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ZNF410 represses fetal globin by singular control of CHD4

Nature Genetics volume 53, pages 719–728 (2021)Cite this article

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Abstract

Known fetal hemoglobin (HbF) silencers have potential on-target liabilities for rational β-hemoglobinopathy therapeutic inhibition. Here, through transcription factor (TF) CRISPR screening, we identify zinc-finger protein (ZNF) 410 as an HbF repressor. ZNF410 does not bind directly to the genes encoding γ-globins, but rather its chromatin occupancy is concentrated solely at CHD4, encoding the NuRD nucleosome remodeler, which is itself required for HbF repression. CHD4 has two ZNF410-bound regulatory elements with 27 combined ZNF410 binding motifs constituting unparalleled genomic clusters. These elements completely account for the effects of ZNF410 on fetal globin repression. Knockout of ZNF410 or its mouse homolog Zfp410 reduces CHD4 levels by 60%, enough to substantially de-repress HbF while eluding cellular or organismal toxicity. These studies suggest a potential target for HbF induction for β-hemoglobin disorders with a wide therapeutic index. More broadly, ZNF410 represents a special class of gene regulator, a conserved TF with singular devotion to regulation of a chromatin subcomplex.

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Fig. 1: ZNF410 is an HbF repressor.
Fig. 2: ZNF410 chromatin occupancy is restricted to two CHD4 elements with densely clustered motifs.
Fig. 3: ZNF410 represses HbF by activating CHD4.
Fig. 4: Zfp410-deficient mice are viable with unremarkable hematology.
Fig. 5: ZNF410-deficient human HSPCs de-repress HbF and retain repopulation potential.

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Data availability

The datasets generated during the current study are available from the indicated repositories when applicable or are included in this article. Datasets generated during the current study are available as follows. (1) RNA-seq of HUDEP-2 cells and CHD4 Δ6.7 kb HUDEP-2 cells edited at ZNF410 were deposited in NCBI’s Gene Expression Omnibus (GEO) and are accessible through GEO series accession number GSE166222. (2) CUT&RUN data for genomic ZNF410 chromatin occupancy are accessible through GEO series accession number GSE166221. (3) ATAC-seq data for HUDEP-2 cells are accessible through GEO series accession number GSE167298. Publicly available datasets referenced in this manuscript are available as follows. (1) Erythroid expression profiling datasets are available under accession numbers GSE53983, GSE54602, GSE22552 and E-MTAB-1035. (2) RNA-seq of HUDEP-2 cells edited at CHD4 is available from the NCBI SRA portal under accession number PRJNA496556, https://www.ncbi.nlm.nih.gov/sra. (3) ZNF410 and CHD4 expression values (TPM) across 54 human tissues were obtained from the GTEx Portal (https://gtexportal.org/home/). (4) Gene dependency scores for 558 cell lines were obtained from the Achilles Avana 20Q2 Public CERES dataset of the DepMap portal74. (5) MEL DNase sequencing data were obtained from the ENCODE project (dataset, ENCSR000CNN; file, ENCFF990ATO). Source data are provided with this paper.

Code availability

The scripts used for analysis of CUT&RUN experiments and motif mapping are provided in the Supplementary Methods. The motif-counting script is available at https://github.com/yao-qiuming/Vinjamur_NG2021.

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Acknowledgements

We thank R. Kurita and Y. Nakamura for sharing HUDEP-2 cells (Department of Research and Development, Central Blood Institute, Blood Service Headquarters, Japanese Red Cross Society and Cell Engineering Division, RIKEN BioResource Research Center, Faculty of Medicine, University of Tsukuba); R. Mathieu and the HSCI-BCH Flow Cytometry Facility, supported by the Harvard Stem Cell Institute and the NIH (U54DK110805) for assistance with flow cytometry; Z. Herbert from the Molecular Biology Core Facilities at the Dana-Farber Cancer Institute for assistance with sequencing; Y. Fujiwara and M. Nguyen from the BCH Mouse Embryonic Stem Cell and Gene Targeting Core (supported by the NIH, NIDDK Center of Excellence in Molecular Hematology (U54DK110805)) for assistance with transgenic mouse generation; J. Doench for assistance with CRISPR screening; S. Henikoff for sharing protein A–MNase for CUT&RUN experiments; J. Bonanno for technical assistance; and S. Orkin, C. Brendel, N. Liu, D. Seruggia, N. Dharia and members of the Bauer laboratory for helpful discussions. D.S.V. was supported by the Cooley’s Anemia Foundation Research Fellowship award; L.P. was supported by the NHGRI (R00HG008399 and R35HG010717); D.E.B. was supported in part by a Sponsored Research Agreement from Sanofi, NHLBI (DP2HL137300 and P01HL032262) and the Burroughs Wellcome Fund.

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Authors and Affiliations

  1. Division of Hematology/Oncology, Boston Children’s Hospital, Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Stem Cell Institute, Broad Institute, Department of Pediatrics, Harvard Medical School, Boston, MA, USA

    Divya S. Vinjamur, Qiuming Yao, Mitchel A. Cole, Connor McGuckin, Chunyan Ren, Jing Zeng, Mir Hossain & Daniel E. Bauer

  2. Molecular Pathology Unit, Center for Cancer Research, Massachusetts General Hospital, Department of Pathology, Harvard Medical School, Boston, MA, USA

    Qiuming Yao & Luca Pinello

  3. Department of Molecular, Cell and Cancer Biology, Li Weibo Institute for Rare Diseases Research, University of Massachusetts Medical School, Worcester, MA, USA

    Kevin Luk & Scot A. Wolfe

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  1. Divya S. Vinjamur
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Contributions

D.E.B. conceived the study. D.E.B., D.S.V. and M.H. designed experiments. D.S.V., C.M., J.Z. and M.H. performed experiments. Q.Y., M.A.C. and L.P. designed computational analyses. D.S.V., Q.Y. and M.A.C. performed data analysis. C.R., K.L. and S.A.W. provided the 3× NLS-Cas9 protein. D.E.B., D.S.V. and Q.Y. wrote the manuscript with input from all authors.

Corresponding author

Correspondence to Daniel E. Bauer.

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Competing interests

D.E.B. and D.S.V. are co-inventors on a patent related to ZNF410 disruption. The authors declare no other competing interests.

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Peer review information Nature Genetics thanks Swee Lay Thein and the other, anonymous, reviewers for their contribution to the peer review of this work.

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

Extended data

Extended Data Fig. 1 ZNF410 targeted HUDEP-2 cells display normal cell growth and elevated γ-globin expression.

HUDEP-2 cells were edited at ZNF410 or AAVS1 (control) using sgRNA:Cas9 RNP electroporation. a, Efficient editing was achieved with all five ZNF410 targeting gRNAs (n = 1 for each gRNA). Cells were cultured in EDM2 for 8 days and (b) cell count and (c) viability data were recorded on alternate days for five individual gRNAs targeting ZNF410 (sg 1-5, n = 3 for sg 1, n = 1 for sg 2-5) in comparison to mock (n = 3) and AAVS1 (control, n = 3) targeted cells. Data are presented as mean values and error bars are standard deviation. d, Robust γ-globin induction was obtained with all five ZNF410 targeting gRNAs (n = 1 for each gRNA) in comparison to mock (n = 1) and AAVS1 (control, n = 1) targeted cells. e, HUDEP-2/Cas9 cells nontransduced (mock, n = 3) or transduced with ZNF410 targeting sgRNA (n = 4) assayed on day 9 of erythroid differentiation with RT-qPCR for HBE1 (p = 0.1426, ns), HBB (p = 0.0353) and HBA (p = 0.0122) expression. All values are relative to Catalase expression (endogenous control) and expressed as fold change relative to mock for each biological replicate. Data are presented as mean values and error bars are standard deviation. Statistically significant differences were determined using paired Student’s t-test comparing ZNF410 targeted cells to mock. f, Three HUDEP-2 ZNF410 biallelic KO clones were generated using paired genomic cleavages that delete the entire coding sequence. Clones were generated in two successive steps. In the first step a HUDEP-2 clone heterozygous for ZNF410 deletion was isolated. The ZNF410 null allele in this clone is designated Null cl. allele 1 and its sequence is shown on the left. In the second step this heterozygous ZNF410 deletion clone was retargeted and three biallelic ZNF410 null clones were isolated with the sequences of the second ZNF410 null allele in each clone shown on the right of the figure.

Extended Data Fig. 2 ZNF410 targeted primary erythroblasts display elevated fetal hemoglobin expression in normal and sickle cell patient derived donor cells.

a, Immunoblot showing protein expression of ZNF410, CHD4, and members of the HbF repressive NuRD subcomplex - GATAD2A, MTA2, MBD2, HDAC2 and RBBP4 - in ZNF410 targeted primary erythroblasts on Day 11 of erythroid culture. GAPDH included as loading control. b, Growth curve of ZNF410 targeted primary erythroblasts (n = 3) compared to mock (n = 3) and safe control (n = 3) targeted cells over 18 days of erythroid differentiation culture. Data are presented as mean values and error bars are standard deviation. c, HBG1/2 (p < 0.05), HBE1 (ns), HBB (p < 0.01) and HBA1/2 (ns) globin gene expression measured by RT-qPCR in ZNF410 targeted (n = 3) compared to Mock (n = 3) and safe control targeted (n = 3) primary erythroblasts. Catalase was used as the endogenous normalization control. Values are displayed relative to the mean of mock samples. Statistical tests compare ZNF410 targeted and mock samples, ns not significant. Data are presented as mean values and error bars are standard deviation. d, ZNF410 targeted (n = 1) by RNP electroporation of Cas9 and sgRNA in CD34 + HSPCs from a sickle cell disease patient and subsequently differentiated to erythroid cells in vitro. At the end of erythroid culture (day 18), HbF level was measured by hemoglobin HPLC and compared to mock (n = 1) and safe control (n = 1) targeted cells.

Source data

Extended Data Fig. 3 Absent ZNF410 chromatin occupancy.

a-c, ɑ-like and -like globin gene clusters and GALNT18 intron 1 with a cluster of 6 ZNF410 motifs indicating absence of ZNF410 occupancy in representative CUT&RUN control IgG (n = 9) and anti-HA (n = 7) in HUDEP-2 cells over-expressing HA-tagged ZNF410, control IgG (n = 1) and anti-ZNF410 (n = 1) in HUDEP-2 cells, and control IgG (n = 2) and anti-ZNF410 (n = 2) in CD34 + HSPC derived erythroid precursors. Positions of ZNF410 motifs (red rectangles), accessible chromatin by representative ATAC-seq in HUDEP-2 cells (gray peaks, n = 3) and DNA sequence conservation by SiPhy rate.

Extended Data Fig. 4 ZNF410 chromatin occupancy.

a, The third most enriched peak for ZNF410 binding (following CHD4 promoter and -6 kb enhancer) by CUT&RUN with anti-HA antibody in HUDEP-2 cells over-expressing ZNF410-HA was at TIMELESS intron 1. Representative CUT&RUN control IgG (n = 9) and anti-HA (n = 7) in HUDEP-2 cells over-expressing HA-tagged ZNF410, control IgG (n = 1) and anti-ZNF410 (n = 1) in HUDEP-2 cells, and control IgG (n = 2) and anti-ZNF410 (n = 2) in CD34 + HSPC derived erythroid precursors. Positions of ZNF410 motifs (red rectangles), accessible chromatin by representative ATAC-seq in HUDEP-2 cells (gray peaks, n = 3), DNA sequence conservation by SiPhy rate, and repetitive elements from RepeatMasker. b, A total of 5 peaks were identified by CUT&RUN with anti-ZNF410 antibody in HUDEP-2 cells. The top 4 peaks were at the CHD4 promoter or -6 kb enhancer, the fifth was at DPY19L3 intron 5. c, A total of 5 peaks were identified by CUT&RUN with anti-ZNF410 antibody in CD34 + HSPC derived erythroid precursors. All 5 peaks were at the CHD4 promoter or -6 kb enhancer. d, Peak of ZNF410 occupancy at DPY19L3 intron 5 in HUDEP-2 cells.

Extended Data Fig. 5 ZNF410 represses HbF by activating CHD4.

a, Comparison of genes downregulated in ZNF410 and CHD4 mutant cells by GSEA (b) LC3-I/II and GAPDH (control) immunoblot in unedited parental and ZNF410 null HUDEP-2 cells (left panel) and mock and ZNF410 targeted primary erythroblasts (c) Correlation of ZNF410 and CHD4 expression across 54 human tissues from GTEx (Pearson r = 0.77, p < 0.0001) (d) CHD4 expression in ZNF410 targeted (n = 3) compared to mock (n = 1) and AAVS1 (n = 1) targeted control HUDEP-2 cells. Data are mean values, error bars are standard deviation. e, Cas9 paired cleavages with CHD4-proximal-gRNA-1 and CHD4-distal-gRNA-1 (CHD4 Δ 6.7 kb) or CHD4-proximal-gRNA-1 and CHD4-distal-gRNA-2 (CHD4 Δ 6.9 kb) were used to generate HUDEP-2 clones with biallelic deletions spanning both of the ZNF410 binding regions upstream of CHD4. Positions of ZNF410 motifs (red rectangles) and accessible chromatin by ATAC-seq (gray peaks) (f) CHD4 expression in CHD4 Δ 6.9 kb clones (n = 3) compared to HUDEP-2 cells (n = 1) (left panel) and HbF level measured by hemoglobin HPLC in CHD4 Δ 6.7 kb (n = 1) and Δ 6.9 kb (#2 and #3, n = 2) clones compared to HUDEP-2 cells (n = 1) (right panel). g, CHD4 Δ 6.9 kb clones and HUDEP-2 cells were subjected to AAVS1 (negative control), ZNF410 and ZBTB7A targeting using RNP electroporation of 3X-NLS-Cas9 and sgRNA. Left panel, editing efficiency measured by indel frequency in HUDEP-2 cells (n = 1) and CHD4 Δ 6.9 kb clones (n = 3) targeted with ZNF410 or ZBTB7A sgRNAs. The shaded portion of the bar represents the percentage of indels resulting in frameshift (fs) alleles. The white portion of the bar represents in-frame indels. Right panel, HBG expression relative to total β-like globin (HBG + HBB) in HUDEP-2 cells (n = 1) and CHD4 Δ 6.9 kb clones (n = 3) targeted with AAVS1 (negative control), ZNF410 or ZBTB7A sgRNAs. h, HBG expression relative to total β-like globin (HBG + HBB) in CHD4 Δ 6.9 kb clone 3 (n = 1) subjected to ZNF410, BCL11A and ZBTB7A targeting using RNP electroporation of 3xNLS-Cas9 and sgRNA compared to mock (n = 1) cells. i, CBX6 expression in mock, AAVS1 and ZNF410 targeted HUDEP-2 cells (n = 2 for mock and control, n = 3 for ZNF410 targeted) and CHD4 Δ 6.7 kb HUDEP-2 cells (n = 3 for mock, control and ZNF410 targeted). Catalase was used as the endogenous normalization control. CBX6 expression in targeted cells is shown relative to expression in mock cells. Data are mean values, error bars are standard deviation.

Source data

Extended Data Fig. 6 Zfp410 is the conserved mouse ortholog of ZNF410.

a, CUT&RUN performed in mouse erythroleukemia (MEL) cells using anti-Zfp410 antibody (n = 3) and IgG control (n = 3). The third most enriched Zfp410 peak (following Chd4 promoter and Chd4 -6 kb enhancer) was at the Hist1h2bl promoter. No Zfp410 motifs were identified at this locus, which overlaps accessible chromatin (DNase-seq, gray peaks). b, Diagram of the Zfp410 gene trap allele. A targeting cassette including splice acceptor site upstream of LacZ was inserted into Zfp410 intron 5 thus disrupting full-length expression. Schema obtained along with mouse ES cells from EuMMCR, Germany. c, Exon and domain structure of mouse Zfp410. d, Mouse embryonic (βh1 and εy) and adult β-major/minor globin gene expression measured by RT-qPCR in Zfp410 Gt/Gt (n = 5) mouse E14.5 fetal liver erythroid cells compared to heterozygous (n = 4) and wildtype (n = 5) control animals. e, Weight was measured at indicated time points over the course of 15 weeks for wildtype male (+/+ (M), n = 1), Zfp410 heterozygous male (+/Gt (M), n = 2), Zfp410 homozygous male (Gt/Gt (M), n = 2), Zfp410 heterozygous female (+/Gt (F), n = 5) and Zfp410 homozygous female (Gt/Gt (F), n = 1) mice. Data are presented as mean values and error bars are standard deviation. f, Peripheral blood hematological parameters for wildtype (n = 1), Zfp410 + /Gt (n = 7) and Zfp410 Gt/Gt (n = 3) mice, with normal ranges for hemoglobin, mean corpuscular volume (MCV), reticulocyte, white blood cell (WBC), neutrophil and platelet count shown by dotted lines.

Extended Data Fig. 7 ZNF410 is dispensable for hematopoietic repopulation and erythropoiesis.

CD34 + HSPCs from donor 3 were edited by RNP electroporation targeting ZNF410, BCL11A or ZBTB7A and infused to NBSGW mice or subject to in vitro erythroid differentiation. a, Indel frequency at ZNF410, BCL11A and ZBTB7A was quantified in input cells 4 days after electroporation, and in engrafted total or sorted cells at bone marrow (BM) harvest. The percentage of frameshift alleles is represented in gray and the percentage of in-frame alleles is represented in white. b, Comparison of engraftment assessed by human CD45 + staining compared to total CD45 + cells in xenografts of ZNF410 (n = 4), BCL11A (n = 3) and ZBTB7A (n = 3) edited and mock control (n = 4) CD34 + HSPCs. Each symbol represents one mouse. c,d, Erythroid maturation, evaluated based on CD71 and CD235a immunophenotype and enucleation frequency, was assessed on day 18 of in vitro erythroid culture in safe control (n = 4), ZNF410 (n = 2), BCL11A (n = 2) and ZBTB7A (n = 2) targeted primary erythroblasts.

Extended Data Fig. 8 Flow cytometry gating strategy for xenograft experiment.

Hierarchy of FACS gates and representative plots for each gate are shown for a representative control (mock) transplanted bone marrow sample. The first gate was plotted to delineate the cell population of interest (POI) and avoid debris. The second and third gates were plotted to exclude doublets. Values in plots are for respective gates.

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Source Data Extended Data Fig. 2

Unprocessed western blots.

Source Data Extended Data Fig. 5

Unprocessed western blots.

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Vinjamur, D.S., Yao, Q., Cole, M.A. et al. ZNF410 represses fetal globin by singular control of CHD4. Nat Genet 53, 719–728 (2021). https://doi.org/10.1038/s41588-021-00843-w

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