scholarly journals Selection of a Dominant Negative Retinoblastoma Protein (RB) Inhibiting Satellite Myoblast Differentiation Implies an Indirect Interaction between MyoD and RB

2000 ◽  
Vol 20 (14) ◽  
pp. 5129-5139 ◽  
Author(s):  
Feng-Qian Li ◽  
Archie Coonrod ◽  
Marshall Horwitz
Keyword(s):  
Myogenic Differentiation ◽  
Retinoblastoma Protein ◽  
Dominant Negative ◽  
Myoblast Differentiation ◽  
Loss Of Function ◽  
Lysosomal Protease ◽  
Dominant Negative Form ◽  
Genes Encoding

ABSTRACT Satellite myoblasts serve as stem cells in postnatal skeletal muscle, but the genes responsible for choosing between growth versus differentiation are largely undefined. We have used a novel genetic approach to identify genes encoding proteins whose dominant negative inhibition is capable of interrupting the in vitro differentiation of C2C12 murine satellite myoblasts. The screen is based on fusion of a library of cDNA fragments with the lysosomal protease cathepsin B (CB), such that the fusion protein intracellularly diverts interacting factors to the lysosome. Among other gene fragments selected in this screen, including those of known and novel sequence, is the retinoblastoma protein (RB) pocket domain. This unique dominant negative form of RB allows us to genetically determine if MyoD and RB associate in vivo. The dominant negative CB-RB fusion produces a cellular phenotype indistinguishable from recessive loss of function RB mutations. The fact that the dominant negative RB inhibits myogenic differentiation in the presence of nonlimiting concentrations of either RB or MyoD suggests that these two proteins do not directly interact. We further show that the dominant negative RB inhibits E2F1 but cannot inhibit a forced E2F1-RB dimer. Therefore, E2F1 is a potential mediator of the dominant negative inhibition of MyoD by CB-RB during satellite cell differentiation. We propose this approach to be generally suited to the investigation of gene function, even when little is known about the pathway being studied.

Regulation of Hox target genes by a DNA bound Homothorax/Hox/Extradenticle complex

Development ◽  
1999 ◽  
Vol 126 (22) ◽  
pp. 5137-5148 ◽  
Author(s):  
H.D. Ryoo ◽  
T. Marty ◽  
F. Casares ◽  
M. Affolter ◽  
R.S. Mann
Keyword(s):  
Nuclear Localization ◽  
Target Genes ◽  
Dominant Negative ◽  
Homeodomain Protein ◽  
Hox Proteins ◽  
Trimeric Complex ◽  
Binding Residue ◽  
Dominant Negative Form

To regulate their target genes, the Hox proteins of Drosophila often bind to DNA as heterodimers with the homeodomain protein Extradenticle (EXD). For EXD to bind DNA, it must be in the nucleus, and its nuclear localization requires a third homeodomain protein, Homothorax (HTH). Here we show that a conserved N-terminal domain of HTH directly binds to EXD in vitro, and is sufficient to induce the nuclear localization of EXD in vivo. However, mutating a key DNA binding residue in the HTH homeodomain abolishes many of its in vivo functions. HTH binds to DNA as part of a HTH/Hox/EXD trimeric complex, and we show that this complex is essential for the activation of a natural Hox target enhancer. Using a dominant negative form of HTH we provide evidence that similar complexes are important for several Hox- and exd-mediated functions in vivo. These data suggest that Hox proteins often function as part of a multiprotein complex, composed of HTH, Hox, and EXD proteins, bound to DNA.

scholarly journals MyoD induced enhancer RNA interacts with hnRNPL to activate target gene transcription during myogenic differentiation

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Yu Zhao ◽  
Jiajian Zhou ◽  
Liangqiang He ◽  
Yuying Li ◽  
Jie Yuan ◽  
...  
Keyword(s):  
Target Gene ◽  
Myogenic Differentiation ◽  
Myoblast Differentiation ◽  
Enhancer Rna ◽  
Super Enhancer ◽  
And Function ◽  
Nuclear Ribonucleoprotein ◽  
Multiple Cells

AbstractEmerging evidence supports roles of enhancer RNAs (eRNAs) in regulating target gene. Here, we study eRNA regulation and function during skeletal myoblast differentiation. We provide a panoramic view of enhancer transcription and categorization of eRNAs. Master transcription factor MyoD is crucial in activating eRNA production. Super enhancer (se) generated seRNA-1 and -2 promote myogenic differentiation in vitro and in vivo. seRNA-1 regulates expression levels of two nearby genes, myoglobin (Mb) and apolipoprotein L6 (Apol6), by binding to heterogeneous nuclear ribonucleoprotein L (hnRNPL). A CAAA tract on seRNA-1 is essential in mediating seRNA-1/hnRNPL binding and function. Disruption of seRNA-1-hnRNPL interaction attenuates Pol II and H3K36me3 deposition at the Mb locus, in coincidence with the reduction of its transcription. Furthermore, analyses of hnRNPL binding transcriptome-wide reveal its association with eRNAs is a general phenomenon in multiple cells. Collectively, we propose that eRNA-hnRNPL interaction represents a mechanism contributing to target mRNA activation.

scholarly journals Haploinsufficiency of autism candidate gene NUAK1 impairs cortical development and behavior

2018 ◽  
Author(s):  
Virginie Courchet ◽  
Amanda J Roberts ◽  
Peggy Del Carmine ◽  
Tommy L. Lewis ◽  
Franck Polleux ◽  
...  
Keyword(s):  
Social Preference ◽  
De Novo ◽  
Sensorimotor Gating ◽  
Learning Task ◽  
Autism Spectrum ◽  
Dominant Negative ◽  
Loss Of Function ◽  
Cortical Connectivity

SUMMARYRecently, numerous rare de novo mutations have been identified in children diagnosed with autism spectrum disorders (ASD). However, despite the predicted loss-of-function nature of some of these de novo mutations, the affected individuals are heterozygous carriers, which would suggest that most of these candidate genes are haploinsufficient and/or that these mutations lead to expression of dominant-negative forms of the protein. Here, we tested this hypothesis with the gene Nuak1, recently identified as a candidate ASD gene and that we previously identified for its role in the development of cortical connectivity. We report that Nuak1 is happloinsufficient in mice in regard to its function in cortical axon branching in vitro and in vivo. Nuak1+/− mice show a combination of abnormal behavioral traits ranging from defective memory consolidation in a spatial learning task, defects in social novelty (but not social preference) and abnormal sensorimotor gating and prepulse inhibition of the startle response. Overall, our results demonstrate that Nuak1 haploinsufficiency leads to defects in the development of cortical connectivity and a complex array of behavorial deficits compatible with ASD, intellectual disability and schizophrenia.

scholarly journals In vivo dissection of Rhoa function in vascular development using zebrafish

2021 ◽  
Author(s):  
Laura M. Pillay ◽  
Joseph J. Yano ◽  
Andrew E. Davis ◽  
Matthew G. Butler ◽  
Keith A. Barnes ◽  
...  
Keyword(s):  
Vascular Dysfunction ◽  
Vascular Development ◽  
Vascular Endothelial Cell ◽  
Fiber Formation ◽  
Dominant Negative ◽  
Loss Of Function ◽  
Vascular Integrity ◽  
Rhoa Activity

Rationale: The small monomeric GTPase RHOA acts as a master regulator of signal transduction cascades by activating effectors of cellular signaling, including the Rho-associated protein kinases ROCK1/2. Previous in vitro cell culture studies suggest that RHOA can regulate many critical aspects of vascular endothelial cell (EC) biology, including focal adhesion, stress fiber formation, and angiogenesis. However, the specific in vivo roles of RHOA during vascular development and homeostasis are still not well understood. Objective: In this study we examine the in vivo functions of RHOA in regulating vascular development and integrity in zebrafish. Methods and Results: We use zebrafish RHOA-ortholog (rhoaa) mutants, transgenic embryos expressing wild type, dominant-negative, or constitutively active forms of rhoaa in ECs, and a pharmacologic inhibitor of ROCK1/2 to study the in vivo consequences of RHOA gain- and loss-of-function in the vascular endothelium. Our findings document roles for RHOA in vascular integrity, developmental angiogenesis, and vascular morphogenesis. Conclusions: Our results indicate that either too much or too little RHOA activity leads to vascular dysfunction in vivo.

scholarly journals Heparan Sulfate-dependent RAGE oligomerization is indispensable for pathophysiological functions of RAGE

2021 ◽  
Author(s):  
Miaomiao Li ◽  
Chih Yean Ong ◽  
Christophe J Langouet-Astrie ◽  
Lisi TAN ◽  
Ashwni Verma ◽  
...  
Keyword(s):  
Heparan Sulfate ◽  
Genetic Model ◽  
Point Mutations ◽  
Active Role ◽  
Dominant Negative ◽  
Unexpected Finding ◽  
Rna Seq ◽  
Dominant Negative Form

RAGE, a druggable inflammatory receptor, is known to function as an oligomer but the exact oligomerization mechanism remains poorly understood. Previously we have shown that heparan sulfate (HS) plays an active role in RAGE oligomerization. To understand the physiological significance of HS-induced RAGE oligomerization in vivo, we generated RAGE knock-in mice (RageAHA/AHA) by introducing point mutations to specifically disrupt HS-RAGE interaction. The RAGE mutant demonstrated normal ligand-binding but impaired capacity of HS-binding and oligomerization. Remarkably, RageAHA/AHA mice phenocopied Rage-/- mice in two different pathophysiological processes, namely bone remodeling and neutrophil-mediated liver injury, which demonstrates that HS-induced RAGE oligomerization is essential for RAGE signaling. Our findings suggest that it should be possible to block RAGE signaling by inhibiting HS-RAGE interaction. To test this, we generated a monoclonal antibody that targets the HS-binding site of RAGE. This antibody blocks RAGE signaling in vitro and in vivo, recapitulating the phenotype of RageAHA/AHA mice. By inhibiting HS-RAGE interaction genetically and pharmacologically, our work validated an alternative strategy to antagonize RAGE. Finally, we have performed RNA-seq analysis of neutrophils and lungs and found that while Rage-/- mice had a broad alteration of transcriptome in both tissues compared to wild-type mice, the changes of transcriptome in RageAHA/AHA mice were much more restricted. This unexpected finding suggests that by preserving the expression of RAGE protein (in a dominant-negative form), RageAHA/AHA mouse might represent a cleaner genetic model to study physiological roles of RAGE in vivo compared to Rage-/- mice.

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 Group I Paks Promote Skeletal Myoblast Differentiation In Vivo and In Vitro

2016 ◽  
Vol 37 (4) ◽  
Author(s):  
Giselle A. Joseph ◽  
Min Lu ◽  
Maria Radu ◽  
Jennifer K. Lee ◽  
Steven J. Burden ◽  
...  
Keyword(s):  
Muscle Development ◽  
Myogenic Differentiation ◽  
Acute Injury ◽  
Myoblast Differentiation ◽  
Differentiation Markers ◽  
Cross Sectional ◽  
Content Type ◽  
Group I

ABSTRACT Skeletal myogenesis is regulated by signal transduction, but the factors and mechanisms involved are not well understood. The group I Paks Pak1 and Pak2 are related protein kinases and direct effectors of Cdc42 and Rac1. Group I Paks are ubiquitously expressed and specifically required for myoblast fusion in Drosophila. We report that both Pak1 and Pak2 are activated during mammalian myoblast differentiation. One pathway of activation is initiated by N-cadherin ligation and involves the cadherin coreceptor Cdo with its downstream effector, Cdc42. Individual genetic deletion of Pak1 and Pak2 in mice has no overt effect on skeletal muscle development or regeneration. However, combined muscle-specific deletion of Pak1 and Pak2 results in reduced muscle mass and a higher proportion of myofibers with a smaller cross-sectional area. This phenotype is exacerbated after repair to acute injury. Furthermore, primary myoblasts lacking Pak1 and Pak2 display delayed expression of myogenic differentiation markers and myotube formation. These results identify Pak1 and Pak2 as redundant regulators of myoblast differentiation in vitro and in vivo and as components of the promyogenic Ncad/Cdo/Cdc42 signaling pathway.

Requirement ofFoxD3-class signaling for neural crest determination inXenopus

Development ◽  
2001 ◽  
Vol 128 (13) ◽  
pp. 2525-2536 ◽  
Author(s):  
Noriaki Sasai ◽  
Kenji Mizuseki ◽  
Yoshiki Sasai
Keyword(s):  
Neural Crest ◽  
Dominant Negative ◽  
Related Factor ◽  
Embryonic Patterning ◽  
Loss Of Function ◽  
Upstream Regulator ◽  
Dominant Negative Form ◽  
Neural Crest Differentiation ◽  
Negative Form

Fox factors (winged-helix transcription factors) play important roles in early embryonic patterning. We show here that FoxD3 (Forkhead 6) regulates neural crest determination in Xenopus embryos. Expression of FoxD3 in the presumptive neural crest region starts at the late gastrula stage in a manner similar to that of Slug, and overlaps with that of Zic-r1. When overexpressed in the embryo and in ectodermal explants, FoxD3 induces expression of neural crest markers. Attenuation of FoxD3-related signaling by a dominant-negative FoxD3 construct (FoxD3delN) inhibits neural crest differentiation in vivo without suppressing the CNS marker Sox2. Interestingly, these loss-of-function phenotypes are reversed by coinjecting Slug. In animal cap explants, neural crest differentiation induced by Slug and Wnt3a is also inhibited by FoxD3delN but not by a dominant-negative form of XBF2. Loss-of-function studies using dominant-negative forms of FoxD3 and Slug indicate that Slug induction by Zic factors requires FoxD3-related signaling, and that FoxD3 and Slug have different requirements in inducing downstream neural crest markers. These data demonstrate that FoxD3 (or its closely related factor) is an essential upstream regulator of neural crest determination.

Abstract 17273: Long Non-Coding RNA Ppp1r1b Regulates Myogenic Differentiation Through Modulating Histone 3 Methylation

Circulation ◽  
2018 ◽  
Vol 138 (Suppl_1) ◽  
Author(s):  
Xuedong Kang ◽  
Yan Zhao ◽  
Marlin Touma
Keyword(s):  
Histone Modification ◽  
Protein Complex ◽  
Myogenic Differentiation ◽  
C2c12 Cells ◽  
Myoblast Differentiation ◽  
Mouse Heart ◽  
Histone 3

Introduction: Long noncoding RNAs (lncRNAs), emerged as critical epigenetic regulators of transcriptome, play important roles in cardiac development and might be targeted to treat human cardiomyocyte dysfunction. In our work, we identified a novel lncRNA that regulates myogenesis. Hypothesis: LncRNA Ppp1r1b regulates myogenesis by modulating Histone 3 methylation Methods: After treated with antisense oligonucleotides (GapmeR) or siRNA against Ppp1r1b-LncRNA, real time PCR and Western blot analyses were performed to examine the expression of myogenic and sarcomere genes. Chromatin immunoprecipitation (CHIP) was used to comparatively analyze gene specific histone modification level. RNA pull-down was employed to identify the protein molecules that interact with Ppp1r1b-LncRNA. Results: By silencing Ppp1r1b-LncRNA with GapmeR, C2C12, a skeletal myoblast cell line, did not develop fully differentiated myotubes, but tend to remain in a quiescent mono-nucleated status. In vivo analysis of GapmeR injected neonatal mouse heart and in vitro siRNA silenced human skeletal myoblasts further confirmed the important role of Ppp1r1b-LncRNA on myogenesis. Members of the MyoD family of muscle-specific transcription factors (MyoD and myogenin) failed to be up-regulated during myogenic differentiation when treated with Ppp1r1b-LncRNA specific GapmeR or siRNA. Key proteins essential for establishing and maintaining normal skeletal muscle architecture, including Tcap and Dystropnin, are also suppressed in Ppp1r1b LncRNA- deficient heart. Analysis of histone modification levels at Myogenin, MyoD1 and Tcap in C2C12 cells revealed more histone tri-methylation at these myogenic and sarcomere structural genes compared to untreated cells. Additional lncRNA- protein complex isolation has further revealed insight into the biological roles of Ppp1r1b-LncRNA. Conclusions: Our results support the role of Ppp1r1b-LncRNA in promoting myogenic differentiation. Ppp1r1b-lncRNA function is mediated by inhibiting histone methylation on promoters of multiple myogenic and sarcomere genes. In particular, the identification of EZH2 in pulled Pp1r1b LncRNA: protein complex implies that Polycomb repressive complex 2 (PRC2) is involved in Ppp1r1b-LncRNA modulated myoblast differentiation.

scholarly journals Myostatin Inhibits Myoblast Differentiation by Down-regulating MyoD Expression

2002 ◽  
Vol 277 (51) ◽  
pp. 49831-49840 ◽  
Author(s):  
Brett Langley ◽  
Mark Thomas ◽  
Amy Bishop ◽  
Mridula Sharma ◽  
Stewart Gilmour ◽  
...  
Keyword(s):  
Culture Media ◽  
Myogenic Differentiation ◽  
Critical Role ◽  
Dominant Negative ◽  
Negative Regulator ◽  
Myoblast Differentiation ◽  
Proliferation And Differentiation ◽  
Smad 3 ◽  
Low Serum

Myostatin, a negative regulator of myogenesis, is shown to function by controlling the proliferation of myoblasts. In this study we show that myostatin is an inhibitor of myoblast differentiation and that this inhibition is mediated through Smad 3.In vitro, increasing concentrations of recombinant mature myostatin reversibly blocked the myogenic differentiation of myoblasts, cultured in low serum media. Western and Northern blot analysis indicated that addition of myostatin to the low serum culture media repressed the levels of MyoD, Myf5, myogenin, and p21 leading to the inhibition of myogenic differentiation. The transient transfection of C2C12myoblasts with MyoD expressing constructs did not rescue myostatin-inhibited myogenic differentiation. Myostatin signaling specifically induced Smad 3 phosphorylation and increased Smad 3·MyoD association, suggesting that Smad 3 may mediate the myostatin signal by interfering with MyoD activity and expression. Consistent with this, the expression of dominant-negativeSmad3rescued the activity of a MyoD promoter-reporter in C2C12myoblasts treated with myostatin. Taken together, these results suggest that myostatin inhibits MyoD activity and expression via Smad 3 resulting in the failure of the myoblasts to differentiate into myotubes. Thus we propose that myostatin plays a critical role in myogenic differentiation and that the muscular hyperplasia and hypertrophy seen in animals that lack functional myostatin is because of deregulated proliferation and differentiation of myoblasts.

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