The role of GA binding protein (GABP) transcription factor in myeloid differentiation and cell cycle progression.

摘要

GABP is an ets transcription factor that regulates transcription of key myeloid genes; it is also known to regulate important cell cycle control genes. GABP consists of two distinct and unrelated proteins that, together, form a functional transcription factor complex. GABPalpha is an ets protein that binds to DNA; it forms a tetrameric complex by recruiting GABPbeta, which contains the transactivation domain. GABPalpha is a single gene in both human and murine genomes and it is the only protein that can recruit GABPbeta to DNA. We cloned GABPalpha from a murine genomic library and prepared a targeting vector in which exon 9 (which encodes the GABPalpha ets domain) was flanked by loxP (floxed) recombination sites. The targeting construct was electroporated into embryonic stem cells, homologous recombinants were implanted into pseudopregnant mice, heterozygous floxed GABPalpha mice were identified, and intercrossing yielded expected Mendelian ratios of wild type, heterozygous, and homozygous floxed GABPalpha mice. Breeding of heterozygous floxed GABPalpha mice to CMV-Cre mice (which express Cre recombinase in all tissues) yielded expected numbers of homozygous mice (only one intact GABPalpha allele), but no nullizygous (GABPalpha-/-) mice, indicating an embryonic lethal defect caused by ablation of GABPalpha. To determine the effect of GABPalpha deletion on myeloid cells, we bred heterozygous and homozygous floxed mice to Mx1Cre mice, which express Cre only dependent upon the injection of synthetic DNA pI-pC. After pI-pC injection these mice had a dramatic decreased complement of Gr1+ or CD11b+ myeloid cells in both bone marrow compartment and in vitro colony assays. We prepared murine embryonic fibroblasts from homozygous floxed mice and efficiently deleted GABPalpha in vitro. We found striking abnormalities in cell proliferation. We used quantitative RT-PCR to identify mechanisms that account for the altered growth of GABPalpha null cells. We found dramatically reduced expression of known GABP target genes that regulate DNA synthesis and cell cycle that appear to account for the proliferative defect. We conclude that GABP is required for growth and maturation of myeloid cells and we identified downstream targets that may account for their failure to proliferate and mature in vitro and in vivo.