Cis-regulatory mechanisms governing stem and progenitor cell transitions

Abstract

Cis-element encyclopedias provide information on phenotypic diversity and disease mechanisms. Although cis-element polymorphisms and mutations are instructive, deciphering function remains challenging. Mutation of an intronic GATA motif (+9.5) in GATA2, encoding a master regulator of hematopoiesis, underlies an immunodeficiency associated with myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Whereas an inversion relocalizes another GATA2 cis-element (-77) to the proto-oncogene EVI1, inducing EVI1 expression and AML, whether this reflects ectopic or physiological activity is unknown. We describe a mouse strain that decouples -77 function from proto-oncogene deregulation. The -77(-/-) mice exhibited a novel phenotypic constellation including late embryonic lethality and anemia. The -77 established a vital sector of the myeloid progenitor transcriptome, conferring multipotentiality. Unlike the +9.5(-/-) embryos, hematopoietic stem cell genesis was unaffected in -77(-/-) embryos. These results illustrate a paradigm in which cis-elements in a locus differentially control stem and progenitor cell transitions, and therefore the individual cis-element alterations cause unique and overlapping disease phenotypes.

Keywords: GATA factor; GATA-2; cis-element; differentiation; hematopoiesis; myeloid; progenitor; stem cell; transcriptome.

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 (Lin⁻Mac1⁺CD41⁻CD48⁻CD150⁺Sca1⁺Kit⁺) and MPPs (Lin⁻Mac1⁺CD41⁻CD48⁻CD150⁻Sca1⁺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 Lin⁻Sca1⁺Kit⁺ and Lin⁻Sca1⁻Kit⁺ cells, CMPs (Lin⁻CD34⁺FcγR^lowKit⁺Sca1⁻), GMPs (Lin⁻CD34⁺FcγR^highKit⁺Sca1⁻), and MEPs (Lin⁻CD34⁻FcγR^lowKit⁺Sca1⁻). (B) Percentages of Lin⁻Sca1⁺Kit⁺ and Lin⁻Sca1⁻Kit⁺ 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 Lin⁻Sca1⁺Kit⁺ and Lin⁻Sca1⁻Kit⁺ 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.