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Differentiation mechanisms and inflammatory functions of neutrophils and macrophages are usually studied by genetic and biochemical approaches that require costly breeding and time-consuming purification to obtain phagocytes for functional analysis. Because Hox oncoproteins enforce self-renewal of factor-dependent myeloid progenitors, we queried whether estrogen-regulated Hoxb8 (ER-Hoxb8) could immortalize macrophage or neutrophil progenitors that would execute normal differentiation and normal innate immune function upon ER-Hoxb8 inactivation. Here we describe methods to derive unlimited quantities of mouse macrophages or neutrophils by immortalizing their respective progenitors with ER-Hoxb8 using different cytokines to target expansion of different committed progenitors. ER-Hoxb8 neutrophils and macrophages are functionally superior to those produced by many other ex vivo differentiation models, have strong inflammatory responses and can be derived easily from embryonic day 13 (e13) fetal liver of mice exhibiting embryonic-lethal phenotypes. Using knockout or small interfering RNA (siRNA) technologies, this ER-Hoxb8 phagocyte maturation system represents a rapid analytical tool for studying macrophage and neutrophil biology.

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Nuclear receptor-binding SET domain protein 1 (NSD1) prototype is a family of mammalian histone methyltransferases (NSD1, NSD2/MMSET/WHSC1, NSD3/WHSC1L1) that are essential in development and are mutated in human acute myeloid leukemia (AML), overgrowth syndromes, multiple myeloma and lung cancers. In AML, the recurring t(5;11)(q35;p15.5) translocation fuses NSD1 to nucleoporin-98 (NUP98). Here, we present the first characterization of the transforming properties and molecular mechanisms of NUP98-NSD1. We demonstrate that NUP98-NSD1 induces AML in vivo, sustains self-renewal of myeloid stem cells in vitro, and enforces expression of the HoxA7, HoxA9, HoxA10 and Meis1 proto-oncogenes. Mechanistically, NUP98-NSD1 binds genomic elements adjacent to HoxA7 and HoxA9, maintains histone H3 Lys 36 (H3K36) methylation and histone acetylation, and prevents EZH2-mediated transcriptional repression of the Hox-A locus during differentiation. Deletion of the NUP98 FG-repeat domain, or mutations in NSD1 that inactivate the H3K36 methyltransferase activity or that prevent binding of NUP98-NSD1 to the Hox-A locus precluded both Hox-A gene activation and myeloid progenitor immortalization. We propose that NUP98-NSD1 prevents EZH2-mediated repression of Hox-A locus genes by colocalizing H3K36 methylation and histone acetylation at regulatory DNA elements. This report is the first to link deregulated H3K36 methylation to tumorigenesis and to link NSD1 to transcriptional regulation of the Hox-A locus.

p6O0, the transforming protein kinase of Rous sarcoma virus, contains the 14-carbon saturated fatty acid, myristic acid, linked through an amide bond to the a-amino group of its NH2-terminal glycine residue. Myristic acid is known to be attached to four other eukaryotic proteins. In each case the fatty acid is also linked through an amide bond to an NH2-terminal glycine. We have used oligonucleotidedirected mutagenesis to examine the amino acid specificity of the enzyme that myristoylates the NH2 terminus of these proteins. Replacement of the NH2-terminal glycine in p6O1 with either alanine or glutamic acid prevented myristoylation completely. This indicates that the myristoylating enzyme may have an absolute specificity for glycine. Strikingly, neither nonmyristoylated mutant src protein induced morphological transformation of infected cells, even though wild-type levels of phosphorylation of cellular proteins on tyrosine were observed in these cells. Since conversion of the NH2-terminal residue from glycine to alanine should have little effect on the conformation of p6O1, the inability of this mutant p6Owr protein to induce morphological transformation suggests that the myristoyl moiety is essential for the transforming activity of the protein.Cellular transformation by Rous sarcoma virus results from the expression of a single viral protein designated p6011 (1). p60src functions as a tyrosine-specific protein kinase (2, 3) in vivo and is also reported to phosphorylate phosphatidylinositol in vitro (4). Tyrosine phosphorylation may be crucial in the control of cellular proliferation. The mitogens epidermal growth factor (5), platelet-derived growth factor (6), and insulin-like growth factor (7) all stimulate tyrosine protein kinase activity when they bind to their cell-surface receptors. Immunofluorescence (8), immunoelectron microscopy (9), and cell fractionation (10-12) all suggest that a significant fraction of the p60'" in transformed cells is associated with the cytoplasmic face of the plasma membrane. p60'1 may therefore deliver an unregulated mitogenic signal through the continuous phosphorylation ofone or more proteins involved in the normal regulation of proliferation.p60src is bound firmly to cellular membranes yet contains no large cluster of hydrophobic amino acids similar to those which are responsible for anchoring membrane-bound proteins such as the HLA and H-2 glycoproteins (human and murine major histocompatibility proteins) to a lipid bilayer. p60s' does, however, contain covalently bound myristic acid, a rare 14-carbon saturated fatty acid (13). This myristic acid moiety is attached by an amide linkage to the a-amino group of the NH2-terminal glycine residue of p6src (14). Consequently, an attractive hypothesis is that the hydrophobic myristoyl group plays a role in binding p6OSrc to membranes.Myristoylation is an uncommon form of protein modification. Nevertheless, the amino acid to which myristic acid is attached has been identified unambiguously in four additional proteins. The c...