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Cis-regulatory mechanisms governing stem and progenitor cell transitions.

Johnson KD, Kong G, Gao X, Chang YI, Hewitt KJ, Sanalkumar R, Prathibha R, Ranheim EA, Dewey CN, Zhang J, Bresnick EH - Sci Adv (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Carbone Cancer Center, Department of Cell and Regenerative Biology, University of Wisconsin School of Medicine and Public Health, Madison, WI 53705, USA. ; University of Wisconsin-Madison Blood Research Program, Madison, WI 53705, USA.

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.

No MeSH data available.


Related in: MedlinePlus

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.
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Figure 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.

Mentions: The +9.5−/− embryos lack fetal liver HSPCs because of an HSC genesis defect in the AGM region (13). Since the −77 resides at Gata2, we asked whether it also controls HSC genesis. We enumerated c-Kit+ HSC clusters in E10.5 AGM of −77+/+ and −77−/− embryos and detected no difference in c-Kit+ cell numbers in dorsal aortas (Fig. 2A). By contrast, the percentage of immunophenotypic HSCs and multipotent progenitors (MPPs) in E13.5 fetal livers significantly increased in −77−/− embryos (Fig. 2B), as did the HSC number per liver. Consistent with elevated HSCs, the percentage of −77−/− HSCs in G0 was significantly lower compared with wild-type HSCs (fig. S1). HSC function was quantitated using a competitive transplantation assay. CD45.1+ recipient mice were lethally irradiated and transplanted with CD45.2+ fetal liver cells from −77+/+, −77+/−, or −77−/− E13.5 embryos and an equal number of wild-type CD45.1+ bone marrow cells. At 4-week intervals, recipient mice were bled, and hematopoietic parameters were quantitated by flow cytometry. Consistent with increased HSCs and MPPs in −77−/− fetal livers, −77−/− donor–derived circulating CD45.2+ cells in recipient mice were higher than those in −77+/+ and −77+/− donor–derived recipient mice (Fig. 2C). By 16 weeks, the −77−/− donor cells contributed significantly more to the myeloid lineage versus the −77+/+ cells; the −77−/− contribution to T cell and B cell lineages was reduced. The functionality of −77−/− donor–derived HSCs was further analyzed by serial transplantation. At all times after secondary transplantation, recipient mice that received −77−/−-derived (CD45.2+) bone marrow HSPCs exhibited higher levels of monocytes versus controls.


Cis-regulatory mechanisms governing stem and progenitor cell transitions.

Johnson KD, Kong G, Gao X, Chang YI, Hewitt KJ, Sanalkumar R, Prathibha R, Ranheim EA, Dewey CN, Zhang J, Bresnick EH - Sci Adv (2015)

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.
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Related In: Results  -  Collection

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Figure 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.
Mentions: The +9.5−/− embryos lack fetal liver HSPCs because of an HSC genesis defect in the AGM region (13). Since the −77 resides at Gata2, we asked whether it also controls HSC genesis. We enumerated c-Kit+ HSC clusters in E10.5 AGM of −77+/+ and −77−/− embryos and detected no difference in c-Kit+ cell numbers in dorsal aortas (Fig. 2A). By contrast, the percentage of immunophenotypic HSCs and multipotent progenitors (MPPs) in E13.5 fetal livers significantly increased in −77−/− embryos (Fig. 2B), as did the HSC number per liver. Consistent with elevated HSCs, the percentage of −77−/− HSCs in G0 was significantly lower compared with wild-type HSCs (fig. S1). HSC function was quantitated using a competitive transplantation assay. CD45.1+ recipient mice were lethally irradiated and transplanted with CD45.2+ fetal liver cells from −77+/+, −77+/−, or −77−/− E13.5 embryos and an equal number of wild-type CD45.1+ bone marrow cells. At 4-week intervals, recipient mice were bled, and hematopoietic parameters were quantitated by flow cytometry. Consistent with increased HSCs and MPPs in −77−/− fetal livers, −77−/− donor–derived circulating CD45.2+ cells in recipient mice were higher than those in −77+/+ and −77+/− donor–derived recipient mice (Fig. 2C). By 16 weeks, the −77−/− donor cells contributed significantly more to the myeloid lineage versus the −77+/+ cells; the −77−/− contribution to T cell and B cell lineages was reduced. The functionality of −77−/− donor–derived HSCs was further analyzed by serial transplantation. At all times after secondary transplantation, recipient mice that received −77−/−-derived (CD45.2+) bone marrow HSPCs exhibited higher levels of monocytes versus controls.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Carbone Cancer Center, Department of Cell and Regenerative Biology, University of Wisconsin School of Medicine and Public Health, Madison, WI 53705, USA. ; University of Wisconsin-Madison Blood Research Program, Madison, WI 53705, USA.

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.

No MeSH data available.


Related in: MedlinePlus