Identification of a new hybrid serum response factor and myocyte enhancer factor 2-binding element in MyoD enhancer required for MyoD expression during myogenesis

MyoD is a critical myogenic factor induced rapidly upon activation of quiescent satellite cells, and required for their differentiation during muscle regeneration. One of the two enhancers of MyoD, the distal regulatory region, is essential for MyoD expression in postnatal muscle. This enhancer contains a functional divergent serum response factor (SRF)-binding CArG element required for MyoD expression during myoblast growth and muscle regeneration in vivo. Electrophoretic mobility shift assay, chromatin immunoprecipitation, and microinjection analyses show this element is a hybrid SRF- and MEF2 Binding (SMB) sequence where myocyte enhancer factor 2 (MEF2) complexes can compete out binding of SRF at the onset of differentiation. As cells differentiate into postmitotic myotubes, MyoD expression no longer requires SRF but instead MEF2 binding to this dual-specificity element. As such, the MyoD enhancer SMB element is the site for a molecular relay where MyoD expression is first initiated in activated satellite cells in an SRF-dependent manner and then increased and maintained by MEF2 binding in differentiated myotubes. Therefore, SMB is a DNA element with dual and stage-specific binding activity, which modulates the effects of regulatory proteins critical in controlling the balance between proliferation and differentiation.     

Myogenin, MyoD, and myosin expression after pharmacologically and surgically induced hypertrophy

The relationshipbetween myogenin or MyoD expression and hypertrophy of the rat soleusproduced either by clenbuterol and 3,3',5-triiodo-L-thyronine(CT) treatment or by surgical overload was examined. Mature female ratswere subjected to surgical overload of the right soleus with the leftsoleus serving as a control. Another group received the same surgicaltreatment but were administered CT. Soleus muscles were harvested 4 wkafter surgical overload and weighed. Myosin heavy chain isoforms wereseparated by using polyacrylamide gel electrophoresis while myogeninand MyoD expression were evaluated by Northern analysis.CT and functional overload increased soleus muscle weight. CT treatmentinduced the appearance of the fast type IIX myosin heavy chain isoform,depressed myogenin expression, and induced MyoD expression. However,functional overload did not alter myogenin or MyoD expression inCT-treated or non-CT-treated rats. Thus pharmacologically andsurgically induced hypertrophy have differing effects on myogenin andMyoD expression, because their levels were associated with changes inmyosin heavy chain composition (especially type IIX) rather thanchanges in muscle mass.

     

A tobacco protein kinase, NPK2, has a domain homologous to a domain found in activators of mitogen-activated protein kinases (MAPKKs)

A cDNA (cNPK2) that encodes a protein of 518 amino acids was isolated from a library prepared from poly(A)⁺ RNAs of tobacco cells in suspension culture. The N-terminal half of the predicted NPK2 protein is similar in amino acid sequence to the catalytic domains of kinases that activate mitogen-activated protein kinases (designated here MAPKKs) from various animals and to those of yeast homologs of MAPKKs. The N-terminal domain of NPK2 was produced as a fusion protein in Escherichia coli, and the purified fusion protein was found to be capable of autophosphorylation of threonine and serine residues. These results indicate that the N-terminal domain of NPK2 has activity of a serine/threonine protein kinase. Southern blot analysis showed that genomic DNAs from various plant species, including Arabidopsis thaliana and sweet potato, hybridized strongly with cNPK2, indicating that these plants also have genes that are closely related to the gene for NPK2. The structural similarity between the catalytic domain of NPK2 and those of MAPKKs and their homologs suggests that tobacco NPK2 corresponds to MAPKKs of other organisms. Given the existence of plant homologs of an MAP kinase and tobacco NPK1, which is structurally and functionally homologous to one of the activator kinases of yeast homologs of MAPKK (MAPKKKs), it seems likely that a signal transduction pathway mediated by a protein kinase cascade that is analogous to the MAP kinase cascades proposed in yeasts and animals, is also conserved in plants.     

功能矫形前伸青春期大鼠下颌后翼外肌MyoD、myogenin 的表达

目的:探讨"应力-生长(改建)"在细胞水平上的体现,为功能矫形治疗和矫治效果的保持提供新思路和实验依据。方法:本实验选用20只4周龄,雄性SD大鼠随机分为8组。其中实验组大鼠经戊巴比妥麻醉后佩戴上颌斜面导板,对照组未佩用。依据时间不同又分为四组:1d,7d,14d,21d。采用RT-PCR技术分析各组大鼠翼外肌组织中肌分化相关基因MyoD、myogenin mRNA的表达变化。结果:未施加功能矫形力的大鼠翼外肌组织MyoD表达伴随其生长发育呈现递减趋势,实验组在第7 d出现表达上调。同时,力学刺激后实验组动物myogenin的表达与对照组相比较在14 d组出现明显上调。结论:功能矫形力作用于翼外肌组织可以诱导MyoD和myogenin的表达上调进而诱导成肌细胞的分化。     

功能矫形前伸青春期大鼠下颌后翼外肌MyoD、myogenin的表达

目的：探讨＂应力-生长（改建）＂在细胞水平上的体现,为功能矫形治疗和矫治效果的保持提供新思路和实验依据。方法：本实验选用20只4周龄,雄性SD大鼠随机分为8组。其中实验组大鼠经戊巴比妥麻醉后佩戴上颌斜面导板,对照组未佩用。依据时间不同又分为四组：1d,7d,14d,21d。采用RT-PCR技术分析各组大鼠翼外肌组织中肌分化相关基因MyoD、myogenin mRNA的表达变化。结果：未施加功能矫形力的大鼠翼外肌组织MyoD表达伴随其生长发育呈现递减趋势,实验组在第7 d出现表达上调。同时,力学刺激后实验组动物myogenin的表达与对照组相比较在14 d组出现明显上调。结论：功能矫形力作用于翼外肌组织可以诱导MyoD和myogenin的表达上调进而诱导成肌细胞的分化。     

A family IIb xylan-binding domain has a similar secondary structure to a homologous family IIa cellulose-binding domain but different ligand specificity.

BACKGROUND: Many enzymes that digest polysaccharides contain separate polysaccharide-binding domains. Structures have been previously determined for a number of cellulose-binding domains (CBDs) from cellulases. RESULTS: The family IIb xylan-binding domain 1 (XBD1) from Cellulomonas fimi xylanase D is shown to bind xylan but not cellulose. Its structure is similar to that of the homologous family IIa CBD from C. fimi Cex, consisting of two four-stranded beta sheets that form a twisted 'beta sandwich'. The xylan-binding site is a groove made from two tryptophan residues that stack against the faces of the sugar rings, plus several hydrogen-bonding polar residues. CONCLUSIONS: The biggest difference between the family IIa and IIb domains is that in the former the solvent-exposed tryptophan sidechains are coplanar, whereas in the latter they are perpendicular, forming a twisted binding site. The binding sites are therefore complementary to the secondary structures of the ligands cellulose and xylan. XBD1 and CexCBD represent a striking example of two proteins that have high sequence similarity but a different function.     

Crystal structure of the ternary complex of a NaV C-terminal domain, a fibroblast growth factor homologous factor, and calmodulin

Voltage-gated Na? (Na(V)) channels initiate neuronal action potentials. Na(V) channels are composed of a transmembrane domain responsible for voltage-dependent Na? conduction and a cytosolic C-terminal domain (CTD) that regulates channel function through interactions with many auxiliary proteins, including fibroblast growth factor homologous factors (FHFs) and calmodulin (CaM). Most ion channel structural studies have focused on mechanisms of permeation and voltage-dependent gating but less is known about how intracellular domains modulate channel function. Here we report the crystal structure of the ternary complex of a human Na(V) CTD, an FHF, and Ca2?-free CaM at 2.2 ?. Combined with functional experiments based on structural insights, we present a platform for understanding the roles of these auxiliary proteins in Na(V) channel regulation and the molecular basis of mutations that lead to neuronal and cardiac diseases. Furthermore, we identify a critical interaction that contributes to the specificity of individual Na(V) CTD isoforms for distinctive FHFs.     

MyoD, Myogenin, and Desmin-nls-lacZ Transgene Emphasize the Distinct Patterns of Satellite Cell Activation in Growth and Regeneration

Although satellite cell differentiation is involved in postnatal myogenesis from growth to posttrauma regeneration, the early stages of this process remain unclear. This study investigatedpHuDes-nls-lacZtransgene activity, as revealed by X-gal staining and the accumulation of MyoD, myogenin, endogenous desmin, and myosin, in order to determine whether satellite cells share the same activation program during growth and regeneration. After birth, skeletal myonuclei in which myogenin expression was limited were briefly characterized by transgene activity. Satellite cells were only evidenced by MyoD and slow myosin accumulation, but failed to initiate transgene expression. After freeze trauma, satellite cell activation led to MyoD, myogenin, and desmin expression. Subsequently, when myosin expression occurred, transgene activation was apparent in regenerating structures, with more intense X-gal staining in mononucleated cells than regenerating myotubes. After the second week posttrauma, only desmin and myogenin expression were maintained in regenerating structures. In culture, the behavior of satellite cells showed that desmin expression was committed before transgene activation occurred, i.e., concurrently with MyoD, myogenin, myosin expression, and the first fusion events. Quantitative analysis confirmed the discrepancy between endogenous desmin and transgene expression and demonstrated the close correlation between transgene activation and the fusion index. Our results strongly suggest that satellite cells promote distinct pathways of myogenic response during growth and regeneration.     

The actin-depolymerizing factor destrin has an actin-stabilizing domain.

Destrin is a 19 kDa actin-depolymerizing protein of the ADF-cofilin family. Destrin was digested with trypsin to a structurally stable 9.2 kDa fragment that contains the actin-binding sequence. The purified 9.2 kDa fragment has an actin filament stabilizing activity, rather than an actin filament depolymerizing activity. The deleted region is probably essential for the actin filament depolymerizing activity of intact destrin. Surprisingly, the 9.2 kDa fragment also has an assembly-promoting activity in the absence of ATP.     

The Gla domain of human prothrombin has a binding site for factor Va

The role of the Gla domain of human prothrombin in interaction with the prothrombinase complex was studied using a peptide with the sequence of the first 46 residues of human prothrombin, PT-(1-46). Intrinsic fluorescence measurements showed that PT-(1-46) undergoes a conformational alteration upon binding calcium; this conclusion is supported by one-dimensional (1)H NMR spectroscopy, which identifies a change in the chemical environment of tryptophan 41. PT-(1-46) binds phospholipid membranes in a calcium-dependent manner with a K(d) of 0.5 microm and inhibits thrombin generation by the prothrombinase complex with a K(i) of 0.8 microm. In the absence of phospholipid membranes, PT-(1-46) inhibits thrombin generation by factor Xa in the presence but not absence of factor Va, suggesting that PT-(1-46) inhibits prothrombin-factor Va binding. The addition of factor Va to PT-(1-46) labeled with the fluorophore sulfosuccinimidyl-7-amino-4-methylcoumarin-3-acetic acid (PT-(1-46)AMCA) caused a concentration-dependent quenching of AMCA fluorescence, providing direct evidence of a PT-(1-46)-factor Va interaction. The K(d) for this interaction was 1.3 microm. These results indicate that the N-terminal Gla domain of human prothrombin is a functional unit that has a binding site for factor Va. The prothrombin Gla domain is important for interaction of the substrate with the prothrombinase complex.     

GGDEF domain is homologous to adenylyl cyclase

The GGDEF domain is detected in many prokaryotic proteins, most of which are of unknown function. Several bacteria carry 12-22 different GGDEF homologues in their genomes. Conducting extensive profile-based searches, we detect statistically supported sequence similarity between GGDEF domain and adenylyl cyclase catalytic domain. From this homology, we deduce that the prokaryotic GGDEF domain is a regulatory enzyme involved in nucleotide cyclization, with the fold similar to that of the eukaryotic cyclase catalytic domain. This prediction correlates with the functional information available on two GGDEF-containing proteins, namely diguanylate cyclase and phosphodiesterase A of Acetobacter xylinum, both of which regulate the turnover of cyclic diguanosine monophosphate. Domain architecture analysis shows that GGDEF is typically present in multidomain proteins containing regulatory domains of signaling pathways or protein-protein interaction modules. Evolutionary tree analysis indicates that GGDEF/cyclase superfamily forms a large diversified cluster of orthologous proteins present in bacteria, archaea, and eukaryotes. Proteins 2001;42:210-216.     

Myogenin expression, cell cycle withdrawal, and phenotypic differentiation are temporally separable events that precede cell fusion upon myogenesis

During terminal differentiation of skeletal myoblasts, cells fuse to form postmitotic multinucleated myotubes that cannot reinitiate DNA synthesis. Here we investigated the temporal relationships among these events during in vitro differentiation of C2C12 myoblasts. Cells expressing myogenin, a marker for the entry of myoblasts into the differentiation pathway, were detected first during myogenesis, followed by the appearance of mononucleated cells expressing both myogenin and the cell cycle inhibitor p21. Although expression of both proteins was sustained in mitogen-restimulated myocytes, 5- bromodeoxyuridine incorporation experiments in serum-starved cultures revealed that myogenin-positive cells remained capable of replicating DNA. In contrast, subsequent expression of p21 in differentiating myoblasts correlated with the establishment of the postmitotic state. Later during myogenesis, postmitotic (p21-positive) mononucleated myoblasts activated the expression of the muscle structural protein myosin heavy chain, and then fused to form multinucleated myotubes. Thus, despite the asynchrony in the commitment to differentiation, skeletal myogenesis is a highly ordered process of temporally separable events that begins with myogenin expression, followed by p21 induction and cell cycle arrest, then phenotypic differentiation, and finally, cell fusion.     

Apoptosis induced by human Fas-associated factor 1, hFAF1, requires its ubiquitin homologous domain, but not the Fas-binding domain

FAF1 is a Fas-binding protein without typical death domain. Instead, FAF1 has several domains found in proteins of ubiquitination pathway. Transient overexpression of hFAF1 in BOSC23 cells caused membrane blebbing and cell body condensation which were characteristics of apoptosis. Subsequent analysis revealed that overexpression of hFAF1 induced nuclear condensation, appearance of phosphatidylserines in the outer leaflet of the cellular membrane, and caspase 3 activation. The apoptotic potential of hFAF1 required downstream ubiquitin homologous domain (UB2) and adjacent nuclear localization signal but not the Fas-binding domain. Our data showed that mere intrinsic overexpression of hFAF1 initiated apoptosis in the absence of any extrinsic death signal in BOSC23 cells.     

Folding topology of the disulfide-bonded dimeric DNA-binding domain of the myogenic determination factor MyoD.

The myogenic determination factor MyoD is a member of the basic-helix-loop-helix (bHLH) protein family. A 68-residue fragment of MyoD encompassing the entire bHLH region (MyoD-bHLH) is sufficient for protein dimerization, sequence-specific DNA binding in vitro, and conversion of fibroblasts into muscle cells. The circular dichroism spectrum of MyoD-bHLH indicates the presence of significant alpha-helical secondary structure; however, the NMR spectrum lacks features of a well-defined tertiary structure. There is a naturally occurring cysteine at residue 135 in mouse MyoD that when oxidized to a disulfide induces MyoD-bHLH to form a symmetric homodimer with a defined tertiary structure as judged by sedimentation equilibrium ultracentrifugation and NMR spectroscopy. Oxidized MyoD-bHLH retains sequence-specific DNA-binding activity, albeit with an apparent 100-1000-fold decrease in affinity. Here, we report the structural characterization of the oxidized MyoD-bHLH homodimer by NMR spectroscopy. Our findings indicate that the basic region is unstructured and flexible, while the HLH region consists of two alpha-helices of unequal length connected by an as yet undetermined loop structure. Qualitative examination of interhelical NOEs suggests several potential arrangements for the two helix 1/helix 2 pairs in the symmetric oxidized dimer. These arrangements were evaluated for whether they could incorporate the disulfide bond, satisfy loop length constraints, and juxtapose the two basic regions. Only a model that aligns helix 1 parallel to helix 1' and antiparallel to helix 2 was consistent with all constraints. Thus, an antiparallel four-helix bundle topology is proposed for the symmetric dimer. This topology is hypothesized to serve as a general model for other bHLH protein domains.     

Monoclonal antibodies identify a possible regulatory domain of MyoD1.

A panel of monoclonal antibodies (mAbs) to murine MyoD1 was generated. One set of mAbs is shown to react with epitope(s) in the cysteine/histidine-rich (C/H) region while another set is shown to react with epitope(s) in the C-terminal portion of MyoD1. One of the mAbs reactive with a C-terminal epitope sensitively detected MyoD1 in whole cell extracts by Western blotting. Time course studies of total protein accumulation during C2C12 myoblast differentiation revealed only subtle changes in the phosphorylation and quantity of MyoD1 protein present in C2C12 cells from induction to 120 hr after induction. These results suggest that modulation of MyoD1 protein or total phosphorylation levels is not tightly associated with the transition of undifferentiated myoblasts to differentiated myocytes. Monoclonal antibodies to the C-terminal epitope produced supershifted bands in gel retardation assays, indicating that these mAbs had no effect on DNA binding. Although the C/H region of MyoD1 does not participate in DNA binding, mAbs reactive with the C/H region neutralized this activity in gel retardation assays. These data suggest that the conserved C/H domain may serve to modulate MyoD1 DNA-binding activity by interacting with another regulator.     

Cloning and characterization of a novel MyoD enhancer-binding factor

Glucocorticoid-induced gene-1 (Gig1) was identified in a yeast one-hybrid screen for factors that interact with the MyoD core enhancer. The Gig1 gene encodes a novel C2H2 zinc finger protein that shares a high degree of sequence similarity with two known DNA binding proteins in humans, Glut4 enhancer factor and papillomavirus binding factor (PBF). The mouse ortholog of PBF was also isolated in the screen. The DNA binding domain of Gig1, which contains TCF-E-tail CR1 and CR2 motifs shown to mediate promoter specificity of TCF-E-tail isoforms, was mapped to a C-terminal domain that is highly conserved in Glut4 enhancer factor and PBF. In mouse embryos, in situ hybridization revealed a restricted pattern of expression of Gig1 that overlaps with MyoD expression. A nuclear-localized lacZ knockin null allele of Gig1 was produced to study Gig1 expression with greater resolution and to assess Gig1 functions. X-gal staining of Gig1(nlacZ) heterozygous embryos revealed Gig1 expression in myotomal myocytes, skeletal muscle precursors in the limb, and in nascent muscle fibers of the body wall, head and neck, and limbs through E14.5 (latest stage examined). Gig1 was also expressed in a subset of Scleraxis-positive tendon precursors/rudiments of the limbs, but not in the earliest tendon precursors of the somite (syndetome) defined by Scleraxis expression. Additional regions of Gig1 expression included the apical ectodermal ridge, neural tube roof plate and floor plate, apparent motor neurons in the ventral neural tube, otic vesicles, notochord, and several other tissues representing all three germ layers. Gig1 expression was particularly well represented in epithelial tissues and in a number of cells/tissues of neural crest origin. Expression of both the endogenous MyoD gene and a reporter gene driven by MyoD regulatory elements was similar in wild-type and homozygous null Gig1(nlacZ) embryos, and mutant mice were viable and fertile, indicating that the functions of Gig1 are redundant with other factors.     

PINCH2 is a new five LIM domain protein,homologous to PINCHand localized to focal adhesions

PINCH is a five LIM domain protein involved in the regulation of integrin-mediated cell adhesion. It has been shown that PINCH interacts with integrin-linked kinase and Nck2. Here we describe a new isoform of PINCH, which we call PINCH2. Therefore, we rename PINCH to PINCH1. PINCH2 has an overall similarity of 92% to PINCH1 and contains five LIM domains like PINCH1. While protein and gene structure of the PINCH homologues are very similar and well conserved during evolution, we observed differential expression pattern of the mRNAs. Based on northern hybridization of mouse embryo RNA, PINCH1 is already detectable at E8.5. It is highly expressed during later stages of development and in all adult mouse tissues analyzed, with the highest levels in heart, lung, bladder, skin, and uterus. In contrast, significant PINCH2 expression starts at E14.5. In adult mice it is widely expressed, similar to PINCH1, but absent from spleen and thymus. In situ hybridization confirmed the Northern data and showed differential expression of PINCH1 and PINCH2 in embryonic intestine. Finally, we demonstrate that PINCH2 localizes to focal adhesions in NIH 3T3 cells and to Z-disks in primary rat cardiomyocytes.