Ethanol exhibits specificity in its effects on differentiation of hematopoietic progenitors.

Ethanol is a known teratogen but the mechanisms by which this simple compound affects fetal development remain unresolved. The goal of the current study was to determine the mechanism by which ethanol affects lymphoid differentiation using an in vitro model of ethanol exposure. Primitive hematopoietic oligoclonal-neonatal-progenitor cells (ONP), with the phenotype Lin(-)HSA(lo)CD43(lo)Sca-1(-)c-Kit(+) that are present in neonatal but not adult bone marrow were sorted from the bone marrow of 2-week-old C57BL/6J mice and cultured under conditions that favor either B cell or myeloid cell differentiation with or without addition of ethanol. The overall growth of the ONP cells was not significantly affected by inclusion of up to 100mM ethanol in the culture medium. However, the differentiation of the progenitor cells along the B-cell pathway was significantly impaired by ethanol in a dose-dependent manner. Exposure of ONP cells to 100mM ethanol resulted in greater than 95% inhibition of B cell differentiation. Conversely, ethanol concentrations up to and including 100mM had no significant effect on differentiation along the myeloid pathway. The effect of ethanol on transcription factor expression was consistent with the effects on differentiation. ONP cells grown in 100mM ethanol failed to upregulate Pax5 and EBF, transcriptional regulators that are necessary for B cell development. However, ethanol had no significant effect on the upregulation of PU.1, a transcription factor that, when expressed in high concentration, favors myeloid cell development. Taken together, these results suggest that ethanol has specificity in its effects on differentiation of hematopoietic progenitors.

In utero exposure to alcohol alters cell fate decisions by hematopoietic progenitors in the bone marrow of offspring mice during neonatal development.

Fetal alcohol syndrome and alcohol related birth defects represent a spectrum of disorders that can result from the consumption of alcohol during pregnancy. Previous studies from this laboratory have shown that alcohol exposure in utero adversely affects hematopoietic progenitors in the bone marrow. Neonatal mice that were exposed in utero to alcohol showed a marked delay in B lymphocyte development. Recent studies have focused on an oligopotential progenitor cell, with the phenotype of HSA(lo)CD43(lo)Lin(-), which yields both B cells and myeloid lineage cells at a high frequency when cultured in vitro with stromal cells and the appropriate cytokines. However, these progenitor cells isolated from neonatal offspring of alcohol fed dams showed a significant decrease in the frequency of B cell formation following in vitro culture. In order to understand the mechanism underlying this defect we examined the expression of key transcription factors (early B cell factor, EBF, and Pax5) in this progenitor pool. Here, we report that >95% of HSA(lo)CD43(lo)Lin(-) cells express EBF and 5% express Pax5. Following liquid culture in the presence of IL-7, these progenitor cells respond by up-regulating Pax5 and the surface expression of CD19 indicating that the cells have committed to the B lineage. By contrast 75% of HSA(lo)CD43(lo)Lin(-) cells isolated from the bone marrow of neonatal animals exposed in utero to alcohol expressed EBF but at a level that was less than 25% the level of cells isolated from control animals. Furthermore, these alcohol-exposed progenitor cells failed to up-regulate Pax5 in response to IL-7 indicating a greatly reduced capacity to expand and differentiate to B lineage cells in liquid cultures. However, the HSA(lo)CD43(lo)Lin(-) cells isolated from the alcohol exposed animals retained the capacity to differentiate to myeloid lineage cells. These results suggest that the interference with the sequential expression of transcription factors in early progenitor cells by in utero alcohol exposure is a potential mechanism for the observed decrease in B lymphocytes in neonatal mice.

Identification of residues in the WD-40 repeat motif of the F-box protein Met30p required for interaction with its substrate Met4p.

The SCF family of ubiquitin-ligases consists of a common core machinery, namelySkp1p, Cdc53p, Hrt1p, and a variable component, the F-box protein that is responsible for substrate recognition. The F-box motif, which consists of approximately 40 amino acids, connects the F-box protein to the core ubiquitin-ligase machinery. Distinct SCF complexes, defined by distinct F-box proteins, target different substrate proteins for proteasome-dependent degradation. As part of the SCF(Met30p) complex, the F-box protein Met30p selects the substrate Met4p, a transcriptional activator for MET biosynthetic genes that mediate sulfur uptake and biosynthesis of sulfur containing compounds. When cells are grown in the absence of methionine, Met4p evades degradation by the SCF(Met30p) complex and activates the MET biosynthetic pathway. However, overproduction of Met30p represses MET gene expression and induces methionine auxotrophy in an otherwise methionine prototrophic strain. Here we demonstrate that overproduction of the C-terminal portion of Met30p, which is composed almost entirely of seven WD-40 repeat motifs, is necessary and sufficient to induce methionine auxotrophy and complement the temperature sensitive (ts) met30-6 mutation. Furthermore, we show that this region of Met30p is important for binding Met4p and that mutations that disrupt this interaction prevent both the induction of methionine auxotrophy and complementation of the met30-6 mutation. These assays have been exploited to identify residues that are important for the interaction of Met30p with its substrate. Since the C-terminal domain of Met30p lacks the F-box and cannot support the ubiquitination of Met4p, our results indicate that the recruitment of Met4p to the SCF(Met30p) complex itself results in inactivation of Met4p, independently of its ubiquitination.

The amino-terminal portion of the F-box protein Met30p mediates its nuclear import and assimilation into an SCF complex.

SCF complexes are a conserved family of ubiquitin ligases composed of a common core of components and a variable component called an F-box protein that defines substrate specificity. The F-box motif links the F-box protein to the core components via its interaction with Skp1p. In yeast, the SCFMet30p complex contains the Met30p F-box protein and regulates Met4p, a transcription factor that mediates sulfur fixation and methionine biosynthesis. Although a nuclear protein, Met30p lacks a definable nuclear localization sequence. Here we show that the entire amino-terminal half of Met30p is required for its proper nuclear localization. Mutations in the F-box, but not mutations in Skp1p, affect Met30p nuclear localization, indicating that the F-box motif plays an important role in Met30p trafficking independent of its interaction with Skp1p binding. Met30p mutants that poorly localize to the nucleus display increased nuclear to cytoplasmic exchange, indicating that the amino terminus mediates nuclear retention in addition to nuclear import. The Met30p F-box motif, residues 180-225, is necessary and sufficient to bind Skp1p; however, mutations upstream of the Met30p F-box inhibit Skp1p binding. We propose that additional factors bind the amino-terminal region of Met30p and mediate its nuclear localization and assimilation into an SCF complex.

Overproduction of polypeptides corresponding to the amino terminus of the F-box proteins Cdc4p and Met30p inhibits ubiquitin ligase activities of their SCF complexes.

Ubiquitin ligases direct the transfer of ubiquitin onto substrate proteins and thus target the substrate for proteasome-dependent degradation. SCF complexes are a family of ubiquitin ligases composed of a common core of components and a variable component called an F-box protein that defines substrate specificity. Distinct SCF complexes, defined by a particular F-box protein, target different substrate proteins for degradation. Although a few have been identified to be involved in important biological pathways, such as the cell division cycle and coordinating cellular responses to changes in environmental conditions, the role of the overwhelming majority of F-box proteins is not clear. Creating inhibitors that will block the in vivo activities of specific SCF ubiquitin ligases may provide identification of substrates of these uncharacterized F-box proteins. Using Saccharomyces cerevisiae as a model system, we demonstrate that overproduction of polypeptides corresponding to the amino terminus of the F-box proteins Cdc4p and Met30p results in specific inhibition of their SCF complexes. Analyses of mutant amino-terminal alleles demonstrate that the interaction of these polypeptides with their full-length counterparts is an important step in the inhibitory process. These results suggest a common means to inhibit specific SCF complexes in vivo.