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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

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.
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Figure 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.

Mentions: The Gata2 expression defect in myeloid progenitors suggested that −77 selectively controls myeloid progenitor cell function, and therefore, our mouse model may provide a unique window into myeloid cell biology/pathology. Colony assays were conducted to quantitate myeloerythroid differentiation potential of −77−/− fetal liver progenitors. The −77−/− fetal livers were greatly impaired in their capacity to form CFU-GEMM (colony-forming unit–granulocyte, erythroid, macrophage, megakaryocyte) and BFU-E (burst-forming unit–erythroid) colonies, whereas myeloid colonies [CFU-GM (colony-forming unit–granulocyte, macrophage)] decreased about two-fold (Fig. 4A). The differentiation potential of flow-sorted immunophenotypic CMPs from −77−/− fetal livers was also enumerated by colony assay. Whereas −77+/+ CMPs generated the full repertoire of colony types (CFU-GEMM, CFU-GM, and BFU-E) (Fig. 4B), −77−/− CMP–derived colonies were smaller, less abundant (Fig. 4C), and strikingly composed almost exclusively of macrophages (Fig. 4D). This gross macrophage bias was also detected in colony assays with −77−/− fetal liver and flow-sorted GMPs (fig. S4). The reduced CFU-GEMM and BFU-E colony-forming potential of −77−/− fetal livers (Fig. 4) and the concomitant increase in CMPs and GMPs suggest that Gata2 stimulates myeloid progenitor cells to generate myeloerythroid progeny. Although reduced Gata2 expression upon −77 deletion blocks differentiation, macrophage generation is maintained. The MEP reduction may or may not reflect impaired CMP activity because MEP generation can bypass classically defined CMPs (30–32). The elevated multipotent cells represent an expected compensatory response, considering the severely defective myeloid progenitor cells and gross anemia of the −77−/− mutant embryos. The long-term repopulating activity of the mutant HSCs reinforces the specificity of −77 function to control myeloid cell function.


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)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4643771&req=5

Figure 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.
Mentions: The Gata2 expression defect in myeloid progenitors suggested that −77 selectively controls myeloid progenitor cell function, and therefore, our mouse model may provide a unique window into myeloid cell biology/pathology. Colony assays were conducted to quantitate myeloerythroid differentiation potential of −77−/− fetal liver progenitors. The −77−/− fetal livers were greatly impaired in their capacity to form CFU-GEMM (colony-forming unit–granulocyte, erythroid, macrophage, megakaryocyte) and BFU-E (burst-forming unit–erythroid) colonies, whereas myeloid colonies [CFU-GM (colony-forming unit–granulocyte, macrophage)] decreased about two-fold (Fig. 4A). The differentiation potential of flow-sorted immunophenotypic CMPs from −77−/− fetal livers was also enumerated by colony assay. Whereas −77+/+ CMPs generated the full repertoire of colony types (CFU-GEMM, CFU-GM, and BFU-E) (Fig. 4B), −77−/− CMP–derived colonies were smaller, less abundant (Fig. 4C), and strikingly composed almost exclusively of macrophages (Fig. 4D). This gross macrophage bias was also detected in colony assays with −77−/− fetal liver and flow-sorted GMPs (fig. S4). The reduced CFU-GEMM and BFU-E colony-forming potential of −77−/− fetal livers (Fig. 4) and the concomitant increase in CMPs and GMPs suggest that Gata2 stimulates myeloid progenitor cells to generate myeloerythroid progeny. Although reduced Gata2 expression upon −77 deletion blocks differentiation, macrophage generation is maintained. The MEP reduction may or may not reflect impaired CMP activity because MEP generation can bypass classically defined CMPs (30–32). The elevated multipotent cells represent an expected compensatory response, considering the severely defective myeloid progenitor cells and gross anemia of the −77−/− mutant embryos. The long-term repopulating activity of the mutant HSCs reinforces the specificity of −77 function to control myeloid cell function.

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