M Y Fiszman

National Institute on Aging, Baltimore, Maryland, United States

Are you M Y Fiszman?

Claim your profile

Publications (64)270.52 Total impact

  • A M Pret · Laurent Balvay · Marc Y. Fiszman
    [Show abstract] [Hide abstract]
    ABSTRACT: Alternative splicing of chicken beta-tropomyosin (beta-TM) pre-mRNAs ensures that in nonmuscle cells, only exon 6A is expressed, whereas in skeletal muscle, exon 6B is utilized preferentially. We have previously shown that efficient splicing of the nonmuscle exon 6A requires two pyrimidine-rich splicing enhancers (S4 and I5Y) that are present in the introns flanking exon 6A. Here, we examined the function of the S4 and I5Y elements by replacing them within beta-TM minigenes by other pyrimidine- and purine-rich sequence elements and analyzing splicing in transfected quail nonmuscle and muscle cells. Several features of these splicing regulatory elements were revealed by this study. First, a wide variety of pyrimidine-rich sequences can replace the intronic S4 splicing enhancer, indicating that pyrimidine composition, rather than sequence specificity, determines activity for this element. Second, one type of purine-rich sequence (GARn), normally found within exons, can also replace the S4 splicing enhancer. Third, the diverse elements tested exhibit differential activation of the splice sites flanking exon 6A and different positional constraints. Fourth, the strength of the S4 splicing enhancer is appropriately set to obtain proper regulation of the transition from exon 6A splicing in myoblasts to exon 6B splicing in myotubes, but this splicing regulatory element is not the target for cell-type-specific splicing factors.
    No preview · Article · Oct 1999 · DNA and Cell Biology
  • Kenneth R. Boheler · Marc Y. Fiszman
    [Show abstract] [Hide abstract]
    ABSTRACT: Today's most urgent problem in transplantation is the lack of suitable donor organs and tissues and as the population ages, demands for organs and tissue therapies will only increase. One alternative to organ transplantation is cell therapy whose aim is to replace, repair or enhance the biological function of damaged tissue or diseased organs. One goal of cellular transplantation thus has been to find a renewable source of cells that could be used in humans. Embryonic stem (ES) cells have the potential to proliferate in vitro in an undifferentiated and pluripotent state. Theoretically, ES cells are capable of unlimited proliferation in vitro. ES cells spontaneously differentiate into derivatives of all three primary germ layers: endoderm, ectoderm and mesoderm, hence providing cells in vitro which can theoretically be isolated and used for transplantation. Furthermore, these pluripotent stem cells can potentially be used to produce large numbers of cells that can be genetically modified in vitro. Once available, this source of cells may obviate some of the critical needs for organ transplantation. Murine ES cells have been extensively studied and all available evidence indicates that all aforementioned expectations are indeed fulfilled by ES cells. ES cells as well as embryonic germ cells have recently been isolated and maintained in culture. The recent descriptions of human ES cells portend the eventual use of allogeneic in vitro differentiated cells for human therapy. This goal, however, is fraught with obstacles. Our aim is first to review the recent advances made with murine ES cells and then to point out potentials and difficulties associated with the use of human ES cells for transplantation.
    No preview · Article · Feb 1999 · Cells Tissues Organs
  • [Show abstract] [Hide abstract]
    ABSTRACT: In mammalian myocardium, relaxation is mainly triggered by the reuptake of calcium from the cytosol to the lumen of the sarcoplasmic reticulum (SR) through the cardiac isoform of the sarco(endo)plasmic reticulum calcium ATPase, SERCA2a. Relaxation abnormalities related to deficient SR Ca(2+)-uptake have been identified in human heart failure and in animal models of cardiac hypertrophy and failure. These alterations have been associated with a reduction in SERCA2a activity and in steady-state SERCA2a protein and mRNA levels. As a first step in the analysis of the mechanisms responsible for this reduction, we have studied a possible down-regulation of the SERCA2 gene transcription during left ventricular hypertrophy (LVH) induced by constriction of the ascending aorta in the rat. Quantifications of the mRNA levels demonstrated no alteration, compared to sham-operated rats, at 5 d after imposition of the pressure overload, whereas a significant decrease was observed at 11 d. Transcription in-vitro experiments (cardiac nuclear run-on assays) performed in isolated cardiomyocytes nuclei showed no changes at 5 d and a 37% reduction of the SERCA2 gene transcription at 11 d. These results strongly suggest that SERCA2 gene expression down-regulation during cardiac hypertrophy occurs, at least in part, at the level of the transcription.
    No preview · Article · Jan 1998 · Comptes Rendus de l Académie des Sciences - Series III - Sciences de la Vie
  • [Show abstract] [Hide abstract]
    ABSTRACT: We have previously characterized the proximal promoter of the mouse IIB myosin heavy chain (MyHC) gene, which is expressed only in fast-contracting glycolytic skeletal muscle fibers. We show here that the substitution into this promoter of a non-canonical TATA sequence from the IgH gene results in inactivity in muscle cells, even though TATA-binding protein (TBP) can bind strongly to this mutated promoter. Chemical foot-printing data show, however, that TBP makes different DNA contacts on this heterologous TATA sequence. The inactivity of such a non-canonical TATA motif in the IIB promoter context appears to be caused by a non-functional conformation of the bound TBP-DNA complex that is incapable of sustaining transcription. The conclusions imply that the precise sequence of the promoter TATA motif needs to be matched with the specific functional class of upstream activator proteins present in a given cell type in order for the gene to be transcriptionally active.
    No preview · Article · Mar 1997 · Journal of Molecular Biology
  • [Show abstract] [Hide abstract]
    ABSTRACT: The sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA2) plays a critical role in regulating Ca2+ movements in myocardium. In cardiac hypertrophy and human heart failure, the decrease in mRNA and protein levels of SERCA2 might account for the reduced diastolic Ca2+ re-uptake seen in these conditions. To investigate the regulation of human SERCA2 gene expression, an 18.6-kb human genomic clone that contains exons 1,2 and 3 of the SERCA2 gene has been isolated, and 13 kb of 5' upstream flanking sequence of which the proximal 2.5 kb of the promoter have been sequenced. Similar to the rabbit gene, the human SERCA2 promoter possesses a TATA-like box (-25 bp), a CAAT-box (-78 bp) and a number of consensus cis-regulatory elements including three Sp1 sites, a CACCC-box, and an OTF-1 binding sequence. No CArG box (present in the rabbit SERCA2 promoter) was identified in the human proximal promoter. Two putative thyroid response elements (TRE) are also present, suggesting that the human SERCA2 gene is also regulated by thyroid hormone as are the rat and rabbit genes. To study transcriptional activity of the human SERCA2 promoter in vitro, luciferase reporter plasmids containing a series of 5' deleted promoter constructs from -2577 bp to +170 bp were transfected into neonatal rat cardiomyocytes and C2C12 myotubes. The results suggest that: (a) the sequences from the transcription start site to -263 bp are necessary to obtain maximal transcriptional activity; (b) sequences from the transcription start site to -125 bp are essential for basal transcriptional activity; (c) at least one positive regulatory element is located between -263 bp and -125 bp; and (d) at least one negative regulatory element is present between -1741 bp and -412 bp.
    No preview · Article · Nov 1996 · Journal of Molecular and Cellular Cardiology
  • Source
    A M Pret · M Y Fiszman
    [Show abstract] [Hide abstract]
    ABSTRACT: Alternative splicing of vertebrate β-tropomyosin transcripts ensures mutually exclusive expression of internal exons 6A and 6B in nonmuscle and skeletal muscle cells, respectively. Recently, we reported that this splicing regulation requires species-specific elements, since the splicing profile for the chicken, rat, and Xenopus β-tropomyosin alternative exons is not reproduced in transfection experiments when heterologous myogenic cells are used. By analyzing the splicing pattern of hybrid chicken/rat β-TM constructions transfected into both quail and mouse cell lines, we demonstrate that chicken β-tropomyosin exon 6A is flanked by stronger splicing signals than rat exon 6A, thus leading to the misregulation of splicing in heterologous cells. We have characterized three splicing signals that contribute to this difference: 1) nonconsensus nucleotide differences at positions +4 and +6 in the donor site downstream of exon 6A, 2) differences in the pyrimidine composition between the branch site and acceptor site upstream of exon 6A, and 3) a pyrimidine-rich intronic exon 6A splicing enhancer present upstream of exon 6A only in the chicken β-TM gene. The functional divergence between splicing signals in two homologous vertebrate genes reveals species-specific strategies for proper modulation of splicing of alternative exons.
    Preview · Article · Jun 1996 · Journal of Biological Chemistry
  • [Show abstract] [Hide abstract]
    ABSTRACT: The human aldolase A gene is expressed in several tissues through the use of three alternative promoters. The activity of one of the promoters, pM, is restricted to skeletal muscle. We reported previously that a proximal 280 bp pM fragment confers tissue-specific expression to a CAT reporter gene in transgenic mice. This small regulatory region directs expression to muscle composed mainly of fast-twitch fibers. Here we show that a minimal promoter fragment from base-pairs -164 to +45 is sufficient to highly active pM during myoblast differentiation in cell culture and demonstrate that two DNA elements play a major role in this activation. These elements consist of a binding site (M1) for unknown ubiquitous proteins and an overlapping binding site for MEF2 and NF1 families of transcription factors. The NF1 factor constitute the main binding activity on the MEF2/NF1 site and, interestingly, some of the DNA-protein complexes that form with muscle nuclear extracts on the NF1 element differ from those that form with non-muscular extracts.
    No preview · Article · Nov 1995 · Journal of Molecular Biology
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The publishers wish to apologise for printing a typegraphical error in the tittle of the above paper. The correct title is as printed above.
    Preview · Article · Feb 1995 · Nucleic Acids Research
  • Source
    L Balvay · A M Pret · D Libri · D M Helfman · M Y Fiszman
    [Show abstract] [Hide abstract]
    ABSTRACT: The diversity of protein isoforms is often generated from single genes by alternative splicing of the primary transcript. Using transfection of beta tropomyosin minigene constructs into homologous and heterologous cell systems, we show that there are differences, among higher vertebrates, in the components of the splicing machinery which control the conserved regulated splicing pattern of two mutually exclusive exons (6A and 6B) present in this gene. These experiments demonstrate that genes which give rise to alternative transcripts may require an appropriate combination of splicing factors which are species-specific, or at least restricted to the same taxonomic subgroup (class). An important practical implication is that the splicing of these genes may be deregulated in heterologous systems in vitro and in vivo, i.e. in transgenic animals.
    Full-text · Article · Sep 1994 · Journal of Biological Chemistry
  • F Edom · V Mouly · J P Barbet · M.Y. Fiszman · G S Butler-Browne
    [Show abstract] [Hide abstract]
    ABSTRACT: In order to test the diversification among satellite cells in man, satellite cells were isolated from human quadriceps and masseter muscles. The growth kinetics and morphological features of these cells were determined in vitro and the expression of the different myosin heavy (embryonic, fetal, fast, and slow) and light chain isoforms was analyzed. In all satellite cell cultures, only the four fast-type light chains (MLC1emb, MLC1F, MLC2F, and MLC3F) were synthesized and no slow myosin light chains were ever detected. However, we found that fused cultures of human satellite cells express both adult fast and slow myosin heavy chains (MHCs), in addition to embryonic and fetal isoforms. In order to determine if distinct fast and slow cell lineages could be detected among the satellite cells, a clonal analysis was carried out on both cell populations. This analysis was first carried out on clonal populations and was confirmed by the analysis of isolated clones. Double-labeling experiments confirmed that all myogenic clones which expressed fast MHC also coexpressed slow MHC. Therefore, we found no evidence for the existence of different fast and slow satellite cell lineages in human postnatal skeletal muscle.
    No preview · Article · Aug 1994 · Developmental Biology
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The chicken beta tropomyosin (beta TM) gene has two alternative transcription start sites (sk and nmCAP sites) which are used in muscle or non muscle tissues respectively. In order to understand the mechanisms involved in the tissue-specific and developmentally-regulated expression of the beta TM gene, we have analyzed the 5' regions associated with each CAP site. Truncated regions 5' to the nmCAP site were inserted upstream to the bacterial chloramphenicol acetyltransferase (CAT) reporter gene and these constructs were transfected into avian myogenic and non myogenic cells. The maximum transcription is driven by the CAT construct (-168/ + 216 nt) in all cell types. Previous deletion analysis of the region 5' to the beta TMskCAP site has indicated that 805 nt confer myotube-specific transcription. In this work, we characterized an enhancer element (-201/-68 nt) which contains an E box (-177), a variant CArG box (-104) and a stretch of 7Cs (-147). Mutation of any of these motifs results in a decrease of the myotube-specific transcriptional activity. Electrophoretic mobility shift assays indicate that these cis-acting sequences specifically bind nuclear proteins. This enhancer functions in an orientation-dependent manner.
    Full-text · Article · Jun 1994 · Nucleic Acids Research
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The chicken β tropomyosin (βTM) gene has two alternative transcription start sites (sk and nmCAP sites) which are used in muscle or non muscle tissues respectively. In order to understand the mechanisms involved in the tissue-specific and developmentally-regulated expression of the βTM gene, we have analyzed the 5′ regions associated with each CAP site. Truncated regions 5′ to the nmCAP site were inserted upstream to the bacterial chloramphenicol acetyltransferase (CAT) reporter gene and these constructs were transfected into avian myogenic and non myogenic cells. The maximum transcription is driven by the CAT construct (− 168/ + 216 nt) in all cell types. Previous deletion analysis of the region 5′ to the βTMskCAP site has indicated that 805 nt confer myotube-specific transcription. In this work, we characterize an enhancer element (− 201/ − 68 nt) which contains an E box (− 177), a variant CArG box (− 104) and a stretch of 7Cs (− 147). Mutation of any of these motifs results in a decrease of the myotube-specific transcriptional activity. Electrophoretic mobility shift assays indicate that these cis-acting sequences specifically bind nuclear proteins. This enhancer functions in an orientation-dependent manner.
    Preview · Article · May 1994 · Nucleic Acids Research
  • L Balvay · M Y Fiszman
    [Show abstract] [Hide abstract]
    ABSTRACT: Tropomyosins are a family of actin filament binding proteins. Like many structural proteins, tropomyosin isoform expression involves the use of multiple genes, but diversity is to a large extent generated by alternative processing of RNA. The tropomyosin family consists of 15 to 20 different protein isoforms which are coded by four genes. Each of these genes code for multiple proteins ranging from two up to as many as nine different isoforms. These genes have been named alpha CTM, alpha FTM, alpha STM and beta TM after to the striated muscle specific subunit of tropomyosin which they code. Their multiple coding potential is based upon the existence of multiple exons associated with initiation of transcription, multiple exons associated with polyadenylation signals and multiple mutually exclusive internal exons which are alternatively spliced. The regulation of this process of alternative splicing have been extensively studied both in the case of exons 2a/2b of the alpha FTM gene and in the case of exons 6a/6b of the beta TM gene. In both cases, one exon is specifically used in one type of muscle tissue, exon 2a is smooth muscle specific and exon 6b is skeletal muscle specific. In both cases, alternative splicing involves a combination of negative regulation, on exon 2b in smooth muscle and on exon 6b in non muscle tissues, and of competition in the alternative situation.
    No preview · Article · Feb 1994 · Comptes rendus des séances de la Société de biologie et de ses filiales
  • M Toutant · M Y Fiszman · M Lemonnier
    [Show abstract] [Hide abstract]
    ABSTRACT: The chick beta tropomyosin (TM) gene has two alternative transcription initiation start sites which are used in muscle or non muscle tissue. A recombinant plasmid containing 805 nucleotides (nt) of the sequence upstream to the muscle CAP site driving the bacterial chloramphenicol acetyltransferase gene is sufficient for muscle specific expression. Of the two E boxes present in this construct, only the E box proximal to the CAP site is functional since deletion or mutation of this E box causes a decrease of CAT activity (about 40%). Separate mutation of Sp1 motifs also reduces the transcription driven by the 805nt fragment. Double mutation of E box and Sp1 motifs show that helix-loop-helix muscle regulatory factors and ubiquitous Sp1 transcription factor are required in the initiation of the transcription of the chick beta TM gene in muscle tissue. Our results also suggest that other factors may participate to this process.
    No preview · Article · Sep 1993 · Comptes Rendus de l Académie des Sciences - Series III - Sciences de la Vie
  • Laurent Balvay · Domenico Libri · Marc Y. Fiszman
    [Show abstract] [Hide abstract]
    ABSTRACT: Nuclear pre-mRNAs must be precisely processed to give rise to mature cytoplasmic mRNAs. This maturation process, known as splicing, involves excision of intron sequences and ligation of the exon sequences. One of the major problems in understanding this process is how splice sites, the sequences which form the boundaries between introns and exons, can be accurately selected. A number of studies have defined conserved sequences within introns which were later shown to interact with small nuclear ribonucleoproteins (snRNPs). However, due to the simplicity of these conserved sequences it has become clear that other elements must be involved and a number of studies have indicated the importance of secondary structures within pre-mRNAs. Using various examples, we shall show that such structures can help to specify splice sites by modifying physical distances within introns or by being involved in the definition of exons and lastly, that they can be part of the regulation of alternative splicing.
    No preview · Article · Mar 1993 · BioEssays
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The dependence of the muscle-specific enhancer of the acetylcholine receptor alpha-subunit gene on other domains of the promoter has been analysed by performing point mutagenesis and modular reconstitution of the enhancer--promoter sequences. The enhancer is inactive in the absence of the proximal region containing an Sp1 binding site and an overlapping G-C homopolymer binding factor site (referred to as GBF). The proximal region can be replaced by an Sp1 binding site from SV40 or an MEF-2 binding site from the muscle creatine kinase gene. Specific mutation of the Sp1 site markedly affects transactivation by CMD1 or myogenin. Mutation of the GBF binding site leads to higher promoter activity in primary cultures of chick myotubes or in quail fibroblasts. In addition, binding of a purified Sp1 protein prevents the binding of GBF in vitro. It is proposed that in the case of the alpha-subunit promoter, the myogenic factors activate transcription in cooperation with Sp1, and that GBF contributes to muscle-specific expression of the promoter by interfering with Sp1 binding in nonmuscle muscle cells or myoblasts.
    Full-text · Article · Mar 1993 · The EMBO Journal
  • Source
    L Balvay · D Libri · M Gallego · M Y Fiszman
    [Show abstract] [Hide abstract]
    ABSTRACT: The chicken beta tropomyosin gene generates three major transcripts by alternative splicing. A pair of internal exons are spliced in a mutually exclusive manner and their utilisation is developmentally regulated. Exon 6A and exon 6B are used respectively in myoblasts and myotubes during the process of differentiation of muscle cells. We have previously reported that, in myoblasts, exon 6B is skipped because of a negative regulation which involves intron as well as exon sequences. In this report, we describe a previously uncharacterized intronic element which is involved in the regulation of the splicing of both exons 6A and 6B. This cis-element is localized 37nt downstream of exon 6A and is approximately 30nt long. Its deletion, as well as modification of its sequence, results in the activation of the use of exon 6B and, at the same time, in the inhibition of the use of exon 6A. The mechanisms by which this region could act are further discussed.
    Full-text · Article · Sep 1992 · Nucleic Acids Research
  • Source
    Laurent Balvay · Domenico Libri · Maria Gallego · Marc Y. Fiszman
    [Show abstract] [Hide abstract]
    ABSTRACT: The chicken β tropomyosin gene generates three major transcripts by alternative splicing. A pair of internal exons are spliced In a mutually exclusive manner and their utilisation is developmentally regulated. Exon 6A and exon 6B are used respectively in myoblasts and myotubes during the process of differentiation of muscle cells. We have previously reported that, in myoblasts, exon 6B is skipped because of a negative regulation which involves intron as well as exon sequences. In this report, we describe a previously uncharacterized intronic element which is involved in the regulation of the splicing of both exons 6A and 6B. This cis-element Is localized 37nt downstream of exon 6A and is approximately 30nt long. Its deletion, as well as modification of its sequence, results in the activation of the use of exon 6B and, at the same time, in the inhibition of the use of exon 6A. The mechanisms by which this region could act are further discussed.
    Full-text · Article · Aug 1992 · Nucleic Acids Research
  • Source
    D Libri · L Balvay · M Y Fiszman
    [Show abstract] [Hide abstract]
    ABSTRACT: The chicken beta tropomyosin gene contains two sets of alternatively spliced, mutually exclusive exons whose utilization is developmentally regulated. Exons 6A and 6B are used in nonmuscle cells (or undifferentiated muscle cells) and skeletal muscle cells, respectively. A complex arrangement of cis-acting sequence elements is involved in alternative splicing regulation. We have performed an extensive mutational analysis on the sequence spanning the region from exon 6A to the constitutive exon 7. A large number of mutant minigenes have been tested in transfection assays of cultured myogenic cells, and the splicing products have been analyzed by cDNA polymerase chain reaction. We demonstrate that in undifferentiated myoblasts, exon 6B is skipped as a result of a negative control on its selection, while exon 6A is spliced as a default choice. We provide evidence that the focal point of such a regulation is localized in the intron upstream of exon 6B and probably involves the blockage of its associated branch point. In differentiated myotubes, in contrast, both exons are accessible to the splicing machinery. We show that the preferential choice of exon 6B in this splicing environment depends on the existence of a competition between the two exons for the flanking constitutive splice sites. We demonstrate that both the donors and the branch points of the two exons are involved in this competition.
    Preview · Article · Aug 1992 · Molecular and Cellular Biology
  • M Y Fiszman · D Libri · L Balvay
    [Show abstract] [Hide abstract]
    ABSTRACT: The beta tropomyosin gene of the chicken contains a pair of alternatively spliced mutually exclusive exons the use of which is developmentally regulated. Exon 6A is used by non muscle and undifferentiated muscle cells (myoblasts) while exon 6B is exclusively used in differentiated skeletal muscle cells. A complex array of cis acting sequence elements are involved in the regulation of this alternative splicing process. Transfection assays of quail muscle cells in culture were used to define these cis acting elements. We show that, in undifferentiated muscle cells, exon 6B is skipped as a result of a negative control on its selection while exon 6A is spliced as a default choice. We provide evidence that this negative control involves a secondary structure of the primary transcript around the 5' end of exon 6B as well as intronic sequence elements located between the branch point and the acceptor splice site of exon 6B. In differentiated muscles, both exons are accessible to the splicing machinery and the preferential use of exon 6B depends on the existence of a competition between the two exons for the selection of the flanking splice sites. In particular, we show that the donor splice site of exon 6A is a weak splice site while the branch point associated with exon 6B is a strong branch point.
    No preview · Article · Feb 1992 · Symposia of the Society for Experimental Biology

Publication Stats

2k Citations
270.52 Total Impact Points

Institutions

  • 1999
    • National Institute on Aging
      • Laboratory of Cardiovascular Science (LCS)
      Baltimore, Maryland, United States
  • 1998
    • Hôpital La Pitié Salpêtrière (Groupe Hospitalier "La Pitié Salpêtrière - Charles Foix")
      Lutetia Parisorum, Île-de-France, France
  • 1996
    • French Institute of Health and Medical Research
      Lutetia Parisorum, Île-de-France, France
  • 1978-1996
    • Institut Pasteur
      Lutetia Parisorum, Île-de-France, France
  • 1994
    • French National Centre for Scientific Research
      Lutetia Parisorum, Île-de-France, France
  • 1989
    • Institut de Génétique et de Biologie Moléculaire et Cellulaire
      Strasburg, Alsace, France