Biomedical subjects

M C Simon

Publications and source records attributed to M C Simon.

At least 55 records · Page 3Linked to original sources

PubMed1996

The myeloid-cell-specific c-fes promoter is regulated by Sp1, PU.1, and a novel transcription factor.

The protein product of the c-fps/fes (c-fes) proto-oncogene has been implicated in the normal development of myeloid cells (macrophages and neutrophils). mRNA for c-fes has been detected exclusively in myeloid cells and vascular endothelial cells in adult mammals. Although a 13-kilobase-pair (kb) human c-fes transgene exhibits high levels of expression in mice, the sequences that confer myeloid-cell-specific expression of the human c-fes gene have not been defined. Transient-transfection experiments demonstrated that plasmids containing 446 bp of c-fes 5'-flanking sequences linked to a luciferase reporter gene were active exclusively in myeloid cells. No other DNA element within the 13-kb human c-fes locus contained positive cis-acting elements, with the exception of a weakly active region within the 3'-flanking sequences. DNase I footprinting assays revealed four distinct sites that bind myeloid nuclear proteins (-408 to -386, -293 to -254, -76 to -65, and -34 to +3). However, the first two footprints resided in sequences that were largely dispensable for transient activity. Plasmids containing 151 bp of 5'-flanking sequences confer myeloid-cell-specific gene expression. Electrophoretic mobility shift analyses demonstrated that the 151-bp region contains nuclear protein binding sites for Sp1, PU.1, and/or Elf-1, and a novel factor. This unidentified factor binds immediately 3' of the PU.1/Elf-1 sites and appears to be myeloid cell specific. Mutation of the PU.1/Elf-1 site or the 3' site (FP4-3') within the context of the c-fes promoter resulted in substantially reduced activity in transient transfections. Furthermore, transient-cotransfection assay demonstrated that PU.1 (and not Elf-1) can transactivate the c-fes promoter in nonmyeloid cell lines. We conclude that the human c-fes gene contains a strong myeloid-cell-specific promoter that is regulated by Sp1, PU.1, and a novel transcription factor.

Animals↗

PubMed1996

Cloning, tissue expression, and chromosomal localization of SUR2, the putative drug-binding subunit of cardiac, skeletal muscle, and vascular KATP channels.

ATP-sensitive inwardly rectifying potassium channels are expressed in a variety of tissues, including heart, skeletal, and smooth muscle, and pancreatic beta-cells. Physiological and pharmacological studies suggest the presence of distinct KATP channels in these tissues. Recently, the KATP channel of beta-cells has been reconstituted in functional form by coexpression of SUR, the sulfonylurea-binding protein, and the inwardly rectifying K+ channel subunit, KIR6.2. In this article, we describe the isolation of cDNAs encoding SUR-like proteins from mouse, SUR2A and SUR2B. Northern blotting showed that the highest expression of the SUR2 isoforms is in the heart and skeletal muscle, with lower levels in all other tissues. By reverse transcription-polymerase chain reaction, SUR2B is ubiquitously expressed, while the apparently alternatively spliced variant, SUR2A, is expressed exclusively in heart. In situ hybridization shows that the SUR2 isoforms are expressed in the parenchyma of the heart and skeletal muscle and in the vascular structures of other tissues. Human SUR2 was localized to chromosome 12, p12.1 by fluorescent in situ hybridization. The structure of the predicted protein and expression pattern of SUR2 suggests that it is the drug-binding channel-modulating subunit of the extrapancreatic KATP channel. Differences in sequence between SUR and between SUR2 isoforms may underlie the tissue-specific pharmacology of the KATP channel.

Amino Acid Sequence↗

PubMed1996

Water quality parameters associated with Aeromonas spp-affected hatcheries.

This study investigates the role of water quality parameters as possible risk or protection factors in Aeromonas spp-affected rainbow trout hatcheries in northeastern Spain. The results revealed an association between oxygen concentration, ammonia concentration and total dissolved solids (TDS) with the prevalence of Aeromonas spp cases on the affected fish farms. The oxygen and ammonia concentrations acted as risk factors when their values were lower than 7.06 mg/L or higher than 0.05 mg/L, respectively, generating odds ratio (OR) values of 2.0 and 2.3. TDS levels, however, acted as a protective factor (OR = 0.39), from an epidemiological point of view but not statistically, when the values were outside the normal range, 199-261 mg/L. On a multivariate level, fish farms exposed to all the associated factors at the same time, had a risk of being affected by Aeromonas spp that was 1.74 times higher than farms with normal quality parameters; the risk increased when TDS had a normal value and it decreased when oxygen and/or ammonia concentrations had normal values.

Aeromonas↗

PubMed1995

PU. 1 is not essential for early myeloid gene expression but is required for terminal myeloid differentiation.

We have previously shown using gene targeting that PU.1 is essential for the development of lymphoid and myeloid lineages during fetal liver hematopoiesis. We now show that PU.1 is required for the maturation of yolk sac-derived myeloid progenitors and for the differentiation of ES cells into macrophages. The role of PU.1 in regulating target genes, thought to be critical in the development of monocytes and granulocytes, has been analyzed. Early genes such as GM-CSFR, G-CSFR, and myeloperoxidase are expressed in PU.1-/- embryos and differentiated PU.1-/- ES cells. However, the expression of genes associated with terminal myeloid differentiation (CD11b, CD64, and M-CSFR) is eliminated in differentiated PU.1-/- ES cells. Development of macrophages is restored with the introduction of a PU.1 cDNA regulated by its own promoter. The PU.1-/- ES cells represent an important model for analyzing myeloid cell development.

Animals↗

PubMed1995

Gotta have GATA.

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Animals↗

PubMed1995

Rescue of GATA-1-deficient embryonic stem cells by heterologous GATA-binding proteins.

Totipotent murine embryonic stem (ES) cells can be differentiated in vitro to form embryoid bodies (EBs) containing hematopoietic cells of multiple lineages, including erythroid cells. In vitro erythroid development parallels that which is observed in vivo. ES cells in which the gene for the erythroid transcription factor GATA-1 has been disrupted fail to produce mature erythroid cells either in vivo or in vitro. With the EB in vitro differentiation assay, constructs expressing heterologous GATA-binding proteins were tested for their abilities to correct the developmental defect of GATA-1-deficient ES cells. The results presented here show that the highly divergent chicken GATA-1 can rescue GATA-1 deficiency to an extent similar to that of murine GATA-1 (mGATA-1), as determined by size and morphology of EBs, presence of red cells, and globin gene expression. Furthermore, GATA-3 and GATA-4, which are normally expressed in different tissues, and a protein consisting of the zinc fingers of GATA-1 fused to the herpes simplex virus VP16 transcription activation domain were able to compensate for the GATA-1 defect. Chimeric molecules in which both zinc fingers of mGATA-1 were replaced with the zinc fingers of human GATA-3 or with the single finger of the fungal GATA factor areA, as well as a construct bearing the zinc finger region alone, displayed rescue activity. These results suggest that neither the transcription activation domains of mGATA-1 nor its zinc fingers impart erythroid cell specificity for its action in vivo. Rather, it appears that specificity is mediated through the cis-acting control regions which determine spatial and temporal expression of the GATA-1 gene. Furthermore, our results demonstrate that the zinc finger region may have a biological function in addition to mediating DNA binding.

Amino Acid Sequence↗

PubMed1995

Development of hematopoietic cells lacking transcription factor GATA-1.

GATA-1 is a zinc-finger transcription factor believed to play an important role in gene regulation during the development of erythroid cells, megakaryocytes and mast cells. Other members of the GATA family, which can bind to the same DNA sequence motif, are co-expressed in several of these hemopoietic lineages, raising the possibility of overlap in function. To examine the specific roles of GATA-1 in hematopoietic cell differentiation, we have tested the ability of embryonic stem cells, carrying a targeted mutation in the X-linked GATA-1 gene, to contribute to various blood cell types when used to produce chimeric embryos or mice. Previously, we reported that GATA-1- mutant cells failed to contribute to the mature red blood cell population, indicating a requirement for this factor at some point in the erythroid lineage (L. Pevny et al., (1991) Nature 349, 257-260). In this study, we have used in vitro colony assays to identify the stage at which mutant erythroid cells are affected, and to examine the requirement for GATA-1 in other lineages. We found that the development of erythroid progenitors in embryonic yolk sacs was unaffected by the mutation, but that the cells failed to mature beyond the proerythroblast stage, an early point in terminal differentiation. GATA-1- colonies contained phenotypically normal macrophages, neutrophils and megakaryocytes, indicating that GATA-1 is not required for the in vitro differentiation of cells in these lineages. GATA-1- megakaryocytes were abnormally abundant in chimeric fetal livers, suggesting an alteration in the kinetics of their formation or turnover. The lack of a block in terminal megakaryocyte differentiation was shown by the in vivo production of platelets expressing the ES cell-derived GPI-1C isozyme. The role of GATA-1 in mast cell differentiation was examined by the isolation of clonal mast cell cultures from chimeric fetal livers. Mutant and wild-type mast cells displayed similar growth and histochemical staining properties after culture under conditions that promote the differentiation of cells resembling mucosal or serosal mast cells. Thus, the mast and megakaryocyte lineages, in which GATA-1 and GATA-2 are co-expressed, can complete their maturation in the absence of GATA-1, while erythroid cells, in which GATA-1 is the predominant GATA factor, are blocked at a relatively early stage of maturation.

Animals↗

PubMed1995

Targeted mutagenesis of the transcription factor GATA-4 gene in mouse embryonic stem cells disrupts visceral endoderm differentiation in vitro.

Transcription factor GATA-4 belongs to a family of zinc finger proteins involved in lineage determination. GATA-4 is first expressed in yolk sac endoderm of the developing mouse and later in cardiac tissue, gut epithelium and gonads. To delineate the role of this transcription factor in differentiation and early development, we studied embryoid bodies derived from mouse embryonic stem (ES) cells in which both copies of the Gata-4 gene were disrupted. Light and electron microscopy demonstrated that embryoid bodies formed from wild-type and heterozygous deficient ES cells were covered with a layer of visceral yolk sac endoderm, whereas no yolk sac endoderm was evident on the surface of the homozygous deficient embryoid bodies. Independently selected homozygous deficient cell lines displayed this distinctive phenotype, suggesting that it was not an artifact of clonal variation. Biochemical markers of visceral endoderm formation, such as alpha-feto-protein, hepatocyte nuclear factor-4 and binding sites for Dolichos biflorus agglutinin, were absent from the homozygous deficient embryoid bodies. Examination of other differentiation markers in the mutant embryoid bodies, studies of ES cell-derived teratocarcinomas and chimeric mouse analysis demonstrated that GATA-4-deficient ES cells have the capacity to differentiate along other lineages. We conclude that, under in vitro conditions, disruption of the Gata-4 gene results in a specific block in visceral endoderm formation. These homozygous deficient cells should yield insights into the regulation of yolk sac endoderm development and the factors expressed by visceral endoderm that influence differentiation of adjoining ectoderm/mesoderm.

Animals↗

PubMed1994

Cloning of ELL, a gene that fuses to MLL in a t(11;19)(q23;p13.1) in acute myeloid leukemia.

To characterize the functions of MLL fusion transcripts, we cloned the gene that fuses to MLL in the translocation t(11;19)(q23;p13.1). This translocation is distinct from another type of 11;19 translocation with a 19p13.3 breakpoint that results in the fusion of MLL to the ENL gene. By PCR screening of a cDNA library prepared from a patient's leukemia cells with this translocation, we obtained a fusion transcript containing exon 7 of MLL and sequence of an unknown gene. The sequence of this gene was amplified and used as a probe to screen a fetal brain cDNA library. On Northern blot analysis, this cDNA detected a 4.4-kb transcript that was abundant in peripheral blood leukocytes, skeletal muscle, placenta, and testis and expressed at lower levels in spleen, thymus, heart, brain, lung, kidney, liver, and ovary. In addition, a 2.8-kb transcript was present in peripheral blood, testis, and placenta. On "zoo blots," this gene was shown to be evolutionarily conserved in 10 mammalian species as well as in chicken, frog, and fish. We have named this gene ELL (for eleven-nineteen lysine-rich leukemia gene). A highly basic, lysine-rich motif of the predicted ELL protein is homologous to similar regions of several proteins, including the DNA-binding domain of poly(ADP-ribose) polymerase. The characterization of the normal functions of ELL as well as its altered function when fused to MLL will be critical to further our understanding of the mechanisms of leukemogenesis.

Acute Disease↗

PubMed1994

Requirement of transcription factor PU.1 in the development of multiple hematopoietic lineages.

The transcription factor PU.1 is a hematopoietic-specific member of the ets family. Mice carrying a mutation in the PU.1 locus were generated by gene targeting. Homozygous mutant embryos died at a late gestational stage. Mutant embryos produced normal numbers of megakaryocytes and erythroid progenitors, but some showed an impairment of erythroblast maturation. An invariant consequence of the mutation was a multilineage defect in the generation of progenitors for B and T lymphocytes, monocytes, and granulocytes. Thus, the developmental programs of lymphoid and myeloid lineages require a common genetic function likely acting at the level of a multipotential progenitor.

Animals↗

PubMed1994

The GATA-4 transcription factor transactivates the cardiac muscle-specific troponin C promoter-enhancer in nonmuscle cells.

The unique contractile phenotype of cardiac myocytes is determined by the expression of a set of cardiac muscle-specific genes. By analogy to other mammalian developmental systems, it is likely that the coordinate expression of cardiac genes is controlled by lineage-specific transcription factors that interact with promoter and enhancer elements in the transcriptional regulatory regions of these genes. Although previous reports have identified several cardiac muscle-specific transcriptional elements, relatively little is known about the lineage-specific transcription factors that regulate these elements. In this report, we demonstrate that the slow/cardiac muscle-specific troponin C (cTnC) enhancer contains a specific binding site for the lineage-restricted zinc finger transcription factor GATA-4. This GATA-4-binding site is required for enhancer activity in primary cardiac myocytes. Moreover, the cTnC enhancer can be transactivated by overexpression of GATA-4 in non-cardiac muscle cells such as NIH 3T3 cells. In situ hybridization studies demonstrate that GATA-4 and cTnC have overlapping patterns of expression in the hearts of postimplantation mouse embryos and that GATA-4 gene expression precedes cTnC expression. Indirect immunofluorescence reveals GATA-4 expression in cultured cardiac myocytes from neonatal rats. Taken together, these results are consistent with a model in which GATA-4 functions to direct tissue-specific gene expression during mammalian cardiac development.

3T3 Cells↗

PubMed1993

Survey of rotavirus infection in a dairy herd: comparison between polyacrylamide gel electrophoresis and two commercial tests.

A survey of rotavirus infection in a dairy herd with a history of neonatal diarrhoea was carried out. Faecal samples taken from 15 cows before and after calving as well as faeces taken from their calves daily from birth to two weeks of life were tested for rotavirus by polyacrylamide gel electrophoresis (PAGE) and compared with an ELISA and a latex agglutination commercial test. Rotavirus excretion was not detected in faeces from cows around parturition by any of the three tests. However, all of their calves shed rotaviruses during the observation period. The onset of rotavirus excretion determined by PAGE ranged from day 2 to day 8 of life (day 4.8 +/- 1.8 on average) and lasted for 4 to 7 days (5.3 +/- 1.1 days on average). Chi-square test showed a significant association (P = 0.0001) between the presence of rotavirus and the altered consistency of calves faeces. All the three tests showed similar results (overall agreement 92.5%) but discrepancies were detected mainly at the beginning or at the end of the rotavirus excretion period. Results obtained with both commercial kits closely paralleled each other and parameters other than sensitivity, specificity, diagnostic accuracy or predictive values have to be considered as selection criteria.

Animals↗

PubMed1993

Mouse GATA-4: a retinoic acid-inducible GATA-binding transcription factor expressed in endodermally derived tissues and heart.

We report the cDNA cloning and characterization of mouse GATA-4, a new member of the family of zinc finger transcription factors that bind a core GATA motif. GATA-4 cDNA was identified by screening a 6.5-day mouse embryo library with oligonucleotide probes corresponding to a highly conserved region of the finger domains. Like other proteins of the family, GATA-4 is approximately 50 kDa in size and contains two zinc finger domains of the form C-X-N-C-(X17)-C-N-X-C. Cotransfection assays in heterologous cells demonstrate that GATA-4 trans activates reporter constructs containing GATA promoter elements. Northern (RNA) analysis and in situ hybridization show that GATA-4 mRNA is expressed in the heart, intestinal epithelium, primitive endoderm, and gonads. Retinoic acid-induced differentiation of mouse F9 cells into visceral or parietal endoderm is accompanied by increased expression of GATA-4 mRNA and protein. In vitro differentiation of embryonic stem cells into embryoid bodies is also associated with increased GATA-4 expression. We conclude that GATA-4 is a tissue-specific, retinoic acid-inducible, and developmentally regulated transcription factor. On the basis of its tissue distribution, we speculate that GATA-4 plays a role in gene expression in the heart, intestinal epithelium, primitive endoderm, and gonads.

Amino Acid Sequence↗

PubMed1993

Transcription factor GATA-1 and erythroid development.

In summary, we derived an experimental system that allows us to dissect the function of GATA-1 in red cell development at a genetic level. We have established the essential nature of GATA-1 during both primitive and definitive erythropoiesis. By ablating the expression of the endogenous GATA-1 gene, we are in a position to introduce a variety of constructs that harbor subtle modifications in flanking or protein-coding sequences. We can now study regulatory regions and functional domains of the protein in the context of a true erythroid environment, experiments that have not been possible heretofore. Although the assay involves the dramatic loss of red cell production, it should be possible to define important regulatory domains that can then be assayed using less stringent systems, such as cell-free extracts for in vitro transcription. The ideal situation would be analyses conducted in GATA-1- erythroid cells. However, these cells have been impossible to generate given the requirement of GATA-1 for Epo receptor expression and red cell viability (C. Simon and S. Orkin, unpublished observations). It may be possible to produce such cells by first expressing the Epo receptor under the influence of a constitutive promoter and then targeting the GATA-1 gene. If GATA-1- red cells were available, the analyses would involve the actual transcription of or chromatin structure surrounding the globin genes. Structure-function studies of the GATA-1 protein could be greatly simplified and a larger number of mutants studied. However, the ES cell system can be used as an alternative until targeted erythroleukemia cells become available. Other applications involve the introduction of other GATA-binding protein family members to determine whether they rescue the mutation. If they cannot, chimeric proteins can be tested to identify which amino acids distinguish the different family members. We feel that these experiments are vital to understanding the function of GATA-1 during erythroid ontogeny. How does GATA-1 regulate red cell genes like globin or the Epo receptor? Once we identify the functional domains of the GATA-binding proteins, we hope to learn what proteins GATA-1 binds to in the basic transcription machinery or in chromatin. Is GATA-1 necessary for globin gene switching? GATA-1 may be modified differently during development so that the locus control region can interact with different globin promoters. We may find that one region of the protein is required for embryonic expression and another for adult globin gene expression.

Amino Acid Sequence↗

PubMed1992

The effect of experimental infection with Corynebacterium pseudotuberculosis on reproduction in adult ewes.

Fifteen ewes were inoculated subcutaneously with 2 x 10(6) cells of Corynebacterium pseudotuberculosis seven days before mating, during the embryonic stage of gestation and during the fetal stage of gestation. The clinical signs, antibody response and the consequences of the infection on reproduction were studied. None of the ewes showed any change in general condition during pregnancy. The effects of experimental infection during gestation were evident after incubation periods that ranged from 25 to 140 days after inoculation and were dependent on the time of the inoculation in relation to the period of gestation. Ewes inoculated before gestation can resolve infection. Ewes inoculated during the embryonic stage were severely affected and some aborted. In others lambs were stillborn or, if born alive, infected. Ewes inoculated during the fetal stage of gestation did not show reproductive disorders although some remained chronically infected.

Abortion, Veterinary↗

PubMed1992

Rescue of erythroid development in gene targeted GATA-1- mouse embryonic stem cells.

Development of definitive (fetal liver-derived) red cells is blocked by a targeted mutation in the gene encoding the transcription factor GATA-1. We used in vitro differentiation of GATA-1- mouse embryonic stem (ES) cells to reveal a requirement for GATA-1 during primitive (yolk sac-derived) erythropoiesis and to establish a rescue assay. We show that the block to development includes primitive, as well as definitive, erythroid cells and is complete at the level of globin RNA expression; that the introduction of a normal GATA-1 gene restores developmental potential both in vivo and in vitro; and that efficient rescue is dependent on a putative autoregulatory GATA-motif in the distal promoter. Use of in vitro differentiated ES cells bridges a gap between conventional approaches to gene function in cell lines and analysis of loss of function mutations in the whole animal.

Animals↗

PubMed1991

Erythroid differentiation in chimaeric mice blocked by a targeted mutation in the gene for transcription factor GATA-1.

The zinc-finger transcription factor GATA-1 (previously known as GF-1, NF-E1 or Eryf 1 binds to GATA consensus elements in regulatory regions of the alpha- and beta-globin gene clusters and other erythroid cell-specific genes. Analysis of the effects of mutations in GATA-binding sites in cell culture and in binding assays in vitro, as well as transactivation studies with GATA-1 expression vectors in heterologous cells, have provided indirect evidence that this factor is involved in the activation of globin and other genes during erythroid cell maturation. GATA-1 is also expressed in megakaryocytes and mast cells, but not in other blood cell lineages or in non-haemopoietic cells. To investigate the role of this factor in haematopoiesis in vivo, we disrupted the X-linked GATA-1 gene by homologous recombination in a male (XY) murine embryonic stem cell line and tested the GATA-1-deficient cells for their ability to contribute to different tissues in chimaeric mice. The mutant embryonic stem cells contributed to all non-haemopoietic tissues tested and to a white blood cell fraction, but failed to give rise to mature red blood cells. This demonstrates that GATA-1 is required for the normal differentiation of erythroid cells, and that other GATA-binding proteins cannot compensate for its absence.

Animals↗

PubMed1990

E1A-dependent trans-activation of the c-fos promoter requires the TATAA sequence.

Previous experiments have demonstrated that transcription of the human c-fos oncogene is activated through the action of the 289-amino acid adenovirus E1A gene product. In this study we have utilized a series of c-fos promoter deletion and substitution mutants to define regulatory sequences that allow the induction by E1A. Although the deletion of upstream promoter sequences has varying degrees of effect on overall promoter activity, these deletions retain inducibility by E1A. This includes the deletion of the serum response element and two elements that bind the ATF transcription factor. In fact, a c-fos promoter deleted to position -53, which leaves the TATA element but no other known functional element, retains inducibility, indicating a role for the TATA element in E1A control. Indeed, substitution of the c-fos TATA element (TATAA) with a TATA sequence from the simian virus 40 early promoter (TATTTAT) abolishes E1A inducibility; this promoter does retain responsiveness to cAMP induction, however, demonstrating that this TATTTAT substitution is functional. We conclude that the E1A-dependent activation of c-fos transcription is mediated through an effect on a TATA-binding protein that has specificity for the TATAA sequence.

Adenovirus Early Proteins↗