Gabp Transcription Factor Is Required for Myeloid Differentiation, In Part, through Its Control of Gfi-1 Expression

Abstract 740

GABP is an ets transcription factor which regulates genes that are required for myeloid differentiation. The tetrameric GABP complex includes GABPα, which binds DNA via its ets domain, and GABPβ, which contains the transcription activation domain. In order to examine the role of Gabp in myeloid differentiation, we created mice in which exons that encode the ets DNA-binding domain are flanked by LoxP recombination sites (Gabpa fl/fl or floxed mice), and bred them to mice that bear Cre recombinase under the control of the pIC-inducible promoter, Mx1. Injection of these mice with pIC induces conditional deletion of Gabpa in hematopoietic tissues, and these mice are referred to as Gabpa knock-out mice (KO). Control mice that carry floxed Gabpa but lack Mx1-Cre were also treated with pIC. KO mice rapidly lost myeloid cells in peripheral blood, bone marrow, and spleen. Bone marrow cells from control and KO mice were sorted for CD11b and Gr1 to distinguish mature granulocytes from immature granulocytes and monocytes. KO mice had few mature myeloid cells, and the residual cells exhibited a dysplastic phenotypic appearance that did not resemble either normal maturing monocytes or granulocytes; instead they exhibited morphologic characteristics of both lineages. These KO myeloid cells simultaneously expressed differentiation-associated monocyte-restricted and granulocyte-restricted genes and markers of immaturity (Ly6C and c-kit). Thus, the patterns of gene and antigen expression by these dysplastic KO myeloid cells did not correspond to either normal monocytes or granulocytes. We recognized that these KO myeloid cells resemble those seen in mice with disruption of the transcriptional repressor, Gfi-1. We isolated granulocyte-monocyte progenitor (GMP) cells from control and KO bone mice, and found that expression of Gfi-1 was significantly reduced in KO GMPs, compared to controls. Chromatin immunoprecipitation demonstrated that Gabp bound the Gfi-1 promoter in vivo and transfection analysis showed that Gabp activated the Gfi-1 promoter. To test the hypothesis that Gabp controls myeloid differentiation through its regulation of Gfi-1, we grew bone marrow cells from both control and KO mice in liquid culture with SCF, Il-3 and Il-6, or GM-CSF. We transduced these cells with retrovirus bearing Gfi-1, and analyzed myeloid colony formation, and expression of CD11b and Gr1. Transduction of KO bone marrow cells with Gfi-1 partially restored myeloid colony formation in semi-solid media, and the percentage of Gr1⁺CD11b⁺ cells in liquid culture. We conclude that GABP plays a non-redundant role in myeloid development, and it controls differentiation of myeloid cells, at least in part, through its regulation of the transcriptional repressor, Gfi-1.