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A Myc enhancer cluster regulates normal and leukaemic haematopoietic stem cell hierarchies

A Correction to this article was published on 16 May 2018

This article has been updated

Abstract

The transcription factor Myc is essential for the regulation of haematopoietic stem cells and progenitors and has a critical function in haematopoietic malignancies1. Here we show that an evolutionarily conserved region located 1.7 megabases downstream of the Myc gene that has previously been labelled as a ‘super-enhancer’2 is essential for the regulation of Myc expression levels in both normal haematopoietic and leukaemic stem cell hierarchies in mice and humans. Deletion of this region in mice leads to a complete loss of Myc expression in haematopoietic stem cells and progenitors. This caused an accumulation of differentiation-arrested multipotent progenitors and loss of myeloid and B cells, mimicking the phenotype caused by Mx1-Cre-mediated conditional deletion of the Myc gene in haematopoietic stem cells3. This super-enhancer comprises multiple enhancer modules with selective activity that recruits a compendium of transcription factors, including GFI1b, RUNX1 and MYB. Analysis of mice carrying deletions of individual enhancer modules suggests that specific Myc expression levels throughout most of the haematopoietic hierarchy are controlled by the combinatorial and additive activity of individual enhancer modules, which collectively function as a ‘blood enhancer cluster’ (BENC). We show that BENC is also essential for the maintenance of MLL–AF9-driven leukaemia in mice. Furthermore, a BENC module, which controls Myc expression in mouse haematopoietic stem cells and progenitors, shows increased chromatin accessibility in human acute myeloid leukaemia stem cells compared to blasts. This difference correlates with MYC expression and patient outcome. We propose that clusters of enhancers, such as BENC, form highly combinatorial systems that allow precise control of gene expression across normal cellular hierarchies and which also can be hijacked in malignancies.

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Figure 1: The BENC enhancer region (15–17), located 1.7 Mb downstream of the Myc locus, is essential for HSC function.
Figure 2: BENC (15–17) directs Myc expression to haematopoietic cells and its deletion closely mimics Mx-Cre-mediated conditional deletion of the Myc gene.
Figure 3: BENC is composed of lineage-specific enhancer modules and the deletion of these modules leads to cell-type-specific downregulation of Myc expression.
Figure 4: BENC is required for maintenance of MLL–AF9 leukaemia in mice and accessibility to module C in human LSCs is linked to patient survival.

Change history

  • 16 May 2018

    In the originally published version of this Letter, the citation after 'TAMERE' in the Methods section should have been to a new reference (Herault et al., 1998; now ref. 49) rather than to ref. 43. This has been corrected online.

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Acknowledgements

We thank members of the Spitz and Trumpp laboratories and colleagues for sharing reagents and helpful comments; A. Przybylla, M. Sohn and M. Neubauer for technical assistance; L. Alfaro and W. Zhang for sample preparation for RNA sequencing; M. Milsom for critical reading of the manuscript; R. Grosschedl and S. Boller for help with B cell development analysis; and the DKFZ Flow Cytometry Core facility and the EMBL and DKFZ Laboratory Animal Resource Facilities. Support was provided by PhD fellowships to V.V.U. (Jeff Schell Darwin Trust); M.P. (EMBL international PhD program), C.B. (Helmholtz International Graduate School for Cancer Research) and post-doctoral fellowship to S.R. (EMBL (EIPOD) under Marie Curie Actions COFUND). The J.E.D. and M.L. laboratories were supported in part by the Medicine by Design program (Toronto University), the Ontario Institute for Cancer Research, Cancer Stem Cell Consortium (OGI-047), the Canadian Institutes of Health Research and the CIHR-Japan Epigenetics in Stem Cells Program, Canadian Cancer Society, Terry Fox Foundation, and a Canada Research Chair to J.E.D. The A.T. laboratory was supported by the SFB 873 and FOR 2674 (Deutsche Forschungsgemeinschaft), the SyTASC consortium (Deutsche Krebshilfe) and the Dietmar Hopp Foundation.

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Authors

Contributions

C.B., L.v.P., V.V.U., A.T. and F.S. designed experiments. C.B., L.v.P., V.V.U., S.R., M.P., K.L., R.S., P.Z. and A.S.B. performed experiments related to mouse data. I.A. contributed to ChIP–seq analysis. A.M. and S.W.N. analysed RNA sequencing data, ATAC-seq and microarray data of patients with AML with conceptual input from C.B., M.L., J.E.D. and A.T. N.T. performed xenograft experiments. P.W.Z., M.L. and J.E.D. led and supervised the human studies. C.B., L.v.P., V.V.U., A.T. and F.S. analysed all other data. A.T. supervised and coordinated all haematopoiesis and leukaemia work as well as gene expression analyses. F.S. supervised the genetic strategies and gene regulation analyses. C.B., L.v.P, F.S. and A.T. wrote the manuscript.

Corresponding authors

Correspondence to Andreas Trumpp or François Spitz.

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Reviewer Information Nature thanks B. Amati and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Figure 1 Chromatin profiles at the Myc locus in different mouse tissues predict position of promoters (H3K4me3), enhancers (H3K27ac) and transcribed genes (H3K36me3).

On the basis of H3K4me3 ChIP–seq data, only the Myc and Pvt1 promoters showed activity in bone marrow cells and in the CH12 lymphoma cell line at the Myc locus. Consistent with the active transcription of these genes, the Myc and Pvt1 gene bodies were marked by H3K36me3. Several strong H3K27ac peaks are specifically present in the BENC region in haematopoietic tissues or derived cell lines (bone marrow, bone-marrow derived macrophages (BMDM), embryonic day (E)14.5 fetal liver (but not in adult liver)), but not in other non-blood-related samples, including brown adipose tissue (BAT), mouse embryonic stem cell line E14 (ES-E14), mouse embryonic fibroblasts (MEF), olfactory bulb (Olfact) and small intestine (Smint). Other putative enhancers, centromeric to Myc or overlapping with Pvt1 are also indicated. Data are from GEO accession number GSE29184 and ref. 6.

Extended Data Figure 2 Enhancer activity of the BENC region located 1.7 Mb downstream of the Myc gene in haematopoietic stem and progenitor cells.

a, Top, schematic representation of the Myc locus on chromosome 15 within its topologically associating domain4 (TAD; brown bar). The position of genes (arrows), predicted promoters (H3K4me3, blue boxes) and enhancers (H3K4me1, H3K27ac, other boxes) are shown. Bottom, representation of the different mouse transposon insertions (top) and Cre-mediated deletions (bottom, red bars) used to identify enhancer regions5. bk, LacZ activity measured by FDG staining in bone marrow derived from LacZ-reporter mice described in a using flow cytometry. b, Representative flow cytometry histograms of the percentage of FDG+ cells (± s.d., vertical) and the geometric mean fluorescence intensity (MFI ± s.e.m.) of HSCs, LSK cells, myeloid cells and lymphoid cells from wild-type, heterozygous 17a and Δ15–17 mice. Data are derived from two independent experiments. c, LacZ activity in HSCs and MPP1–4 cells isolated from wild-type (Non-tg), heterozygous 3a, 17a and Δ15–17 mice and shown as geometric mean fluorescence intensity (MFI ± s.e.m.) values. d, e, LacZ activity in HSCs and MPP1–4 cells from wild-type (Non-tg), heterozygous 3a, 17a and Δ15–17 mice shown as geometric mean fluorescence intensity (MFI ± s.e.m.). fh, Representative histograms showing lacZ activity in HSC and MPP (f), progenitor (g) and differentiated cell (h) populations of bone marrow from wild-type (Non-tg) as well as heterozygous 17a and 14b mice. EB, erythroblast. Data are mean percentage of FDG+ cells (± s.d.) from two independent experiments. ik, LacZ activity in HSPCs (LSK), myeloid committed progenitor (LSK) and differentiated cell (Lin+) populations of heterozygous mice carrying the indicated insertions or deletions measured by FDG staining. Data are geometric mean fluorescence intensity (MFI ± s.e.m.) and mean percentage of FDG+ cells ( ± s.d., vertical); representative data from two independent experiments are shown. The sample size is as follows: n = 3 mice per group.

Source data

Extended Data Figure 3 The enhancer region (15–17) is critical for HSC function and interacts with the Myc promoter.

ad, Comparison of control and homozygous MycΔ15–17/Δ15–17 mice. Body weight (a), bone marrow cellularity normalized to body weight (b), number of LSK cells (c) and representative flow cytometry profiles using indicated markers for differentiated cell populations (d) are shown. e, f, Transplantation of homozygous MycΔ15–17/Δ15–17 bone marrow cells in a competitive setting. Representative flow cytometry profiles showing the peripheral blood (PB) chimerism of transplanted CD45.2+ (either homozygous MycΔ15–17/Δ15–17 or control) cells as indicated (e) and of HSC and MPP1–4 cells derived from the bone marrow of competitively transplanted mice 16 weeks after transplantation (f). g, h, Transplantation of LSK combined with LSK cells derived from homozygous MycΔ15–17/Δ15–17 or controls into T-cell-deficient NSG mice. Distribution of CD4- and CD8-expressing mature T cell populations (g) and thymic progenitors (h) derived from transplanted homozygous MycΔ15–17/Δ15–17 or control cells. i, j, Physical proximity between Myc and BENC revealed by DNA FISH in HSPCs. i, Schematic representation of the locus, including the position of the three BACs used in DNA FISH and their relative distances. j, Two-dimensional projection images and three-dimensional reconstruction of double-staining DNA FISH for the BACs indicated for representative nuclei of LSK cells. k, Three-dimensional distance measurement of the BACs are in micrometre scale. The box plot shows the 3rd quartile, median and the 1st quartile. The whiskers of the box plot extend to the data points less than 1.5× the interquartile range from the 1st and the 3rd quartile. The number of measurements for each double staining (n) is indicated. Sample sizes are as follows: n = 5 mice per group from two independent experiments (ac); n = 3 mice per group from one experiment (g, h); n = 62 cells (397P6_22L9), n = 55 (22L9_207P4), n = 34 cells (397P6_207P4) from one experiment (k). P values shown are from an unpaired two-tailed t-test.

Source data

Extended Data Figure 4 The MycΔ15–17 deletion is allelic to the MycΔORF deletion and the bone marrow phenotype of MycΔ15–17 mice closely mimics Mx-Cre-mediated conditional deletion of Myc.

a, Number of colonies obtained in colony-forming assays using bone marrow cells from the indicated genotypes. Mean values (± s.e.m.) of two biological replicates (dots) with technical duplicates are shown. b, Pictures of representative dishes (left) and colonies (right) of one experiment with two biological duplicates and technical duplicates. c, Total cell numbers of differentiated cell types present in different compound heterozygous mice isolated from legs, hips and spine. d, Relative Myc expression in haematopoietic and non-haematopoietic tissues obtained from homozygous MycΔ15–17/Δ15–17 and control mice. Data are mean ± s.e.m. of three mice. e, Relative Mycn mRNA expression in haematopoietic bone marrow cell populations derived from indicated mutant mice. All data are mean ± s.e.m. fn, Comparison of adult mice with poly(I:C)-induced Mx-Cre-mediated deletion of the Myc gene with ones carrying a homozygous MycΔ15–17/Δ15–17 allele. Bone marrow cellularity normalized to body weight (f), number of LSK cells (g), number of committed progenitor populations (h), number of differentiated cells (i), thymus cellularity (j), number of thymic mature T cells and progenitors (k) and thymic double negative (DN) populations (l), MEPs and erythroid progenitors (m) is shown. Bone marrow cell numbers refer to cells isolated from legs, hips and spine. n, Representative flow cytometry profiles of bone marrow cells derived from mouse mutants indicated on the left and gated as indicated at the top stained with indicated cell surface markers. All data are mean ± s.e.m. Sample sizes are as follows: n = 2 mice per group analysed each in technical duplicates (a); n = 4 mice per group from one experiment (c); n = 3 mice per group from one experiment (d); MycWT/flox (n = 7 mice for all populations, except MPP1 (n = 6 mice), MEP (n = 4 mice), CD8+ T (n = 6 mice), megakaryocytes (Mgk) (n = 6 mice)); MycΔORF/WT (n = 6 mice for all populations, except HSCs (n = 5 mice), CMP (n = 5 mice), GMP (n = 3 mice), MEP (n = 2 mice), CLP (n = 3 mice), RBC (n = 5 mice)); MycΔ15–17/flox (n = 6 mice for all populations, except MPP1 (n = 5 mice), MPP3 (n = 5 mice), MEP (n = 5 mice), RBC (n = 5 mice), megakaryocytes (n = 5)); MycΔ15–17/ΔORF (n = 8 mice for all populations, except MPP1 (n = 7 mice), MPP3 (n = 7 mice), GMP (n = 5 mice), CLP (n = 7 mice), CD8+ T (n = 7 mice)), data from two independent experiments (e); n = 17 mice for Mycflox/flox and MycΔMx, n = 6 mice for control, n = 12 mice for MycΔ15–17/Δ15–17, from two independent experiments (f, g); n = 8 mice for Mycflox/flox and MycΔMx, n = 4 mice for control, n = 7 mice for MycΔ15–17/Δ15–17, from two independent experiments (h); n = 17 mice for Mycflox/flox and MycΔMx, n = 6 mice for control and n = 11 mice for MycΔ15–17/Δ15–17, from two independent experiments (i); n = 9 mice for Mycflox/flox, n = 8 mice for MycΔMx, n = 5 mice for control and n = 6 mice for MycΔ15–17/Δ15–17 from one experiment (j); n = 7 mice for Mycflox/flox, n = 8 mice for MycΔMx, n = 5 mice for control and n = 6 mice for MycΔ15–17/Δ15–17, from two independent experiments (k, l); n = 7 (MEP) and n = 17 (all other populations) mice for Mycflox/flox and MycΔMx, n = 4 (MEP) and n = 6 mice (all other populations) for control, n = 7 (MEP) and n = 11 (all other populations) mice for MycΔ15–17/Δ15–17, from two independent experiments (m); two independent experiments, except for the thymus (one experiment) (n). P values are from unpaired two-tailed t-test.

Source data

Extended Data Figure 5 BENC is a multi-modular enhancer and recruits haematopoietic transcription factors to its constituents in a cell-type-specific manner.

a, Overview of ATAC-seq profiles from various thymic progenitor populations in the Myc locus (data from ImmGen repository48). The Notch-responsive enhancer described in ref. 9 is highlighted in yellow and BENC in grey. b, Top, overview of the Myc locus and adjacent regions including BENC. The ATAC-seq profile of LSK cells and H3K27ac profiles for various haematopoietic cell populations are shown. BENC is clearly marked in a cell-type-specific manner by H3K27ac and the chromatin accessible in LSK cells as measured by ATAC-seq. Bottom, same graphic as shown in Fig. 3a. ATAC-seq and H3K27ac profiles of various haematopoietic cell types. Both the accessibility and deposition of H3K27ac change in a cell-type-specific manner. c, Genomic coordinates of BENC modules in the mouse genome (genome assembly mm9). d, Top, ATAC-seq profiles reveal an open chromatin configuration at the Myc promoter in all blood cells tested, whereas chromatin accessibility at the different BENC modules is much more dynamic. For example, ATAC peaks in LSK cells were present at the C, D and G modules, whereas MEPs showed a double peak at module I and natural killer (NK) cells showed a double peak at module D. Bottom, chromatin immunoprecipitation followed by sequencing (ChIP–seq) profiles of transcription factors aligned to the Myc promoter and BENC modules A–I. Several important haematopoietic transcriptional regulators are not detected at the Myc promoter or only bind faintly to it (MEIS1, FLI1, PU.1, RUNX1, SCL (also known as TAL1), MYB), whereas they bind strongly to BENC modules in a differential manner. The ChIP–seq data were extracted from different sources outlined in Extended Data Table 1. e, ChIP–seq profiles for GATA1, -2, -3 in different haematopoietic cells (see Extended Data Table 1 for sources). Ery-P, erythrocyte progenitors; MPP, multipotent progenitor; HPC, haematopoietic progenitor cell. f, ChIP–seq profiles of PU.1 showing preferential occupancy at the A and B modules but weak signals at the other BENC modules.

Extended Data Figure 6 Consequences of deletion of individual BENC modules for bone marrow populations and during B cell development.

a, Copy of Fig. 3c with exact P values from unpaired two-tailed t-tests included in the heat map tiles. b, Changes in cell numbers of various bone marrow haematopoietic cell populations in MycΔmodule/Δ15–17 mice. Data are shown as log2-transformed mean values of the ratio of MycΔmodule/Δ15–17 mice to the respective controls. c, Myc expression in HSCs in homozygous MycΔ15–17/Δ15–17 and MycΔmodule/Δ15–17 mice. d, Flow cytometric gating strategy used to quantify PreProB (B220+CD24CD43+), ProB PreB (B220+CD24+CD43IgMIgD), transitional B (B220+CD24+CD43IgM+IgD), and mature B (B220+CD24+CD43IgD+) cells. e, f, Representative expression profiles of homozygous MycΔ15–17/Δ15–17 (e) and MycΔD/Δ15–17 (f) mice analysed by flow cytometry. Data show a reduction in the fraction of B220+ cells as well as an accumulation of PreProB cells in both mutants. g, h, Cell frequencies and Myc expression during early B cell development in MycΔmodule/Δ15–17 mice showing effects of module A–B, C, G–H and I deletion. g, Quantification of PrePro B, ProB PreB, transitional B and mature B cells in mice with the indicated BENC module deletions (MycΔmodule/Δ15–17) shown as the frequencies of B220+ cells. h, Relative Myc mRNA levels in early B cell developmental stages in mice with BENC module deletions. Data are mean ± s.e.m. i, ATAC-seq profiles of BENC in CLPs and B cell progenitors obtained from the Immgen48 repository. Sample sizes are as follows. a, See sample sizes for Fig. 3c in the Methods. b, MycΔ15–17/WT control for MycΔ15–17/Δ15–17 mice (n = 4 mice for all other populations, except HSC, MPP1, MPP3, MEP (n = 3 mice), MycΔ15–17/Δ15–17 mice (n = 3 mice for all populations), MycΔ15–17/WT control for MycΔ15–17/ΔA–B mice (n = 10 mice), MycΔ15–17/ΔA–B mice (n = 10 mice), MycΔ15–17/WT control for MycΔ15–17/ΔC mice (n = 14 for all populations, except HSCs, MPP1, MPP2, MPP3 and MPP4 (n = 12 mice)), MycΔ15–17/ΔC mice (n = 14 mice for all populations, except B, granulocytes, RBC, megakaryocytes, CD4+ T and CD8+ T cells (n = 13 mice)), MycΔ15–17/WT control for MycΔ15–17/ΔD mice (n = 14 mice for all populations, except HSCs, MPP1, MPP2, MPP3, MPP4 (n = 12 mice)), MycΔ15–17/ΔD mice (n = 9 mice for all populations, except HSCs, MPP1, MPP2, MPP3, MPP4 (n = 6 mice), MycΔ15–17/WT control for MycΔ15–17/ΔG–H mice (n = 8 mice for all populations, except HSCs, MPP1, MPP2, MPP3, MPP4, CMP, GMP, MEP, CLP (n = 6 mice)), MycΔ15–17/ΔG–H mice (n = 12 mice for all populations, except HSCs, MPP1, MPP2, MPP3, MPP4, CMP, GMP, MEP, CLP (n = 7 mice)), MycWT/WT control for MycΔI/ΔI mice (n = 9 mice for all populations), MycΔI/ΔI mice (n = 9 mice) from two to three independent experiments. c, See sample sizes for Fig. 3c in the Methods. e, f, See sample sizes for Fig. 3e in the Methods. g, MycΔ15–17/WT control for MycΔ15–17/ΔA–B mice (n = 3 mice), MycΔ15–17/ΔA–B mice (n = 3 mice), MycΔ15–17/WT control for MycΔ15–17/ΔC mice (n = 5 mice), MycΔ15–17/ΔC mice (n = 5 mice), MycΔ15–17/WT control for MycΔ15–17/ΔG–H mice (n = 3 mice), MycΔ15–17/ΔG–H mice (n = 7 mice), MycWT/WT control for MycΔI/ΔI mice (n = 4 mice), MycΔI/ΔI mice (n = 4 mice), data from one experiment. h, MycΔ15–17/WT control for MycΔ15–17/ΔA–B mice (n = 3 mice), MycΔ15–17/ΔA–B mice (n = 3 mice), MycΔ15–17/WT control for MycΔ15–17/ΔC mice (n = 5 mice), MycΔ15–17/ΔC mice (n = 5 mice for all populations, except mature B cells (n = 4 mice)), MycΔ15–17/WT control for MycΔ15–17/ΔG–H mice (n = 3 mice), MycΔ15–17/ΔG–H mice (n = 7 mice for all populations, except PrePro B and ProB PreB (n = 6 mice)), MycWT/WT control for MycΔI/ΔI mice (n = 4 mice), MycΔI/ΔI mice (n = 4 mice), data from one experiment. P values are from an unpaired two-tailed t-test.

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Extended Data Figure 7 The BENC enhancer modules overlap with a mouse leukaemia super-enhancer and loss of BENC in an MLL–AF9 leukaemia mouse model prolongs survival.

a, Comparison of BENC modules from normal haematopoietic tissues (see also Extended Data Fig. 1) with super-enhancer elements as defined previously2. Localization of BENC modules is shown above ChIP–seq tracks and super-enhancer elements defined by Brd4 occupancy as well as broad distribution of H3K27ac marks are indicated at the bottom. b, c, Loss of BENC in MLL–AF9-mediated leukaemias (for experimental setup, see Fig. 4a) in response to poly(I:C) injections (dotted lines) leads to a delay in AML progression (b) and to an increased survival of the mice that initially had leukaemia (c). In order to induce Cre expression from the Mx1 promoter, mice transplanted with leukaemic cells were subjected to four injections of poly(I:C) starting eight days after transplantation. In contrast to the experiment shown in Fig. 4b, c, mice were thereafter not injected with additional rounds of poly(I:C). As a consequence, leukaemic cells that had not recombined (genomic escapees) survived and expanded as Myc-expressing Mx-Cre;MycΔ15–17/flox cells, causing the death of the recipient mice. Together with the data presented in Fig. 4b, c, in which continuous injections of poly(I:C) result in the clearance of leukaemic cells from the peripheral blood in some mice, this argues for an insufficient deletion of the conditional Myc allele in this experiment and demonstrates that BENC is essential for maintenance of leukaemia. d, Loss of BENC in MLL–AF9 leukaemic cells isolated from the peripheral blood results in a significant reduction in Myc expression. e, f, Upregulation of myeloid differentiation markers on blast cells in the peripheral blood after Mx-Cre-mediated deletion of BENC. e, Three representative histogram plots of Gr1 and CD11b expression for leukaemic blasts from peripheral blood of n = 11 mice (Mx-Cre;MycWT/flox) or n = 12 mice (Mx-Cre;MycΔ15–17/flox). f, Quantification of Gr1 and CD11b expression of blasts. MFI is shown as mean ± s.e.m. Sample sizes are as follows: Mx-Cre;MycWT/flox (n = 12 mice for 8, 20 and 25 days, n = 2 mice for 35 days), Mx-Cre;MycΔ15–17/flox (n = 13 mice for 8 days, n = 12 mice for 20 days, n = 10 mice for 25 days, n = 9 mice for 35 days, n = 3 mice for 54 days) (b); n = 12 mice for Mx-Cre;MycWT/flox and n = 13 mice for Mx-Cre;MycΔ15–17/flox (c); n = 4 mice for Mx-Cre;MycWT/flox and n = 8 mice for Mx-Cre;MycΔ15–17/flox (d); n = 11 mice for Mx-Cre;MycWT/flox and n = 12 mice for Mx-Cre;MycΔ15–17/flox (f). P values are from an unpaired two-tailed t-test (b, d, f) or two-tailed Wilcoxon rank-sum test (c).

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Extended Data Figure 8 The BENC structure is conserved in the human genome and module C is differentially regulated in LSCs and its accessibility correlates with overall patient survival.

a, ATAC-seq analysis of human haematopoietic cell types in the BENC region. Risk single-nucleotide polymorphisms that are associated with haematological traits and focal amplifications in patients with AML are shown at the top22,23,24,25. b, Genomic coordinates of BENC modules in the human genome (hg19). c, Display of the 100 longest in cis interactions of promoters with enhancers and enhancer clusters in CD34+ cells measured by promoter capture high-resolution chromosome conformation capture (Hi-C)14. Highlighted in red are the interactions mapped to the BENC region and interactions within BENC modules are labelled accordingly. d, ATAC-seq profiles of module C in primary human AML samples divided into LSC+ (red) and LSC (blue) fractions, ranked from high to low. e, PERT model estimates of relative proportions of cell-type-specific transcriptional programs (MONO, monocytes; ETP, early T cell progenitor; GRAN, granulocytes; PROB, pro-B cells) composing the global gene expression of fractions. Spearman’s rank correlation between these estimated cell-specific proportions of transcriptional programs and module C peaks of fractions were determined. f, Gene set enrichment analysis of MYC target signatures in LSC fractions stratified according to ATAC-seq peak height in module C. g, Correlation between the maximum peak height in module C (bold) and the other modules in LSCs and patient survival. Hazard ratios and P values from the Wald test are shown. Module C is the only module that is more accessible in LSC+ cells compared to LSC cells (see Fig. 4d) and that shows a correlation with patient survival. h, Correlation between the ATAC-seq peak height in module C and either white blood cell counts (WBC) or percentage of bone marrow blast counts (%BM-Blast). ik, Correlation of ATAC-seq signals in immunophenotypic pre-leukaemic HSCs (pHSCs), LSCs and blasts with overall survival using the GSE74912 dataset29. i, ATAC-seq signal in module C in CD34+ cord blood cells (CD34+ CB), CD34+ bone marrow cells (CD34+ BM), pHSCs, LSCs and blasts. j, k, Kaplan–Meier representation of overall survival according to ATAC-seq signal in module C in pre-leukaemic HSC (j) and LSC (k) fractions. For this stratification, the patient cohort was split according to the median of the maximum ATAC-seq peak height in module C. Sample sizes are as follows: n = 1 patient (CD34+ cord blood cells), n = 2 bone marrow samples (CD34+ bone marrow cells), n = 16 bone marrow samples (pre-leukaemic HSCs), n = 8 bone marrow samples (LSCs), n = 15 bone marrow samples (blast) (i); n = 15 patients (j); n = 8 patients (k). P values in (i) from unpaired two-tailed t-test.

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Extended Data Table 1 Sources of publically available ChIP–seq, ATAC-seq and capture Hi-C datasets
Extended Data Table 2 Cell-surface phenotypes of analysed cell populations

Supplementary information

Supplementary Figure 1

This file contains the flow cytometric gating Strategies for hematopoietic cell populations (11 gating strategies). (PDF 3683 kb)

Life Sciences Reporting Summary (PDF 97 kb)

Supplementary Table 1

A list of insertion and deletion mouse lines. (XLSX 46 kb)

Supplementary Table 2

The percentage of MLL-AF9-IRES-GFP positive cells in the peripheral blood of leukemia mice. (XLSX 42 kb)

Supplementary Table 3

AML patient characteristics. (XLSX 40 kb)

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Bahr, C., von Paleske, L., Uslu, V. et al. A Myc enhancer cluster regulates normal and leukaemic haematopoietic stem cell hierarchies. Nature 553, 515–520 (2018). https://doi.org/10.1038/nature25193

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