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Biomedical subjects

M C Simon

Publications and source records attributed to M C Simon.

At least 37 records · Page 2Linked to original sources
PubMed1999

PU.1 and Spi-B are required for normal B cell receptor-mediated signal transduction.

PU.1 and Spi-B have previously been implicated in the regulation of genes encoding B cell receptor (BCR) signaling components. Spi-B-/- B lymphocytes respond poorly to BCR stimulation; PU.1-/- mice, however, lack B cells, precluding an analysis of BCR responses. We now show that PU.1+/- Spi-B-/- B cells exhibit more extensive defects than Spi-B-/- B cells, indicating that both PU.1 and Spi-B are required for normal BCR signaling. Strikingly, BCR cross-linking results in substantially reduced protein tyrosine phosphorylation in mutant B cells. Further analysis shows that Igalpha is phosphorylated and syk is recruited and becomes phosphorylated but that BLNK and PLCgamma phosphorylation are defective in mutant cells. Our data support the existence of a novel component coupling syk to downstream targets.

Animals↗
PubMed1998

Mitochondrial reactive oxygen species trigger hypoxia-induced transcription.

Transcriptional activation of erythropoietin, glycolytic enzymes, and vascular endothelial growth factor occurs during hypoxia or in response to cobalt chloride (CoCl2) in Hep3B cells. However, neither the mechanism of cellular O2 sensing nor that of cobalt is fully understood. We tested whether mitochondria act as O2 sensors during hypoxia and whether hypoxia and cobalt activate transcription by increasing generation of reactive oxygen species (ROS). Results show (i) wild-type Hep3B cells increase ROS generation during hypoxia (1. 5% O2) or CoCl2 incubation, (ii) Hep3B cells depleted of mitochondrial DNA (rho0 cells) fail to respire, fail to activate mRNA for erythropoietin, glycolytic enzymes, or vascular endothelial growth factor during hypoxia, and fail to increase ROS generation during hypoxia; (iii) rho0 cells increase ROS generation in response to CoCl2 and retain the ability to induce expression of these genes; and (iv) the antioxidants pyrrolidine dithiocarbamate and ebselen abolish transcriptional activation of these genes during hypoxia or CoCl2 in wild-type cells, and abolish the response to CoCl2 in rho degrees cells. Thus, hypoxia activates transcription via a mitochondria-dependent signaling process involving increased ROS, whereas CoCl2 activates transcription by stimulating ROS generation via a mitochondria-independent mechanism.

Adenosine Triphosphate↗
PubMed1998

Stabilization of wild-type p53 by hypoxia-inducible factor 1alpha.

Although hypoxia (lack of oxygen in body tissues) is perhaps the most physiological inducer of the wild-type p53 gene, the mechanism of this induction is unknown. Cells may detect low oxygen levels through a haem-containing sensor protein. The hypoxic state can be mimicked by using cobalt chloride and the iron chelator desferrioxamine: like hypoxia, cobalt chloride and desferrioxamine activate hypoxia-inducible factor 1alpha (HIF-1alpha), which stimulates the transcription of several genes that are associated with hypoxia. Here we show that these treatments induce accumulation of wild-type p53 through HIF-1alpha-dependent stabilization of p53 protein. Induction of p53 does not occur in either a mutant hepatoma cell line that is unable to induce HIF-1alpha or embryonic stem cells derived from mice lacking HIF-1beta. HIF-1alpha is found in p53 immunoprecipitates from MCF7 cells that express wild-type p53 and are either hypoxic or have been exposed to desferrioxamine. Similarly, anti-haemagglutinin immunoprecipitates from lysates of normoxic PC3M cells that had been co-transfected with haemagglutinin-tagged HIF-1alpha and wild-type p53 also contain p53. Transfection of normoxic MCF7 cells with HIF-1alpha stimulates a co-transfected p53-dependent reporter plasmid and increases the amount of endogenous p53. Our results suggest that hypoxic induction of transcriptionally active wild-type p53 is achieved as a result of the stabilization of p53 by its association with HIF-1alpha.

Animals↗
PubMed1998

PU.1 and hematopoiesis: lessons learned from gene targeting experiments.

The advent of gene targeting technology in mouse embryonic stem cells has revolutionized the study of the development of organ systems in mammals. Hematopoietic transcription factors play a critical role in blood cell development. Targeted mutagenesis of the murine PU.1 locus has revealed the pivotal role this protein plays in blood cell differentiation at all stages of hematopoiesis (yolk sac, fetal liver and bone marrow). PU.1 has been disrupted by two independent research groups and both strains of PU.1-deficient mice exhibit abnormalities in B cell, T cell, monocyte and neutrophil development. The independent mutations have yielded some differences in phenotype suggesting that the different strategies for gene targeting have resulted in the beginning of an 'allelic series' at the PU.1 locus. Both strains of PU.1-/- mice provide exciting reagents for future study of the role of this factor in blood lineage specification.

Animals↗
PubMed1998

Oxygen, genes, and development: an analysis of the role of hypoxic gene regulation during murine vascular development.

Development of the mammalian cardiovascular system is a complex process guided by both genetic and environmental components. Significant advances in the genetics of vascular development have been accomplished most recently by the analysis of multiple "knockout" and transgenic mice which exhibit varying degrees of impaired vascularity. This review focuses on the potential of the environment of the developing embryo to affect its development. In particular we analyze the evidence implicating the ability of physiological parameters such as oxygen and glucose concentrations within and surrounding the embryo to affect the expression of genes critical for vascular development. We conclude that the vascularization of a developing mammalian embryo is a plastic process dependent on the dynamic interaction between fundamental genetic and physiological factors.

Animals↗
PubMed1998

Normal myeloid development requires both the glutamine-rich transactivation domain and the PEST region of transcription factor PU.1 but not the potent acidic transactivation domain.

Gene targeting of transcription factor PU.1 results in an early block to fetal hematopoiesis, with no detectable lymphoid or myeloid cells produced in mouse embryos. Furthermore, PU.1(-/-) embryonic stem (ES) cells fail to differentiate into Mac-1(+) and F4/80(+) macrophages in vitro. We have previously shown that a PU.1 transgene under the control of its own promoter restores the ability of PU. 1(-/-) ES cells to differentiate into macrophages. In this study, we take advantage of our PU.1(-/-) ES cell rescue system to genetically test which previously identified PU.1 functional domains are necessary for the development of mature macrophages. PU.1 functional domains include multiple N-terminal acidic and glutamine-rich transactivation domains, a PEST domain, several serine phosphorylation sites, and a C-terminal Ets DNA binding domain, all delineated and characterized by using standard biochemical and transactivational assays. By using the production of mature macrophages as a functional readout in our assay system, we have established that the glutamine-rich transactivation domain, a portion of the PEST domain, and the DNA binding domain are required for myelopoiesis. Deletion of three acidic domains, which exhibit potent transactivation potential in vitro, had no effect on the ability of PU.1 to promote macrophage development. Furthermore, mutagenesis of four independent sites of serine phosphorylation also had no effect on myelopoiesis. Collectively, our results indicate that PU.1 interacts with important regulatory proteins during macrophage development via the glutamine-rich and PEST domains. The PU.1(-/-) ES cell rescue system represents a powerful, in vitro strategy to functionally map domains of PU.1 essential for normal hematopoiesis and the generation of mature macrophages.

3T3 Cells↗
PubMed1997

Defective B cell receptor-mediated responses in mice lacking the Ets protein, Spi-B.

Spi-B is a hematopoietic-specific Ets family transcription factor closely related to PU.1. Previous gene targeting experiments have shown that PU.1 is essential for the production of both lymphocytes and monocytes. We have now generated mice with a null mutation at the Spi-B locus. Unlike PU.1 mutant mice, Spi-B-/- mice are viable, fertile and possess mature B and T lymphocytes. However, Spi-B-/- mice exhibit severe abnormalities in B cell function and selective T cell-dependent humoral immune responses. First, although Spi-B-/- splenic B cells respond normally to lipopolysaccharide stimulation in vitro, these B cells proliferate poorly and die in response to B cell receptor (surface IgM) cross-linking. Secondly, Spi-B-/- mice display abnormal T-dependent antigenic responses in vivo and produce low levels of antigen-specific IgG1, IgG2a and IgG2b after immunization. Finally, Spi-B-/- mice show a dramatic defect in germinal center formation and maintenance. In contrast to wild-type animals, germinal centers in Spi-B-/- mice are smaller and short-lived with significantly increased numbers of apoptotic B cells. Taken together, these results demonstrate that Spi-B is essential for antigen-dependent expansion of B cells, T-dependent immune responses and maturation of normal germinal centers in vivo.

Animals↗
PubMed1997

Expression of two myeloid cell-specific genes requires the novel transcription factor, c-fes expression factor.

The protein product of the c-fes proto-oncogene has been implicated in the normal development of myeloid cells (macrophages and granulocytes). We have previously shown that 151 base pairs of c-fes 5'-flanking sequences are sufficient for myeloid cell-specific expression and include functional binding sites for Sp1, PU.1, and a novel nuclear factor (Heydemann, A., Juang, G., Hennessy, K., Parmacek, M. S., and Simon, M. C. (1996) Mol. Cell. Biol. 16, 1676-1686). This novel hematopoietic transcription factor, termed FEF (c-fes expression factor), binds to a cis-acting element that is located at nucleotides -9 to -4 of the c-fes promoter between two Ets binding sites (at -19 to -15 and -4 to +1) which bind PU.1. We now show that a FEF binding site exists in the myeloid cell-specific regulatory region of a second gene, the -2.7-kilobase pair enhancer of chicken lysozyme. The lysozyme FEF site is immediately 5' to a PU. 1 site, analogous to their arrangement in the c-fes promoter, and allows the formation of a preliminary FEF consensus site, 5'-GAAT(C/G)A-3'. This consensus site does not match any sites for known transcription factors. Importantly, although PU.1 binds immediately 3' of the FEF site in both the c-fes promoter and the chicken lysozyme enhancer (CLE), we show that they bind independently. The FEF sites are required for high levels of transcription by both the CLE and the c-fes promoter in transient transfection experiments. Importantly, elimination of the CLE FEF site abolishes all transcriptional activity of this enhancer element. Mutation of the adjacent PU.1 site in either the c-fes promoter or the CLE, reduces activity by approximately 50%. Therefore, transcription of both lysozyme and fes in myeloid cells requires FEF and PU.1. UV cross-linking experiments show that the FEF binding activity consists of a single 70-kDa protein in both human and murine cell lines. FEF binding activity is not affected by antibodies that specifically recognize a number of cloned transcription factors. Collectively, these data indicate that we have identified a novel transcription factor that is functionally important for the expression of at least two myeloid cell-specific genes.

Animals↗
PubMed1997

Abnormal angiogenesis and responses to glucose and oxygen deprivation in mice lacking the protein ARNT.

The arylhydrocarbon-receptor nuclear translocator (ARNT) is a member of the basic-helix-loop-helix-PAS family of heterodimeric transcription factors which includes the arylhydrocarbon receptor (AHR), hypoxia-inducible factor-1alpha (HIF-1alpha) and the Drosophila single-minded protein (Sim). ARNT forms heterodimeric complexes with the arylhydrocarbon receptor, HIF-1alpha, Sim and the PAS protein Per. In response to environmental pollutants, AHR-ARNT heterodimers regulate genes involved in the metabolism of xenobiotics, whereas ARNT-HIF-1alpha heterodimers probably regulate those involved in the response to oxygen deprivation. By generating a targeted disruption of the Arnt locus in the mouse, we show here that Arnt-/- embryonic stem cells fail to activate genes that normally respond to low oxygen tension. Arnt-/- ES cells also failed to respond to a decrease in glucose concentration, indicating that ARNT is crucial in the response to hypoxia and to hypoglycaemia. Arnt-/- embryos were not viable past embryonic day 10.5 and showed defective angiogenesis of the yolk sac and branchial arches, stunted development and embryo wasting. The defect in blood vessel formation in Arnt-/- yolk sacs is similar to the angiogenic abnormalities reported for mice deficient in vascular endothelial growth factor or tissue factor. On the basis of these findings, we propose a model in which increasing tissue mass during organogenesis leads to the formation of hypoxic/nutrient-deprived cells, the subsequent activation of ARNT, and a concomitant increase in the expression of genes (including that encoding vascular endothelial growth factor) that promote vascularization of the developing yolk sac and solid tissues.

Animals↗
PubMed1997

Developmental analysis and subcellular localization of the murine homologue of ELL.

The ELL gene was first identified by its involvement with MLL in the translocation (11;19)(q23;p13.1) in acute myeloid leukemia. To date, nine other MLL partner genes have been cloned, but their precise functions have yet to be determined. To characterize the functions of ELL further, we have cloned the murine homologue of ELL and have found that the gene is highly conserved at the nucleotide and amino acid level. The open reading frame of the murine homologue contains 602 aa, slightly smaller than the 621 aa in the human gene. With Northern blot analysis, a 3.4-kb transcript is detected in all tissues examined with greatest levels of expression in the liver. Unlike human ELL, only a single transcript can be detected with either murine coding sequence or 3' untranslated region probes. To examine the spatial and temporal pattern of expression in murine development, in situ hybridization studies were performed with sense and antisense riboprobes from the 3' untranslated region of murine Ell. Ell is expressed diffusely by embryonic day 7.5 (E7.5). In addition, high levels of expression can be detected in maternally derived decidual tissue. At E14.5, Ell is expressed diffusely throughout the embryo. However by E16.5, specific expression in the liver and gastrointestinal tract becomes prominent and remains so in both neonates and adults. To determine the subcellular localization of ELL, we developed a polyclonal antiserum to ELL that was used for immunofluorescence studies in COS-7, HeLa, NIH 3T3, and A7r5 cells. The ELL protein was localized to the nucleus but excluded from nucleoli in all cell lines examined. Recently, the gene product of ELL was found to function as an RNA polymerase II elongation factor, an activity that is consistent with our immunofluorescence data. Thus, these studies extend our understanding of the normal functions of ELL and provide additional insight into its aberrant function when fused to MLL in acute myeloid leukemia.

Acute Disease↗
PubMed1997

A Candida albicans genome project: cosmid contigs, physical mapping, and gene isolation.

A new project to map the genome of the pathogenic fungus, Candida albicans, has been started. The entire genome was cloned as 5088 cosmids, stored in individual microtiter plate wells. DNA was prepared and fingerprinted using restriction digestion, fluorescent labeling, and analysis on an ABI sequencer. These data are being used to construct contigs of the genome. Simultaneously, a DNA pooling system has been set up, suitable for PCR-based isolation of cosmids containing any known gene. Ultimately, these approaches will lead to the creation of a physically based map of the C. albicans genome, providing the means to localize precisely all the genes, act as a substrate for genome sequencing projects, and provide probes for future studies of genome rearrangement and comparative genomics.

Candida albicans↗
PubMed1997

PU.1 functions in a cell-autonomous manner to control the differentiation of multipotential lymphoid-myeloid progenitors.

Transcription factor PU.1 is required for the development of lymphoid and myeloid progenitors during fetal hematopoiesis. By generating chimeric animals using PU.1-/- ES cells or PU.1(-/-) hematopoietic progenitors, we demonstrate that PU.1 functions in an exclusively cell-autonomous manner to regulate the development of the lymphoid-myeloid system. Multipotential lymphoid-myeloid progenitors (AA4.1+, Lin-) are significantly reduced in PU.1(-/-) embryos and fail to differentiate into B lymphoid or myeloid cells in vitro. These results suggest that the lymphoid and myeloid lineages develop in the fetal liver from a common hematopoietic progenitor not shared with erythrocytes and megakaryocytes. Finally, the Ikaros gene is expressed in PU.1 mutant embryos, suggesting that PU.1 and Ikaros are independently required for specification of embryonic lymphoid cell fates.

Animals↗
PubMed1996

The Ets protein Spi-B is expressed exclusively in B cells and T cells during development.

Spi-B and PU.1 are hematopoietic-specific transcription factors that constitute a subfamily of the Ets family of DNA-binding proteins. Here we show that contrary to previous reports, PU.1 and Spi-B have very different expression patterns. PU.1 is expressed at high levels in B cells, mast cells, megakaryocytes, macrophages, neutrophils, and immature erythroid cells and at lower levels in mature erythrocytes. PU.1 is completely absent from peripheral T cells and most T cell lines based on sensitive RT-PCR assays. In contrast, Spi-B is expressed exclusively in lymphoid cells and can be detected in early fetal thymus and spleen. In situ hybridizations of adult murine tissues demonstrate Spi-B mRNA in the medulla of the thymus, the white pulp of the spleen, and the germinal centers of lymph nodes. Spi-B expression is very abundant in B cells and both Spi-B mRNA and protein are detected in some T cells. In situ hybridization and Northern blot analysis suggest that Spi-B gene expression increases during B cell maturation and decreases during T cell maturation. Gel-retardation experiments show that Spi-B can bind to all putative PU.1 binding sites, but do not reveal any preferred Spi-B binding site. Finally, both PU.1 and Spi-B function as transcriptional activators of the immunoglobulin light-chain enhancer E lambda 2.4 when coexpressed with Pip (PU.1-interaction partner) in NIH-3T3 cells. Taken together, these data suggest that differences in patterns of expression between Spi-B and PU.1 distinguish the function of each protein during development of the immune system.

B-Lymphocytes↗
PubMed1996

Characterization of a murine Ahr null allele: involvement of the Ah receptor in hepatic growth and development.

The Ah receptor (AHR) is a ligand-activated transcription factor that mediates a pleiotropic response to environmental contaminants such as benzo[a]pyrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin. In an effort to gain insight into the physiological role of the AHR and to develop models useful in risk assessment, gene targeting was used to inactivate the murine Ahr gene by homologous recombination. Ahr-/- mice are viable and fertile but show a spectrum of hepatic defects that indicate a role for the AHR in normal liver growth and development. The Ahr-/- phenotype is most severe between 0-3 weeks of age and involves slowed early growth and hepatic defects, including reduced liver weight, transient microvesicular fatty metamorphosis, prolonged extramedullary hematopoiesis, and portal hypercellularity with thickening and fibrosis.

Alleles↗
PubMed1996

Targeted disruption of the mouse Stat1 gene results in compromised innate immunity to viral disease.

The STAT1 transcription factor is activated in response to many cytokines and growth factors. To study the requirement for STAT1 in vivo, we disrupted the Stat1 gene in embryonic stem (ES) cells and in mice. Stat1(-1-)ES cells were unresponsive to interferon (IFN), but retained responsiveness to leukemia inhibitory factor (LIF) and remained LIF dependent for undifferentiated growth. Stat1(-1-1) animals were born at normal frequencies and displayed no gross developmental defects. However, these animals failed to thrive and were extremely susceptible to viral disease. Cells and tissues from Stat1(-1-) mice were unresponsive to IFN, but remained responsive to all other cytokines tested. Thus, STAT1 appears to be specific for IFN pathways that are essential for viability in the face of otherwise innocuous pathogens.

Animals↗
PubMed1996

DNA Extraction and PCR Methods for the Detection of Listeria monocytogenes in Cold-Smoked Salmon.

Protocols for the specific detection of Listeria monocytogenes in cold-smoked salmon were developed. PCR was used as the method of detection. Inhibitors of PCR present in the food samples were removed by ether extraction or column purification, or their effect was overcome by the use of Tween 20 as an enhancer. These protocols are many times more rapid than conventional detection methodologies and also have the potential for automation.

Journal Article↗