Bismuth-oxide nanoparticles: study in a beam and as deposited.

Bi2O3 is a promising material for solid-oxide fuel cells (SOFC) due to the high ionic conductivity of some phases. The largest value is reached for its δ-phase, but it is normally stable at temperatures too high for SOFC operation, while nanostructured oxide is believed to have more suitable stabilization temperature. However, to manufacture such a material with a controlled chemical composition is a challenging task. In this work, we investigated the fabrication of nanostructured Bi2O3 films formed by deposition of free Bi-oxide nanoparticles created in situ. The particle-production method was based on reactive sputtering and vapour aggregation. Depending on the fabrication conditions, the nanoparticles contained either a combination of Bi-metal and Bi-oxide, or only Bi-oxide. Prior to deposition, the free particles were probed in the beam - by synchrotron-based photoelectron spectroscopy (PES), which allowed assessing their composition "on the-fly". The nanoparticle films obtained after deposition were studied by PES, scanning electron microscopy, transmission electron microscopy, and electron diffraction. The films' chemical composition, grain dimensions, and crystal structure were probed. Our analysis suggests that our method produced Bi-oxide films in more than one polymorph of Bi2O3.

Tin-oxide nanoparticles deposited from a beam: what happens to the composition?

The debate around the oxidation states occurring in laboratory-prepared tin-oxide samples has been for a long time an obstacle for an unambiguous assignment of characterization studies performed on such samples. In particular the changes in the Sn core-level energies caused by oxidation - i.e. the chemical shifts - as measured by photoelectron spectroscopy (PES) have been under discussion. The assignment problem is especially pronounced for nanoscale structures, which are important for photovoltaics, electronics, catalysis, and gas sensing. The reasons for the difficulties lie both in the natural properties of tin oxides, which can have substantial deficiencies of oxygen and tin in the lattice, and in the shortcomings of the fabrication and PES-characterization procedures themselves. Our recent PES study on tin-oxide nanoparticles fabricated by vapour-aggregation gave a chemical shift two times larger than earlier reported for Sn(iv) oxide for the Sn 4d level. The implemented fabrication technique forms an in-vacuum beam of particles whose composition can be both controlled and characterized by PES. In the present work SnO and SnO2 nanoparticles fabricated this way were deposited from the beam and probed by PES directly, as well as after exposure to air. The deposited nanoparticle films were also imaged by TEM (Transmission Electron Microscopy). The effects of the deposition process and exposure to air on the chemical composition were studied. The PES study of deposited SnO2 nanoparticles in the Sn 4d and Sn 3d core-level regions revealed the same core level shift as for unsupported nanoparticles, indicating that the chemical composition is preserved in the deposition process. The TEM study demonstrated a crystalline structure of separate SnO2 particles with lattice constants close to the macroscopic Sn(iv)-oxide. The PES study on the particles exposed to air showed changes in the composition. For the film of initially SnO particles a higher intermediate oxide was created. For the SnO2 nanoparticle film a lower, but strong, intermediate oxide was observed, likely at the surface.

Metal-passivated PbS nanoparticles: fabrication and characterization.

Organic-shell-free PbS nanoparticles have been produced in the size range relevant for quantum-dot solar cells (QDSCs) by a vapor aggregation method involving magnetron reactive sputtering. This method creates a beam of free 5-10 nm particles in a vacuum. The dimensions of the particles were estimated after their deposition on a substrate by imaging them using ex situ SEM and HRTEM electron microscopy. The particle structure and chemical composition could be deduced "on the fly", prior to deposition, using X-ray photoelectron spectroscopy (XPS) with tunable synchrotron radiation. Our XPS results suggest that under certain conditions it is possible to fabricate particles with a semiconductor core and 1 to 2 monolayer shells of metallic lead. For this case the absolute energy of the highest occupied molecular orbital (HOMO) in PbS has been determined to be (5.0 ± 0.5) eV below the vacuum level. For such particles deposited on a substrate HRTEM has confirmed the XPS-based conclusions on the crystalline PbS structure of the semiconductor core. Absorption spectroscopy on the deposited film has given a value of ∼1 eV for the lowest exciton. Together with the valence XPS results this has allowed us to reconstruct the energy level scheme of the particles. The results obtained are discussed in the context of the properties of PbS QDSCs.

Embryonic lethality and impairment of haematopoiesis in mice heterozygous for an AML1-ETO fusion gene.

Acute myeloid leukaemia (AML) is a major haematopoietic malignancy characterized by the proliferation of a malignant clone of myeloid progenitor cells. A reciprocal translocation, t(8;21)(q22;q22), observed in the leukaemic cells of approximately 40% of patients with the M2 subtype of AML disrupts both the AML1 (CBFA2) gene on chromosome 21 and the ETO (MTG8) gene on chromosome 8 (refs 3-5). A chimaeric protein is synthesized from one of the derivative chromosomes that contains the N terminus of the AML1 transcription factor, including its DNA-binding domain, fused to most of ETO, a protein of unknown function. We generated mice that mimic human t(8;21) with a "knock-in' strategy. Mice heterozygous for an AML1-ETO allele (AML1-ETO/+) die in midgestation from haemorrhaging in the central nervous system and exhibit a severe block in fetal liver haematopoiesis. This phenotype is very similar to that resulting from homozygous disruption of the AML1 (Cbfa2) or Cbfb genes, indicating that AML1-ETO blocks normal AML1 function. However, yolk sac cells from AML1-ETO/+ mice differentiated into macrophages in haematopoietic colony forming unit (CFU) assays, unlike Cbfa2-/- or Cbfb-/-cells, which form no colonies in vitro. This indicates that AML1-ETO may have other functions besides blocking wild-type AML1, a property that may be important in leukaemogenesis.

The macrophage transcription factor PU.1 directs tissue-specific expression of the macrophage colony-stimulating factor receptor.

The macrophage colony-stimulating factor (M-CSF) receptor is expressed in a tissue-specific fashion from two distinct promoters in monocytes/macrophages and the placenta. In order to further understand the transcription factors which play a role in the commitment of multipotential progenitors to the monocyte/macrophage lineage, we have initiated an investigation of the factors which activate the M-CSF receptor very early during the monocyte differentiation process. Here we demonstrate that the human monocytic M-CSF receptor promoter directs reporter gene activity in a tissue-specific fashion. Since one of the few transcription factors which have been implicated in the regulation of monocyte genes is the macrophage- and B-cell-specific PU.1 transcription factor, we investigated whether PU.1 binds and activates the M-CSF receptor promoter. Here we demonstrate that both in vitro-translated PU.1 and PU.1 from nuclear extracts bind to a specific site in the M-CSF receptor promoter just upstream from the major transcription initiation site. Mutations in this site which eliminate PU.1 binding decrease M-CSF receptor promoter activity significantly in macrophage cell lines only. Furthermore, PU.1 transactivates the M-CSF receptor promoter in nonmacrophage cells. These results suggest that PU.1 plays a major role in macrophage gene regulation and development by directing the expression of a receptor for a key macrophage growth factor.

Multiple functional domains of AML1: PU.1 and C/EBPalpha synergize with different regions of AML1.

Control elements of many genes are regulated by multiple activators working in concert to confer the maximal level of expression, but the mechanism of such synergy is not completely understood. The promoter of the human macrophage colony-stimulating factor (M-CSF) receptor presents an excellent model with which we can study synergistic, tissue-specific activation for two reasons. First, myeloid-specific expression of the M-CSF receptor is regulated transcriptionally by three factors which are crucial for normal hematopoiesis: PU.1, AML1, and C/EBPalpha. Second, these proteins interact in such a way as to demonstrate at least two examples of synergistic activation. We have shown that AML1 and C/EBPalpha activate the M-CSF receptor promoter in a synergistic manner. As we report here, AML1 also synergizes, and interacts physically, with PU. 1. Detailed analysis of the physical and functional interaction of AML1 with PU.1 and C/EBPalpha has revealed that the proteins contact one another through their DNA-binding domains and that AML1 exhibits cooperative DNA binding with C/EBPalpha but not with PU.1. This difference in DNA-binding abilities may explain, in part, the differences observed in synergistic activation. Furthermore, the activation domains of all three factors are required for synergistic activation, and the region of AML1 required for synergy with PU.1 is distinct from that required for synergy with C/EBPalpha. These observations present the possibility that synergistic activation is mediated by secondary proteins contacted through the activation domains of AML1, C/EBPalpha, and PU.1.

CCAAT enhancer-binding protein (C/EBP) and AML1 (CBF alpha2) synergistically activate the macrophage colony-stimulating factor receptor promoter.

Transcription factors play a key role in the development and differentiation of specific lineages from multipotential progenitors. Identification of these regulators and determining the mechanism of how they activate their target genes are important for understanding normal development of monocytes and macrophages and the pathogenesis of a common form of adult acute leukemia, in which the differentiation of monocytic cells is blocked. Our previous work has shown that the monocyte-specific expression of the macrophage colony-stimulating factor (M-CSF) receptor is regulated by three transcription factors interacting with critical regions of the M-CSF receptor promoter, including PU.1 and AML1.PU.1 is essential for myeloid cell development, while the AML1 gene is involved in several common leukemia-related chromosome translocations, although its role in hematopoiesis has not been fully identified. Along with AML1, a third factor, Mono A, interacts with a small region of the promoter which can function as a monocyte-specific enhancer when multimerized and linked to a heterologous basal promoter. Here, we demonstrate by electrophoretic mobility shift assays with monocytic nuclear extracts, COS-7 cell-transfected factors, and specific antibodies that the monocyte-enriched factor Mono A is CCAAT enhancer-binding protein (C/EBP). C/EBP has been shown previously to be an important transcription factor involved in hepatocyte and adipocyte differentiation; in hematopoietic cells, C/EBP is specifically expressed in myeloid cells. In vitro binding analysis reveals a physical interaction between C/EBP and AML1. Further transfection studies show that C/EBP and AML1 in concert with the AML1 heterodimer partner CBF beta synergistically activate M-CSF receptor by more then 60 fold. These results demonstrate that C/EBP and AML1 are important factors for regulating a critical hematopoietic growth factor receptor, the M-CSF receptor, suggesting a mechanism of how the AML1 fusion protein could contribute to acute myeloid leukemia. Furthermore, they demonstrate physical and functional interactions between AML1 and C/EBP transcription factor family members.

Identification of a region which directs the monocytic activity of the colony-stimulating factor 1 (macrophage colony-stimulating factor) receptor promoter and binds PEBP2/CBF (AML1).

The receptor for the macrophage colony-stimulating factor (or colony-stimulating factor 1 [CSF-1]) is expressed from different promoters in monocytic cells and placental trophoblasts. We have demonstrated that the monocyte-specific expression of the CSF-1 receptor is regulated at the level of transcription by a tissue-specific promoter whose activity is stimulated by the monocyte/B-cell-specific transcription factor PU.1 (D.-E. Zhang, C.J. Hetherington, H.-M. Chen, and D.G. Tenen, Mol. Cell. Biol. 14:373-381, 1994). Here we report that the tissue specificity of this promoter is also mediated by sequences in a region II (bp -88 to -59), which lies 10 bp upstream from the PU.1-binding site. When analyzed by DNase footprinting, region II was protected preferentially in monocytic cells. Electrophoretic mobility shift assays confirmed that region II interacts specifically with nuclear proteins from monocytic cells. Two gel shift complexes (Mono A and Mono B) were formed with separate sequence elements within this region. Competition and supershift experiments indicate that Mono B contains a member of the polyomavirus enhancer-binding protein 2/core-binding factor (PEBP2/CBF) family, which includes the AML1 gene product, while Mono A is a distinct complex preferentially expressed in monocytic cells. Promoter constructs with mutations in these sequence elements were no longer expressed specifically in monocytes. Furthermore, multimerized region II sequence elements enhanced the activity of a heterologous thymidine kinase promoter in monocytic cells but not other cell types tested. These results indicate that the monocyte/B-cell-specific transcription factor PU.1 and the Mono A and Mono B protein complexes act in concert to regulate monocyte-specific transcription of the CSF-1 receptor.

PU.1 (Spi-1) autoregulates its expression in myeloid cells.

PU.1 (Spi-1), a member of the Ets transcription factor family, is predominantly expressed in myeloid (granulocytes, monocytes and macrophages) and B cells. PU.1 is upregulated early during commitment of multipotential progenitors to the myeloid lineages and inhibition of PU.1 function in human CD34+ progenitors prior to this upregulation blocks myeloid colony formation. Since PU.1 expression appears to play a role in hematopoietic development, we characterized the PU.1 promoter. Here we report that the murine PU.1 promoter, as well as the human promoter, demonstrate tissue-specific reporter gene expression in myeloid cell lines but not in T cells and HeLa (non-hematopoietic cells) cells. Deletion analysis of the PU.1 promoter indicates that tissue-specific functional elements are encoded in the -61 to -39 bp and -7 to +34 bp regions. The first region contains a functional octamer (Oct) site at -54 bp and an Sp1 site at -39 bp. The second contains a binding site at +20 bp for both PU.1 itself and the related ets family member Spi-B. In vivo footprinting assays demonstrate that a hypersensitive band was detected at the PU.1 site in myeloid cells but not in HeLa. A mutation of the PU.1 site which abolished PU.1 binding caused a significant decrease in promoter activity. Mutation of the Oct and/or Sp1 site results in a lesser decrease of promoter activity in myeloid cells. Co-transfection of PU.1 or Spi-B in cells lacking PU.1 and Spi-B specifically transactivated a minimal promoter containing the PU.1 binding site, indicating that PU.1 can activate its own promoter elements in an autoregulatory loop. Positive autoregulation of the PU.1 promoter may play an important role in the function of PU.1 in myeloid cells.

Function of PU.1 (Spi-1), C/EBP, and AML1 in early myelopoiesis: regulation of multiple myeloid CSF receptor promoters.

Our studies of the promoters of the myeloid CSF receptors (M, GM, and G) in cell lines have led to the findings that the promoters are small, and are all activated by the PU.1 and C/EBP proteins. To date, we have only found evidence for involvement of C/EBP alpha, although further experiments will be needed to exclude the role of C/EBP beta and C/EBP delta in receptor gene expression. These studies suggest a model of hematopoiesis (Fig. 2) in which the lineage commitment decisions of multipotential cells are made by the alternative patterns of expression of certain transcription factors, which then activate growth factor receptors which allow those cells to respond to the appropriate growth factor to proliferate and survive. For example, expression of GATA-1 activates its own expression, as well as that of the erythropoietin receptor, inducing these cells to be capable of responding to erythropoietin. Similarly, expression of PU.1 activates its own promoter, and turns on the three myeloid CSF receptors (M, GM, and G), pushing these cells along the pathway of myeloid differentiation. C/EBP proteins, particularly C/EBP alpha, are also critical for myeloid receptor promoter function, and may also act via autoregulatory mechanisms. Murine C/EBP alpha has a C/EBP binding site in its own promoter. Human C/EBP alpha autoregulates its own expression in adipocytes by activating the USF transcription factor. Myeloid genes expressed later during differentiation, such as CD11b, are also activated by PU.1, which is expressed at highest levels in mature myeloid cells, but not by C/EBP alpha, which is downregulated in a differentiated murine myeloid cell line. Consistent with this model are the findings that overexpression of PU.1 in erythroid cells blocks erythroid differentiation, leading to erythroleukemia, and overexpression of GATA-1 in a myeloid line blocks myeloid differentiation. While these findings have provided some framework for understanding myeloid gene regulation, there are a number of critical questions to be addressed in the near future: What is the pattern of expression of the C/EBP proteins during the course of myeloid differentiation and activation of human CD34+ cells? What is the effect of targeted disruption and other mutations of the C/EBP and AML1 proteins on myeloid development and receptor expression? What are the interactions among these three different types of factors (ets, basic region-zipper, and Runt domain proteins) to activate the promoters? What is the effect of translocations, mutations, and alterations in expression of these factors, particularly in different forms of AML?

Imaging columns of the light elements carbon, nitrogen and oxygen with sub Angstrom resolution.

It is reported that lattice imaging with a 300 kV field emission microscope in combination with numerical reconstruction procedures can be used to reach an interpretable resolution of about 80 pm for the first time. A retrieval of the electron exit wave from focal series allows for the resolution of single atomic columns of the light elements carbon, nitrogen, and oxygen at a projected nearest neighbor spacing down to 85 pm. Lens aberrations are corrected on-line during the experiment and by hardware such that resulting image distortions are below 80 pm. Consequently, the imaging can be aberration-free to this extent. The resolution enhancement results from increased electrical and mechanical stability of the instrument coupled with a low spherical aberration coefficient of 0.595 + 0.005 mm.

Sub-Angstrom high-resolution transmission electron microscopy at 300 keV.

Sub-Angstrom transmission electron microscopy has been achieved at the National Center for Electron Microscopy (NCEM) by a one-Angstrom microscope (OAM) project using software and enhanced hardware developed within a Brite-Euram project (Ultramicroscopy 64 (1996) 1). The NCEM OAM provides materials scientists with transmission electron microscopy at a resolution better than 1 A by using extensive image reconstruction to exploit the significantly higher information limit of an FEG-TEM over its Scherzer resolution limit. Reconstruction methods chosen used off-axis holograms and focal series of underfocused images. Measured values of coherence parameters predict an information limit of 0.78 A. Images from a [1 1 0] diamond test specimen show that sub-Angstrom resolution of 0.89 A has been achieved with the OAM using focal series reconstruction.

CCAAT/enhancer-binding protein activates the CD14 promoter and mediates transforming growth factor beta signaling in monocyte development.

Transcription factors from the CCAAT/enhancer-binding protein (C/EBP) family play important roles in myeloid cell differentiation. CD14 is a monocyte/macrophage differentiation marker and is strongly up-regulated during monocytic cell differentiation. Here, we report the direct binding of C/EBP to the monocyte-specific promoter of CD14. Transactivation analyses demonstrate that C/EBP family members significantly activate the CD14 promoter. These data indicate that C/EBP is directly involved in the regulation of CD14 gene expression. When myelomonoblastic U937 cells are treated with vitamin D(3) and TGF-beta, they differentiate toward monocytic cells. Using specific antibodies against different C/EBP family members in electrophoretic mobility shift assays and Western blot assays, we have identified a specific increase in the DNA binding and the expression of C/EBPalpha and C/EBPbeta during U937 monocytic cell differentiation, and we found C/EBPalpha and C/EBPbeta bind to the promoter in heterodimer. Furthermore, with stably transfected cell lines, we demonstrate that the C/EBP binding site in the CD14 promoter plays a critical role for mediating TGF-beta signaling in the synergistic activation of CD14 expression by vitamin D(3) and TGF-beta during U937 differentiation. This may indicate that C/EBPs have important functions in the process of TGF-beta signal transduction during monocyte differentiation.

Hepatocytes contribute to soluble CD14 production, and CD14 expression is differentially regulated in hepatocytes and monocytes.

CD14 presents as a glycosylphosphatidylinositol-linked membrane protein on the surface of monocytes/macrophages and as a soluble protein in the serum. Our previous studies have shown that an 80-kilobase pair (kb) genomic DNA fragment containing the human CD14 gene is sufficient to direct CD14 expression in a monocyte-specific manner in transgenic mice. In addition, we discovered that human CD14 is highly expressed in hepatocytes. Here, we report the generation of transgenic mice with either a 24- or 33-kb human CD14 genomic DNA fragment. Data from multiple transgenic lines show that neither the 24- nor the 33-kb transgenic mice express human CD14 in monocytes/macrophages. However, human CD14 is highly expressed in the liver of the 33-kb transgenic mice. These results demonstrate that human CD14 expression is regulated differently in monocytes and hepatocytes. Furthermore, we identified an upstream regulatory element beyond the 24-kb region, but within the 33-kb region of the human CD14 gene, which is critical for CD14 expression in hepatocytes, but not in monocytes/macrophages. Most importantly, the data demonstrate that the liver is one of the major organs for the production of soluble CD14. These transgenic mice provide an excellent system to further explore the functions of soluble CD14.

Cloning and characterization of a novel human ubiquitin-specific protease, a homologue of murine UBP43 (Usp18).

The ubiquitin-specific proteases (UBP) are a family of enzymes that cleave ubiquitin from ubiquitinated protein substrates. We have recently cloned UBP43, a novel member of this family from AML1-ETO knock-in mice. To analyze the role of UBP43 in hematopoiesis and leukemogenesis, we have cloned a full-length human UBP43 cDNA by screening a human monocytic cDNA library as well as by 5'- and 3'-rapid amplification of cDNA ends analyses. This cDNA encodes a polypeptide of 372 amino acids with all of the structural motifs of a deubiquitinating enzyme. The human UBP43 mRNA is strongly expressed in human liver and thymus. Transfection analysis has demonstrated that UBP43 is a nuclear protein. Interestingly, the gene encoding human UBP43 maps to chromosome 22q11.2. This region, known as DiGeorge syndrome critical region, contains a minimal area of 2 Mb and is consistently deleted in DiGeorge syndrome and related disorders. The syndrome is marked by thymic aplasia or hypoplasia, parathyroid hypoplasia, or congenital cardiac abnormalities. Taken together, our results broaden the understanding of a new human ubiquitin-specific protease, UBP43, and suggest that this gene may also be related to DiGeorge syndrome.

Electron microscopy observations on the role of twinning in the evolution of microstructures.

Twinning plays an important role in phase transformations and can have significant effects on microstructural evolution. Different roles of twinning in the development of microstructures during precipitation and phase transformations are reviewed and illustrated with examples from investigations by high-resolution electron microscopy, including the effect of multiple twinning on the development of Ge precipitates in Al-Ge and Ag-Ge alloys, the twin dissociation of grain boundaries in Au, the formation of hexagonal Si at twin intersections and the effect of twin boundaries on the equilibrium shape of Pb inclusions in Al.

AML1 (CBFalpha2) cooperates with B cell-specific activating protein (BSAP/PAX5) in activation of the B cell-specific BLK gene promoter.

AML1 plays a critical role during hematopoiesis and chromosomal translocations involving AML1 are commonly associated with different forms of leukemia, including pre-B acute lymphoblastic leukemia. To understand the function of AML1 during B cell differentiation, we analyzed regulatory regions of B cell-specific genes for potential AML1-binding sites and have identified a putative AML1-binding site in the promoter of the B cell-specific tyrosine kinase gene, blk. Gel mobility shift assays and transient transfection assays demonstrate that AML1 binds specifically to this site in the blk promoter and this binding site is important for blk promoter activity. Furthermore, in vitro binding analysis revealed that the AML1 runt DNA-binding domain physically interacts with the paired DNA-binding domain of BSAP, a B cell-specific transcription factor. BSAP has been shown previously to be important for B cell-specific regulation of the blk gene. Physical interaction of AML1 with BSAP correlates with functional cooperativity in transfection studies where AML1 and BSAP synergistically activate blk promoter transcription by more than 50-fold. These results demonstrate physical and functional interactions between AML1 and BSAP and suggest that AML1 is an important factor for regulating a critical B cell-specific gene, blk.

Octamer binding factors and their coactivator can activate the murine PU.1 (spi-1) promoter.

PU.1 (spi-1), a member of the Ets transcription factor family, is predominantly expressed in myeloid and B cells, activates many B cell and myeloid genes, and is critical for development of both of these lineages. Our previous studies (Chen, H. M., Ray-Gallet, D., Zhang, P., Hetherington, C. J., Gonzalez, D. A., Zhang, D.-E., Moreau-Gachelin, F., and Tenen, D. G. (1995) Oncogene 11, 1549-1560) demonstrate that the PU.1 promoter directs cell type-specific reporter gene expression in myeloid cell lines, and that PU.1 activates its own promoter in an autoregulatory loop. Here we show that the murine PU.1 promoter is also specifically and highly functional in B cell lines as well. Oct-1 and Oct-2 can bind specifically to a site at base pair -55 in vitro, and this site is specifically protected in B cells in vivo. We also demonstrate that two other sites contribute to promoter activity in B cells; an Sp1 binding site adjacent to the octamer site, and the PU.1 autoregulatory site. Finally, we show that the B cell coactivator OBF-1/Bob1/OCA-B is only expressed in B cells and not in myeloid cells, and that OBF-1/Bob1/OCA-B can transactivate the PU.1 promoter in HeLa and myeloid cells. This B cell restricted coactivator may be responsible for the B cell specific expression of PU.1 mediated by the octamer site.

Absence of granulocyte colony-stimulating factor signaling and neutrophil development in CCAAT enhancer binding protein alpha-deficient mice.

Transcription factors are master regulatory switches of differentiation, including the development of specific hematopoietic lineages from stem cells. Here we show that mice with targeted disruption of the CCAAT enhancer binding protein alpha gene (C/EBP alpha) demonstrate a selective block in differentiation of neutrophils. Mature neutrophils and eosinophils are not observed in the blood or fetal liver of mutant animals, while other hematopoietic lineages, including monocytes, are not affected. Instead, most of the white cells in the peripheral blood of mutant mice had the appearance of myeloid blasts. We also observed a selective loss of expression of a critical gene target of CCAAT enhancer binding protein alpha, the granulocyte colony-stimulating factor receptor. As a result, multipotential myeloid progenitors from the mutant fetal liver are unable to respond to granulocyte colony-stimulating factor signaling, although they are capable of forming granulocyte-macrophage and macrophage colonies in methylcellulose in response to other growth factors. Finally, we demonstrate that the lack of granulocyte development results from a defect intrinsic to the hematopoietic system; transplanted fetal liver from mutant mice can reconstitute lymphoid but not neutrophilic cells in irradiated recipients. These studies suggest a model by which transcription factors can direct the differentiation of multipotential precursors through activation of expression of a specific growth factor receptor, allowing proliferation and differentiation in response to a specific extracellular signal. In addition, the c/ebp alpha -/- mice may be useful in understanding the mechanisms involved in acute myelogenous leukemia, in which a block in differentiation of myeloid precursors is a key feature of the disease.

Regulation of CD14 expression during monocytic differentiation induced with 1 alpha,25-dihydroxyvitamin D3.

CD14, a monocyte/macrophage receptor for the complex of LPS and LPS binding protein, is a differentiation marker for the monocyte/macrophage lineage. We have analyzed the regulation of CD14 expression during 1 alpha,25-dihydroxyvitamin D3 (VitD3)-induced monocytic differentiation. Using FACS, Northern blotting, and nuclear run-on analyses, we demonstrate that the up-regulation of CD14 expression during monocytic cell maturation is regulated mainly at the level of gene transcription, and that new protein synthesis is required for CD14 induction. We have recently cloned the CD14 5' upstream sequence and demonstrated its tissue-specific promoter activity. Using stable transfection of the monocytoid U937 cell line with a series of deletion mutants of the CD14 5' upstream sequence coupled to a reporter gene construct, we show that bp -128 to -70 is the critical region for the induction of CD14 expression. This region contains two binding sites for the Sp1 transcription factor. A 3-bp mutation at the distal Sp1-binding site not only eliminates Sp1 interaction, but also abolishes most of the VitD3 induction of CD14 expression. Electrophoretic mobility shift analysis does not detect a direct interaction of the CD14 distal Sp1-binding site with the vitamin D3 receptor and its partner, the retinoid X receptor. These data demonstrate that VitD3 induces CD14 indirectly through some intermediary factor, and suggest a critical role for Sp1 in this process.