Research ArticleHEALTH AND MEDICINE

Cis-regulatory mechanisms governing stem and progenitor cell transitions

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Science Advances  04 Sep 2015:
Vol. 1, no. 8, e1500503
DOI: 10.1126/sciadv.1500503
  • Fig. 1 −77 leukemogenic, long-range enhancer is essential for embryogenesis and hematopoiesis.

    (A) Vertebrate conservation plot of −77 showing positions of GATA motifs (G; WGATAR) and E-box (E; CANNTG) within the deleted region. (B) Genotypes of −77+/+, −77+/−, and −77−/− NeoR- embryos at timed developmental stages and genotypes of NeoR and NeoR+ pups at time of weaning. (C) Representative E13.5 +9.5−/− embryo exhibiting anemia, hemorrhage, and edema. (D) Representative E15.5 −77+/+, −77+/−, and −77−/− littermates. (E) Representative E15.5 fetal livers from −77+/+ and −77−/− littermates. (F and G) E15.5 peripheral blood quantitation [−77+/+ (n = 3) and −77−/− (n = 3)] and representative Wright-Giemsa staining. (H) Relative expression of fetal and adult β-globin mRNA in peripheral blood at E15.5. (I) Total cells [−77+/+ (n = 10), −77+/− (n = 24), and −77−/− (n = 13)] and Ter119+ cells [−77+/+ (n = 7), −77+/− (n = 17), and −77−/− (n = 5)] in E13.5 fetal livers. (J) Representative flow cytometric analysis and quantitation of E13.5 fetal livers for CD71+Ter119 R1 and R2 erythroid progenitors [−77+/+ (n = 7), −77+/− (n = 17), and −77−/− (n = 5)]. Graphs show means ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001.

  • Fig. 2 Long-term repopulating HSC generation and function do not require −77.

    (A) Whole-mount immunostaining of E10.5 −77+/+ and −77−/− embryos showing CD31+ cells (magenta) and c-Kit+ cells (green) within the dorsal aorta (DA). Scale bars, 100 μm. Quantitation of c-Kit+ cells within the whole dorsal aorta [−77+/+ (n = 3) and −77−/− (n = 3)] is shown on the right. (B) Representative flow cytometric analysis of E13.5 fetal livers for HSCs (LinMac1+CD41CD48CD150+Sca1+Kit+) and MPPs (LinMac1+CD41CD48CD150Sca1+Kit+). Quantitation of HSCs and MPPs is presented both as a percentage of total fetal liver cells (top) and as the number of cells per liver (bottom) [−77+/+ (n = 5), −77+/− (n = 6), and −77−/− (n = 4)]. (C) Contribution of −77+/− and −77−/− versus −77+/+ fetal liver cells in a competitive transplantation assay [−77+/+ (4 livers; 10 recipients), −77+/− (6 livers; 12 recipients), and −77−/− (3 livers; 11 recipients)]. After 20 weeks, secondary transplants were performed with bone marrow from fetal liver–transplanted −77−/− versus −77+/+ mice (2 bone marrow donors for each genotype; 8 recipient mice). The peripheral blood of recipient mice was analyzed for CD45.2 expression by flow cytometry at 4-week intervals after transplantation. Flow cytometric analysis of the proportions of CD45.2+ monocytes (Mac1+Gr), granulocytes (Mac1+Gr+), B cells (CD19+), and T cells (Thy1.2+) in the peripheral blood of recipient mice. Graphs show means ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001.

  • Fig. 3 Selective loss of Gata2 expression in −77−/− myeloid progenitors disrupts homeostasis.

    (A) Representative flow cytometric analysis of E13.5 fetal livers for LinSca1+Kit+ and LinSca1Kit+ cells, CMPs (LinCD34+FcγRlowKit+Sca1), GMPs (LinCD34+FcγRhighKit+Sca1), and MEPs (LinCD34FcγRlowKit+Sca1). (B) Percentages of LinSca1+Kit+ and LinSca1Kit+ cells [top; −77+/+ (n = 7), −77+/− (n = 17), and −77−/− (n = 6)] and CMPs, GMPs, and MEPs [bottom; −77+/+ (n = 8), −77+/− (n = 6), and −77−/− (n = 9)]. (C) Quantitation of Gata2 mRNA levels in LinSca1+Kit+ and LinSca1Kit+ cells [−77+/+ (n = 5) and −77−/− (n = 7)] and CMPs, GMPs, and MEPs [−77+/+ (n = 4) and −77−/− (n = 4)]. (D) Profiles for chromatin accessibility via the assay for transposase-accessible chromatin (ATAC) and histone H3K4 monomethylation mined from existing data (53). Graphs show means ± SEM; **P < 0.01, ***P < 0.001.

  • Fig. 4 Limited differentiation potential of −77−/− fetal liver progenitors.

    (A) Colony-forming activity of hematopoietic progenitors from E14.5 fetal livers [−77+/+ (n = 12), −77+/− (n = 17), and −77−/− (n = 3)]. (B) Colony-forming activity of CMPs and GMPs sorted from E13.5 fetal livers [−77+/+ (n = 3) and −77−/− (n = 3)]. (C and D) Representative colonies and Wright-Giemsa–stained cells obtained from plating of CMPs. Scale bars, 2 mm. Mac, macrophage; Ery, erythroblast; Neu, neutrophil; Mye, myeloid. Graphs show means ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001.

  • Fig. 5 −77 establishes a functionally vital sector of the myeloid progenitor cell transcriptome.

    (A) Quantitative gene expression analysis in CMPs, GMPs, and MEPs from −77+/+ and 77−/− E13.5 fetal livers [−77+/+ (n = 4) and −77−/− (n = 4)]. (B) MA plot of RNA-seq–based comparison of CMP transcriptomes from −77+/+ and −77−/− E13.5 fetal livers [−77+/+ (n = 3) and −77−/− (n = 3)]. Red points indicate down- or up-regulated genes [false discovery rate (FDR) <0.05]. (C) Heatmap depicting statistically significant genes down- or up-regulated by >1.5-fold. (D) Venn diagram depicting the extent of overlap between genes regulated by +9.5 in AGM (13) and −77 in CMPs. Genes shared between data sets are indicated in red in (C). (E) Evidence for direct GATA-2 regulation of −77 target genes. ChIP-seq profiles of GATA-2 and Scl/TAL1 in HPC-7 cells (58) and quantitative gene expression analysis in CMPs from −77+/+ and −77−/− E13.5 fetal livers [−77+/+ (n = 4) and −77−/− (n = 4)]. Graphs show means ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001.

  • Fig. 6

    Selective control of stem and progenitor cell transitions by distinct cis-elements within a genetic locus. The model depicts differential +9.5 and −77 functions to control critical stages of hematopoiesis. Disruption of −77 and +9.5 in humans causes acute myeloid leukemia and primary immunodeficiency, respectively. Whereas both enhancers are essential for GATA-2 regulation, they operate in distinct cellular contexts, and genomic analyses indicate major differences in their target gene ensembles. Prog, myeloerythroid progenitor; Mye, myeloid progenitor; Ery, erythroid; DA, dorsal aorta.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/1/8/e1500503/DC1

    Table S1. Sequences of primers used for qPCR.

    Fig. S1. Reduced quiescence of −77−/− HSCs.

    Fig. S2. Expansion of HSPC compartment in −77−/− fetal livers.

    Fig. S3. Distinct dynamic chromatin signatures at the −77 and +9.5 during hematopoiesis.

    Fig. S4. Macrophage bias of −77−/− myeloid progenitors.

    Fig. S5. −77 target genes define myeloerythroid, megakaryocyte, and nonhematopoietic protein interaction networks.

    Fig. S6. −77 controls a cohort of genes with hematopoietic function.

    Fig. S7. Selective myeloerythroid expression patterns of −77 target genes.

    Fig. S8. A minority of −77-regulated genes are GATA-1–regulated.

  • Supplementary Materials

    This PDF file includes:

    • Table S1. Sequences of primers used for qPCR.
    • Fig. S1. Reduced quiescence of −77−/− HSCs.
    • Fig. S2. Expansion of HSPC compartment in −77−/− fetal livers.
    • Fig. S3. Distinct dynamic chromatin signatures at the −77 and +9.5 during hematopoiesis.
    • Fig. S4. Macrophage bias of −77−/− myeloid progenitors.
    • Fig. S5. −77 target genes define myeloerythroid, megakaryocyte, and nonhematopoietic protein interaction networks.
    • Fig. S6. −77 controls a cohort of genes with hematopoietic function.
    • Fig. S7. Selective myeloerythroid expression patterns of −77 target genes.
    • Fig. S8. A minority of −77-regulated genes are GATA-1–regulated

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