scholarly journals Muscle Enriched Lamin Interacting Protein (Mlip) Binds Chromatin and Is Required for Myoblast Differentiation

Cells ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 615
Author(s):  
Elmira Ahmady ◽  
Alexandre Blais ◽  
Patrick G. Burgon
Keyword(s):  
Protein Complexes ◽  
Myogenic Differentiation ◽  
Direct Interaction ◽  
Myoblast Differentiation ◽  
C2c12 Myoblast ◽  
Mobility Shift ◽  
Interacting Protein ◽  
Gel Mobility Shift ◽  
Genomic Regions ◽  
Myogenic Program

Muscle-enriched A-type lamin-interacting protein (Mlip) is a recently discovered Amniota gene that encodes proteins of unknown biological function. Here we report Mlip’s direct interaction with chromatin, and it may function as a transcriptional co-factor. Chromatin immunoprecipitations with microarray analysis demonstrated a propensity for Mlip to associate with genomic regions in close proximity to genes that control tissue-specific differentiation. Gel mobility shift assays confirmed that Mlip protein complexes with genomic DNA. Blocking Mlip expression in C2C12 myoblasts down-regulates myogenic regulatory factors (MyoD and MyoG) and subsequently significantly inhibits myogenic differentiation and the formation of myotubes. Collectively our data demonstrate that Mlip is required for C2C12 myoblast differentiation into myotubes. Mlip may exert this role as a transcriptional regulator of a myogenic program that is unique to amniotes.

Regulation of yeast COX6 by the general transcription factor ABF1 and separate HAP2- and heme-responsive elements

1992 ◽  
Vol 12 (5) ◽  
pp. 2302-2314
Author(s):  
J D Trawick ◽  
N Kraut ◽  
F R Simon ◽  
R O Poyton
Keyword(s):  
Transcription Factor ◽  
Carbon Source ◽  
Protein Binding ◽  
Glucose Repression ◽  
Protein Complexes ◽  
Carbon Sources ◽  
Major Protein ◽  
Mobility Shift ◽  
Gel Mobility Shift ◽  
In Cells

Transcription of the Saccharomyces cerevisiae COX6 gene is regulated by heme and carbon source. It is also affected by the HAP2/3/4 transcription factor complex and by SNF1 and SSN6. Previously, we have shown that most of this regulation is mediated through UAS6, an 84-bp upstream activation segment of the COX6 promoter. In this study, by using linker scanning mutagenesis and protein binding assays, we have identified three elements within UAS6 and one element downstream of it that are important. Two of these, HDS1 (heme-dependent site 1; between -269 and -251 bp) and HDS2 (between -228 and -220 bp), mediate regulation of COX6 by heme. Both act negatively. The other two elements, domain 2 (between -279 and -269 bp) and domain 1 (between -302 and -281 bp), act positively. Domain 2 is required for optimal transcription in cells grown in repressing but not derepressing carbon sources. Domain 1 is essential for transcription per se in cells grown on repressing carbon sources, is required for optimal transcription in cells grown on a derepressing carbon source, is sufficient for glucose repression-derepression, and is the element of UAS6 at which HAP2 affects COX6 transcription. This element contains the major protein binding sites within UAS6. It has consensus binding sequences for ABF1 and HAP2. Gel mobility shift experiments show that domain 1 binds ABF1 and forms different numbers of DNA-protein complexes in extracts from cells grown in repressing or derepressing carbon sources. In contrast, gel mobility shift experiments have failed to reveal that HAP2 or HAP3 binds to domain 1 or that hap3 mutations affect the complexes bound to it. Together, these findings permit the following conclusions: COX6 transcription is regulated both positively and negatively; heme and carbon source exert their effects through different sites; domain 1 is absolutely essential for transcription on repressing carbon sources; ABF1 is a major component in the regulation of COX6 transcription; and the HAP2/3/4 complex most likely affects COX6 transcription indirectly.

scholarly journals Regulation of yeast COX6 by the general transcription factor ABF1 and separate HAP2- and heme-responsive elements.

1992 ◽  
Vol 12 (5) ◽  
pp. 2302-2314 ◽  
Author(s):  
J D Trawick ◽  
N Kraut ◽  
F R Simon ◽  
R O Poyton
Keyword(s):  
Transcription Factor ◽  
Carbon Source ◽  
Protein Binding ◽  
Glucose Repression ◽  
Protein Complexes ◽  
Carbon Sources ◽  
Major Protein ◽  
Mobility Shift ◽  
Gel Mobility Shift ◽  
In Cells

Transcription of the Saccharomyces cerevisiae COX6 gene is regulated by heme and carbon source. It is also affected by the HAP2/3/4 transcription factor complex and by SNF1 and SSN6. Previously, we have shown that most of this regulation is mediated through UAS6, an 84-bp upstream activation segment of the COX6 promoter. In this study, by using linker scanning mutagenesis and protein binding assays, we have identified three elements within UAS6 and one element downstream of it that are important. Two of these, HDS1 (heme-dependent site 1; between -269 and -251 bp) and HDS2 (between -228 and -220 bp), mediate regulation of COX6 by heme. Both act negatively. The other two elements, domain 2 (between -279 and -269 bp) and domain 1 (between -302 and -281 bp), act positively. Domain 2 is required for optimal transcription in cells grown in repressing but not derepressing carbon sources. Domain 1 is essential for transcription per se in cells grown on repressing carbon sources, is required for optimal transcription in cells grown on a derepressing carbon source, is sufficient for glucose repression-derepression, and is the element of UAS6 at which HAP2 affects COX6 transcription. This element contains the major protein binding sites within UAS6. It has consensus binding sequences for ABF1 and HAP2. Gel mobility shift experiments show that domain 1 binds ABF1 and forms different numbers of DNA-protein complexes in extracts from cells grown in repressing or derepressing carbon sources. In contrast, gel mobility shift experiments have failed to reveal that HAP2 or HAP3 binds to domain 1 or that hap3 mutations affect the complexes bound to it. Together, these findings permit the following conclusions: COX6 transcription is regulated both positively and negatively; heme and carbon source exert their effects through different sites; domain 1 is absolutely essential for transcription on repressing carbon sources; ABF1 is a major component in the regulation of COX6 transcription; and the HAP2/3/4 complex most likely affects COX6 transcription indirectly.

scholarly journals Ganoderma microsporum immunomodulatory protein, GMI, promotes C2C12 myoblast differentiation in vitro via upregulation of Tid1 and STAT3 acetylation

PLoS ONE ◽  
2020 ◽  
Vol 15 (12) ◽  
pp. e0244791
Author(s):  
Wan-Huai Teo ◽  
Jeng-Fan Lo ◽  
Yu-Ning Fan ◽  
Chih-Yang Huang ◽  
Tung-Fu Huang
Keyword(s):  
Myogenic Differentiation ◽  
Atp Synthesis ◽  
C2c12 Cells ◽  
Myoblast Differentiation ◽  
C2c12 Myoblast ◽  
Muscle Loss ◽  
C2c12 Myoblasts ◽  
Immunomodulatory Protein ◽  
Pre Treatment

Ageing and chronic diseases lead to muscle loss and impair the regeneration of skeletal muscle. Thus, it’s crucial to seek for effective intervention to improve the muscle regeneration. Tid1, a mitochondrial co-chaperone, is important to maintain mitochondrial membrane potential and ATP synthesis. Previously, we demonstrated that mice with skeletal muscular specific Tid1 deficiency displayed muscular dystrophy and postnatal lethality. Tid1 can interact with STAT3 protein, which also plays an important role during myogenesis. In this study, we used GMI, immunomodulatory protein of Ganoderma microsporum, as an inducer in C2C12 myoblast differentiation. We observed that GMI pretreatment promoted the myogenic differentiation of C2C12 myoblasts. We also showed that the upregulation of mitochondria protein Tid1 with the GMI pre-treatment promoted myogenic differentiation ability of C2C12 cells. Strikingly, we observed the concomitant elevation of STAT3 acetylation (Ac-STAT3) during C2C12 myogenesis. Our study suggests that GMI promotes the myogenic differentiation through the activation of Tid1 and Ac-STAT3.

Activation of the NF-κB Pathway by Inflammatory Stimuli in Human Neutrophils

Blood ◽  
1997 ◽  
Vol 89 (9) ◽  
pp. 3421-3433 ◽  
Author(s):  
Patrick P. McDonald ◽  
Anette Bald ◽  
Marco A. Cassatella
Keyword(s):  
Cell Activation ◽  
Protein Complexes ◽  
Binding Activity ◽  
Human Neutrophils ◽  
Resting Cells ◽  
Mobility Shift ◽  
Dna Binding Activity ◽  
Cytokines And Chemokines ◽  
Inflammatory Stimuli ◽  
Gel Mobility Shift

Abstract Activated neutrophils have the ability to upregulate the expression of many genes, in particular those encoding cytokines and chemokines, and to subsequently release the corresponding proteins. Although little is known to date concerning the regulation of gene transcription in neutrophils, it is noteworthy that many of these genes depend on the activation of transcription factors, such as NF-κB, for inducible expression. We therefore investigated whether NF-κB/Rel proteins are expressed in human neutrophils, as well as their fate on cell activation. We now report that dimers consisting of p50 NFκB1, p65 RelA, and/or c-Rel are present in neutrophils and that the greater part of these protein complexes is physically associated with cytoplasmic IκB-α in resting cells. Following neutrophil stimulation with proinflammatory agonists (such as lipopolysaccharide [LPS], tumor necrosis factor-α [TNF-α], and fMet-Leu-Phe) that induce the production of cytokines and chemokines in these cells, NF-κB/Rel proteins translocated to nuclear fractions, resulting in a transient induction of NF-κB DNA binding activity, as determined in gel mobility shift assays. The onset of both processes was found to be closely paralleled by, and dependent on, IκB-α degradation. Proinflammatory neutrophil stimuli also promoted the accumulation of IκB-α mRNA transcripts, resulting in the reexpression of the IκB-α protein. To our knowledge, this constitutes the first indication that NF-κB activation may underlie the action of proinflammatory stimuli towards human neutrophil gene expression and, as such, adds a new facet to our understanding of neutrophil biology.

scholarly journals Mutant DMPK 3′-UTR transcripts disrupt C2C12 myogenic differentiation by compromising MyoD

2002 ◽  
Vol 159 (3) ◽  
pp. 419-429 ◽  
Author(s):  
Jeffrey D. Amack ◽  
Shannon R. Reagan ◽  
Mani S. Mahadevan
Keyword(s):  
Cell Culture ◽  
Myogenic Differentiation ◽  
Myoblast Differentiation ◽  
C2c12 Myoblast ◽  
Cell Culture Model ◽  
Rna Molecules ◽  
Protein Levels ◽  
Culture Model ◽  
Dystrophia Myotonica Protein Kinase ◽  
E Boxes

Myotonic dystrophy (DM) is caused by two similar noncoding repeat expansion mutations (DM1 and DM2). It is thought that both mutations produce pathogenic RNA molecules that accumulate in nuclear foci. The DM1 mutation is a CTG expansion in the 3′ untranslated region (3′-UTR) of dystrophia myotonica protein kinase (DMPK). In a cell culture model, mutant transcripts containing a (CUG)200 DMPK 3′-UTR disrupt C2C12 myoblast differentiation; a phenotype similar to what is observed in myoblast cultures derived from DM1 patient muscle. Here, we have used our cell culture model to investigate how the mutant 3′-UTR RNA disrupts differentiation. We show that MyoD protein levels are compromised in cells that express mutant DMPK 3′-UTR transcripts. MyoD, a transcription factor required for the differentiation of myoblasts during muscle regeneration, activates differentiation-specific genes by binding E-boxes. MyoD levels are significantly reduced in myoblasts expressing the mutant 3′-UTR RNA within the first 6 h under differentiation conditions. This reduction correlates with blunted E-box–mediated gene expression at time points that are critical for initiating differentiation. Importantly, restoring MyoD levels rescues the differentiation defect. We conclude that mutant DMPK 3′-UTR transcripts disrupt myoblast differentiation by reducing MyoD levels below a threshold required to activate the differentiation program.

Wnt4 activates the canonical β-catenin pathway and regulates negatively myostatin: functional implication in myogenesis

2011 ◽  
Vol 300 (5) ◽  
pp. C1122-C1138 ◽  
Author(s):  
Henri Bernardi ◽  
Stephanie Gay ◽  
Yann Fedon ◽  
Barbara Vernus ◽  
Anne Bonnieu ◽  
...  
Keyword(s):  
Cell Fate ◽  
Satellite Cells ◽  
Myogenic Differentiation ◽  
Expression Profiles ◽  
C2c12 Cells ◽  
Myoblast Differentiation ◽  
Strong Increase ◽  
C2c12 Myoblast ◽  
Wnt Proteins ◽  
Myogenic Activity

Expression of Wnt proteins is known to be important for developmental processes such as embryonic pattern formation and determination of cell fate. Previous studies have shown that Wn4 was involved in the myogenic fate of somites, in the myogenic proliferation, and differentiation of skeletal muscle. However, the function of this factor in adult muscle homeostasis remains not well understood. Here, we focus on the roles of Wnt4 during C2C12 myoblasts and satellite cells differentiation. We analyzed its myogenic activity, its mechanism of action, and its interaction with the anti-myogenic factor myostatin during differentiation. Established expression profiles indicate clearly that both types of cells express a few Wnts, and among these, only Wnt4 was not or barely detected during proliferation and was strongly induced during differentiation. As attested by myogenic factors expression pattern analysis and fusion index determination, overexpression of Wnt4 protein caused a strong increase in satellite cells and C2C12 myoblast differentiation leading to hypertrophic myotubes. By contrast, exposure of satellite and C2C12 cells to small interfering RNA against Wnt4 strongly diminished this process, confirming the myogenic activity of Wnt4. Moreover, we reported that Wnt4, which is usually described as a noncanonical Wnt, activates the canonical β-catenin pathway during myogenic differentiation in both cell types and that this factor regulates negatively the expression of myostatin and the regulating pathways associated with myostatin. Interestingly, we found that recombinant myostatin was sufficient to antagonize the differentiation-promoting activities of Wnt4. Reciprocally, we also found that the genetic deletion of myostatin renders the satellite cells refractory to the hypertrophic effect of Wnt4. These results suggest that the Wnt4-induced decrease of myostatin plays a functional role during hypertrophy. We propose that Wnt4 protein may be a key factor that regulates the extent of differentiation in satellite and C2C12 cells.

scholarly journals Hes6 regulates myogenic differentiation

Development ◽  
2002 ◽  
Vol 129 (9) ◽  
pp. 2195-2207
Author(s):  
Judy Cossins ◽  
Ann E. Vernon ◽  
Yun Zhang ◽  
Anna Philpott ◽  
Philip H. Jones
Keyword(s):  
Protein Interactions ◽  
Kinase Inhibitor ◽  
Myogenic Differentiation ◽  
C2c12 Cells ◽  
Myoblast Differentiation ◽  
C2c12 Myoblast ◽  
Xenopus Embryos ◽  
Enhancer Of Split

Hes6 is a basic helix-loop-helix transcription factor homologous to Drosophila Enhancer of Split (EoS) proteins. It is known to promote neural differentiation and to bind to Hes1, a related protein that is part of the Notch signalling pathway, affecting Hes1-regulated transcription. We show that Hes6 is expressed in the murine embryonic myotome and is induced on C2C12 myoblast differentiation in vitro. Hes6 binds DNA containing the Enhancer of Split E box (ESE) motif, the preferred binding site of Drosophila EoS proteins, and represses transcription of an ESE box reporter. When overexpressed in C2C12 cells, Hes6 impairs normal differentiation, causing a decrease in the induction of the cyclin-dependent kinase inhibitor, p21Cip1, and an increase in the number of cells that can be recruited back into the cell cycle after differentiation in culture. In Xenopus embryos, Hes6 is co-expressed with MyoD in early myogenic development. Microinjection of Hes6 RNA in vivo in Xenopus embryos results in an expansion of the myotome, but suppression of terminal muscle differentiation and disruption of somite formation at the tailbud stage. Analysis of Hes6 mutants indicates that the DNA-binding activity of Hes6 is not essential for its myogenic phenotype, but that protein-protein interactions are. Thus, we demonstrate a novel role for Hes6 in multiple stages of muscle formation.

scholarly journals BVES is a novel interactor of ANO5 and regulates myoblast differentiation

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Haiwen Li ◽  
Li Xu ◽  
Yandi Gao ◽  
Yuanbojiao Zuo ◽  
Zuocheng Yang ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Myoblast Differentiation ◽  
C2c12 Myoblast ◽  
Interacting Proteins ◽  
Cellular Functions ◽  
C2c12 Myoblasts ◽  
Interacting Protein ◽  
C Terminus ◽  
N Terminus ◽  
Labeling Approach

Abstract Background Anoctamin 5 (ANO5) is a membrane protein belonging to the TMEM16/Anoctamin family and its deficiency leads to the development of limb girdle muscular dystrophy R12 (LGMDR12). However, little has been known about the interactome of ANO5 and its cellular functions. Results In this study, we exploited a proximal labeling approach to identify the interacting proteins of ANO5 in C2C12 myoblasts stably expressing ANO5 tagged with BioID2. Mass spectrometry identified 41 unique proteins including BVES and POPDC3 specifically from ANO5-BioID2 samples, but not from BioID2 fused with ANO6 or MG53. The interaction between ANO5 and BVES was further confirmed by co-immunoprecipitation (Co-IP), and the N-terminus of ANO5 mediated the interaction with the C-terminus of BVES. ANO5 and BVES were co-localized in muscle cells and enriched at the endoplasmic reticulum (ER) membrane. Genome editing-mediated ANO5 or BVES disruption significantly suppressed C2C12 myoblast differentiation with little impact on proliferation. Conclusions Taken together, these data suggest that BVES is a novel interacting protein of ANO5, involved in regulation of muscle differentiation.

scholarly journals NDD1, a High-Dosage Suppressor ofcdc28-1N, Is Essential for Expression of a Subset of Late-S-Phase-Specific Genes in Saccharomyces cerevisiae

1999 ◽  
Vol 19 (5) ◽  
pp. 3312-3327 ◽  
Author(s):  
Chong Jin Loy ◽  
David Lydall ◽  
Uttam Surana
Keyword(s):  
Binding Sites ◽  
Nuclear Division ◽  
Protein Complexes ◽  
Essential Gene ◽  
Promoter Sequence ◽  
S Phase ◽  
Restrictive Temperature ◽  
Mobility Shift ◽  
Dna Fragment ◽  
Gel Mobility Shift

ABSTRACT cdc28-1N mutants progress through the G1and S phases normally at the restrictive temperature but fail to undergo nuclear division. We have isolated a gene, NDD1, which at a high dosage suppresses the nuclear-division defect ofcdc28-1N. NDD1 (nuclear division defective) is an essential gene. Its expression during the cell cycle is tightly regulated such that NDD1 RNA is most abundant during the S phase. Cells lacking the NDD1 gene arrest with an elongated bud, a short mitotic spindle, 2N DNA content, and an undivided nucleus, suggesting that its function is required for some aspect of nuclear division. We show that overexpression of Ndd1 results in the upregulation of bothCLB1 and CLB2 transcription, suggesting that the suppression of cdc28-1N by NDD1 may be due to an accumulation of these cyclins. Overproduction of Ndd1 also enhances the expression of SWI5, whose transcription, like that of CLB1 and CLB2, is activated in the late S phase. Ndd1 is essential for the expression of CLB1,CLB2, and SWI5, since none of these genes are transcribed in its absence. Both CLB2 expression and its upregulation by NDD1 are mediated by a 240-bp promoter sequence that contains four MCM1-binding sites. However, Ndd1 does not appear to be a component of any of the protein complexes assembled on this DNA fragment, as indicated by gel mobility shift assays. Instead, overexpression of NDD1 prevents the formation of one of the complexes whose appearance correlates with the termination of CLB2 expression in G1. The inability of GAL1 promoter-driven CLB2 to suppress the lethality of NDD1 null mutant suggests that, in addition to CLB1 and CLB2, NDD1may also be required for the transcription of other genes whose functions are necessary for G2/M transition.

scholarly journals Rbm24 displays dynamic functions required for myogenic differentiation during muscle regeneration

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Raphaëlle Grifone ◽  
Audrey Saquet ◽  
Manon Desgres ◽  
Claudia Sangiorgi ◽  
Caterina Gargano ◽  
...  
Keyword(s):  
Muscle Regeneration ◽  
Muscle Development ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Myogenic Differentiation ◽  
Regulation Of Gene Expression ◽  
Repair Process ◽  
Myoblast Differentiation ◽  
C2c12 Myoblast ◽  
Adult Mice

AbstractSkeletal muscle has a remarkable capacity of regeneration after injury, but the regulatory network underlying this repair process remains elusive. RNA-binding proteins play key roles in the post-transcriptional regulation of gene expression and the maintenance of tissue homeostasis and plasticity. Rbm24 regulates myogenic differentiation during early development, but its implication in adult muscle is poorly understood. Here we show that it exerts multiple functions in muscle regeneration. Consistent with its dynamic subcellular localization during embryonic muscle development, Rbm24 also displays cytoplasm to nucleus translocation during C2C12 myoblast differentiation. In adult mice, Rbm24 mRNA is enriched in slow-twitch muscles along with myogenin mRNA. The protein displays nuclear localization in both slow and fast myofibers. Upon injury, Rbm24 is rapidly upregulated in regenerating myofibers and accumulates in the myonucleus of nascent myofibers. Through satellite cell transplantation, we demonstrate that Rbm24 functions sequentially to regulate myogenic differentiation and muscle regeneration. It is required for myogenin expression at early stages of muscle injury and for muscle-specific pre-mRNA alternative splicing at late stages of regeneration. These results identify Rbm24 as a multifaceted regulator of myoblast differentiation. They provide insights into the molecular pathway orchestrating the expression of myogenic factors and muscle functional proteins during regeneration.

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