Molecular mechanism of growth and differentiation of neutrophilic granulocyte mediated by G-CSF

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Our goal is to understand the molecular mechanism of growth and differentiation of neutrophilic granulocytes, which are mediated by granulocyte colony-stimulating factor (G-CSF). For this purpose, we have investigated the myeloid-specific zinc-finger protein MZF-2 that plays a role in granulocyte differentiation. Mutational analysis of mouse MZF-2 demonstrated that MZF-2 protein carries a transcription-inhibitory region at the N-terminus, a transactivation domain (TA domain) in the middle of the molecule, and a DNA-binding zinc-finger domains in the C-terminal region. We also found that the TA domain was phosphorylated by ERK MAP kinase. To determine sites phosphorylated by ERK, we constructed GST-MZF-2 fusion proteins with various alanine substitution of putative ERK phosphorylation sites. An in vitro phosphorylation analysis using the mutant substrates revealed that ERK phosphorylated three serine residues (Ser257, Ser275 and Ser295) in the TA domain. A mutant MZF-2 protein containin … More g alanine substitutions of the these serine residues showed higher transactivation activity than the wild-type protein when expressed in LGM-1 myeloid cells, suggesting that ERK MAP kinase negatively regulates the transcriptional activity of MZF-2 in vivo. To identify nuclear factors involved in the MZF-2-dependent gene activation, we screened mouse CDNA library for MZF-2 binding protein by using the ras recruitment two-hybrid system, and isolated a CDNA encoding a huge (3035 amino acid residues) protein, which showed a significant homology to. Drosophila Domino protein. The cloned protein, designated as mammalian Domino (mDomino), has a SWI/SNF-type ATP /helicase structure with a domain containing two polyglutamine tracts in the C-terminal region. Mutational analyzes using deletion mutants of mDomino revealed that MZF-2 interacted with the C-terminal, glutamine-rich domain. Moreover, the MZF-2-dependent reporter activation was enhanced when the mDomino C-terminal domain was co-expressed. These results suggest that chromatin remodeling caused by mDomino/MZF-2 complex may regulate myeloid-specific gene activation in granulocyte development. Less