Enforced expression of the GATA-2 transcription factor blocks normal hematopoiesis.
The zinc finger transcription factor GATA-2 is highly expressed in immature hematopoietic cells and declines with blood cell maturation. To investigate its role in normal adult hematopoiesis, a bicistronic retroviral vector encoding GATA-2 and the green fluorescent protein (GFP) was used to maintain the high levels of GATA-2 that are normally present in primitive hematopoietic cells. Coexpression of the GFP marker facilitated identification and quantitation of vector-expressing cells. Bone marrow cells transduced with the GATA-2 vector expressed GFP as judged by flow cytometry and GATA-2 as assessed by immunoblot analysis. A 50% to 80% reduction in hematopoietic progenitor-derived colony formation was observed with GATA-2/GFP-transduced marrow, compared with marrow transduced with a GFP-containing vector lacking the GATA-2 cDNA. Culture of purified populations of GATA-2/GFP-expressing and nonexpressing cells confirmed a specific ablation of the colony-forming ability of GATA-2/GFP-expressing progenitor cells. Similarly, loss of spleen colony-forming ability was observed for GATA-2/GFP-expressing bone marrow cells. Despite enforced GATA-2 expression, marrow cells remained viable and were negative in assays to evaluate apoptosis. Although efficient transduction of primitive Sca-1(+) Lin- cells was observed with the GATA-2/GFP vector, GATA-2/GFP-expressing stem cells failed to substantially contribute to the multilineage hematopoietic reconstitution of transplanted mice. Additionally, mice transplanted with purified, GATA-2/GFP-expressing cells showed post-transplant cytopenias and decreased numbers of total and gene-modified bone marrow Sca-1(+) Lin- cells. Although Sca-1(+) Lin- bone marrow cells expressing the GATA-2/GFP vector were detected after transplantation, no appreciable expansion in their numbers occurred. In contrast, control GFP-expressing Sca-1(+) Lin- cells expanded at least 40-fold after transplantation. Thus, enforced expression of GATA-2 in pluripotent hematopoietic cells blocked both their amplification and differentiation. There appears to be a critical dose-dependent effect of GATA-2 on blood cell differentiation in that downregulation of GATA-2 expression is necessary for stem cells to contribute to hematopoiesis in vivo.
Persons
D A
DA
Division of Experimental Hematology, Department of Hematology/Oncology, St Jude Children's Research Hospital, Memphis, TN 38105, USA.
Allay
J A
JA
Allay
E R
ER
Ashmun
R A
RA
Orlic
D
D
Jane
S M
SM
Cunningham
J M
JM
Nienhuis
A W
AW
D016475:3T3 Cells; D000818:Animals; D017209:Apoptosis; D001854:Bone Marrow Cells / Q000378:metabolism*; D016026:Bone Marrow Transplantation; D002453:Cell Cycle; D002454:Cell Differentiation; D002455:Cell Division; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000502:physiology; D050989:GATA2 Transcription Factor; D015870:Gene Expression; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006410:Hematopoiesis; D008164:Luminescent Proteins / Q000235:genetics; D051379:Mice; D012190:Retroviridae / Q000235:genetics; D013154:Spleen / Q000166:cytology / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics* / Q000502:physiology; D014162:Transfection
D004268:DNA-Binding Proteins; D050989:GATA2 Transcription Factor; C494712:Gata2 protein, mouse; D008164:Luminescent Proteins; D014157:Transcription Factors; D049452:Green Fluorescent Proteins