37 results on '"Olson, E. N."'
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2. Functional receptors for transforming growth factor-beta are retained by biochemically differentiated C2 myocytes in growth factor-deficient medium containing EGTA but down-regulated during terminal differentiation.
- Author
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Hu, J S, primary and Olson, E N, additional
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- 1990
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3. Muscle creatine kinase isoenzyme expression in adult human brain.
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Hamburg, R J, primary, Friedman, D L, additional, Olson, E N, additional, Ma, T S, additional, Cortez, M D, additional, Goodman, C, additional, Puleo, P R, additional, and Perryman, M B, additional
- Published
- 1990
- Full Text
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4. Different members of the jun proto-oncogene family exhibit distinct patterns of expression in response to type beta transforming growth factor.
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Li, L, primary, Hu, J S, additional, and Olson, E N, additional
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- 1990
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5. Cloning and characterization of a novel Kruppel-associated box family transcriptional repressor that interacts with the retinoblastoma gene product, RB.
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Skapek, S X, Jansen, D, Wei, T F, McDermott, T, Huang, W, Olson, E N, and Lee, E Y
- Abstract
The retinoblastoma gene product, RB, seems to function as a key tumor suppressor by repressing the expression of genes activated by members of the E2F family of transcription factors. In order to accomplish this, RB has been proposed to interact with a transcriptional repressor. However, no genuine transcriptional repressors have been identified by virtue of interaction with RB. By using the yeast two-hybrid system, we have identified a novel member of a known family of transcriptional repressors that contain zinc fingers of the Kruppel type and a portable transcriptional repressor motif known as the Kruppel-associated box (KRAB). The mouse and human forms of the novel RB-associated KRAB protein (RBaK) are widely expressed. The amino acid motif that links the KRAB domain and zinc fingers appears to be required for interaction with RB in vitro. Human RBaK ectopically expressed in fibroblasts is an 80-kDa protein that is localized to the nucleus. The expression of either RB or RBaK in 10T1/2 fibroblasts represses the activation of an E2F-dependent promoter and decreases DNA synthesis to a similar degree. However, a mutant form of RBaK that cannot interact with RB in vitro is unable to prevent DNA synthesis. We present a model in which RB physically interacts with the novel transcriptional repressor RBaK to repress E2F-dependent genes and prevent DNA synthesis.
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- 2000
6. Stimulation of slow skeletal muscle fiber gene expression by calcineurin in vivo.
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Naya, F J, Mercer, B, Shelton, J, Richardson, J A, Williams, R S, and Olson, E N
- Abstract
Adult skeletal muscle fibers can be categorized into fast and slow twitch subtypes based on specialized contractile and metabolic properties and on distinctive patterns of muscle gene expression. Muscle fiber-type characteristics are dependent on the frequency of motor nerve stimulation and are thought to be controlled by calcium-dependent signaling. The calcium, calmodulin-dependent protein phosphatase, calcineurin, stimulates slow fiber-specific gene promoters in cultured skeletal muscle cells, and the calcineurin inhibitor, cyclosporin A, inhibits slow fiber gene expression in vivo, suggesting a key role of calcineurin in activation of the slow muscle fiber phenotype. Calcineurin has also been shown to induce hypertrophy of cardiac muscle and to mediate the hypertrophic effects of insulin-like growth factor-1 on skeletal myocytes in vitro. To determine whether activated calcineurin was sufficient to induce slow fiber gene expression and hypertrophy in adult skeletal muscle in vivo, we created transgenic mice that expressed activated calcineurin under control of the muscle creatine kinase enhancer. These mice exhibited an increase in slow muscle fibers, but no evidence for skeletal muscle hypertrophy. These results demonstrate that calcineurin activation is sufficient to induce the slow fiber gene regulatory program in vivo and suggest that additional signals are required for skeletal muscle hypertrophy.
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- 2000
7. Cooperative transcriptional activation by serum response factor and the high mobility group protein SSRP1.
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Spencer, J A, Baron, M H, and Olson, E N
- Abstract
Serum response factor (SRF) is a MADS box transcription factor that controls a wide range of genes involved in cell proliferation and differentiation. The MADS box mediates homodimerization and binding of SRF to the consensus sequence CC(A/T)6GG, known as a CArG box, which is found in the control regions of numerous serum-inducible and muscle-specific genes. Using a modified yeast one-hybrid screen to identify potential SRF cofactors, we found that SRF interacts with the high mobility group factor SSRP1 (structure-specific recognition protein). This interaction, which occurs in yeast and mammalian cells, is mediated through the MADS box of SRF and a basic region of SSRP1 encompassing amino acids 489-542, immediately adjacent to the high mobility group domain. SSRP1 does not bind the CArG box, but interaction of SSRP1 with SRF dramatically increases the DNA binding activity of SRF, resulting in synergistic transcriptional activation of native and artificial SRF-dependent promoters. These results reveal an important role for SSRP1 as a coregulator of SRF-dependent transcription in mammalian cells.
- Published
- 1999
8. Identification of a Novel Fatty Acylated Protein That Partitions between the Plasma Membrane and Cytosol and Is Deacylated in Response to Serum and Growth Factor Stimulation*
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James, G and Olson, E N
- Abstract
Several proteins involved in transmembrane signaling have been shown previously to be modified covalently by long-chain fatty acids. Using the BC3H1 cell line, which contains a broad array of fatty acylated proteins, we have examined the possibility that acylation of certain proteins is modulated in response to mitogenic stimulation. In the present study, we describe a 64-kDa palmitoylated protein, referred to as p64, that is deacylated following stimulation of quiescent cells with fetal bovine serum, fibroblast growth factor, and phorbol dibutyrate. Western blot analysis of membrane and soluble fractions using a polyclonal antibody against p64 revealed that approximately 70% of p64 in unstimulated cells is present in the cytosol in a non-acylated form, whereas palmitoylated p64 is found exclusively in the membrane fraction. Extraction of membranes with 0.5 M sodium chloride, 0.2 M sodium pyrophosphate, or 0.2 M sodium carbonate failed to release p64, suggesting that the acylated form of this protein is tightly associated with membranes. Pulse labeling of proteins in quiescent cells with [3H] palmitate and subsequent chasing in medium containing 20% fetal bovine serum, fibroblast growth factor, or phorbol dibutyrate revealed that the fatty acid associated with p64 undergoes mitogen-stimulated turnover, whereas turnover of fatty-acid on other acylated proteins is not observed. Palmitate is the predominant fatty acid associated with p64; however, small amounts of covalent myristate are also detected. Both fatty acids are attached post-translationally to p64 through a hydroxylamine-sensitive linkage, suggesting that acylation of this protein is catalyzed by a palmitoyl transferase with relaxed specificity for fatty acid substrates. Together, these results suggest that palmitoylation may participate in the association of p64 with the plasma membrane and that mitogen-dependent deacylation might alter interactions between this protein and other membrane components.
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- 1989
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9. Myristoylation, Phosphorylation, and Subcellular Distribution of the 80-kDa Protein Kinase C Substrate in BC3H1 Myocytes*
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James, G and Olson, E N
- Abstract
Numerous reports have described a phosphoprotein with an apparent molecular mass of 68-87 kDa, often referred to as the 80K protein, which serves as a major specific substrate for protein kinase C in a wide variety of cell types. This protein has been shown to be myristoylated in macrophages, apparently in a stimulus-dependent manner. In the present study, we have defined the kinetics for myristoylation of the 80K protein in BC3H1 myocytes and have examined the subcellular distribution of the [3H]myristate and 32P-labeled forms of the protein before and after activation of protein kinase C by phorbol dibutyrate (PDBu). The 80K protein was identified in BC3H1 myocytes by apparent molecular mass of 68 kDa (consistent with the previously reported size of the murine homologue), isoelectric point of 4.6-4.8, PDBu-inducible phosphorylation, peptide mapping, and labeling with [3H]myristate. Incorporation of [3H]myristate by this protein occurred through an amide linkage and was abolished completely by cycloheximide. Pulse labeling of quiescent cells with [3H]myristate revealed no alteration in myristoylation of the 80K protein in either the crude membrane or soluble fractions after PDBu-induced phosphorylation. The subcellular distribution of this protein (approximately 80% membrane, approximately 20% cytosol) also was the same in control and PDBu-stimulated cells. Phosphorylation of both the membrane-bound and soluble forms was increased approximately 6-fold upon stimulation of cultures with PDBu; the soluble form was phosphorylated to a 4-fold higher stoichiometry than its membrane-bound counterpart. Together, these data demonstrate that the 80K protein is myristoylated cotranslationally in BC3H1 cells and that protein kinase C-dependent phosphorylation of the 80K protein does not alter its subcellular distribution or degree of myristoylation. The fact that 20% of total myristoylated 80K protein resides in the cytosol also indicates that myristoylation alone is not sufficient to target this protein to the plasma membrane.
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- 1989
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10. Overexpression of a single helix-loop-helix-type transcription factor, scleraxis, enhances aggrecan gene expression in osteoblastic osteosarcoma ROS17/2.8 cells.
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Liu, Y, Watanabe, H, Nifuji, A, Yamada, Y, Olson, E N, and Noda, M
- Abstract
Cell differentiation is determined by a certain set of transcription factors such as MyoD in myogenesis. However, transcription factors that play a positive role in phenotypic gene expression in skeletal cells are largely unknown, except the recently identified CBFA1. Scleraxis is a helix-loop-helix-type transcription factor whose transcripts are expressed in sclerotome and in a certain set of skeletal cells; however, nothing is known about its function with regard to the regulation of cell function. To examine possible roles of scleraxis, we overexpressed scleraxis in osteoblastic ROS17/2.8 cells, which express low levels of scleraxis. Scleraxis overexpression enhanced expression of the aggrecan gene, which is not normally expressed at high levels in these osteoblastic cells. Overexpression of scleraxis also increased mRNA levels of type II collagen and osteopontin while suppressing expression of osteoblast phenotype-related genes encoding type I collagen and alkaline phosphatase. Transient transfection experiments indicated that scleraxis enhanced the chloramphenicol acetyltransferase activity of the reporter construct AgCAT-8, which contained an 8-kilobase pair (kb) fragment of the aggrecan gene including both the promoter and its first intron. Deletion analysis identified a 1-kb region that is responsive to scleraxis within the aggrecan gene. This region contains two adjacent E-box sequences. A 29-base pair DNA fragment (AgE) containing these E-box sequences bound to proteins in the ROS17/2.8 cell nuclear extracts as well as to in vitro translated scleraxis. This binding was competed with unlabeled AgE, but not with a mutated E-box DNA sequence (mAgE), indicating the specificity of the binding activity. The AgE binding activity in the ROS17/2.8 cell nuclear extracts was enhanced in the cells overexpressing scleraxis and was supershifted by the antiserum raised against scleraxis. Furthermore, AgE, but not mAgE, conferred responsiveness to scleraxis overexpression to a heterologous promoter. Finally, replacement mutation of the AgE sequence within the 2.5-kb AgCAT-1 construct significantly reduced its responsiveness to scleraxis. These results indicate that overexpression of a single helix-loop-helix-type transcription factor, scleraxis, enhances aggrecan gene expression via binding to the E-box-containing AgE sequence in ROS17/2.8 cells.
- Published
- 1997
11. Phosphorylation of the MADS-Box transcription factor MEF2C enhances its DNA binding activity.
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Molkentin, J D, Li, L, and Olson, E N
- Abstract
Members of the myocyte enhancer factor-2 (MEF2) family of transcription factors activate muscle gene expression by binding an A/T-rich DNA sequence in the control regions of muscle-specific genes. There are four MEF2 factors in vertebrates, MEF2A-D, which share homology in an amino-terminal MADS domain and an adjacent region known as the MEF2 domain, that together mediate DNA binding and dimerization. We show that serine 59 located between the MADS and MEF2 domains of MEF2C is phosphorylated in vivo and can be phosphorylated in vitro by casein kinase-II (CKII). Phosphorylation of this site enhanced the DNA binding and transcriptional activity of MEF2C by increasing its DNA binding activity 5-fold. In vivo 32P labeling experiments showed that serine 59 is the only phosphorylation site in the MADS and MEF2 domains. Mutagenesis of this serine to an aspartic acid resulted in an increase in DNA binding and transcriptional activity of MEF2C comparable to that observed when this site was phosphorylated, suggesting that phosphorylation augments DNA binding activity by introducing negative charge. This phosphorylation site, which corresponds to a CKII recognition site, is conserved in all known MEF2 factors in organisms ranging from flies to humans, consistent with its importance for the functions of MEF2C.
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- 1996
12. Regulation of creatine phosphokinase expression during differentiation of BC3H1 cells.
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Olson, E N, Caldwell, K L, Gordon, J I, and Glaser, L
- Abstract
The intracellular mechanisms involved in the regulation of creatine phosphokinase expression in the BC3H1 muscle-like cell line have been examined under conditions of enzyme induction and repression. In the presence of low serum concentrations, BC3H1 cells cease to grow and synthesize high levels of creatine phosphokinase. When differentiated BC3H1 cultures are exposed to media containing high serum concentrations, cell division is reinitiated and further induction of creatine phosphokinase is inhibited. Accumulation of creatine phosphokinase-mRNA appears to be intimately coupled to the state of growth of BC3H1 cells. Log phase cells do not contain detectable levels of translatable creatine phosphokinase-mRNA; however, following cessation of growth, creatine phosphokinase-mRNA accumulates in approximate proportion to the increase in creatine phosphokinase activity. Reinitiation of cell division in quiescent differentiated cultures results in the arrest of further accumulation of creatine phosphokinase-mRNA but does not inhibit the translation of pre-existing creatine phosphokinase-mRNA. Under conditions of enzyme repression, however, the newly synthesized creatine phosphokinase appears to be enzymatically inactive. These results indicate that the expression of the muscle phenotype in BC3H1 cells is regulated by components present in serum and that myogenic differentiation is at least partially reversible following re-entry of quiescent cells into the cell cycle.
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- 1983
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13. The myogenic regulatory factor MRF4 represses the cardiac alpha-actin promoter through a negative-acting N-terminal protein domain.
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Moss, J B, Olson, E N, and Schwartz, R J
- Abstract
Cardiac alpha-actin is activated early during the development of embryonic skeletal muscle and cardiac myocytes. The gene product remains highly expressed in adult striated cardiac muscle yet is dramatically reduced in skeletal muscle. Activation and repression of cardiac alpha-actin gene activity in developing skeletal muscle correlates with changes in the relative content of the four myogenic regulatory factors. Cardiac alpha-actin promoter activity, assessed in primary chick myogenic cultures, was activated by endogenous myogenic regulatory factors but was inhibited in the presence of co-expressed MRF4. By exchanging N- and C-terminal domains of MRF4 and MyoD, the N terminus of MRF4 was identified as the mediator of repressive activity, revealing a novel negative regulatory role for MRF4. The relative ratios of myogenic regulatory factors may have fundamental roles in selecting specific muscle genes for activation and/or repression.
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- 1996
14. Specific interaction of the PDZ domain protein PICK1 with the COOH terminus of protein kinase C-alpha.
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Staudinger, J, Lu, J, and Olson, E N
- Abstract
PICK1 is a protein kinase C (PKC) alpha-binding protein initially identified using the yeast two-hybrid system. Here we report that PICK1 contains a PDZ domain that interacts specifically with a previously unidentified PDZ-binding domain (QSAV) at the extreme COOH terminus of PKCalpha and that mutation of a putative carboxylate-binding loop within the PICK1 PDZ domain abolishes this interaction. The PDZ-binding domain in PKCalpha is absent from other PKC isoforms that do not interact with PICK1. We also demonstrate that PICK1 can homooligomerize through sequences that are distinct from the carboxylate-binding loop, suggesting that self-association and PKCalpha binding are not mutually exclusive. A Caenorhabditis elegans PICK1-like protein is also able to bind to PKCalpha, suggesting a conservation of function through evolution. Association of PKCalpha with PICK1 provides a potential mechanism for the selective targeting of PKCalpha to unique subcellular sites.
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- 1997
15. Expression of acetylcholine receptor alpha-subunit mRNA during differentiation of the BC3H1 muscle cell line.
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Olson, E N, Glaser, L, Merlie, J P, and Lindstrom, J
- Abstract
The accumulation of translatable acetylcholine receptor alpha-subunit mRNA was examined in the BC3H1 muscle cell line in response to serum and cell growth. Relative amounts of alpha-subunit mRNA were quantitated during differentiation by cell-free translation and immunoprecipitation with an alpha-subunit-specific monoclonal antibody. Logarithmically growing cells do not possess cell surface acetylcholine receptors; however, a significant amount of alpha-subunit mRNA is detectable in cells under these conditions. Furthermore, alpha-subunit is synthesized in growing undifferentiated cells at a rate similar to that of differentiated cultures. Following growth arrest of BC3H1 cells, surface receptors are induced to levels greater than 100-fold above that of growing cells. The relative level of translatable alpha-subunit mRNA in differentiated cells, however, is only approximately 4-fold greater than in growing cultures. Induction of alpha-subunit mRNA appears to be reversible since reinitiation of growth in quiescent differentiated BC3H1 cells results in a reduction in relative abundance of this mRNA species to levels comparable to that of undifferentiated cells and the concomitant loss of surface receptors. These results indicate that receptor expression during differentiation is regulated both post-translationally and at the level of receptor subunit mRNA accumulation.
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- 1984
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16. MEF2B is a component of a smooth muscle-specific complex that binds an A/T-rich element important for smooth muscle myosin heavy chain gene expression.
- Author
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Katoh, Y, Molkentin, J D, Dave, V, Olson, E N, and Periasamy, M
- Abstract
To understand smooth muscle-specific gene expression, we have focused our studies on the smooth muscle myosin heavy chain (SMHC) gene, a smooth muscle-specific marker. In this study, we demonstrate that the SMHC promoter region (-1594 to -1462 base pairs) containing the A/T-rich element can activate the heterologous thymidine kinase promoter in smooth muscle cells, but not in fibroblasts. Mutations of this A/T-rich element decreased SMHC promoter activity significantly. Both gel mobility shift assays and DNase I footprinting revealed that this region binds to specific protein complexes from smooth muscle nuclear extracts, whereas nuclear extracts from skeletal muscle and fibroblasts produced a different binding pattern. We also demonstrate that the protein complex obtained from smooth muscle nuclear extract reacts with MEF2B-specific antibody, but not with antibodies specific to MEF2A, MEF2C, or MEF2D, suggesting that only MEF2B protein binds to the A/T-rich element. Furthermore, MEF2B overexpression in smooth muscle cells up-regulated the SMHC promoter, suggesting that MEF2B is important for SMHC gene regulation. This is the first report demonstrating a role for MEF2 factors in smooth muscle-specific gene expression.
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- 1998
17. Structure, expression, and properties of an atypical protein kinase C (PKC3) from Caenorhabditis elegans. PKC3 is required for the normal progression of embryogenesis and viability of the organism.
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Wu, S L, Staudinger, J, Olson, E N, and Rubin, C S
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Little is known about differential expression, functions, regulation, and targeting of "atypical" protein kinase C (aPKC) isoenzymes in vivo. We have cloned and characterized a novel cDNA that encodes a Caenorhabditis elegans aPKC (PKC3) composed of 597 amino acids. In post-embryonic animals, a 647-base pair segment of promoter/enhancer DNA directs transcription of the 3.6-kilobase pair pkc-3 gene and coordinates accumulation of PKC3 protein in approximately 85 muscle, epithelial, and hypodermal cells. These cells are incorporated into tissues involved in feeding, digestion, excretion, and reproduction. Mammalian aPKCs promote mitogenesis and survival of cultured cells. In contrast, C. elegans PKC3 accumulates in non-dividing, terminally differentiated cells that will not undergo apoptosis. Thus, aPKCs may control cell functions that are independent of cell cycle progression and programmed cell death. PKC3 is also expressed during embryogenesis. Ablation of PKC3 function by microinjection of antisense RNA into oocytes yields disorganized, developmentally arrested embryos. Thus, PKC3 is essential for viability. PKC3 is enriched in particulate fractions of disrupted embryos and larvae. Immunofluorescence microscopy revealed that PKC3 accumulates near cortical actin cytoskeleton/plasma membrane at the apical surface of intestinal cells and in embryonic cells. A candidate anchoring/targeting protein, which binds PKC3 in vitro, has been identified.
- Published
- 1998
18. Decrease in transforming growth factor-beta binding and action during differentiation in muscle cells.
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Ewton, D Z, Spizz, G, Olson, E N, and Florini, J R
- Abstract
We report here the effects of differentiation on the binding and action of transforming growth factor-beta (TGF-beta) in three lines of myogenic cells. In two lines (L6-A1 and C2) which irreversibly differentiate by fusing to form postmitotic myotubes, there is a virtual disappearance of TGF-beta binding sites as differentiation occurs. Analyses of the binding curves by the method of Scatchard indicates that there is little or no change in affinity but a substantial decrease in the number of binding sites. In L6-A1 cells, responsiveness to TGF-beta decreases in parallel to the loss of receptors. The decreases in TGF-beta binding and activity with differentiation are not paralleled by similar changes in another growth factor, insulin-like growth factor-I, which exhibits little change in binding and only a modest decrease in activity as L6-A1 myoblasts differentiate to form myotubes. In a third cell line (BC3H1), which exhibits reversible differentiation without fusion, there is little or no change in TGF-beta binding as the cells differentiate. Comparisons with reported decreases in binding of fibroblast and epidermal growth factors indicates that there are substantial differences in growth factor binding and actions as muscle cells differentiate, but it is not possible to make the simple generalization that differentiation is accompanied by a decrease in binding of all growth factors.
- Published
- 1988
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19. Regulation of differentiation of the BC3H1 muscle cell line through cAMP-dependent and -independent pathways.
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Hu, J S and Olson, E N
- Abstract
Serum mitogens, fibroblast growth factor (FGF), and type beta transforming growth factor (TGF-beta) suppress differentiation of the mouse muscle cell line BC3H1; however, the signal transduction pathways whereby these growth factors exert their effects on this system are unknown. The goal of this study was to determine whether the program for differentiation of BC3H1 cells was susceptible to negative regulation by signaling pathways involving cAMP or protein kinase C and whether these intracellular effectors participate in the mechanism by which growth factors prevent establishment of the myogenic phenotype. Exposure of BC3H1 cells to dibutyryl cAMP, 8-bromo-cAMP, or compounds that stimulate adenylate cyclase, i.e. forskolin, prostaglandin E1, and cholera toxin, prevented up-regulation of muscle-specific gene products following growth arrest in mitogen-deficient medium. Conversely, addition of cAMP to differentiated BC3H1 myocytes caused down-regulation of muscle-specific mRNAs. In contrast to the ability of cAMP to block differentiation, chronic exposure to O-tetradecanoylphorbol-13-acetate, the potent activator of protein kinase C, exhibited no apparent effects on expression of muscle-specific gene products. The proto-oncogenes c-myc and c-fos were up-regulated rapidly by cAMP in a manner similar to that observed previously by serum, FGF, and TGF-beta. However, these growth factors failed to increase intracellular cAMP levels, and they did not induce ornithine decarboxylase, which was subject to positive regulation by cAMP and O-tetradecanoyl-13-acetate. Together, these data indicate that differentiation of BC3H1 cells is subject to negative regulation through a cAMP-dependent pathway and that serum mitogens, FGF, and TGF-beta inhibit differentiation through a mechanism independent of cAMP or protein kinase C.
- Published
- 1988
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20. Serum and fibroblast growth factor inhibit myogenic differentiation through a mechanism dependent on protein synthesis and independent of cell proliferation.
- Author
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Spizz, G, Roman, D, Strauss, A, and Olson, E N
- Abstract
Myogenesis is accompanied by the withdrawal of proliferating myoblasts from the cell cycle, their fusion to form myotubes, and the coordinate expression of a variety of muscle-specific gene products, such as the muscle isoenzyme of creatine kinase (MCK). In the present study we used the nonfusing muscle cell line, BC3H1, to examine the mechanisms involved in regulation of MCK mRNA expression. Proliferating BC3H1 cells, in media with 20% fetal calf serum, had undetectable levels of MCK mRNA. Exposure of undifferentiated cells to media containing 0.5% serum resulted in withdrawal of cells from the cell cycle and in a several hundred-fold increase in the steady state level of MCK mRNA. Induction of this muscle-specific mRNA could be rapidly reversed by exposure of quiescent differentiated cells to media containing either 20% serum or pituitary fibroblast growth factor. The decline in the steady state level of MCK mRNA following mitogenic stimulation was not dependent upon reentry of cells into the cell cycle, but it did require protein synthesis. Together, these data indicate that fibroblast growth factor can specifically inhibit muscle-specific gene expression through a mechanism independent of cell proliferation. The finding that MCK mRNA was down-regulated by a mechanism that required protein synthesis suggests that mitogen-inducible early gene products may be involved in regulation of muscle gene expression.
- Published
- 1986
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21. Regulation of surface expression of acetylcholine receptors in response to serum and cell growth in the BC3H1 muscle cell line.
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Olson, E N, Glaser, L, Merlie, J P, Sebanne, R, and Lindstrom, J
- Abstract
The regulation of acetylcholine receptor synthesis, assembly, and cell surface expression in response to serum and cell growth in the BC3H1 muscle-like cell line have been examined. In the presence of media containing low concentrations of serum, BC3H1 cells withdraw from the cell cycle and express high levels of cell surface acetylcholine receptors. Exposure of quiescent, differentiated BC3H1 cells to high concentrations of serum results in re-entry of cells into the cell cycle and a loss of surface acetylcholine receptors by a first order process (t1/2 = 8.6 h) which appears to be initiated within 1 h following serum addition. Under these conditions, the rate of synthesis of acetylcholine receptor alpha-subunits is not significantly reduced; however, the efficiency of assembly of alpha-subunits into functional acetylcholine receptors is significantly less than that observed in quiescent cells. The receptors which are assembled following serum stimulation of quiescent cells are not transported to the cell surface, nor do they accumulate within the cells in a functional form (defined by the ability to bind alpha-bungarotoxin) in the cells. We conclude that reinitiation of cell division in quiescent cultures of BC3H1 cells results in alterations in several post-translational processing steps required for the surface expression of acetylcholine receptors.
- Published
- 1983
- Full Text
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22. Specificity of fatty acid acylation of cellular proteins.
- Author
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Olson, E N, Towler, D A, and Glaser, L
- Abstract
Labeling of the BC3H1 muscle cell line with [3H] palmitate and [3H]myristate results in the incorporation of these fatty acids into a broad spectrum of different proteins. The patterns of proteins which are labeled with palmitate and myristate are distinct, indicating a high degree of specificity of fatty acylation with respect to acyl chain length. The protein-linked [3H]palmitate is released by treatment with neutral hydroxylamine or by alkaline methanolysis consistent with a thioester linkage or a very reactive ester linkage. In contrast, only a small fraction of the [3H]myristate which is attached to proteins is released by treatment with hydroxylamine or alkaline methanolysis, suggesting that myristate is linked to proteins primarily through amide bonds. The specificity of fatty acid acylation has also been examined in 3T3 mouse fibroblasts and in PC12 cells, a rat pheochromacytoma cell line. In both cells, palmitate is primarily linked to proteins by a hydroxylamine-labile linkage while the major fraction of the myristic acid (60-70%) is linked to protein via amide linkage and the remainder via an ester linkage. Major differences were noted in the rate of fatty acid metabolism in these cells; in particular in 3T3 cells only 33% of the radioactivity incorporated from myristic acid into proteins is in the form of fatty acids. The remainder is presumably the result of conversion of label to amino acids. In BC3H1 cells, palmitate- and myristate-containing proteins also exhibit differences in subcellular localization. [3H]Palmitate-labeled proteins are found almost exclusively in membranes, whereas [3H]myristate-labeled proteins are distributed in both the soluble and membrane fractions. These results demonstrate that fatty acid acylation is a covalent modification common to a wide range of cellular proteins and is not restricted solely to membrane-associated proteins. The major acylated proteins in the various cell lines examined appear to be different, suggesting that the acylated proteins are concerned with specialized cell functions. The linkages through which fatty acids are attached to proteins also appear to be highly specific with respect to the fatty acid chain length.
- Published
- 1985
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23. Cooperative transcriptional activation by the neurogenic basic helix-loop-helix protein MASH1 and members of the myocyte enhancer factor-2 (MEF2) family.
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Black, B L, Ligon, K L, Zhang, Y, and Olson, E N
- Abstract
Establishment of skeletal muscle and neural cell types is controlled by families of myogenic and neurogenic basic helix-loop-helix (bHLH) proteins, respectively. Myogenic bHLH proteins have been shown to activate skeletal muscle transcription in collaboration with members of the myocyte enhancer factor-2 (MEF2) family of MCM1-agamous-deficiens-serum response factor (MADS)-box transcription factors, which are expressed in differentiated myocytes and neurons. Here, we show that the neurogenic bHLH protein MASH1 interacts with members of the MEF2 family and that this interaction, mediated by the DNA binding and dimerization domains of these factors, results in synergistic activation of transcription through either the MASH1 or the MEF2 DNA binding site. Consistent with their involvement in activation of neuronal gene expression, members of the MEF2 family are expressed in P19 embryonal carcinoma cells that have been induced to form neurons following treatment with retinoic acid. These results suggest that members of the MEF2 family perform similar roles in synergistic activation of transcription in myogenic and neurogenic lineages by serving as cofactors for cell type-specific bHLH proteins.
- Published
- 1996
24. Expression of the smooth muscle cell calponin gene marks the early cardiac and smooth muscle cell lineages during mouse embryogenesis.
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Miano, J M and Olson, E N
- Abstract
Although several genes are considered markers for vascular smooth muscle cell (SMC) differentiation, few have been rigorously tested for SMC specificity in mammals, particularly during development where considerable overlap exists between different muscle gene programs. Here we describe the temporospatial expression pattern of the SMC calponin gene (formerly h1 or basic calponin) during mouse embryogenesis and in adult mouse tissues and cell lines. Whereas SMC calponin mRNA expression is restricted exclusively to SMCs in adult tissues, during early embryogenesis, SMC calponin transcripts are expressed throughout the developing cardiac tube as well as in differentiating SMCs. Transcription of the SMC calponin gene initiates at two closely juxtaposed sites in the absence of a consensus TATAA or initiator element. Transient transfection assays in cultured SMC demonstrated that high level SMC calponin promoter activity required no more than 549 nucleotides of 5 sequence. In contrast to the strict cell type-specificity of SMC calponin mRNA expression, the SMC calponin promoter showed activity in several cell lines that do not express the endogenous SMC calponin gene. These results demonstrate that SMC calponin responds to cardiac and smooth muscle gene regulatory programs and suggest that the cardiac and smooth muscle cell lineages may share a common gene regulatory program early in embryogenesis, which diverges as the heart matures. The finding that the isolated SMC calponin promoter is active in a wider range of cells than the endogenous SMC calponin gene also suggests that long-range repression or higher order regulatory mechanism(s) are involved in cell-specific regulation of SMC calponin expression.
- Published
- 1996
25. The mouse MRF4 promoter is trans-activated directly and indirectly by muscle-specific transcription factors.
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Black, B L, Martin, J F, and Olson, E N
- Abstract
MRF4 is a member of the basic helix-loop-helix (bHLH) family of muscle-specific transcription factors, which also includes MyoD, myogenin, and myf5. The myocyte enhancer binding factor 2 (MEF2) proteins also serve as important muscle-specific transcription factors. In addition to activating the expression of many muscle-specific structural genes, various members of these two classes of proteins activate their own expression and the expression of each other in a complex transcriptional network that results in the establishment and maintenance of the muscle phenotype. To begin to determine how the expression of MRF4 is regulated by other muscle-specific transcription factors, we have isolated a region of the MRF4 gene that confers muscle-specific expression and have analyzed this promoter region for cis-acting elements involved in trans-activation by the myogenic bHLH and MEF2 transcription factors. Here, we show that in 10T1/2 fibroblasts the MRF4 promoter is trans-activated by myogenin, MyoD, myf5, and by the MEF2 factors, but that MRF4 does not activate expression of its own promoter. Myogenin activated the MRF4 promoter directly by an E box-dependent mechanism, while MEF2 factors activated the promoter through an indirect pathway. The E box-dependent regulation of the MRF4 promoter is in contrast to the regulation of the myogenin and MyoD promoters and may represent a mechanism for the differential expression of these factors during myogenesis.
- Published
- 1995
26. Alpha and beta subunits of the nicotinic acetylcholine receptor contain covalently bound lipid.
- Author
-
Olson, E N, Glaser, L, and Merlie, J P
- Abstract
Labeling of the BC3H1 muscle-like cell line with [3H] palmitate, followed by immunoprecipitation of the acetylcholine receptor, indicated that the alpha and beta subunits of the receptor contain covalently bound fatty acid. After acid hydrolysis, fatty acid methyl esters could be recovered from the isolated [3H]palmitate-labeled alpha subunit. Treatment of differentiated BC3H1 cells with cerulenin, an inhibitor of fatty acid and sterol synthesis and fatty acid acylation of proteins, resulted in a 50% inhibition in expression of the acetylcholine receptor on the cell surface under conditions where there was minimal inhibition of protein synthesis. We conclude that this previously undetected post-translational modification may play a role in assembly and/or surface expression of the acetylcholine receptor.
- Published
- 1984
- Full Text
- View/download PDF
27. Fatty acylation of cellular proteins. Temporal and subcellular differences between palmitate and myristate acylation.
- Author
-
Olson, E N and Spizz, G
- Abstract
Previous studies demonstrated that palmitate and myristate are covalently linked to distinct sets of cellular proteins and that the linkages through which these fatty acids are attached to the polypeptide chains are different (Olson, E. N., Towler, D. A., and Glaser, L. (1985) J. Biol. Chem. 260, 3784-3790). In the present study, the kinetics and subcellular sites of acylation of proteins with palmitate and myristate were examined in the BC3H1 muscle cell line. Acylation with myristate was an extremely early modification that appeared to take place cotranslationally or shortly thereafter for a variety of soluble and membrane-bound proteins. In contrast, acylation of proteins with palmitate was a post-translational event that occurred exclusively on membrane proteins. To begin to understand the intracellular pathways that acyl proteins follow during their maturation, the degree of glycosylation, and the nature of the interaction of these proteins with membranes were examined. The majority of acyl proteins were tightly associated with membranes and could not be removed by conditions that release peripheral proteins from membranes. However, only a minor fraction of acylated proteins were N-glycosylated. These data suggest that the acyltransferases that attach palmitate and myristate to proteins are present in different subcellular locations and demonstrate that these fatty acids are attached to newly synthesized acyl proteins at different times during their maturation. The lack of carbohydrate on the majority of integral membrane acyl proteins suggests that these proteins may follow intracellular pathways that are different from those followed by cell surface glycoproteins.
- Published
- 1986
- Full Text
- View/download PDF
28. Muscle specificity encoded by specific serum response factor-binding sites.
- Author
-
Chang PS, Li L, McAnally J, and Olson EN
- Subjects
- Actins genetics, Animals, Base Sequence, Embryo, Mammalian, Genes, fos, Mice, Mice, Transgenic, Molecular Sequence Data, Organ Specificity, Recombinant Fusion Proteins chemistry, Recombinant Fusion Proteins genetics, Serum Response Factor, Transcription Factors metabolism, beta-Galactosidase genetics, DNA-Binding Proteins metabolism, Microfilament Proteins genetics, Muscle Proteins genetics, Muscle, Skeletal metabolism, Nuclear Proteins metabolism, Promoter Regions, Genetic
- Abstract
Serum response factor (SRF) is a MADS box transcription factor that regulates muscle-specific and growth factor-inducible genes by binding the consensus sequence CC(A/T)6GG, known as a CArG box. Because SRF expression is not restricted solely to muscle, its expression alone cannot account for the muscle specificity of some of its target genes. To understand further the role of SRF in muscle-specific transcription, we created transgenic mice harboring lacZ transgenes linked to tandem copies of different CArG boxes with flanking sequences. CArG boxes from the SM22 and skeletal alpha-actin promoters directed highly restricted expression in developing smooth, cardiac, and skeletal muscle cells during early embryogenesis. In contrast, the CArG box and flanking sequences from the c-fos promoter directed expression throughout the embryo, with no preference for muscle cells. Systematic swapping of the core and flanking sequences of the SM22 and c-fos CArG boxes revealed that cell type specificity was dictated in large part by sequences immediately flanking the CArG box core. Sequences that directed widespread embryonic expression bound SRF more strongly than those that directed muscle-restricted expression. We conclude that sequence variations among CArG boxes influence cell type specificity of expression and account, at least in part, for the ability of SRF to distinguish between growth factor-inducible and muscle-specific genes in vivo.
- Published
- 2001
- Full Text
- View/download PDF
29. Association of COOH-terminal-binding protein (CtBP) and MEF2-interacting transcription repressor (MITR) contributes to transcriptional repression of the MEF2 transcription factor.
- Author
-
Zhang CL, McKinsey TA, Lu JR, and Olson EN
- Subjects
- Alcohol Oxidoreductases, Amino Acid Motifs, Amino Acid Sequence, Animals, Carrier Proteins chemistry, Carrier Proteins genetics, Cell Line, DNA-Binding Proteins chemistry, Histone Deacetylases chemistry, Histone Deacetylases classification, Histone Deacetylases metabolism, MEF2 Transcription Factors, Macromolecular Substances, Mice, Molecular Sequence Data, Mutation, Myogenic Regulatory Factors, Phosphoproteins chemistry, Phosphoproteins genetics, Precipitin Tests, Protein Binding, Repressor Proteins chemistry, Repressor Proteins genetics, Transcription Factors metabolism, Two-Hybrid System Techniques, Carrier Proteins metabolism, DNA-Binding Proteins genetics, DNA-Binding Proteins metabolism, Gene Expression Regulation, Phosphoproteins metabolism, Repressor Proteins metabolism, Transcription Factors genetics
- Abstract
The class II histone deacetylases (HDACs) 4, 5, and 7 share a common structural organization, with a carboxyl-terminal catalytic domain and an amino-terminal extension that mediates interactions with members of the myocyte enhancer factor-2 (MEF2) family of transcription factors. Association of these HDACs with MEF2 factors represses transcription of MEF2 target genes. MEF2-interacting transcription repressor (MITR) shares homology with the amino-terminal extensions of class II HDACs and also acts as a transcriptional repressor, but lacks a histone deacetylase catalytic domain. This suggests that MITR represses transcription by recruiting other corepressors. We show that the amino-terminal regions of MITR and class II HDACs interact with the transcriptional corepressor, COOH-terminal-binding protein (CtBP), through a CtBP-binding motif (P-X-D-L-R) conserved in MITR and HDACs 4, 5, and 7. Mutation of this sequence in MITR abolishes interaction with CtBP and impairs, but does not eliminate, the ability of MITR to inhibit MEF2-dependent transcription. The residual repressive activity of MITR mutants that fail to bind CtBP can be attributed to association with other HDAC family members. These findings reveal CtBP-dependent and -independent mechanisms for transcriptional repression by MITR and show that MITR represses MEF2 activity through recruitment of multicomponent corepressor complexes that include CtBP and HDACs.
- Published
- 2001
- Full Text
- View/download PDF
30. Homeodomain protein MHox and MADS protein myocyte enhancer-binding factor-2 converge on a common element in the muscle creatine kinase enhancer.
- Author
-
Cserjesi P, Lilly B, Hinkley C, Perry M, and Olson EN
- Subjects
- Animals, Base Sequence, Binding Sites, Cell Line, DNA metabolism, HeLa Cells, Humans, MEF2 Transcription Factors, Molecular Sequence Data, Mutagenesis, Myogenic Regulatory Factors, Oligonucleotide Probes, Polymerase Chain Reaction, Trans-Activators metabolism, Transcription, Genetic, Transfection, Creatine Kinase genetics, DNA-Binding Proteins metabolism, Enhancer Elements, Genetic, Gene Expression Regulation, Enzymologic, Homeodomain Proteins, Muscles enzymology, Transcription Factors metabolism
- Abstract
MHox is a mesoderm-specific homeodomain protein that binds an A/T-rich element that is essential for activity of the muscle creatine kinase (MCK) enhancer. The MHox binding site also binds the ubiquitous homeodomain protein Oct-1 as well as myocyte enhancer-binding factor-2 (MEF2), which belongs to the MADS superfamily of transactivators. To determine which of these proteins activates MCK transcription through the A/T element, we mutated this sequence such that it would selectively bind MHox, MEF2, or Oct-1 and tested the activities of the mutant enhancers in skeletal muscle cells. These mutant enhancers revealed that only MEF2 is able to activate the MCK enhancer through the A/T element. The convergence of homeodomain and MADS proteins on the A/T element in the MCK enhancer provides a mechanism through which a single DNA sequence can mediate positive and negative regulation of gene transcription and is reminiscent of the roles of these two classes of transcription factors in the control of other cell-specific genes.
- Published
- 1994
31. Myogenin and acetylcholine receptor alpha gene promoters mediate transcriptional regulation in response to motor innervation.
- Author
-
Merlie JP, Mudd J, Cheng TC, and Olson EN
- Subjects
- Aging metabolism, Animals, Chloramphenicol O-Acetyltransferase genetics, Enhancer Elements, Genetic, Helix-Loop-Helix Motifs physiology, Kinetics, Mice, Mice, Inbred C57BL, Mice, Inbred CBA, Mice, Transgenic, Motor Neurons physiology, Muscle Development, Muscles innervation, Restriction Mapping, Time Factors, Transcription, Genetic, Chloramphenicol O-Acetyltransferase biosynthesis, Gene Expression Regulation, Muscle Denervation, Muscles metabolism, Myogenin genetics, Promoter Regions, Genetic, Receptors, Nicotinic genetics
- Abstract
Several genes expressed in skeletal muscle are transcriptionally repressed by electrical activity arising from motor innervation and are rapidly induced following denervation. Among these are genes encoding the subunits of the nicotinic acetylcholine receptor (AChR) and the myogenic helix-loop-helix protein myogenin, which activates muscle-specific genes. To understand how electrical activity arising from motor innervation is converted into a transcriptional response, we have attempted to localize cis-acting sequences in the AChR alpha subunit and myogenin genes sufficient to direct activity-dependent transcription. Here we show that an 111-base pair and a 335-base pair region from the promoters of the AChR alpha subunit and myogenin genes, respectively, can confer activity-dependent regulation to a linked reporter gene in transgenic mice. The presence of binding sites for myogenic helix-loop-helix proteins in both of these regulatory regions is consistent with the hypothesis that these myogenic regulators serve as nuclear targets for the signaling cascade through which motor innervation leads to changes in gene transcription in skeletal muscle.
- Published
- 1994
32. Role of myocyte-specific enhancer-binding factor (MEF-2) in transcriptional regulation of the alpha-cardiac myosin heavy chain gene.
- Author
-
Adolph EA, Subramaniam A, Cserjesi P, Olson EN, and Robbins J
- Subjects
- Animals, Base Sequence, Chloramphenicol O-Acetyltransferase genetics, DNA, DNA-Binding Proteins genetics, MEF2 Transcription Factors, Mice, Mice, Transgenic, Molecular Sequence Data, Mutagenesis, Site-Directed, Myogenic Regulatory Factors, Promoter Regions, Genetic, Transcription Factors genetics, Transcription, Genetic, DNA-Binding Proteins physiology, Gene Expression Regulation, Myocardium metabolism, Myosins genetics, Transcription Factors physiology
- Abstract
The intergenic region between the mouse alpha-cardiac myosin heavy chain and beta-myosin heavy chain genes has previously been shown to direct expression of the bacterial chloramphenicol acetyltransferase reporter gene in transgenic mice in a tissue-specific manner. Sequence analyses located a putative myocyte-specific enhancer-binding factor (MEF-2) site situated in the regulatory region of this gene proximal to the start site of transcription. The role of this element in directing the cardiac compartment-specific expression of the transgene was assessed. The polymerase chain reaction was used to perform substitution mutagenesis of the MEF-2 binding site, and lack of MEF-2 binding was confirmed by gel retardation assays. The resultant construct was used to generate transgenic mice. Surprisingly, transgene expression was not down-regulated, but was significantly increased in the hearts of the MEF-2 mutant mice. In addition, cardiac-specific expression of the transgene was perturbed with significant levels of ectopic expression occurring in the aorta.
- Published
- 1993
33. Interactions among vertebrate helix-loop-helix proteins in yeast using the two-hybrid system.
- Author
-
Staudinger J, Perry M, Elledge SJ, and Olson EN
- Subjects
- Amino Acid Sequence, Animals, Base Sequence, Binding Sites, DNA, DNA-Binding Proteins chemistry, DNA-Binding Proteins genetics, Fungal Proteins genetics, Fungal Proteins metabolism, Inhibitor of Differentiation Protein 2, Mice, Molecular Sequence Data, Muscle Proteins metabolism, MyoD Protein, Protein Conformation, Recombinant Fusion Proteins metabolism, T-Lymphocytes metabolism, TCF Transcription Factors, Transcription Factor 7-Like 1 Protein, DNA-Binding Proteins metabolism, Repressor Proteins, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae Proteins, Transcription Factors
- Abstract
The helix-loop-helix (HLH) motif is contained in a rapidly growing family of transcription factors and has been shown to mediate dimerization among heterologous HLH-containing proteins. E12 is a widely expressed HLH protein that preferentially forms heterodimers with cell type-specific HLH proteins such as MyoD, myogenin, and the achaete-scute gene products. As a first step toward screening for novel cell type-specific partners of E12, we used a modification of the two-hybrid assay for detection of protein-protein interactions in vivo to determine whether dimerization of HLH proteins with E12 can occur in yeast. Using the GAL4 DNA-binding domain fused to the E12 HLH motif and the GAL4 transcription activation domain fused to MyoD, we show that E12 and MyoD can efficiently dimerize in yeast and reconstruct a hybrid transcription factor that activates reporter genes linked to the GAL4 DNA-binding site. The GAL4 DNA-binding domain fused to E12 was used to screen a mouse T-cell cDNA library in which the cDNA was fused to the GAL4 activation domain. Several cDNA clones encoding proteins that interact with E12 were isolated, one of which corresponded to the HLH protein Id-2. Given the ability of E12 to dimerize preferentially with cell type-specific HLH proteins, this strategy should be useful for cloning novel partners for E12 from a variety of cell types.
- Published
- 1993
34. Helix-loop-helix proteins as regulators of muscle-specific transcription.
- Author
-
Edmondson DG and Olson EN
- Subjects
- Amino Acid Sequence, Animals, Cell Line, DNA-Binding Proteins genetics, HeLa Cells, Humans, Molecular Sequence Data, Multigene Family, Muscle Proteins genetics, Muscles cytology, MyoD Protein, Protein Structure, Secondary, Sequence Homology, Amino Acid, Transcription Factors genetics, Vertebrates, DNA-Binding Proteins physiology, Gene Expression Regulation, Muscle Proteins physiology, Muscles physiology, Transcription Factors physiology, Transcription, Genetic
- Published
- 1993
35. Analysis of the oligomerization of myogenin and E2A products in vivo using a two-hybrid assay system.
- Author
-
Chakraborty T, Martin JF, and Olson EN
- Subjects
- Adenovirus Early Proteins, Animals, Cell Line, Chloramphenicol O-Acetyltransferase genetics, Chloramphenicol O-Acetyltransferase metabolism, Growth Substances pharmacology, HeLa Cells, Humans, Kinetics, Macromolecular Substances, Muscle Proteins genetics, Myogenin, Oncogene Proteins, Viral genetics, Plasmids, Recombinant Proteins metabolism, Transcription, Genetic, Transcriptional Activation, Transfection, Muscle Proteins metabolism, Oncogene Proteins, Viral metabolism, Trans-Activators metabolism
- Abstract
Members of the helix-loop-helix (HLH) family of proteins bind DNA and activate transcription as homo- and heterodimers. Myogenin is a muscle-specific HLH protein that binds DNA in vitro as a heterodimer with several widely expressed HLH proteins, such as the E2A gene products E12 and E47. We describe a method for detection of protein-protein interactions among HLH proteins in vivo in which dimerization through the HLH motif reconstructs a hybrid transcription factor containing the DNA-binding domain of yeast GAL4 linked to one HLH motif and the activation domain of VP-16 linked to another. We have used this assay to investiagate whether myogenin forms homomeric or heteromeric complexes in vivo and to determine whether growth factors and oncogenes that inhibit myogenesis influence myogenin's ability to dimerize. The results show that myogenin heterodimerizes with E12 and E47 in vivo, but it does not homodimerize to a measurable extent. Peptide growth factors, as well as the immediate early gene products c-Jun, v-Fos, and c-Myc, inhibit the activity of myogenin through a mechanism independent of its association with E2A products.
- Published
- 1992
36. Repression of myogenin function by TGF-beta 1 is targeted at the basic helix-loop-helix motif and is independent of E2A products.
- Author
-
Martin JF, Li L, and Olson EN
- Subjects
- Cells, Cultured, Chloramphenicol O-Acetyltransferase metabolism, DNA metabolism, Muscle Proteins genetics, Mutation, MyoD Protein, Myogenin, Protein Processing, Post-Translational, TCF Transcription Factors, Transcription Factor 7-Like 1 Protein, Transcription, Genetic, Transfection, DNA-Binding Proteins genetics, Muscle Proteins antagonists & inhibitors, Transcription Factors genetics, Transforming Growth Factor beta pharmacology
- Abstract
The muscle-specific helix-loop-helix (HLH) proteins myogenin, MyoD, myf5, and MRF4 form hetero-oligomers with ubiquitous HLH proteins encoded by the E2A gene and activate muscle transcription by binding to a DNA sequence known as an E-box (CANNTG). Transforming growth factor-beta (TGF-beta) can inhibit muscle differentiation by silencing the transcription-activating potential of myogenic HLH proteins without affecting their ability to bind DNA. We show that repression by TGF-beta is directed at the basic-HLH motif of myogenin and is independent of E2A products. Using a series of reporter genes as targets for trans-activation by myogenin, transcriptional repression by TGF-beta is also shown to map to the E-box motif and to not require heterologous DNA sequence elements. These results demonstrate that TGF-beta represses muscle-specific transcription through a post-translational mechanism that renders the basic-HLH regions of the myogenic regulators nonfunctional. The selective repression of myogenic HLH proteins by TGF-beta indicates that the TGF-beta signaling system can discriminate between different classes of HLH proteins and implies that myogenic HLH proteins activate muscle-specific transcription through a unique mechanism.
- Published
- 1992
37. Mitogenic repression of myogenin autoregulation.
- Author
-
Edmondson DG, Brennan TJ, and Olson EN
- Subjects
- Animals, Blotting, Northern, Cell Line, Homeostasis, Kinetics, Methionine metabolism, Mice, Muscle Proteins biosynthesis, Myogenin, Plasmids, RNA genetics, RNA isolation & purification, Sulfur Radioisotopes, Trans-Activators genetics, Transfection, Muscle Proteins genetics, Muscles metabolism
- Abstract
The muscle-specific helix-loop-helix proteins MyoD and myogenin have been shown to positively autoregulate their own expression and to cross-activate one another's expression following transfection into a variety of nonmyogenic cell lines. We show that the ability of an exogenous myogenin expression vector to activate the endogenous myogenin allele in 10T1/2 fibroblasts is dependent on serum withdrawal. Repression of myogenin autoregulation by serum can be observed with a reporter gene linked to myogenin upstream sequences, indicating that repression occurs at the level of transcription. Like MyoD, myogenin exhibits a short half-life (approximately 20 min), which may serve to maintain the autoregulatory loop without allowing excess accumulation of the protein.
- Published
- 1991
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