The Transcriptional Repressor GFI-1 Antagonizes PU.1 Activity through Protein-Protein Interaction

Richard Dahl,Sangeeta R Iyer,Kristin S Owens,Dorothy D Cuylear,M Celeste Simon

doi:10.1074/jbc.m607613200

Abstract

Mice lacking the zinc finger transcriptional repressor protein GFI-1 are neutropenic. These mice generate abnormal immature myeloid cells exhibiting characteristics of both macrophages and granulocytes. Furthermore, Gfi-1(-/-) mice are highly susceptible to bacterial infection. Interestingly, Gfi-1(-/-) myeloid cells overexpress target genes of the PU.1 transcription factor such as the macrophage colony-stimulating factor receptor and PU.1 itself. We therefore determined whether GFI-1 modulates the transcriptional activity of PU.1. Our data demonstrate that GFI-1 physically interacts with PU.1, repressing PU.1-dependent transcription. This repression is functionally significant, as GFI-1 blocked PU.1-induced macrophage differentiation of a multipotential hematopoietic progenitor cell line. Retroviral expression of GFI-1 in primary murine hematopoietic progenitors increased granulocyte differentiation at the expense of macrophage differentiation. We interbred Gfi-1(+/-) and PU.1(+/-) mice and observed that heterozygosity at the PU.1 locus partially rescued the Gfi-1(-/-) mixed myeloid lineage phenotype, but failed to restore granulocyte differentiation. Our data demonstrate that GFI-1 represses PU.1 activity and that lack of this repression in Gfi-1(-/-) myeloid cells contributes to the observed mixed lineage phenotype.

Highlights

Institute, the Howard Hughes Medical Institute, the Leukemia Research Foundation, and the dedicated health research funds of the University of New Mexico School of Medicine
We observed a significant increase in colony-forming unitmacrophage (CFU-M; p Ͻ 0.05) and colony-forming unit-granulocyte (CFU-G; p Ͻ 0.02) colonies obtained from Gfi-1Ϫ/Ϫ
Because PU.1 target genes are overexpressed in myeloid cells obtained from Gfi-1Ϫ/Ϫ mice [9, 10], we examined whether the essential granulocyte transcription factor GFI-1 antagonizes PU.1 activity

Summary

The abbreviations used are

M-CSFR, macrophage colony-stimulating factor receptor; C/EBP␣, CCAAT/enhancer binding protein-␣; rv, retroviral; GFP, green fluorescent protein; GST, glutathione S-transferase; Znf, zinc finger; IL, interleukin; G-CSF, granulocyte colony-stimulating factor; OHT, hydroxytamoxifen; ChIP, chromatin immunoprecipitation; FACS, fluorescence-activated cell sorter; RT, reverse transcription; CFU-M, colony-forming unit-macrophage; CFU-G, colony-forming unit-granulocyte. GFI-1 Antagonizes PU. uncommitted hematopoietic progenitors are important in determining cell fate decisions [15, 19, 20]. GFI-1 blocks PU.1-induced macrophage differentiation of a myeloid cell line, and exogenous GFI-1 expression in primary hematopoietic progenitors increases granulocyte differentiation. We examined bone marrow from Gfi-1Ϫ/ϪPU.1ϩ/Ϫ mice and observed that decreasing PU. dosage reduced the mixed myeloid lineage phenotype of Gfi-1-deficient cells. Our data suggest that GFI-1 antagonism of PU. is critical for downregulation of macrophage gene expression in developing granulocytes

EXPERIMENTAL PROCEDURES

Findings

DISCUSSION