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Transcription factor competition at the γ-globin promoters controls hemoglobin switching

Nature Genetics volume 53, pages 511–520 (2021)Cite this article

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An Author Correction to this article was published on 17 March 2021

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Abstract

BCL11A, the major regulator of fetal hemoglobin (HbF, α2γ2) level, represses γ-globin expression through direct promoter binding in adult erythroid cells in a switch to adult hemoglobin (HbA, α2β2). To uncover how BCL11A initiates repression, we used CRISPR–Cas9, dCas9, dCas9-KRAB and dCas9-VP64 screens to dissect the γ-globin promoters and identified an activator element near the BCL11A-binding site. Using CUT&RUN and base editing, we demonstrate that a proximal CCAAT box is occupied by the activator NF-Y. BCL11A competes with NF-Y binding through steric hindrance to initiate repression. Occupancy of NF-Y is rapidly established following BCL11A depletion, and precedes γ-globin derepression and locus control region (LCR)–globin loop formation. Our findings reveal that the switch from fetal to adult globin gene expression within the >50-kb β-globin gene cluster is initiated by competition between a stage-selective repressor and a ubiquitous activating factor within a remarkably discrete region of the γ-globin promoters.

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Fig. 1: dCas9 dense perturbation reveals an activator element at the γ-globin promoters.
Fig. 2: NF-Y activates γ-globin through direct binding to the proximal CCAAT.
Fig. 3: Base editing of the NF-Y motif reduces γ-globin expression.
Fig. 4: NF-Y rapidly activates γ-globin after acute depletion of BCL11A.
Fig. 5: NF-Y binding is affected by steric hindrance at the γ-globin promoters.
Fig. 6: A simplified model for hemoglobin switching.

Data availability

All raw and processed CRISPR screen, CUT&RUN, ChIP–seq, PRO-seq and ATAC-seq data have been deposited in the NCBI Gene Expression Omnibus under accession number GSE150530. All unprocessed immunoblot gels for Fig. 4a and Extended Data Figs. 2a, 4a,c and 5f can be found in Source data provided with this paper.

Code availability

We made use of publically available software for processing high-throughput sequencing raw data. For single-locus CUT&RUN footprinting, the code can be found at https://bitbucket.org/qzhudfci/cutruntools/src/master/. Code for deconvolution of CRISPR screen data can be found at https://github.com/pinellolab. Custom codes used in this study can be found at https://github.com/yao-qiuming/Nan_NG2020.

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Acknowledgements

We thank S. Henikoff at the Fred Hutchinson Cancer Research Center for pA-MNase, T. Muir at Princeton University Department of Chemistry for split-intein cDNA and B. Huang at UCSF Department of Pharmaceutical Chemistry for split-mNG2 cDNA. We thank Y. Nakamura at the Cell Engineering Division of RIKEN BioResource Center for HUDEP-1 and HUDEP-2 cell lines. We thank Z. Herbert, M. Berkeley and A. Caruso at the Molecular Biology Core Facilities for high-throughput DNA sequencing, S. Goldman at the Nascent Transcriptomics Core for generation of PRO-seq libraries and performing analyses, and all members at the Hematologic Neoplasia Flow Cytometry core for sorting cells. We also thank M. Cole, M. Canver and C. Smith for critical input and experimental contributions, A. Bowker for technical assistance and members of the Orkin, Bauer and Yuan laboratories for input. D.E.B. was supported by the Burroughs Wellcome Fund and NHLBI (nos. DP2HL137300 and P01HL032262). S.H.O. is an Investigator of the Howard Hughes Medical Institute, supported by both NHLBI (nos. R01HL032259 and P01HL032262) and the Doris Duke Charitable Foundation. G.-C.Y. was supported by NHGRI (no. R01HG009663). L.P. was supported by NHGRI Genomic Innovator Award (no. R35HG010717). N.L. was supported by NIDDK (no. K99DK120925).

Author information

Author notes

  1. Guo-Cheng Yuan

    Present address: Department of Genetics and Genomic Medicine, Icahn School of Medicine at Mount Sinai, New York, NY, USA

  2. These authors contributed equally: Nan Liu, Shuqian Xu.

Authors and Affiliations

  1. Cancer and Blood Disorders Center, Dana-Farber Cancer Institute and Boston Children’s Hospital, Harvard Medical School, Boston, MA, USA

    Nan Liu, Shuqian Xu, Qiuming Yao, Phraew Sakon, Daniel E. Bauer & Stuart H. Orkin

  2. Department of Hematology, Qilu Hospital, Cheeloo College of Medicine, Shandong University, Jinan, China

    Shuqian Xu

  3. Molecular Pathology Unit & Center for Cancer Research, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA

    Qiuming Yao, Jonathan Y. Hsu & Luca Pinello

  4. Department of Pediatric Oncology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA, USA

    Qian Zhu, Yan Kai & Guo-Cheng Yuan

  5. Howard Hughes Medical Institute, Boston, MA, USA

    Stuart H. Orkin

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Contributions

N.L. and S.H.O. conceived the study. N.L. designed and performed all experiments, except those detailed below. D.E.B. conceived the Cas9 and dCas9 dense perturbation screens. S.X. performed the screens. Q.Y. analyzed the screen data. J.Y.H. and L.P. performed CRISPR–SURF deconvolution. S.X., Q.Y. and N.L. performed dCas9 disruption validation. Q.Y. and N.L. analyzed PRO-seq data. N.L., Q.Z. and Y.K. analyzed CUT&RUN data under the supervision of G.-C.Y. P.S. assisted with immunoblotting and RT–qPCR. N.L. and S.H.O. wrote the manuscript with input from all authors.

Corresponding author

Correspondence to Stuart H. Orkin.

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The authors declare no competing interests.

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Peer review information Nature Genetics thanks Emery Bresnick, Douglas Higgs and Sjaak Philipsen for their contribution to the peer review of this work. Peer reviewer reports are available.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Dense perturbation of the β-globin locus.

a, Flow chart of the dense perturbation experiment design. b, Zoomed in view of the dense perturbation results at HBB and HBD genes. gRNAs that target the exons are enriched in Cas9 experiment. HbF raw score is enrichment of individual gRNAs in HbF-high compared to unsorted population at end of erythroid maturation, plotted as log2 fold change. HbF score shows deconvoluted underlying genomic regulatory signal with corresponding p-values shown on -log10 scale. c, Zoomed in view of the dense perturbation results at HBG1 gene. Not that gRNAs that target the exons are depleted in Cas9 experiment. d, Zoomed in view of the dCas9 dense perturbation result at HS3 of the LCR aligned to PhastCons46way scores. The four regions highlighted in green contain GATA1 or GATA1-TAL1 composite motifs (CTG[N8-9]GATA), with the sequences shown below. e, RT-qPCR showing that dCas9/sgRNA binding at -115 of γ-globin promoters reduced γ-globin expression in HUDEP-2 cells. Note that the γ-globin is only expressed at a basal level in cells expressing AAVS1 control sgRNA. The result is shown as mean (SD) of three technical replicates. Statistical tests of the beta coefficients were performed empirically through bootstrapping and two-tailed tests. Multiple hypothesis testing was accounted for with the Benjamini-Hochberg (BH) procedure.

Extended Data Fig. 2 NFYA binds to γ-globin promoters and is required for LCR-γ-globin interaction.

a, Left, western blot gel showing validation of NFYA knockdown efficiency (cropped). All three shRNAs tested showed efficient depletion of NFYA. Right, validation of NFYA knockdown efficiency at mRNA level using RT-qPCR. shRNA3 exhibited efficient knockdown of NFYA mRNA in all three cells tested and was used thereafter. The result is shown as mean (SD) of two technical replicates. b, ChIP-seq tracks of NFYA in HUDEP-2, HUDEP-1, BCL11A KO HUDEP-2 cells with or without NFYA knockdown. c, ChIP-qPCR validation of NFYA binding at the γ-globin promoters in HUDEP-1 and BCL11A KO HUDEP-2 cells. No strong binding was detected in HUDEP-2 cells which does not express γ-globin. The result is shown as mean (SD) of three technical replicates. d, Chromosome Conformation Capture qPCR in BCL11A KO HUDEP-2 cells with or without NFYA knockdown. EcoRI fragment encompassing HS2-4 of the LCR was used as anchor point to evaluate LCR-globin interaction. The result is shown as mean (SD) of three technical replicates.

Source data

Extended Data Fig. 3 NF-Y binds to the proximal CCAAT in the γ-globin promoters.

a, Upper panel, heatmap comparison of NFYA ChIP-seq in HUDEP-2 cells, NFYA CUT&RUN in primary human CD34+ derived erythroid cells with or without NFYA knockdown. Lower panel, comparing the signal of the above three experiments at a representative genomic region. b, Venn diagram showing the overlap between NFYA CUT&RUN and ChIP-seq peaks. c, Motif analysis from 5000 random peaks of NFYA CUT&RUN identifies CCAAT as the highest ranked motif. E-value is reported by MEME. d, Zoomed in view of BCL11A CUT&RUN in HUDEP-2 and NFYA CUT&RUN in BCL11A KO HUDEP-2 cells at the γ-globin promoters. Distal (−118 to −113) indicates the distal TGACCA motif that BCL11A binds, and proximal (−88 to −84) indicates the proximal CCAAT motif. e, Single locus footprint of NF-Y at the CCNB1 promoter (upper) and CDK1 promoter (lower). Both CCAAT motifs show strong NF-Y footprints in the two promoters. f, Single locus footprint of NF-Y at the γ-globin promoters in HUDEP-1 (upper), BCL11A KO adult CD34+ derived erythroid cells (middle) and cord blood CD34+ derived erythroid cells. Only the proximal motif shows NF-Y footprint.

Extended Data Fig. 4 Base editing of the BCL11A and NF-Y motif.

a, Left, split-intein mediated ligation of Cas9NG-Intein-N and Intein-C-AID, producing full-length Target-AID-NG. Blue arrow indicates the ligation sites. Right, immunoblot validating the expression of each component and the ligation products. The ligation is incomplete, but the level of ligated product is much higher than the original vector (cropped). b, NFYA binding at the γ-promoters diminished in all the NF-Y motif-edited clones (red), and increased in all the BCL11A motif-edited clones (orange), as revealed by NFYA CUT&RUN. NF-Y motif editing was carried out in BCL11A KO HUDEP-2 cells while BCL11A motif editing was carried out in wild-type HUDEP-2 cells. c, Upper, RT-qPCR analysis of γ-globin expression after acute depletion of C/EBPβ, C/EBPγ, CDP, NFIA and NFIC. Lower, immunoblot validating protein depletion (cropped). BCL11A KO HUDEP-2 cells were differentiated for 3 days after nucleofection. The result is shown as mean (SD) of three technical replicates. d, Flow cytometry analysis of HbF levels for BCL11A base-edited clones at day 7 and 10. Longer editing resulted in higher base editing rate (Fig. 3e) and higher percentage of HbF positive cells. e, A control base editing experiment in which a nucleotide 9 bp away from the BCL11A motif was edited. Sanger sequencing confirmed C-T conversion. f, Left, FACS of BCL11A motif base-edited bulk cells into high and low HbF populations. The C-T conversion rate of BCL11A motif in each population was measured by Sanger sequencing and quantified with TIDER. HbF high cells show 87% conversion and HbF low cells show only 7.4% conversion. g, Left, flow cytometry analysis of HbF level in individual clones derived from BCL11A motif base editing. Data is showed as mean (SD) of multiple independent clones. Nonedit: n = 23, base edited: n = 30. Right, gating strategy. h, Single locus footprint of NF-Y at the γ-promoters in clone A9d, a BCL11A motif-edited clone.

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Extended Data Fig. 5 Acute depletion of BCL11A leads to rapid binding of NF-Y.

a, Schematic diagram of primary human CD34+ differentiation and acute depletion of BCL11A using CRISPR/Cas9. b, Pairwise correlation of PRO-seq experiments. All the experiments in each time point showed high degree of correlation, indicating very minor transcriptional fluctuation upon BCL11A depletion. c, Average PRO-seq signal at -200 to +600 bp relative to TSS exhibited promoter pausing of PolII. d, Quantification of PRO-seq reads on HBG1/2 and HBB genes after 32 or 72 hrs of BCL11A acute depletion. The y-axis shows Reads Per Million (RPM) for HBG1+HBG2 or HBB. The result is shown as mean (SD) of two biologically independent samples (independent cell cultures and CRISPR KO). e, CUT&RUN of TBP in CD34+ cells undergoing erythroid differentiation after 32 or 72 hrs of BCL11A acute depletion. The result shown is representative of two biological replicates. Quantification of KO/Ctrl and the corresponding p-values are reported by MAnorm. f, Western blot for BCL11A and NFYA in adult primary human CD34+ derived erythroid cells upon KO of NFYA, BCL11A or both (cropped). g, RT-qPCR analysis of γ-globin expression in adult primary human CD34+ derived erythroid cells upon KO of NFYA, BCL11A or both. Knockout of NFYA after 72 hours decreases γ-globin expression. The result is shown as mean (SD) of three technical replicates. h, Chromosome Conformation Capture qPCR in adult primary human CD34+ derived erythroid cells, comparing BCL11A KO and BCL11A/NF-Y double KO. EcoRI fragment encompassing HS2-4 of the LCR was used as anchor point to evaluate LCR-globin interaction. The result is shown as mean (SD) of three technical replicates.

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Unprocessed immunoblots.

Source Data Extended Data Fig. 2

Unprocessed immunoblots.

Source Data Extended Data Fig. 4

Unprocessed immunoblots.

Source Data Extended Data Fig. 5

Unprocessed immunoblots.

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Liu, N., Xu, S., Yao, Q. et al. Transcription factor competition at the γ-globin promoters controls hemoglobin switching. Nat Genet 53, 511–520 (2021). https://doi.org/10.1038/s41588-021-00798-y

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