Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 20
Filter
Add more filters










Publication year range
1.
Bone ; 120: 411-422, 2019 03.
Article in English | MEDLINE | ID: mdl-30529011

ABSTRACT

The two SIBLING (Small Integrin Binding Ligand N-linked Glycoproteins), bone sialoprotein (BSP) and osteopontin (OPN) are expressed in osteoblasts and osteoclasts. In mature BSP knockout (KO, -/-) mice, both bone formation and resorption as well as mineralization are impaired. OPN-/- mice display impaired resorption, and OPN is described as an inhibitor of mineralization. However, OPN is overexpressed in BSP-/- mice, complicating the understanding of their phenotype. We have generated and characterized mice with a double KO (DKO) of OPN and BSP, to try and unravel their respective contributions. Despite the absence of OPN, DKO bones are still hypomineralized. The SIBLING, matrix extracellular phosphoglycoprotein with ASARM motif (MEPE) is highly overexpressed in both BSP-/- and DKO and may impair mineralization through liberation of its ASARM (Acidic Serine-Aspartate Rich MEPE associated) peptides. DKO mice also display evidence of active formation of trabecular, secondary bone as well as primary bone in the marrow-ablation repair model. A higher number of osteoclasts form in DKO marrow cultures, with higher resorption activity, and DKO long bones display a localized and conspicuous cortical macroporosity. High bone formation and resorption parameters, and high cortical porosity in DKO mice suggest an active bone modeling/remodeling, in the absence of two key regulators of bone cell performance. This first double KO of SIBLING proteins thus results in a singular, non-trivial phenotype leading to reconsider the interpretation of each single KO, concerning in particular matrix mineralization and the regulation of bone cell activity.


Subject(s)
Bone Remodeling/physiology , Bone and Bones/physiopathology , Calcification, Physiologic/physiology , Gene Deletion , Integrin-Binding Sialoprotein/deficiency , Osteopontin/deficiency , Animals , Biomarkers/metabolism , Bone Marrow/pathology , Bone Matrix/physiopathology , Cancellous Bone/physiopathology , Cell Differentiation , Extracellular Matrix Proteins/metabolism , Gene Expression Regulation , Integrin-Binding Sialoprotein/metabolism , Mice, Knockout , Osteoblasts/metabolism , Osteoclasts/metabolism , Osteogenesis , Osteopontin/metabolism , Reproducibility of Results
2.
Nat Commun ; 9(1): 1133, 2018 03 19.
Article in English | MEDLINE | ID: mdl-29556040

ABSTRACT

In genome editing with CRISPR-Cas9, transgene integration often remains challenging. Here, we present an approach for increasing the efficiency of transgene integration by homology-dependent repair (HDR). CtIP, a key protein in early steps of homologous recombination, is fused to Cas9 and stimulates transgene integration by HDR at the human AAVS1 safe harbor locus. A minimal N-terminal fragment of CtIP, designated HE for HDR enhancer, is sufficient to stimulate HDR and this depends on CDK phosphorylation sites and the multimerization domain essential for CtIP activity in homologous recombination. HDR stimulation by Cas9-HE, however, depends on the guide RNA used, a limitation that may be overcome by testing multiple guides to the locus of interest. The Cas9-HE fusion is simple to use and allows obtaining twofold or more efficient transgene integration than that with Cas9 in several experimental systems, including human cell lines, iPS cells, and rat zygotes.


Subject(s)
Carrier Proteins/metabolism , Nuclear Proteins/metabolism , Recombinational DNA Repair , Animals , Base Sequence , CRISPR-Cas Systems , Carrier Proteins/chemistry , Carrier Proteins/genetics , Cell Line , DNA Breaks, Double-Stranded , Endodeoxyribonucleases , Enhancer Elements, Genetic , Female , HCT116 Cells , HEK293 Cells , Homologous Recombination , Humans , INDEL Mutation , Induced Pluripotent Stem Cells/metabolism , Nuclear Proteins/chemistry , Nuclear Proteins/genetics , Oocytes/metabolism , Phosphorylation , Protein Multimerization , RNA, Guide, Kinetoplastida/genetics , Rats , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Transgenes , Virus Integration/genetics , Zygote/metabolism
3.
Oligonucleotides ; 17(2): 258-63, 2007.
Article in English | MEDLINE | ID: mdl-17638529

ABSTRACT

Single-stranded oligonucleotides (SSOs) mediate gene repair of punctual chromosomal mutations at a low frequency. We hypothesized that enhancement of DNA binding affinity of SSOs by intercalating agents may increase the number of corrected cells. Several biochemical modifications of SSOs were tested for their capability to correct a chromosomally integrated and mutated GFP reporter gene in human 293 cells. SSOs of 25 nucleotide length conjugated with acridine at their 5' end increased the efficiency of gene correction up to 10-fold compared to nonmodified SSOs. Acridine and psoralen conjugates were both evaluated, and acridine-modified SSOs were the most effective. Conjugation with acridine at the 3' end of the SSO inhibited gene correction, whereas flanking the SSO by acridine on both sides provided an intermediate level of correction. These results suggest that increasing the stability of hybridization between SSO and its target without hampering a 3' extension improves gene targeting, in agreement with the "annealing-integration" model of DNA repair.


Subject(s)
Acridines/metabolism , Intercalating Agents/metabolism , Oligonucleotides/genetics , Oligonucleotides/metabolism , Targeted Gene Repair/methods , Ficusin/metabolism , Genetic Therapy , Humans , Oligonucleotides, Antisense/genetics , Oligonucleotides, Antisense/metabolism , Transfection
4.
Dev Biol ; 210(2): 277-87, 1999 Jun 15.
Article in English | MEDLINE | ID: mdl-10357891

ABSTRACT

Left-right (LR) asymmetry of the heart in vertebrates is regulated by early asymmetric signals in the embryo, including the secreted signal Sonic hedgehog (Shh), but less is known about LR asymmetries of visceral organs. Here we show that Shh also specifies asymmetries in visceral precursors in the zebrafish and that cardiac and visceral sidedness are independent. The transcription factors fli-1 and Nkx-2.5 are expressed asymmetrically in the precardiac mesoderm and subsequently in the heart; an Eph receptor, rtk2, and an adhesion protein, DM-GRASP, mark early asymmetries in visceral endoderm. Misexpression of shh mRNA, or a dominant negative form of protein kinase A, on the right side reverses the expression of these asymmetries in precursors of both the heart and the viscera. Reversals in the heart and gut are uncoordinated, suggesting that each organ interprets the signal independently. Misexpression of Bone Morphogenetic Protein (BMP4) on the right side reverses the heart, but visceral organs are unaffected, consistent with a function for BMPs locally in the heart field. Zebrafish mutants with midline defects show independent reversals of cardiac and visceral laterality. Thus, hh signals influence the development of multiple organ asymmetries in zebrafish and different organs appear to respond to a central cascade of midline signaling independently, which in the heart involves BMP4.


Subject(s)
Body Patterning , Bone Morphogenetic Proteins/physiology , Embryo, Nonmammalian/physiology , Embryonic Induction , Proteins/physiology , Proto-Oncogene Proteins , Transcription Factors , Xenopus Proteins , Zebrafish/embryology , Animals , Bone Morphogenetic Protein 4 , Bone Morphogenetic Proteins/genetics , DNA-Binding Proteins/genetics , Embryo, Nonmammalian/cytology , Gene Expression Regulation, Developmental , Heart/embryology , Hedgehog Proteins , Homeobox Protein Nkx-2.5 , Homeodomain Proteins/genetics , Mesoderm/physiology , Proteins/genetics , Proto-Oncogene Protein c-fli-1 , Signal Transduction , Trans-Activators/genetics , Viscera/embryology , Zebrafish Proteins
5.
J Biol Chem ; 274(12): 7941-5, 1999 Mar 19.
Article in English | MEDLINE | ID: mdl-10075690

ABSTRACT

Activation of NF-kappaB transcription factors requires phosphorylation and ubiquitin-proteasome-dependent degradation of IkappaB proteins. We provide evidence that a human F-box protein, h-betaTrCP, a component of Skp1-Cullin-F-box protein (SCF) complexes, a new class of E3 ubiquitin ligases, is essential for inducible degradation of IkappaBalpha. betaTrCP associates with Ser32-Ser36 phosphorylated, but not with unmodified IkappaBalpha or Ser32-Ser36 phosphorylation-deficient mutants. Expression of a F-box-deleted betaTrCP inhibits IkappaBalpha degradation, promotes accumulation of phosphorylated Ser32-Ser36 IkappaBalpha, and prevents NF-kappaB-dependent transcription. Our findings indicate that betaTrCP is the adaptor protein required for IkappaBalpha recognition by the SCFbetaTrCP E3 complex that ubiquitinates IkappaBalpha and makes it a substrate for the proteasome.


Subject(s)
Cell Cycle Proteins/metabolism , Cysteine Endopeptidases/metabolism , DNA-Binding Proteins/metabolism , GTP-Binding Proteins/metabolism , I-kappa B Proteins , Multienzyme Complexes/metabolism , Peptide Synthases/metabolism , HeLa Cells , Humans , Models, Chemical , NF-KappaB Inhibitor alpha , NF-kappa B/biosynthesis , NF-kappa B/genetics , Phosphorylation , Proteasome Endopeptidase Complex , S-Phase Kinase-Associated Proteins , SKP Cullin F-Box Protein Ligases , Serine/metabolism , Transcription, Genetic , beta-Transducin Repeat-Containing Proteins
6.
Dev Biol ; 208(1): 14-29, 1999 Apr 01.
Article in English | MEDLINE | ID: mdl-10075838

ABSTRACT

Hedgehog signalling has been implicated in a variety of processes in vertebrate development, and in each case, the activity of Hh proteins is thought to be mediated by their interaction with a large multipass transmembrane protein encoded by the patched (ptc) gene. In this study we present the full-length coding sequence and describe the wild-type expression pattern of a second ptc gene in zebrafish, Zf-ptc2. We find that at the sequence level Zf-ptc2 is more closely related than Zf-ptc1 to the ptc genes initially characterised in other vertebrate species. We also show that transcription of Zf-ptc2, like Zf-ptc1, is dependent upon Hh signalling and present evidence that it is activated in response to lower levels of Hh activity than is Zf-ptc1. In addition we find no evidence for any specificity in the regulatory interactions between the various Hh proteins and the two ptc genes in the zebrafish.


Subject(s)
Membrane Proteins/genetics , Proteins/genetics , Trans-Activators , Zebrafish Proteins , Zebrafish/genetics , Amino Acid Sequence , Animals , Cloning, Molecular , Embryonic Development , Gene Expression Regulation, Developmental/genetics , Hedgehog Proteins , Membrane Proteins/chemistry , Microinjections , Molecular Sequence Data , Mutation , Patched Receptors , Patched-1 Receptor , Phylogeny , RNA, Antisense/genetics , RNA, Messenger/metabolism , Receptors, Cell Surface , Sequence Alignment , Sequence Homology, Amino Acid , Signal Transduction/genetics , Transcription, Genetic/genetics
7.
Curr Biol ; 9(4): 207-10, 1999 Feb 25.
Article in English | MEDLINE | ID: mdl-10074433

ABSTRACT

Defects in beta-catenin regulation contribute to the neoplastic transformation of mammalian cells. Dysregulation of beta-catenin can result from missense mutations that affect critical sites of phosphorylation by glycogen synthase kinase 3beta (GSK3beta). Given that phosphorylation can regulate targeted degradation of beta-catenin by the proteasome, beta-catenin might interact with an E3 ubiquitin ligase complex containing an F-box protein, as is the case for certain cell cycle regulators. Accordingly, disruption of the Drosophila F-box protein Slimb upregulates the beta-catenin homolog Armadillo. We reasoned that the human homologs of Slimb - beta-TrCP and its isoform beta-TrCP2 (KIAA0696) - might interact with beta-catenin. We found that the binding of beta-TrCP to beta-catenin was direct and dependent upon the WD40 repeat sequences in beta-TrCP and on phosphorylation of the GSK3beta sites in beta-catenin. Endogenous beta-catenin and beta-TrCP could be coimmunoprecipitated from mammalian cells. Overexpression of wild-type beta-TrCP in mammalian cells promoted the downregulation of beta-catenin, whereas overexpression of a dominant-negative deletion mutant upregulated beta-catenin protein levels and activated signaling dependent on the transcription factor Tcf. In contrast, beta-TrCP2 did not associate with beta-catenin. We conclude that beta-TrCP is a component of an E3 ubiquitin ligase that is responsible for the targeted degradation of phosphorylated beta-catenin.


Subject(s)
Cytoskeletal Proteins/metabolism , GTP-Binding Proteins/metabolism , Trans-Activators , Animals , Cadherins/metabolism , Carrier Proteins/metabolism , Cell Line , Cytoskeletal Proteins/chemistry , Cytoskeletal Proteins/genetics , Drosophila , GTP-Binding Proteins/chemistry , Genes, Reporter , HeLa Cells , Humans , Phosphorylation , Protein Isoforms/chemistry , Protein Isoforms/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Repetitive Sequences, Amino Acid , Transfection , Ubiquitin-Protein Ligases , beta Catenin , beta-Transducin Repeat-Containing Proteins
8.
Proc Natl Acad Sci U S A ; 95(24): 14220-5, 1998 Nov 24.
Article in English | MEDLINE | ID: mdl-9826681

ABSTRACT

Myogenin, one of the MyoD family of proteins, is expressed early during somitogenesis and is required for myoblast fusion in vivo. Previous studies in transgenic mice have shown that a 184-bp myogenin promoter fragment is sufficient to correctly drive expression of a beta-galactosidase transgene during embryogenesis. We show here that mutation of one of the DNA motifs present in this region, the MEF3 motif, abolished correct expression of this beta-galactosidase transgene. We have found that the proteins that bind to the MEF3 site are homeoproteins of the Six/sine oculis family. Antibodies directed specifically against Six1 or Six4 proteins reveal that each of these proteins is present in the embryo when myogenin is activated and constitutes a muscle-specific MEF3-binding activity in adult muscle nuclear extracts. Both of these proteins accumulate in the nucleus of C2C12 myogenic cells, and transient transfection experiments confirm that Six1 and Six4 are able to transactivate a reporter gene containing MEF3 sites. Altogether these results establish Six homeoproteins as a family of transcription factors controlling muscle formation through activation of one of its key regulators, myogenin.


Subject(s)
Embryonic and Fetal Development , Gene Expression Regulation, Developmental , Homeodomain Proteins/metabolism , Muscle, Skeletal/metabolism , Myogenin/genetics , Trans-Activators , Amino Acid Sequence , Animals , Base Sequence , Binding Sites , Cell Line , Cell Nucleus/metabolism , Conserved Sequence , Fetal Heart/metabolism , Liver/metabolism , Mice , Mice, Transgenic , Muscle, Skeletal/embryology , Mutagenesis, Site-Directed , Myocardium/metabolism , Nerve Tissue Proteins/metabolism , Oligodeoxyribonucleotides , Spleen/metabolism , Transfection , beta-Galactosidase/biosynthesis , beta-Galactosidase/genetics
9.
J Biol Chem ; 271(51): 32529-37, 1996 Dec 20.
Article in English | MEDLINE | ID: mdl-8955077

ABSTRACT

Hypoxia-inducible factor 1 (HIF-1) is a basic helix-loop-helix transcription factor which is expressed when mammalian cells are subjected to hypoxia and which activates transcription of genes encoding erythropoietin, vascular endothelial growth factor, and other proteins that are important for maintaining oxygen homeostasis. Previous studies have provided indirect evidence that HIF-1 also regulates transcription of genes encoding glycolytic enzymes. In this paper we characterize hypoxia response elements in the promoters of the ALDA, ENO1, and Ldha genes. We demonstrate that HIF-1 plays an essential role in activating transcription via these elements and show that although absolutely necessary, the presence of a HIF-1 binding site alone is not sufficient to mediate transcriptional responses to hypoxia. Analysis of hypoxia response elements in the ENO1 and Ldha gene promoters revealed that each contains two functionally-essential HIF-1 sites arranged as direct and inverted repeats, respectively. Our data establish that functional hypoxia-response elements consist of a pair of contiguous transcription factor binding sites at least one of which contains the core sequence 5'-RCGTG-3' and is recognized by HIF-1. These results provide further evidence that the coordinate transcriptional activation of genes encoding glycolytic enzymes which occurs in hypoxic cells is mediated by HIF-1.


Subject(s)
DNA-Binding Proteins/physiology , Fructose-Bisphosphate Aldolase/genetics , Hypoxia/genetics , L-Lactate Dehydrogenase/genetics , Nuclear Proteins/physiology , Phosphopyruvate Hydratase/genetics , Promoter Regions, Genetic , Transcription Factors , Base Sequence , Cells, Cultured , DNA-Binding Proteins/metabolism , Gene Expression Regulation, Enzymologic , Helix-Loop-Helix Motifs , Humans , Hypoxia-Inducible Factor 1 , Hypoxia-Inducible Factor 1, alpha Subunit , Molecular Sequence Data , Mutagenesis, Site-Directed , RNA, Messenger/genetics , Restriction Mapping , Transcription, Genetic , Transfection
10.
Development ; 122(9): 2835-46, 1996 Sep.
Article in English | MEDLINE | ID: mdl-8787757

ABSTRACT

Signalling by members of the Hedgehog family of secreted proteins plays a central role in the development of vertebrate and invertebrate embryos. In Drosophila, transduction of the Hedgehog signal is intimately associated with the activity of protein kinase A and the product of the segment polarity gene patched. We have cloned a homologue of patched from the zebrafish Danio rerio and analysed the spatiotemporal regulation of its transcription during embryonic development in both wild-type and mutant animals. We find a striking correlation between the accumulation of patched1 transcripts and cells responding to sonic hedgehog activity both in the neurectoderm and mesoderm, suggesting that like its Drosophila counterpart, patched1 is regulated by sonic hedgehog activity. Consistent with this interpretation, mis-expression of sonic hedgehog results in ectopic activation of patched1 transcription. Using dominant negative and constitutively active forms of the protein kinase A subunits, we also show that expression of patched1 as well as of other sonic hedgehog targets, is regulated by protein kinase A activity. Taken together, our findings suggest that the mechanism of signalling by Hedgehog family proteins has been highly conserved during evolution.


Subject(s)
Central Nervous System/embryology , Cyclic AMP-Dependent Protein Kinases/metabolism , Drosophila Proteins , Embryonic Induction , Gene Expression Regulation, Developmental , Insect Hormones/genetics , Membrane Proteins/genetics , Proteins/metabolism , Trans-Activators , Zebrafish/embryology , Amino Acid Sequence , Animals , Cloning, Molecular , Conserved Sequence , DNA Primers , Drosophila/genetics , Evolution, Molecular , Hedgehog Proteins , In Situ Hybridization , Molecular Sequence Data , Proteins/genetics , Receptors, Cell Surface , Sequence Homology, Amino Acid , Signal Transduction , Transcription, Genetic , Zebrafish/genetics
12.
Mol Cell Biol ; 14(10): 6797-808, 1994 Oct.
Article in English | MEDLINE | ID: mdl-7935397

ABSTRACT

The expression of the human aldolase A gene is controlled by three alternative promoters. In transgenic mice, pN and pH are active in all tissues whereas pM is activated specifically in adult muscles composed mainly of fast, glycolytic fibers. To detect potential regulatory regions involved in the fast-muscle-specific activation of pM, we analyzed DNase I hypersensitivity in a 4.3-kbp fragment from the 5' end of the human aldolase A gene. Five hypersensitive sites were located near the transcription initiation site of each promoter in those transgenic-mouse tissues in which the corresponding promoter was active. Only one muscle-specific hypersensitive site was detected, mapping near pM. To functionally delimit the elements required for muscle-specific activity of pM, we performed a deletion analysis of the aldolase A 5' region in transgenic mice. Our results show that a 280-bp fragment containing 235 bp of pM proximal upstream sequences together with the noncoding M exon is sufficient for tissue-specific expression of pM. When a putative MEF-2-binding site residing in this proximal pM region is mutated, pM is still active and no change in its tissue specificity is detected. Furthermore, we observed a modulation of pM activity by elements lying further upstream and downstream from pM. Interestingly, pM was expressed in a tissue-specific way in all transgenic mice in which the 280-bp region was present (32 lines and six founder animals). This observation led us to suggest that the proximal pM region contains elements that are able to override to some extent the effects of the surrounding chromatin.


Subject(s)
Fructose-Bisphosphate Aldolase/genetics , Gene Expression Regulation , Muscles/enzymology , Promoter Regions, Genetic/genetics , Animals , Base Sequence , Chloramphenicol O-Acetyltransferase/biosynthesis , Chloramphenicol O-Acetyltransferase/genetics , Deoxyribonuclease I/metabolism , Fructose-Bisphosphate Aldolase/biosynthesis , Humans , Mice , Mice, Transgenic , Molecular Sequence Data , Multigene Family/genetics , RNA, Messenger/biosynthesis , Recombinant Fusion Proteins/biosynthesis , Tissue Distribution , Transcription, Genetic
13.
Nature ; 369(6475): 19-20, 1994 May 05.
Article in English | MEDLINE | ID: mdl-8164733
14.
Dev Suppl ; : 43-51, 1994.
Article in English | MEDLINE | ID: mdl-7579523

ABSTRACT

The segment polarity gene hedgehog plays a central role in cell patterning during embryonic and post-embryonic development of the dipteran, Drosophila melanogaster. Recent studies have identified a family of hedgehog related genes in vertebrates; one of these, Sonic hedgehog is implicated in positional signalling processes that show interesting similarities with those controlled by its Drosophila homologue.


Subject(s)
Drosophila Proteins , Drosophila/growth & development , Genes, Insect , Proteins/genetics , Vertebrates/growth & development , Amino Acid Sequence , Animals , Chick Embryo , Drosophila/embryology , Drosophila/genetics , Embryonic Induction/genetics , Extremities/embryology , Gene Expression , Hedgehog Proteins , Mice , Molecular Sequence Data , Morphogenesis/genetics , Sequence Alignment , Vertebrates/embryology , Vertebrates/genetics
15.
Cell ; 75(7): 1431-44, 1993 Dec 31.
Article in English | MEDLINE | ID: mdl-8269519

ABSTRACT

The segment polarity gene hedgehog (hh) encodes a novel signaling protein that mediates local cell-cell interactions in the developing Drosophila embryo. Here we describe the existence of an hh-related gene family in the zebrafish, Brachydanio rerio. One of these genes, sonic hedgehog (shh), is expressed in the notochord, floor plate, and posterior fin mesoderm, tissues associated with polarizing activities in various vertebrate embryos. The pattern of shh expression in zebra-fish mutants affecting axial structures, together with the consequences of its ectopic expression in normal embryos, is consistent with a role for shh in floor plate induction. By expressing shh in transgenic Drosophila embryos, we also demonstrate a strong functional conservation between the fish and fly hh genes.


Subject(s)
Drosophila Proteins , Proteins/genetics , Zebrafish/embryology , Amino Acid Sequence , Animals , Base Sequence , Cloning, Molecular , DNA Primers/chemistry , Female , Gene Expression , Genes , Hedgehog Proteins , Male , Molecular Sequence Data , Morphogenesis , RNA, Messenger/genetics , Sequence Alignment , Sequence Homology, Amino Acid
16.
Am J Physiol ; 265(3 Pt 1): C626-9, 1993 Sep.
Article in English | MEDLINE | ID: mdl-8214018

ABSTRACT

Thymic involution occurs in young adult male Wistar rats that have performed two runs to exhaustion (RTE) on a treadmill, separated by a 24-h rest period, but not after a single RTE. We were interested in determining whether programmed cell death (or apoptosis) is responsible for the corresponding decrease in T-cell numbers in the thymus. DNA fragmentation, which is an early feature of apoptosis and easily detected by agarose gel electrophoresis, was found in rat thymocytes after the second RTE (the duration of 1 RTE was approximately 5 h). It was also detected after a single RTE or after 2.5 h of running only, and the levels of DNA fragmentation were always roughly similar. In addition, DNA fragmentation was decreased in RU-486 vs. vehicle-treated rats that had run for 2.5 h. These results indicate that physical stress induces glucocorticoid receptor-mediated apoptosis of rat thymocytes. Because apoptosis is induced to similar levels during mild and severe physical stresses, some additional events must be associated to provoke thymic involution.


Subject(s)
Apoptosis , Physical Exertion , Thymus Gland/cytology , Animals , Apoptosis/drug effects , Male , Mifepristone/pharmacology , Rats , Rats, Wistar
17.
Mol Cell Biol ; 13(1): 9-17, 1993 Jan.
Article in English | MEDLINE | ID: mdl-8417365

ABSTRACT

The human aldolase A gene is transcribed from three different promoters, pN, pM, and pH, all of which are clustered within a small 1.6-kbp DNA domain. pM, which is highly specific to adult skeletal muscle, lies in between pN and pH, which are ubiquitous but particularly active in heart and skeletal muscle. A ubiquitous enhancer, located just upstream of pH start sites, is necessary for the activity of both pH and pN in transient transfection assays. Using transgenic mice, we studied the sequence controlling the muscle-specific promoter pM and the relations between the three promoters and the ubiquitous enhancer. A 4.3-kbp fragment containing the three promoters and the ubiquitous enhancer showed an expression pattern consistent with that known in humans. In addition, while pH was active in both fast and slow skeletal muscles, pM was active only in fast muscle. pM activity was unaltered by the deletion of a 1.8-kbp region containing the ubiquitous enhancer and the pH promoter, whereas pN remained active only in fast skeletal muscle. These findings suggest that in fast skeletal muscle, a tissue-specific enhancer was acting on both pN and pM, whereas in other tissues, the ubiquitous enhancer was necessary for pN activity. Finally, a 2.6-kbp region containing the ubiquitous enhancer and only the pH promoter was sufficient to bring about high-level expression of pH in cardiac and skeletal muscle. Thus, while pH and pM function independently of each other, pN, remarkably, shares regulatory elements with each of them, depending on the tissue. Importantly, expression of the transgenes was independent of the integration site, as originally described for transgenes containing the beta-globin locus control region.


Subject(s)
Fructose-Bisphosphate Aldolase/genetics , Muscles/physiology , Promoter Regions, Genetic , Animals , Cell Differentiation , DNA, Recombinant , Enhancer Elements, Genetic , Gene Expression Regulation, Enzymologic , Globins/genetics , Humans , Mice , Mice, Transgenic , Muscles/cytology , RNA, Messenger/genetics
18.
Nucleic Acids Res ; 19(15): 4173-80, 1991 Aug 11.
Article in English | MEDLINE | ID: mdl-1651479

ABSTRACT

The human aldolase A gene is transcribed from three different promoters, which are all clustered within a 1.6 kbp DNA domain. Two of these, PN and PH, are ubiquitous and seem to be co-regulated in most tissues while the third one, PM, is specific to adult skeletal muscle. We investigated the sequences involved in the ubiquitous activity of the PN and PH promoters of the human aldolase A gene. Deletion analysis, performed by transient expression assays of chloramphenicol acetyltransferase reporter genes in human HepG2 hepatoma cells, indicated that PH activity results from the interaction of an upstream activating region with two distinct core promoters. The upstream activating region was able to stimulate transcription from the HSV tk promoter as efficiently as the SV40 enhancer in all cell types tested. It appears, therefore, to be a strong ubiquitous enhancer. DNAsel footprinting revealed protections covering sequences scattered along the enhancer, including Sp1 and AP1 motifs. Importantly, we found that this enhancer was also necessary to activity of the other ubiquitous promoter of the aldolase A gene, PN. These studies demonstrate that expression of the human aldolase A gene is mediated by a complex interplay of enhancer and promoter elements.


Subject(s)
Enhancer Elements, Genetic/physiology , Fructose-Bisphosphate Aldolase/genetics , Promoter Regions, Genetic/physiology , Base Sequence , Chromosome Deletion , Enhancer Elements, Genetic/genetics , Gene Expression Regulation/physiology , Humans , Molecular Sequence Data , Plasmids/genetics , Promoter Regions, Genetic/genetics , Simian virus 40/genetics , Thymidine Kinase/genetics , Transfection/genetics , Tumor Cells, Cultured
19.
Biochem Biophys Res Commun ; 178(2): 553-7, 1991 Jul 31.
Article in English | MEDLINE | ID: mdl-1650193

ABSTRACT

Illegitimate transcription corresponds to the low level presence of specific transcripts in nonspecific cells. This phenomenon allows to analyse any tissue-specific disease transcript in any easily accessible cell. We demonstrate here that the start sites of transcription are the same in specific and non-specific cells, which indicates that illegitimate transcription is due to a low level activity of the normal promoter. In addition, it is possible to increase about 10 fold the abundance of illegitimate transcripts through the use of cycloheximide. This treatment should, therefore, facilitate detection and qualitative analysis of illegitimate transcripts.


Subject(s)
Promoter Regions, Genetic , Transcription, Genetic , Base Sequence , Blotting, Southern , Carcinoma, Hepatocellular , Cell Line , Cell Transformation, Viral , Herpesvirus 4, Human/genetics , Humans , Liver Neoplasms , Molecular Sequence Data , Muscles/physiology , Oligonucleotide Probes , Polymerase Chain Reaction/methods
20.
Proc Natl Acad Sci U S A ; 86(8): 2617-21, 1989 Apr.
Article in English | MEDLINE | ID: mdl-2495532

ABSTRACT

Using in vitro amplification of cDNA by the polymerase chain reaction, we have detected spliced transcripts of various tissue-specific genes (genes for anti-Müllerian hormone, beta-globin, aldolase A, and factor VIIIc) in human nonspecific cells, such as fibroblasts, hepatoma cells, and lymphoblasts. In rats, erythroid- and liver-type pyruvate kinase transcripts were also detected in brain, lung, and muscle. The abundance of these "illegitimate" transcripts is very low; yet, their existence and the possibility of amplifying them by the cDNA polymerase chain reaction provide a powerful tool to analyze pathological transcripts of any tissue-specific gene by using any accessible cell.


Subject(s)
Glycoproteins , Growth Inhibitors , Transcription, Genetic , Animals , Anti-Mullerian Hormone , Factor VIII/genetics , Fibroblasts/physiology , Fructose-Bisphosphate Aldolase/genetics , Gene Amplification , Gene Expression Regulation , Globins/genetics , Humans , Liver Neoplasms, Experimental/genetics , Lymphocytes/physiology , Pyruvate Kinase/genetics , RNA Splicing , RNA, Messenger/genetics , Rats , Testicular Hormones/genetics
SELECTION OF CITATIONS
SEARCH DETAIL
...