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Heterochromatin rewiring and domain disruption-mediated chromatin compaction during erythropoiesis

Nature Structural & Molecular Biology volume 30, pages 463–474 (2023)Cite this article

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Abstract

Mammalian erythropoiesis involves progressive chromatin compaction and subsequent enucleation in terminal differentiation, but the mechanisms underlying the three-dimensional chromatin reorganization remain obscure. Here, we systematically analyze the higher-order chromatin in purified populations of primary human erythroblasts. Our results reveal that heterochromatin regions undergo substantial compression, with H3K9me3 markers relocalizing to the nuclear periphery and forming a significant number of long-range interactions, and that ~58% of the topologically associating domain (TAD) boundaries are disrupted, while certain TADs enriched for markers of the active transcription state and erythroid master regulators, GATA1 and KLF1, are selectively maintained during terminal erythropoiesis. Finally, we demonstrate that GATA1 is involved in safeguarding selected essential chromatin domains during terminal erythropoiesis. Our study therefore delineates the molecular characteristics of a development-driven chromatin compaction process, which reveals transcription competence as a key indicator of the selected domain maintenance to ensure appropriate gene expression during the extreme compaction of chromatin.

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Fig. 1: Dynamic chromatin architecture during progressive nuclear condensation of terminal erythropoiesis.
Fig. 2: Chromatin condensation in terminal erythropoiesis is coupled with relocalization and establishment of long-range interactions of heterochromatin.
Fig. 3: A vast number of chromatin domain boundaries are disrupted in terminal erythropoiesis.
Fig. 4: Domain maintenance in the Ortho-E stage is determined by chromatin activity and erythroid gene expression.
Fig. 5: Erythroid master regulators GATA1 and KLF1 are strongly associated with maintained chromatin domains.
Fig. 6: GATA1 is essential in safeguarding selected chromatin domains in terminal erythropoiesis.

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Data availability

The data in this manuscript have been deposited in the Genome Sequence Archive of the National Genomics Data Center. The assigned accession numbers of the submission are HRA001026 and HRA002092. H3K27ac HiChIP data were downloaded from the Gene Expression Omnibus (accession code GSE131055). The public database of the hg19 genome and annotation files (v27lift37) are available from the GENCODE portal (https://www.gencodegenes.org). Source data are provided with this paper. The large source data files are available from https://github.com/LeeSlab/Code-for-Li-et-al.

Code availability

The code is available from https://github.com/LeeSlab/Code-for-Li-et-al.

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Acknowledgements

We thank the Flow Cytometry Core and Imaging Core of the National Center for Protein Sciences at Peking University. We thank the High-Performance Computing Platform of the Center for Life Sciences (Peking University) for supporting data analysis. We are grateful to H. Lyu, F. Wang, Y. Guo and C. Shan for technical support. We thank Z. Wang at the Chinese Academy of Medical Sciences and Peking Union Medical College for helping with the immunofluorescence assay and proofreading the manuscript. We thank X. Ji, Y. Jiang and R. Wang at Peking University for helping with the Hi-C method. We thank C. Li, W. Tao and C. Zhang at Peking University for advice on data analysis. This project was supported by the National Key Research & Development Program of China (2022YFA1103300), the National Natural Science Foundation of China (81970110) and the Peking-Tsinghua Center for Life Sciences and School of Life Sciences at Peking University (to H.-Y.L.). This work was supported by the National Key Research and Development Program (2017YFA0104500) and the Foundation for Innovative Research Groups of the National Natural Science Foundation of China (81621001) (to X.-J.H.).

Author information

Author notes

  1. These authors contributed equally: Dong Li, Fan Wu.

Authors and Affiliations

  1. Ministry of Education Key Laboratory of Cell Proliferation and Differentiation, School of Life Sciences, Peking University, Beijing, China

    Dong Li, Fan Wu, Shuo Zhou & Hsiang-Ying Lee

  2. Peking-Tsinghua Center for Life Sciences, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China

    Dong Li, Fan Wu, Shuo Zhou, Xiao-Jun Huang & Hsiang-Ying Lee

  3. Joint Graduate Program of Peking-Tsinghua-NIBS, School of Life Sciences, Tsinghua University, Beijing, China

    Dong Li

  4. Peking University People’s Hospital, Peking University Institute of Hematology, National Clinical Research Center for Hematologic Disease, Beijing Key Laboratory of Hematopoietic Stem Cell Transplantation, Collaborative Innovation Center of Hematology, Peking University, Beijing, China

    Xiao-Jun Huang & Hsiang-Ying Lee

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Contributions

D.L., F.W., X.-J.H. and H.-Y.L. conceptualized this project and designed the experiments. D.L. and S.Z. performed the experiments. F.W. and D.L. conducted the bioinformatics analyses. D.L., F.W., X.-J.H. and H.-Y.L. wrote the manuscript. All authors discussed the results and commented on the manuscript.

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Correspondence to Hsiang-Ying Lee.

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Nature Structural & Molecular Biology thanks Daria Onichtchouk and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editors: Sara Osman and Dimitris Typas, in collaboration with the Nature Structural & Molecular Biology team.

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Extended data

Extended Data Fig. 1 Purification and validation of human erythroblasts at distinct terminal stages.

a, Fluorescence-activated cell sorting (FACS) scheme for isolating primary human terminal erythroblasts of distinct stages from the ex vivo CD34+ erythroid differentiation system. To avoid the effects of the cell cycle, G0/G1 cells were gated based on the Hoechst 33342 signal. CD235a+ CD71+ CD45low CD36+ CD117high CD105high cells are pro-erythroblasts (Pro-Es), CD235a+ CD71+ CD45low CD36+ CD117dim CD105dim cells are basophilic erythroblasts (Baso-Es), CD235a+ CD71+ CD45low CD36+ CD117− CD105-FSChigh cells are polychromatic erythroblasts (Poly-Es), and CD235a+ CD71+ CD45low CD36+ CD117− CD105− FSClow cells are orthochromatic erythroblasts (Ortho-Es) (n = 3). b, Representative images of purified primary Pro-Es, Baso-Es, Poly-Es and Ortho-Es by benzidine-Giemsa staining (3 independent experiments). c, Pellets of 1 million cells at the indicated stages demonstrating the accumulation of hemoglobin along with erythroid differentiation. d, Nuclear diameter and area of sorted Pro-Es, Baso-Es, Poly-Es and Ortho-Es (n = 20, the median is shown at the center line, and the whisker indicate the min and max value). e, Cell cycle profiles of Pro-Es, Baso-Es, Poly-Es and Ortho-Es without gating for specific cell cycle stages. f, Proliferation assay of FACS-purified Pro-Es, Baso-Es, Poly-Es and Ortho-Es (starting from 1 million cells/sample, mean ± SD, n = 5). g, Expression of CD105 and CD117 (c-Kit) on the cell surface of uncultured (Day 0) and four-day cultured (Day 4) Pro-Es (n = 3). h, Cell size (indicated by forward scatter, FSC) of uncultured (Day 0) and four-day cultured (Day 4) Pro-Es (n = 3). i, Hoechst 33342 staining of uncultured (Day 0) and four-day cultured (Day 4) Pro-Es. The arrow indicates enucleated reticulocytes (3 independent experiments). j, Representative image of four-day cultured (Day 4) Pro-Es by Giemsa staining. (arrows indicate enucleated reticulocytes) (3 independent experiments).

Source data

Extended Data Fig. 2 Overview of chromatin architecture during terminal erythropoiesis.

a, Hi-C contact probability as a function of distance of non-cell-cycle-specific Pro-Es and Ortho-Es. P(s)~S−0.5 indicates the mitotic state, and ~S−1 indicates the fractal globule state. b, Estimation of nucleosomal-repeat length (NRL) based on MNase-seq in Pro-Es and Ortho-Es. c, Compartment analysis in Pro-Es and Ortho-Es based on the PC1 value of the Hi-C assay (AA: compartment A in Pro-Es, remaining compartment A in Ortho-Es; BB: compartment B in Pro-Es, remaining compartment B in Ortho-Es; AB: compartment A in Pro-E, switching to compartment B in Ortho-Es; BA: compartment B in Pro-Es, switching to compartment A in Ortho-Es). d-f, Normalized signal of RNA-seq (d), normalized H3K27ac signal (e) and H3K9me3 signal (f) by the CUT&RUN assay in compartment A (Pro-E: n = 11,794; Ortho-E: n = 13,512) or B (Pro-E: n = 16,632; Ortho-E: n = 14,912) of Pro-Es and Ortho-Es (res = 500kb). TPM, transcripts per kilobase million. Boxplot central line, median; upper and lower limits, 75th and 25th percentiles; whiskers, box height represents interquartile range (IQR) and whiskers are 1.5 × IQR. g, (Top) Formula for quantification of compartment strength; (bottom) compartment strength in Pro-Es and Ortho-Es. h, Schematic description of interchromosomal fraction (ICF). i-k, ICF for chromatin interactions in the whole genome (i), compartment A (j) and compartment B (k) of Pro-Es and Ortho-Es. Boxplot data points represent each 5kb bin in the whole genome (n = 565,693), compartment A (n = 210,215) and compartment B (n = 355,478). Boxplot central line, median; upper and lower limits, 75th and 25th percentiles; whiskers, box height represents interquartile range (IQR) and whiskers are 1.5 × IQR. The p values were calculated by the two-sided Wilcoxon signed-rank test (the exact p values are shown at the top of the plots).

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Extended Data Fig. 3 Characterization of heterochromatin rewiring in terminal erythroid differentiation.

a, (Left) Immunofluorescence images of the heterochromatin marker H3K9me3 (red) in Pro-Es; DNA was counterstained with DAPI (blue). (Right) Signal distribution of H3K9me3 across the corresponding line of cells indicated (n = 21 cells examined over 3 independent experiments). b, (Left) Immunofluorescence images of the heterochromatin marker H3K9me3 (red) in Ortho-Es; DNA was counterstained with DAPI (blue). (Right) Signal distribution of H3K9me3 across the corresponding line of cells indicated (n = 21 cells examined over 3 independent experiments). c, Compartment (PC1 value of Hi-C) and H3K9me3 (CUT&RUN) distribution of Pro-Es and Ortho-Es at Chr2: 0–60 Mb. Red: compartment A; blue: compartment B. d, Immunoblotting of H3K9me3 and H3K9me2 in Pro-Es, Baso-Es, Poly-Es and Ortho-Es (2 independent experiments; densitometry quantifications were performed twice, and the mean values are shown beneath the corresponding bands). e, FISH images using probes of site 2 and site 3 (genomic locations indicated in Fig. 2g.) to demonstrate intercompartment interaction between A (site 3) and B (site 2) at the single-nucleus level in Pro-Es (left) and Ortho-Es (right); Probe 2: red, Probe 3: green (3 independent experiments). f, The overlap percentage of Probe 2 and Probe 3 in Pro-Es and Ortho-Es demonstrates the frequency of A-B intercompartment interactions (n = 50 cells examined over 3 independent experiments).

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Extended Data Fig. 4 Dynamic features of TAD and domain boundary during terminal erythropoiesis.

a, TAD numbers called by different algorithms in Pro-Es and Ortho-Es. G0/G1 samples were used for analysis unless otherwise noted. b, TAD length distribution in Pro-Es and Ortho-Es. c, The average insulation score at TADs and the neighboring regions (±0.5 TAD length) of Pro-Es (cyan, purple) and Ortho-Es (red, green). The analysis is based on merged TADs of Pro-Es and Ortho-Es. d, Average insulation scores of the disrupted, maintained and de novo-formed TAD boundaries in Pro-Es and Ortho-Es. e&f, Volcano plot of differential and constant binding of CTCF (e) and SMC3 (f) between Pro-Es and Ortho-Es. The differentially peaks are called by the diffbind R package. Increased, reduced and stable peaks are plotted as orange, blue and grey dots, respectively (fold change > 2, FDR < = 0.05). g, Immunoblotting of CTCF, SMC3, RAD21, SNF2H, BRG1, CHD4, ACTB and histone H3 in the Pro-E, Baso-E, Poly-E and Ortho-E stages. (Quantification of protein level was normalized based on total protein staining for each sample using the entire lane; quantifications were performed twice, and the mean values are shown beneath the corresponding bands mean). h, Quantification of ATP levels in Pro-E, Baso-E, Poly-E and Ortho-E stages (mean ± SD, n = 4). i, Nucleosomal signal (based on MNase-seq) at the sites with CTCF loss from Pro-Es to Ortho-Es.

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Extended Data Fig. 5 Domain strength dynamics in terminal erythroid development.

a, Heatmaps showing the normalized average interaction frequencies for TADs and the neighboring ±0.5 TAD regions in 10 TAD groups (defined in Methods). The stability of TADs decreased gradually from group 1 through group 10. b, Distribution of CTCF and SMC3 signals by CUT&RUN assay in TADs and the neighboring ±0.5 Mb regions within the stable (Groups 1 & 2) vs. disrupted (Groups 9 & 10) TADs. c, (Left) Top ten GO terms of upregulated genes in TAD regions of Groups 1 & 2; (right) top ten GO terms of downregulated genes in TAD regions of Groups 9 & 10. d, Schematic of FISH probe design demonstrating a promoter-enhancer interaction in a stable TAD region. Promoter (P) probe: GATA1 promoter region (chrX:48,642,195–48,646,409), and enhancer (E) probe (chrX:48,656,933–48,660,281). The H3K27ac-HiChIP track was generated by analyzing the GSE131055 dataset. e, FISH image of the GATA1 promoter probe (green) and enhancer probe (red) demonstrates the interaction between the GATA1 promoter and its enhancer at the single-nucleus level in Pro-Es (left) and Ortho-Es (right) (3 independent experiments). f, The overlap percentage of promoter probe and enhancer probe in Pro-Es and Ortho-Es demonstrates the frequency of E-P interactions (n = 50 cells examined over 3 independent experiments).

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Extended Data Fig. 6 Analysis of chromatin architecture in expelled nuclei and that in Ortho-Es under transcription inhibition.

a, Experimental scheme of transcription inhibition (Ti; DRB + triptolide) in Ortho-Es (n = 2). b, Western blotting of Pol II and GAPDH after 4 hours of Ti treatment in Ortho-Es (n = 2). c, TAD numbers in indicated cell populations and expelled nuclei (mean, n = 2). d, Boxplot showing the TAD boundary strength in Ortho-Es (4 h control or Ti) and nuclei. Boxplot data points represent each boundary of indicated cells or nuclei (4 h control: n = 1,280; 4 h Ti, n = 1,188; nuclei, n = 835). Boxplot central line, median; upper and lower limits, 75th and 25th percentiles; whiskers, box height represents interquartile range (IQR) and whiskers are 1.5 × IQR. The p values were calculated by the two-sided Wilcoxon signed-rank test (the p values are shown in the figure). e, Representative heatmap showing the relatively stable chromatin structure at Chr3: 9–11.5 Mb in Ortho-Es (4 h control or Ti) and expelled nuclei. f, Heatmaps showing the normalized average interaction frequencies for the three types of boundaries (referring to boundaries in Fig. 3a) and their neighboring regions (±0.5 TAD length) in Ortho-Es (4 h control or Ti) and expelled nuclei. g, The ratio of trans (interchromosomal) and cis (intrachromosomal) interactions to total chromatin interactions in Pro-Es, Ortho-Es (0 h, 4 h control or Ti), and nuclei (mean, n = 2). h, Compartment analysis in Ortho-Es (4 h control or Ti) based on the PC1 value of the Hi-C assay. (AA: compartment A in control, remaining compartment A in Ti; BB: compartment B in control, remaining compartment B in Ti; AB: compartment A in control, switching to compartment B in Ti; BA: compartment B in control, switching to compartment A in Ti). i, Boxplot showing the TAD score distribution of TADs in Ortho-Es (4 h control or Ti) and expelled nuclei. TAD regions are defined in the 4 h control stage (n = 1,263). Boxplot central line, median; upper and lower limits, 75th and 25th percentiles; whiskers, box height represents interquartile range (IQR) and whiskers are 1.5 × IQR. The p values were calculated by the two-sided Wilcoxon signed-rank test (the p values are shown in the figure).

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Extended Data Fig. 7 GATA1 and KLF1 motif accessibility and chromatin binding in terminal erythroblasts.

a, Heatmap showing the motif enrichment score based on the ATAC-seq assay. Peaks of differential chromatin accessibility in Pro-Es vs. Ortho-Es were ranked by the fold change and then grouped into ~1500 peaks/bin. (Each bin is shown as a square in the plot.) The bins from left to right were arranged from the chromatin accessible sites that are most enriched in Pro-Es to those that are most enriched in Ortho-Es. A de novo motif search was conducted in the peaks of the same bin. The factors of enriched motifs are listed on the right of the plot. b, GATA1- or KLF1-associated intra-TAD interaction frequency within TAD groups 1~10. Each group has n = 298 TADs. Boxplot central line, median; upper and lower limits, 75th and 25th percentiles; whiskers, box height represents interquartile range (IQR) and whiskers are 1.5 × IQR.

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Extended Data Fig. 8 Functional assays of GATA1 in chromatin domain maintenance.

a, Schematic description of GATA1 and KLF1 knockout strategies using CRISPR/Cas9 in purified primary human Pro-E cells. b, Immunoblotting of GATA1 after knockout by control, KLF1 or GATA1 sgRNA for 48 h in Pro-E. Histone H3 is shown as the loading control (3 independent experiments). c, Genotyping of KLF1 loci after knockout with control or KLF1 sgRNA for 48 h. (The arrow indicates the rejoined DNA after complete fragment removal by paired gRNAs, 3 independent experiments) d, Images of Pro-Es after knockout by control, GATA1 or KLF1 sgRNA for 72 h by benzidine-Giemsa staining (3 independent experiments). e, Cell viability after knockout by control, GATA1 or KLF1 sgRNA for 48 h or 72 h, measured by flow cytometry (percentage of DAPI-negative cells, mean ± SD, n = 3). f&g, (Top) Hi-C contact matrices of Pro-Es after knockout by control or GATA1 sgRNA for 72 h at Chr5: 111.5–113 Mb (f) and Chr6: 134–136 Mb (g). (Bottom) Corresponding CUT&RUN signal of GATA1 in Pro-E and Ortho-E, HiChIP of GATA1 in day 12 terminal erythroblasts, and CUT&RUN tracks of GATA1 in KO-Control and KO-GATA1 samples. Juicebox parameters were balanced and normalized at 5 kb resolution. The dashed triangle shows the disrupted TAD resulting from GATA1 knockout. h & i, Hi-C contact matrices of Pro-Es after knockout by control or KLF1 sgRNA for 72 h at Chr2: 60.5–63 Mb (h) and Chr3: 182–184 Mb (i). Juicebox parameters were balanced and normalized at 5 kb resolution. j, Density maps showing corresponding SMC3 and CTCF CUT&RUN signals at the GATA1 peaks in Pro-Es and Ortho-Es.

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Extended Data Fig. 9 Model of chromatin compaction and domain disruption in terminal erythropoiesis.

During terminal erythroid differentiation, chromatin is multidimensionally rearranged to achieve nuclear compaction to 20–30% of the original volume. a, Heterochromatin reorganizes by establishing long-range interactions and aggregating at the subnuclear membrane region. (NE: nuclear envelope; A: compartment A; B: compartment B). b, A large number of TADs undergo disruption, while selected TADs enriched for transcription competence markers are maintained until the end of erythroid differentiation. c, The early erythroblasts (Pro-Es) contain sufficient energy to sustain the nuclear structure relapse between normal cell cycles; the levels of ATP, CTCF and chromatin remodeling complex (CRC) decrease as terminal differentiation progresses, and therefore, the late erythroblasts (Ortho-Es) can maintain the chromatin architecture only in selected essential regions, which are enriched for transcription activity and/or the binding of critical transcription factors such as GATA1.

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Li, D., Wu, F., Zhou, S. et al. Heterochromatin rewiring and domain disruption-mediated chromatin compaction during erythropoiesis. Nat Struct Mol Biol 30, 463–474 (2023). https://doi.org/10.1038/s41594-023-00939-3

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