Loss of genomic methylation causes p53-dependent apoptosis and epigenetic deregulation.

Cytosine methylation of mammalian DNA is essential for the proper epigenetic regulation of gene expression and maintenance of genomic integrity. To define the mechanism through which demethylated cells die, and to establish a paradigm for identifying genes regulated by DNA methylation, we have generated mice with a conditional allele for the maintenance DNA methyltransferase gene Dnmt1. Cre-mediated deletion of Dnmt1 causes demethylation of cultured fibroblasts and a uniform p53-dependent cell death. Mutational inactivation of Trp53 partially rescues the demethylated fibroblasts for up to five population doublings in culture. Oligonucleotide microarray analysis showed that up to 10% of genes are aberrantly expressed in demethylated fibroblasts. Our results demonstrate that loss of Dnmt1 causes cell-type-specific changes in gene expression that impinge on several pathways, including expression of imprinted genes, cell-cycle control, growth factor/receptor signal transduction and mobilization of retroelements.

Chick myotendinous antigen. I. A monoclonal antibody as a marker for tendon and muscle morphogenesis.

Extracellular matrix components are likely to be involved in the interaction of muscle with nonmuscle cells during morphogenesis and in adult skeletal muscle. With the aim of identifying relevant molecules, we generated monoclonal antibodies that react with the endomysium, i.e., the extracellular matrix on the surface of single muscle fibers. Antibody M1, which is described here, specifically labeled the endomysium of chick anterior latissimus dorsi muscle (but neither the perimysium nor, with the exception of blood vessels and perineurium, the epimysium ). Endomysium labeling was restricted to proximal and distal portions of muscle fibers near their insertion points to tendon, but absent from medial regions of the muscle. Myotendinous junctions and tendon fascicles were intensely labeled by M1 antibody. In chick embryos, " myotendinous antigen" (as we tentatively call the epitope recognized by M1 antibody) appeared first in the perichondrium of vertebrae and limb cartilage elements, from where it gradually extended to the premuscle masses. Around day 6, tendon primordia were clearly labeled. The other structures labeled by M1 antibody in chick embryos were developing smooth muscle tissues, especially aorta, gizzard, and lung buds. In general, tissues labeled with M1 antibody appeared to be a subset of the ones accumulating fibronectin. In cell cultures, M1 antibody binds to fuzzy, fibrillar material on the substrate and cell surfaces of living fibroblast and myogenic cells, which confirms an extracellular location of the antigenic site. The appearance of myotendinous antigen during limb morphogenesis and its distribution in adult muscle and tendon are compatible with the idea that it might be involved in attaching muscle fibers to tendon fascicles. Its biochemical characterization is described in the accompanying paper ( Chiquet , M., and D. Fambrough , 1984, J. Cell Biol. 98:1937-1946).

Chick myotendinous antigen. II. A novel extracellular glycoprotein complex consisting of large disulfide-linked subunits.

This report describes the biochemical characterization of a novel extracellular matrix component, " myotendinous antigen," which appears early in chick limb morphogenesis at sites connecting developing muscle fibers, tendons, and bone ( Chiquet , M., and D. Fambrough , 1984; J. Cell Biol., 98:1926-1936). This extracellular matrix antigen is a major component of the secretory proteins released into the medium by fibroblast and muscle cultures; the soluble form is characterized here. This form of myotendinous antigen is a large glycoprotein complex consisting of several disulfide linked subunits (Mr approximately 150,000-240,000). The differently sized antigen subunits are related, since they yielded very similar proteolytic cleavage patterns. M1 antibody can bind to the denatured subunits. The antigen subunits, as well as a Mr approximately 80,000 pepsin-resistant antigenic domain derived from them, are resistant to bacterial collagenase. Despite possessing subunits similar in size to fibronectin, myotendinous antigen appears to be both structurally and antigenically unrelated to fibronectin or to other known extracellular matrix components. About seven times more M1 antigen per cell nucleus was released into the medium in fibroblast as compared to muscle cultures. In muscle conditioned medium, myotendinous antigen is noncovalently complexed to very high molecular weight material that could be heavily labeled by [3H]glucosamine and [35S]sulfate. This material is sensitive to chondroitinase ABC and hence appears to contain sulfated glycosaminoglycans. We speculate that myotendinous antigen might interact with proteoglycans on the surface of muscle fibers, thereby acting as a link to tendons.

Synthesis, insertion into the plasma membrane, and turnover of alpha-bungarotoxin receptors in chick sympathetic neurons.

alpha-Bungarotoxin was used to identify an integral membrane protein in the plasma membrane of chick sympathetic neurons. The synthesis, insertion into the plasma membrane, and turnover of the alpha-bungarotoxin receptor were studied using isotopically labeled amino acids (2H, 13C, 15N) to directly label receptor molecules. Neurons incubated in medium containing dense amino acids continued to insert unlabeled receptors from a pool of previously synthesized molecules for 2 h. Density-labeled receptors began to appear in the plasma membrane after this 2-h period. Synthesis of receptors, but not insertion into the surface, was blocked by cycloheximide (100 microgram/ml). Neither colchicine (0.05 microgram/ml) of actinomycin D (5 microgram/ml) has any effect on alpha-bungarotoxin receptor synthesis or insertion. Autoradiographic studied revealed that receptors occur on growth cones, axons, and cell bodies of single neurons and explanted ganglia. The rate of insertion of newly synthesized receptors into the plasma membrane of axons extending from explanted sympathetic ganglia was approximately the same as that into the cell body portion of the ganglion. Cytochalasin B (2 microgram/ml) rapidly distrupted growth cones but had no effect on receptor insertion. These experiments suggested that the growth cone is not the sole or even the primary site for insertion of this membrane protein. The kinetics of turnover of the alpha-bungarotoxin receptor were a first-order exponential with t 1/2 = 11 h. Neurons that had their surface receptors labeled with 125I-alpha-bungarotoxin produced [125I]iodotyrosine. This process was inhibited by low temperature (23 degrees C) and also by a metabolic inhibitor. This is interpreted as evidence that receptors turn over by a mechanism in which they are internalized and then proteolytically degraded.

Fluidity of the surface of cultured muscle fibers. Rapid lateral diffusion of marked surface antigens.

Fluorescent antibody fragments of anti-muscle plasma membrane antibody bound as small fluorescent spots when applied by micropipetting to cultured myotubes. The spots were observed to enlarge with time. The rate of enlargement of fluorescent spots was greater when fragments were applied than when divalent antibody was used. It was also greater at 23 degrees -25 degrees C than at 0 degrees -4 degrees C. With glutaraldehyde-fixed cells no increase in the size of the spots was seen. The observations are consistent with the spread of fluorescent spots due to diffusion of surface protein antigens within the plane of a fluid membrane. From measurements of spot size against time, a diffusion constant of 1-3 x 10(-9) cm(2) s(-1) can be calculated for muscle plasma membrane proteins of mol wt approximately 200,000. This value is consistent with other observations on the diffusion of surface antigens and of labeled lipid molecules in synthetic and natural membranes.

Different steady state subcellular distributions of the three splice variants of lysosome-associated membrane protein LAMP-2 are determined largely by the COOH-terminal amino acid residue.

The extensively glycosylated lysosome-associated membrane proteins (LAMP)-2a, b, and c are derived from a single gene by alternative splicing that produces proteins with differences in the transmembrane and cytosolic domains. The lysosomal targeting signals reside in the cytosolic domain of these proteins. LAMPs are not restricted to lysosomes but can also be found in endosomes and at the cell surface. We investigated the subcellular distribution of chimeras comprised of the lumenal domain of avian LAMP-1 and the alternatively spliced domains of avian LAMP-2. Chimeras with the LAMP-2c cytosolic domain showed predominantly lysosomal distribution, while higher levels of chimeras with the LAMP-2a or b cytosolic domain were present at the cell surface. The increase in cell surface expression was due to differences in the recognition of the targeting signals and not saturation of intracellular trafficking machinery. Site-directed mutagenesis defined the COOH-terminal residue of the cytosolic tail as critical in governing the distributions of LAMP-2a, b, and c between intracellular compartments and the cell surface.

Newly synthesized acetylcholine receptors are located in the Golgi apparatus.

Chick skeletal muscle cells in tissue culture were fixed and treated with saponin to allow [125I]alpha-bungarotoxin access into the cells while preserving ultrastructure. The kinetics of binding of iodinated alpha-bungarotoxin to intracellular acetylcholine (ACh) receptors and to surface A Ch receptors were comparable. About half of the intracellular ACh receptors are newly synthesized and in the pathway leading to incorporation into the plasma membrane. Correlated electron microscope autoradiographic and kinetic studies of this receptor population suggest that a substantial fraction of the newly synthesized ACh receptors are located in the Golgi apparatus, where they reside for approx. 2 h.

Aggregates of acetylcholine receptors are associated with plaques of a basal lamina heparan sulfate proteoglycan on the surface of skeletal muscle fibers.

Hybridoma techniques have been used to generate monoclonal antibodies to an antigen concentrated in the basal lamina at the Xenopus laevis neuromuscular junction. The antibodies selectively precipitate a high molecular weight heparan sulfate proteoglycan from conditioned medium of muscle cultures grown in the presence of [35S]methionine or [35S]sulfate. Electron microscope autoradiography of adult X. laevis muscle fibers exposed to 125I-labeled antibody confirms that the antigen is localized within the basal lamina of skeletal muscle fibers and is concentrated at least fivefold within the specialized basal lamina at the neuromuscular junction. Fluorescence immunocytochemical experiments suggest that a similar proteoglycan is also present in other basement membranes, including those associated with blood vessels, myelinated axons, nerve sheath, and notochord. During development in culture, the surface of embryonic muscle cells displays a conspicuously non-uniform distribution of this basal lamina proteoglycan, consisting of large areas with a low antigen site-density and a variety of discrete plaques and fibrils. Clusters of acetylcholine receptors that form on muscle cells cultured without nerve are invariably associated with adjacent, congruent plaques containing basal lamina proteoglycan. This is also true for clusters of junctional receptors formed during synaptogenesis in vitro. This correlation indicates that the spatial organization of receptor and proteoglycan is coordinately regulated, and suggests that interactions between these two species may contribute to the localization of acetylcholine receptors at the neuromuscular junction.

(Na+ + K+)-ATPase correlated with a major group of intramembrane particles in freeze-fracture replicas of cultured chick myotubes.

Immunofluorescence microscopy with a fluorescein-labeled monoclonal antibody was used to map the distribution of sodium- and potassium-ion stimulated ATPase [( Na,K]-ATPase) on the surface of tissue-cultured chick skeletal muscle. At this level of resolution it appeared that the (Na,K)-ATPase molecules were distributed nearly uniformly over the plasma membrane. These molecules could be cross-linked by use of the monoclonal antibody followed by a second antibody directed against the monoclonal antibody; the resulting fluorescent pattern was a set of small dots (patches) on the muscle surface. This pattern was stable over several hours, and there was little evidence of interiorization or of coalescence of the patches. Myotubes labeled with immunofluorescence were fixed in glutaraldehyde, cryoprotected with glycerin, then fractured and replicated by standard methods. Replicas of the immunofluorescence-labeled myotubes revealed clusters of intramembrane particles (IMP) only when the immunofluorescent images indicated a patching of the (Na,K)-ATPase molecules. Double antibody cross-linking of antigenic sites on myotubes with each of three other monoclonal antibodies to plasma membrane antigens likewise resulted in patched patterns of immunofluorescence, but in none of these cases were clusters of intramembrane particles found in freeze-fracture replicas. In each case it was shown that the (Na,K)-ATPase molecules were not patched. Other control experiments showed that patching of (Na,K)-ATPase molecules did not cause co-patching of one of the other plasma membrane proteins defined by a monoclonal antibody and did not cause detectable co-clustering of acetylcholine receptors. Detailed mapping showed that there was a one-to-one correspondence between immunofluorescent patches related to the (Na,K)-ATPase and clusters of IMP in a freeze-fracture replica of the same cell. We conclude that the intramembrane particles patched by double antibody cross-linkage of the (Na,K)-ATPase are caused by (Na,K)-ATPase molecules in the fracture plane. Quantification of the IMP indicated that the (Na,K)-ATPase-related particles account for up to 50% of particles evident in the replicas, or up to about 400 particles/micrometers2 of plasma membrane. Particles related to the (Na,K)-ATPase were similar to the average particle size and were as heterodisperse in size as the total population of IMP.(ABSTRACT TRUNCATED AT 400 WORDS)

Lysosomal membrane dynamics: structure and interorganellar movement of a major lysosomal membrane glycoprotein.

The biochemistry and intracellular transit of an integral membrane glycoprotein of chicken fibroblast lysosomes were studied with monoclonal antibody techniques. The glycoprotein had an apparent molecular weight of 95,000-105,000. Structural analysis involving metabolic labeling with [35S]methionine and cleavage with glycosidases revealed the presence of numerous oligosaccharide chains N-linked to a core polypeptide of apparent molecular weight 48,000. A primary localization of the glycoprotein to lysosomes was demonstrated by the coincidence of antibody binding sites with regions of acridine orange uptake, electron immunocytochemical labeling on the inner surface of lysosome-like vacuolar membranes, and preferential association of the glycoprotein with lysosome-enriched subcellular fractions from Percoll gradients. In addition, small quantities of the glycoprotein were detected on endocytic vesicle and plasma membranes. To study the intracellular pathway of the glycoprotein, we used a monoclonal antibody whose binding to the glycoprotein at the cell surface had no effect on the number or subcellular distribution of antigen molecules. Incubation of chicken fibroblasts with monoclonal antibody at 37 degrees C led to the rapid uptake and subsequent delivery of antibody to lysosomes, where antibody was degraded. This process continued undiminished for many hours on cells continuously exposed to the antibody and was not blocked by the addition of cycloheximide. The rate at which antigen sites were replenished in the plasma membrane of cells prelabeled with antibody (t1/2 = 2 min) was essentially equivalent to the rate of internalization of antibody bound to cell surfaces. These results suggest that there is a continuous and rapid exchange of this glycoprotein between plasma membrane and the membranes of endosomes and/or lysosomes.

Acetylcholine receptor turnover in membranes of developing muscle fibers.

[125I mono-iodo-alpha-bungarotoxin is used as a specific marker in a description of acetylcholine receptor metabolism. It is concluded that acetylcholine receptors in the surface membranes of chick and rat myotubes developing in cell cultures have a half-life of 22-24 h. Alpha-bungarotoxin (bound to a receptor which is removed from the membrane) is degraded to monoiodotyrosine which appears in the medium. Several observations are consistent with a model in which receptors or alpha-bungarotoxin-receptor complexes are internalized and then degraded: (a) the rate of appearance of iodotyrosine does not reach its maximal rate until 90 min after alpha-bungarotoxin is bound to the surface receptors; (b) 2,4-dinitrophenol, reduced temperature, and cell disruption all inhibit the degradation process. The degradation of surface receptors is not coupled to the process by which receptors are incorporated into the membrane. Evidence suggest that receptors are incorporated into the surface membrane from a presynthesized set of receptors containing about 10% as many alpha-bungarotoxin binding sites as does the surface. Additionally, a third set of acetylcholine receptors is described containing about 30% as amny binding sites as does the surface. These "hidden" recptors are not precursors yet are not readily accessible for binding of extracellular alpha-bungarotoxin. These findings are discussed in relation to both plasma membrane biosynthesis and control of chemosensitivity in developing and denervated skeletal muscle.

Fibronectin expression during myogenesis.

The biosynthesis and localization of fibronectin during chick muscle differentiation are described. This study employed two monoclonal antibodies, one that selectively killed mononucleated cells and one specific for avian fibronectin. These antibodies allowed precise analyses of fibronectin expression in well-defined cultures of myoblasts or myotubes and avoided the complications of exogenous fibronectin and contamination by fibroblasts or unfused myoblasts. Fibronectin synthesis, as a fraction of total protein synthesis, remains constant at 0.3-0.4% before and after myoblast fusion, suggesting that the absolute rate of fibronectin synthesis may increase somewhat when myotubes synthesize and accumulate myofibrillar proteins. The pattern of fibronectin arrangement does change during myogenesis. In myotube cultures, the appearance of pulse-labeled fibronectin at the cell surface and its secretion into the medium begin after a 2-3-h lag period, in contrast to the 30-min lag period observed in fibroblast cultures. This lag between polypeptide biosynthesis and the exteriorization of the new protein is thus a characteristic of each cell type rather than the protein. All of the major secretory proteins of myogenic cells, including fibronectin and collagenous components, share this 2-3-h intracellular transit time.

Extracellular matrix organization in developing muscle: correlation with acetylcholine receptor aggregates.

Monoclonal antibodies recognizing laminin, heparan sulfate proteoglycan, fibronectin, and two apparently novel connective tissue components have been used to examine the organization of extracellular matrix of skeletal muscle in vivo and in vitro. Four of the five monoclonal antibodies are described for the first time here. Immunocytochemical experiments with frozen-sectioned muscle demonstrated that both the heparan sulfate proteoglycan and laminin exhibited staining patterns identical to that expected for components of the basal lamina. In contrast, the remaining matrix constituents were detected in all regions of muscle connective tissue: the endomysium, perimysium, and epimysium. Embryonic muscle cells developing in culture elaborated an extracellular matrix, each antigen exhibiting a unique distribution. Of particular interest was the organization of extracellular matrix on myotubes: the build-up of matrix components was most apparent in plaques overlying clusters of an integral membrane protein, the acetylcholine receptor (AChR). The heparan sulfate proteoglycan was concentrated at virtually all AChR clusters and showed a remarkable level of congruence with receptor organization; laminin was detected at 70-95% of AChR clusters but often was not completely co-distributed with AChR within the cluster; fibronectin and the two other extracellular matrix antigens occurred at approximately 20, 8, and 2% of the AChR clusters, respectively, and showed little or no congruence with AChR. From observations on the distribution of extracellular matrix components in tissue cultured fibroblasts and myogenic cells, several ideas about the organization of extracellular matrix are suggested. (a) Congruence between AChR clusters and heparan sulfate proteoglycan suggests the existence of some linkage between the two molecules, possibly important for regulation of AChR distribution within the muscle membrane. (b) The qualitatively different patterns of extracellular matrix organization over myotubes and fibroblasts suggest that each of these cell types uses somewhat different means to regulate the assembly of extracellular matrix components within its domain. (c) The limited co-distribution of different components within the extracellular matrix in vitro and the selective immune precipitation of each antigen from conditioned medium suggest that each extracellular matrix component is secreted in a form that is not complexed with other matrix constituents.

The pathway and targeting signal for delivery of the integral membrane glycoprotein LEP100 to lysosomes.

A complete set of chimeras was made between the lysosomal membrane glycoprotein LEP100 and the plasma membrane-directed vesicular stomatitis virus G protein, combining a glycosylated lumenal or ectodomain, a single transmembrane domain, and a cytosolic carboxyl-terminal domain. These chimeras, the parent molecules, and a truncated form of LEP100 lacking the transmembrane and cytosolic domains were expressed in mouse L cells. Only LEP100 and chimeras that included the cytosolic 11 amino acid carboxyl terminus of LEP100 were targeted to lysosomes. The other chimeras accumulated in the plasma membrane, and truncated LEP100 was secreted. Chimeras that included the extracellular domain of vesicular stomatitis G protein and the carboxyl terminus of LEP100 were targeted to lysosomes and very rapidly degraded. Therefore, in chimera-expressing cells, virtually all the chimeric molecules were newly synthesized and still in the biosynthesis and lysosomal targeting pathways. The behavior of one of these chimeras was studied in detail. After its processing in the Golgi apparatus, the chimera entered the plasma membrane/endosome compartment and rapidly cycled between the plasma membrane and endosomes before going to lysosomes. In pulse-expression experiments, a large population of chimeric molecules was observed to appear transiently in the plasma membrane by immunofluorescence microscopy. Soon after protein synthesis was inhibited, this surface population disappeared. When lysosomal proteolysis was inhibited, chimeric molecules accumulated in lysosomes. These data suggest that the plasma membrane/early endosome compartment is on the pathway to the lysosomal membrane. This explains why mutations that block endocytosis result in the accumulation of lysosomal membrane proteins in the plasma membrane.

Structure of LEP100, a glycoprotein that shuttles between lysosomes and the plasma membrane, deduced from the nucleotide sequence of the encoding cDNA.

LEP100, a membrane glycoprotein that has the unique property of shuttling from lysosomes to endosomes to plasma membrane and back, was purified from chicken brain. Its NH2-terminal amino acid sequence was determined, and an oligonucleotide encoding part of this sequence was used to clone the encoding cDNA. The deduced amino acid sequence consists of 414 residues of which the NH2-terminal 18 constitute a signal peptide. The sequence includes 17 sites for N-glycosylation in the NH2-terminal 75% of the polypeptide chain followed by a region lacking N-linked oligosaccharides, a single possible membrane-spanning segment, and a cytoplasmic domain of 11 residues, including three potential phosphorylation sites. Eight cysteine residues are spaced in a regular pattern through the lumenal (extracellular) domain, while a 32-residue sequence rich in proline, serine, and threonine occurs at its midpoint. Expression of the cDNA in mouse L cells resulted in targeting of LEP100 primarily to the mouse lysosomes.

An endogenous MDCK lysosomal membrane glycoprotein is targeted basolaterally before delivery to lysosomes.

Using surface immunoprecipitation at 37 degrees C to "catch" the transient apical or basolateral appearance of an endogenous MDCK lysosomal membrane glycoprotein, the AC17 antigen, we demonstrate that the bulk of newly synthesized AC17 antigen is polarly targeted from the Golgi apparatus to the basolateral plasma membrane or early endosomes and is then transported to lysosomes via the endocytic pathway. The AC17 antigen exhibits very similar properties to members of the family of lysosomal-associated membrane glycoproteins (LAMPs). Parallel studies of an avian LAMP, LEP100, transfected into MDCK cells revealed colocalization of the two proteins to lysosomes, identical biosynthetic and degradation rates, and similar low levels of steady-state expression on both the apical (0.8%) and basolateral (2.1%) membranes. After treatment of the cells with chloroquine, newly synthesized AC17 antigen, while still initially targeted basolaterally, appears stably in both the apical and basolateral domains, consistent with the depletion of the AC17 antigen from lysosomes and its recycling in a nonpolar fashion to the cell surface.

Acetylcholine sensitivity of muscle fiber membranes: mechanism of regulation by motoneurons.

Inhibitors of RNA and protein synthesis prevent the development of acetylcholine supersensitivity in organ-cultured rat diaphragm muscle but do not affect established acetylcholine sensitivity. The restriction of this sensitivity in innervated muscle apparently involves muscle fibers.

Neuromuscular junction in myasthenia gravis: decreased acetylcholine receptors.

The number of acetylcholine receptors was determined in the neuromuscular junctions of eight patients with typical myasthenia gravis and in five controls, by means of (125)1-labeled alpha-bungarotoxin binding. The junctional acetylcholine receptors were reduced in the myasthenic muscles as compared with the controls. This reduction in receptors may account for the defect in neuromuscular transmission in myasthenia gravis.

Acetylcholine receptors: number and distribution at neuromuscular junctions in rat diaphragm.

The number of acetylcholine receptors per motor end plate in the rat diaphragm, measured by the binding of [(125)1]alpha-bungarotoxin, varies directly with rat size and is (4.0 +/- 0.2) x 10(7) for full-grown male rats. Autoradiographic analysis of single fibers labeled with this substance reveals that virtually all of these receptors are localized in the end plate.

The Biology of Isolated Chromatin: Chromosomes, biologically active in the test tube, provide a powerful tool for the study of gene action.

The isolated chromatin of higher organisms possesses several properties characteristic of the same chromatin in life. These include the presence of histone bound to DNA, the state of repression of the genetic material, and the ability to serve as template for the readout of the derepressed portion of the genome by RNA polymerase. The important respect in which isolated chromatin differs from the material in vivo, fragmentation of DNA into pieces shorter (5 x 10(6) to 20 x 10(6) molecular weight) than the original, does not appear to importantly alter such transcription. The study of isolated chromatin has already revealed the material basis of the restriction of template activity; it is the formation of a complex between histone and DNA. Chromatin isolated by the methods now available, together with the basis provided by our present knowledge of chromatin biochemistry and biophysics, should make possible and indeed assure rapid increase in our knowledge of chromosomal structure and of all aspects of the control of gene activity and hence of developmental processes.