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EBF1-deficient bone marrow stroma elicits persistent changes in HSC potential

Abstract

Crosstalk between mesenchymal stromal cells (MSCs) and hematopoietic stem cells (HSCs) is essential for hematopoietic homeostasis and lineage output. Here, we investigate how transcriptional changes in bone marrow (BM) MSCs result in long-lasting effects on HSCs. Single-cell analysis of Cxcl12-abundant reticular (CAR) cells and PDGFRα+Sca1+ (PαS) cells revealed an extensive cellular heterogeneity but uniform expression of the transcription factor gene Ebf1. Conditional deletion of Ebf1 in these MSCs altered their cellular composition, chromatin structure and gene expression profiles, including the reduced expression of adhesion-related genes. Functionally, the stromal-specific Ebf1 inactivation results in impaired adhesion of HSCs, leading to reduced quiescence and diminished myeloid output. Most notably, HSCs residing in the Ebf1-deficient niche underwent changes in their cellular composition and chromatin structure that persist in serial transplantations. Thus, genetic alterations in the BM niche lead to long-term functional changes of HSCs.

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Fig. 1: Ebf1-deficient MSCs show impaired function.
Fig. 2: Reduced numbers of HSPCs and myeloid cells in mice with a Prx1Cre-mediated deletion of Ebf1 in the BM microenvironment.
Fig. 3: Decreased numbers of HSPCs in animals with LepRCre and tamoxifen-inducible NG2Cre-ER-mediated deletion of Ebf1 in stromal cells.
Fig. 4: Transcriptome changes in CAR and PαS cells upon Ebf1 deletion.
Fig. 5: Transplantation of HSCs from Ebf1-deficient niche to wild-type recipients does not reverse reduced myeloid output.
Fig. 6: Niche-dependent changes in HSC chromatin accessibility and transcriptome.
Fig. 7: Niche-dependent changes in HSC composition.

Data availability

The data that support the findings of this study are available from the corresponding authors upon request. Source data for Extended Data Fig. 1d are provided with the paper. All high-throughput data in this study were deposited at the Gene Expression Omnibus (GEO) under series GSE127970 (CAR, PαS RNA-seq and ATAC-seq), series GSE128089 (HSC RNA-seq and ATAC-seq), and series GSE128743 (single-cell RNA-seq). A web application for scRNA-seq data visualization is available at https://hematopoietic-niche-regulation-by-ebf1.ie-freiburg.mpg.de.

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Acknowledgements

We are grateful to E. Trompouki for critical reading of the manuscript and M. Rott for assistance with the manuscript preparation. We thank S. Muellerke, I. Falk, K. Schlegel, D. Komandin and F. Ludin for technical assistance and members of the R. Grosschedl laboratory for discussions. We thank the Deep Sequencing, Imaging, FACS and Animal facilities of the Max Planck Insitute of Immunobiology and Epigenetics. This work was supported by the funds from the Max Planck Society and German Research Foundation. D. Grün was supported by the Max Planck Society, the German Research Foundation (DFG) (GR4980/1-1, GR4980/3-1, and GRK2344 MeInBio) and by the DFG under Germany’s Excellence Strategy (CIBSS-EXC-2189 Project ID 390939984).

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Contributions

M.D. designed and performed experiments. S.R. and P.C. performed bioinformatics analysis. J.S.H. performed and analyzed single-cell RNA-seq experiments and created a shiny web application. D.G. supervised J.S.H. E.L. performed tail vein injections. R.G. conceived and supervised the study. R.G. and M.D. wrote the manuscript with input from all authors.

Corresponding authors

Correspondence to Marta Derecka or Rudolf Grosschedl.

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The authors declare no competing interests.

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Peer review information L. A. Dempsey was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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

Extended data

Extended Data Fig. 1 Ebf1 deficiency alters MSC composition and gene expression.

a, Representative gating strategy used to identify and isolate CAR and PaS cells. b, Heat map depicting log2-transformed normalized expression of signature genes (rows) across the cells in each cluster (columns). Key genes are indicated on the right. Color code of clusters corresponds to Fig.1a. All clusters were compared in a pairwise fashion against each other and differentially expressed genes (adjusted P-value<0.05) appearing in at least 3 comparisons. Data represent 912 single-sorted wild-type cells from 3 independent experiments (n=4 mice). Negative binomial distributions reflecting the gene expression variability within each subgroup were inferred, based on a background model for the expected transcript count variability, a P-value for the observed difference in transcript counts between the two subgroups is computed as described for DESeq2. The P-values were corrected for multiple testing via the Benjamini–Hochberg method. c, t-SNE maps showing normalized expression of Ptprc, Rag1 and J chain in sequences CAR (CD45CD31LinPDGFRα+Sca1) and PαS (CD45CD31LinPDGFRα+Sca1+) cells. d, Representative immunoblot analysis of EBF1 protein level in total cells extracts from 1x104 sorted cells (n=2 independent experiments). Actin was used as a loading control. EBF1 band is marked with an arrow. e, t-SNE map showing clusters of cells with similar transcriptomes within CAR and PαS populations, derived by RaceID3 algorithm. Data represent 1777 single-sorted wild-type and knockout cells from 3 independent experiments (n=4 mice per genotype). f, t-SNE map showing distribution of CAR and PαS cells from Ebf1+/+Prx1Cre mice (black) and from Ebf1fl/flPrx1Cre mice (blue) within established clusters. g, MA plot showing differential gene expression between Ebf1+/+Prx1Cre and Ebf1fl/flPrx1Cre cells from scRNA-seq analysis. Upregulated genes are shown in red and downregulated genes are shown in blue. Source data.

Extended Data Fig. 2 Ebf1 deficiency alters MSC in vitro differentiation.

Integrated analysis of the scRNA-seq dataset from CAR and PαS cells from Ebf1+/+Prx1Cre and Ebf1fl/flPrx1Cre mice with data published by Tikhonova et al. and Baryawno et al. a, UMAP plot depicting cells collored by used technology CEL-Seq2 (blue) utilized by Derecka et al. study or 10x (red) utlized by Tikhonova et al. study. b, UMAP plot depicting cells collored by used technology CEL-Seq2 (blue) utilized by Derecka et al. study or 10x (red) utlized by Baryawno et al. study. c, Percentage of Annexin V positive CAR and PαS cells in the BM. Data represent the mean ± s.e.m, for 3 independent experiments, n=13 Ebf1+/+Prx1Cre mice and n=12 Ebf1fl/flPrx1Cre mice. Percentage of CAR and PαS cells within each cell cycle phase. Data represent the mean ± s.e.m, for 3 independent experiments, n=11 (CAR) and n=14 (PαS) Ebf1+/+Prx1Cre mice and n=11 (CAR) and n=12 (PαS) Ebf1fl/flPrx1Cre mice (*P=0.02 for G0, *P=0.04 for G1, *P=0.01 for s/G2/M). d, Histomorphometric analysis of tibiae from n=12 Ebf1+/+Prx1Cre and n=12 Ebf1fl/flPrx1Cre mice showing bone volume as a percentage of tissue volume (BV/TV) in the trabecular area of the bone (*P=0.01) and osteoblast surface as a percentage of bone surface (Ob.S/BS) in the trabecular (*P=0.03) and endocortical area of the bone. e, Quantification of CFU-F colonies from sorted CAR and PaS cells. Data are presented as mean ± s.e.m for 3 independent experiments, n=9 for Ebf1+/+Prx1Cre and n=10 for Ebf fl/flPrx1Cre mice (*p=0.03, ***p=0.0003). f, Representative images showing multilineage differetiation of CFU-F colonies from PαS cells from Ebf1+/+Prx1Cre and Ebf1fl/flPrx1Cre mice (n=3 independent experiments). Adipocytes were stained with Oild Red O, osteoblasts with Alizarin Red S and chondrocytes with Toluidine Blue. g, Relative mRNA expression levels of mesenchymal lineage-specific markers from PαS-derived CFU-F colonies sub-cloned into secondary cultures and subjected to adipogenic, osteogenic and chondrogenic in vitro differentiation. Data are represented as mean fold change ± s.e.m over Ebf1+/+Prx1Cre cells set as 1, for 3 independent experiments, n=26 clones from Ebf1+/+Prx1Cre mice and n=35 clones from Ebf1fl/flPrx1Cre mice (Adipogenic differentiation); n=32 clones for each genotype (Osteogenic fifferentiation); n=38 clones from Ebf1+/+Prx1Cre mice and n=48 clones from Ebf1fl/flPrx1Cre mice (Chondrogenic differentiation). Statistical analysis in c, d, e, g was performed using two-tailed unpaired Student’s t-test.

Extended Data Fig. 3 Prx1Cre mediated deletion of Ebf1.

a, Total BM cellularity. Data represent the mean ± s.e.m, n=22 Ebf1+/+Prx1Cre mice and n=20 Ebf1fl/flPrx1Cre mice. b, Quantification of CFU colonies from the spleen and peripheral blood. Data represent the mean ± s.e.m, for 3 independent experiments, n=12 Ebf1+/+Prx1Cre mice and n=10 Ebf1fl/flPrx1Cre mice. SP-spleen, PB-peripheral blood. c, Frequency of neutrophils (CD11b+CD11cLy6G+), monocytes (CD11b+CD11cLy6Chi) and macrophages (MHC-II+CD11b+F4/80+) in the BM. Data represent the mean ± s.e.m, n=10 Ebf1+/+Prx1Cre mice and n=12 Ebf1fl/flPrx1Cre mice (*P=0.03, **P=0.001). d, Absolute numbers of B cells (CD19+B220+) with n=14 mice per group, T cells (CD3+) with n=16 Ebf1+/+Prx1Cre mice and n=15 Ebf1fl/flPrx1Cre mice and erythroid cells (Ter119+) with n=15 Ebf1+/+Prx1Cre mice and n=14 Ebf1fl/flPrx1Cre mice, in the BM. e, Quantification of early B cell subsets: common lymphoid progenitors-CLP (LinSca1loc-kitloIL-7Rα+CD135+) with n=10 mice per group, pro-B cells (B220+CD43+), pre-B cells (B220loCD43) and rec-B cells (B220hiCD43) with n=14 mice per group, in the BM. Statistical analysis in a-e was performed using two-tailed unpaired Student’s t-test.

Extended Data Fig. 4 LepRCre and NG2Cre-ERTM dependent deletion of Ebf1.

a, Total BM cellularity. Data represent the mean ± s.e.m, n=22 Ebf1+/+LepRCre mice and n=20 Ebf1fl/flLepRCre mice. b, Absolute numbers of MPP3 (CD135CD48+CD150LSK) and MPP4 (CD135+ CD150LSK) in the BM. Data represent the mean ± s.e.m, n=17 Ebf1+/+LepRCre mice and n=20 Ebf1fl/flLepRCre mice (*P=0.02). c, Numbers of CLPs (LinSca1loc-kitloIL-7Rα+CD135+) in the BM. Data represent the mean ± s.e.m, n=12 Ebf1+/+LepRCre mice and n=13 Ebf1fl/flLepRCre mice (*P=0.03). d, Frequency of neutrophils (CD11b+CD11cLy6G+) (*P=0.03), monocytes (CD11b+CD11cLy6Chi) and macrophages (MHC-II+CD11b+F4/80+) (*P=0.01) in the BM. Data represent the mean ± s.e.m, n=8 mice per group. e, Absolute numbers of T cells (CD3+) and erythroid cells (Ter119+) in the BM. Data represent the mean ± s.e.m, n=22 (T cells) and n=19 (erythroid) Ebf1+/+LepRCre mice and n=19 Ebf1fl/flLepRCre mice. f, Numbers of B lineage cells (CD19+B220+) with n=13 mice per group, including pro-B cells (B220+CD43+), pre-B cells (B220loCD43) and rec-B cells (B220hiCD43) with n=12 mice per grop, in the BM of Ebf1fl/flLepRCre and Ebf1+/+LepRCre mice. Data represent the mean ± s.e.m (**p=0.03). g, Total BM cellularity. Data represent the mean ± s.e.m, n=16 Ebf1+/+ NG2Cre-ERTM mice and n=17 Ebf1fl/flNG2Cre-ERTM mice. h, Absolute numbers of MPP3 (CD135CD48+CD150LSK) and MPP4 (CD135+ CD150LSK) in the BM. Data represent the mean ± s.e.m, n=16 mice per group. I, Numbers of B cells (CD19+B220+), T cells (CD3+) and erythroid cells (Ter119+) in the BM of Ebf1fl/flNG2Cre-ERTM and Ebf1+/+NG2Cre-ERTM mice. Data represent the mean ± s.e.m, n=12 mice per group. Statistical analysis in a-i was performed using two-tailed unpaired Student’s t-test.

Extended Data Fig. 5 Chromatin accessibility in CAR and PαS cells from Ebf1+/+ and Ebf1–/– mice.

a, Frequency and absolute number of HSCs in the spleens of Ebf1+/+Prx1Cre and Ebf1fl/flPrx1Cre mice after G-CSF treatment. G-CSF was administered subcutaneously once a day for six consecutive days. Mice were analyzed on day 7 from the start of G-CSF treatment. Data represent the mean ± s.e.m, for 3 independent experiments, n=7 mice per group (*p=0.03, two-tailed unpaired Student’s t-test). b, Venn diagram showing the numbers of identified ATAC peaks in CAR and PαS cells from Ebf1+/+ and Ebf1/ mice. c, Percentage of ATAC peaks in different genomic regions in CAR and PαS cells. d, Motifs identified in clusters presented in Fig.4g. e, Venn diagrams comparing differentially expressed genes with differential ATAC peaks containing EBF motif in CAR and PαS cells from Ebf1+/+ and Ebf1/ mice. Upregulated genes are in red and downregulated genes are in green. All genes marked by black circle in WT only cluster (blue box) show loss of ATAC signal within EBF motif in Ebf1/ (KO) cells. Genes marked by black circle in common cluster show loss of EBF1-specific footprint in Ebf1-/- cells. f, Digital genomic footprinting analysis showing average normalized Tn5 insertion profiles around EBF footprinted motifs in merged ATAC peaks. Insertions on the forward and reverse strands are indicated in red and blue, respectively.

Extended Data Fig. 6. Transplantation of HSCs from Ebf1-deficient niche to wild-type recipients.

a, Percent of chimerism in peripheral blood (PB) during the course of experiment. Percentage of CD45.2+ donor-derived cells in PB within myeloid cells (Mac1+) b, B cells (CD19+) c, and T cells (CD3+) d over time. Data represent the mean ± s.e.m with n=18 recipients of HSCs from Ebf1+/+Prx1Cre mice and n=18 recipients of HSCs from Ebf1fl/flPrx1Cre mice in the primary bone marrow transplantation and n=20 recipients of HSCs from Ebf1+/+Prx1Cre mice and n=29 recipients of HSCs from Ebf1fl/flPrx1Cre mice in the secondary bone marrow transplantation.

Extended Data Fig. 7 Niche-dependent changes in HSCs chromatin accessibility and transcriptome.

a, ATAC signal ±3 Kb around the center of the peak. b, Motifs identified in clusters with lost and gained accessibility in HSCs from Ebf1+/+Prx1Cre and Ebf1fl/flPrx1Cre mice. c, Heatmaps showing footprinted motif co-occurrence enrichment clustering. d, GSEA plot showing enrichments of myeloid cells differentiation signature in HSCs derived from wild-type niche. The Normalized Enrichment Score (NES) was computed using a Kolmogorov–Smirnov statistic. Significance was assessed using a two-tailed t-test comparing to a null distribution computed via 1000 gene-set permutations. Correction for multiple testing was performed using FDR adjustment, n=183. e, Representative tracks showing differential merged ATAC signal in HSCs from Ebf1+/+Prx1Cre and Ebf1fl/flPrx1Cre mice correlated with genes differentially expressed between cluster 2 and 3 (Fig 6e). Region on chromosome 8 with Pcm1 and Asah1 serves as a control without differential ATAC signal. The scale in the y-axis represents RPKM.

Extended Data Fig. 8 Niche-dependent changes in HSCs composition.

a, t-SNE maps highlighting normalized expression of Mki67 in sequenced HSCs. b, Representative tracks showing differential merged ATAC signal in HSCs from Ebf1+/+Prx1Cre and Ebf1fl/flPrx1Cre mice correlated with genes differentially expressed between cluster 2 and 3 (Fig. 7e). Region on chromosome 1 with Fbxo36 and Cab39 serves as a control without differential ATAC signal. The scale in the y-axis represents RPKM. c, t-SNE map showing clusters of cells with similar transcriptomes within LSK (LinSca-1+c-kit+) populations, derived by RaceID3 algorithm. Data represent 2844 single-sorted wild-type and knockout cells from 2 independent experiments (n=5 mice for Ebf1+/+Prx1Cre group and n=6 mice for Ebf1fl/flPrx1Cre group). d, t-SNE map displaying clusters with statistically significant enrichment of wild-type cells (cluster 6) or Ebf1-deficient cells (cluster 3 and 10) within LSK population. Data represent 2844 single-sorted wild-type and knockout cells from 2 independent experiments (n=5 mice for Ebf1+/+Prx1Cre group and n=6 mice for Ebf1fl/flPrx1Cre group). Fisher’s exact test was used for statistics (exact P values and cell numbers for each cluster are provided in Supplementary Table 5). e, t-SNE maps showing normalized expression of Mpo in sequenced LSKs. f, t-SNE maps showing normalized expression of Ctsg in sequenced LSKs. g, MA plot showing differential gene expression between cluster 10 and cluster 11 from scRNA-seq analysis of LSKs. Genes upregulated in cluster 11 are shown in red and downregulated genes are shown in blue.

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Derecka, M., Herman, J.S., Cauchy, P. et al. EBF1-deficient bone marrow stroma elicits persistent changes in HSC potential. Nat Immunol 21, 261–273 (2020). https://doi.org/10.1038/s41590-020-0595-7

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