advanced Genome Engineering and Flow Cytometry Shared Resource Laboratories are supported by NIH grant P30CA21765 and by St. Jude/ALSAC.

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Contributions

P.A.D., R.F., S.N.P., H.W.B., M.C., S.M.P.-M., Y.C. and M.J.W designed experiments. P.A.D., R.F. and H.W.B. performed experiments and analyzed data. P.A.D., S.N.P., Y.L. and L.E.P. performed sequencing and associated analyses. M.C., S.M.P.-M. and Y.C. provided conceptual advice and technical expertise. M.J.W. supervised the study. P.A.D. and M.J.W. wrote the manuscript. All authors discussed the results and assisted in the preparation of the manuscript.

Corresponding author

Correspondence to Mitchell J. Weiss.

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Competing interests

M.J.W. is on advisory boards for Cellarity, Graphite Bio, Novartis and Forma Therapeutics, and is an equity owner of Beam Therapeutics. All other authors declare no competing interests.

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Peer review information Nature Genetics thanks Sjaak Philipsen and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

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-2Δεγδβ/GγΑγ 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-2Δεγδβ, and HUDEP-2Δεγδβ/GγΑγ cells using a 117.8 kb probe for chr11 (green, shown in panel a) and a 5.2 kb probe from the deleted region between HBG1 and HBG2 (HBG; red, shown in panel b) in wild-type HUDEP-2, HUDEP-2Δεγδβ, and HUDEP-2Δεγδβ/GγΑγ cells. Results are from a single experiment. Scale bar = 5 μm.

Extended Data Fig. 2 Characterization of the HUDEP-2Δεγδβ/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-2Δεγδβ/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-2Δεγδβ/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.

Source data

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-2Δεγδβ/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-2Δεγδβ/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-2Δεγδβ/GγAγ cells. The intersection represents the fraction of shared boundaries between the HUDEP-2 and HUDEP-2Δεγδβ/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-2Δεγδβ/GγAγ and HUDEP-2Δεγδβ/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 proerythroblasts79. 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 data80.

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-2Δεγδβ/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-2Δεγδβ/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.

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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.

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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.

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Doerfler, P.A., Feng, R., Li, Y. et al. Activation of γ-globin gene expression by GATA1 and NF-Y in hereditary persistence of fetal hemoglobin. Nat Genet 53, 1177–1186 (2021). https://doi.org/10.1038/s41588-021-00904-0

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