Msx1 antagonizes the myogenic activity of Pax3 in migrating limb muscle precursors

The migration of myogenic precursors to the vertebrate limb exemplifies a common problem in development - namely, how migratory cells that are committed to a specific lineage postpone terminal differentiation until they reach their destination. Here we show that in chicken embryos, expression of the Msx1 homeobox gene overlaps with Pax3 in migrating limb muscle precursors, which are committed myoblasts that do not express myogenic differentiation genes such as MyoD. We find that ectopic expression of Msx1 in the forelimb and somites of chicken embryos inhibits MyoD expression as well as muscle differentiation. Conversely, ectopic expression of Pax3 activates MyoD expression, while co-ectopic expression of Msx1 and Pax3 neutralizes their effects on MyoD. Moreover, we find that Msx1 represses and Pax3 activates MyoD regulatory elements in cell culture, while in combination, Msx1 and Pax3 oppose each other's trancriptional actions on MyoD. Finally, we show that the Msx1 protein interacts with Pax3 in vitro, thereby inhibiting DNA binding by Pax3. Thus, we propose that Msx1 antagonizes the myogenic activity of Pax3 in migrating limb muscle precursors via direct protein-protein interaction. Our results implicate functional antagonism through competitive protein-protein interactions as a mechanism for regulating the differentiation state of migrating cells.

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Transducing positional information to the Hox genes: critical interaction of cdx gene products with position-sensitive regulatory elements

Development ◽

10.1242/dev.125.22.4349 ◽

1998 ◽

Vol 125 (22) ◽

pp. 4349-4358 ◽

Cited By ~ 4

Author(s):

J. Charite ◽

W. de Graaff ◽

D. Consten ◽

M.J. Reijnen ◽

J. Korving ◽

...

Keyword(s):

Binding Sites ◽

Hox Genes ◽

Regulatory Element ◽

Ectopic Expression ◽

Positional Information ◽

Regulatory Elements ◽

Gene Products ◽

Anteroposterior Patterning

Studies of pattern formation in the vertebrate central nervous system indicate that anteroposterior positional information is generated in the embryo by signalling gradients of an as yet unknown nature. We searched for transcription factors that transduce this information to the Hox genes. Based on the assumption that the activity levels of such factors might vary with position along the anteroposterior axis, we devised an in vivo assay to detect responsiveness of cis-acting sequences to such differentially active factors. We used this assay to analyze a Hoxb8 regulatory element, and detected the most pronounced response in a short stretch of DNA containing a cluster of potential CDX binding sites. We show that differentially expressed DNA binding proteins are present in gastrulating embryos that bind to these sites in vitro, that cdx gene products are among these, and that binding site mutations that abolish binding of these proteins completely destroy the ability of the regulatory element to drive regionally restricted expression in the embryo. Finally, we show that ectopic expression of cdx gene products anteriorizes expression of reporter transgenes driven by this regulatory element, as well as that of the endogenous Hoxb8 gene, in a manner that is consistent with them being essential transducers of positional information. These data suggest that, in contrast to Drosophila Caudal, vertebrate cdx gene products transduce positional information directly to the Hox genes, acting through CDX binding sites in their enhancers. This may represent the ancestral mode of action of caudal homologues, which are involved in anteroposterior patterning in organisms with widely divergent body plans and modes of development.

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Cell heterogeneity upon myogenic differentiation: down-regulation of MyoD and Myf-5 generates ‘reserve cells’

Journal of Cell Science ◽

10.1242/jcs.111.6.769 ◽

1998 ◽

Vol 111 (6) ◽

pp. 769-779 ◽

Cited By ~ 24

Author(s):

N. Yoshida ◽

S. Yoshida ◽

K. Koishi ◽

K. Masuda ◽

Y. Nabeshima

Keyword(s):

Cell Cycle ◽

Myogenic Differentiation ◽

Significant Proportion ◽

Ectopic Expression ◽

Terminal Differentiation ◽

Growth Conditions ◽

Down Regulation ◽

Undifferentiated Cells ◽

Myoblast Cells ◽

Myod Expression

When a proliferating myoblast culture is induced to differentiate by deprivation of serum in the medium, a significant proportion of cells escape from terminal differentiation, while the rest of the cells differentiate. Using C2C12 mouse myoblast cells, this heterogeneity observed upon differentiation was investigated with an emphasis on the myogenic regulatory factors. The differentiating part of the cell population followed a series of well-described events, including expression of myogenin, p21(WAF1), and contractile proteins, permanent withdrawal from the cell cycle and cell fusion, whereas the rest of the cells did not initiate any of these events. Interestingly, the latter cells showed an undetectable or greatly reduced level of MyoD and Myf-5 expression, which had been originally expressed in the undifferentiated proliferating myoblasts. When these undifferentiated cells were isolated and returned to the growth conditions, they progressed through the cell cycle and regained MyoD expression. These cells demonstrated identical features with the original culture on the deprivation of serum. They produced both MyoD-positive differentiating and MyoD-negative undifferentiated populations once again. Thus the undifferentiated cells in the serum-deprived culture were designated ‘reserve cells’. Upon serum deprivation, MyoD expression rapidly decreased as a result of down-regulation in approximately 50% of the cells. After this heterogenization, MyoD positive cells expressed myogenin, which is the earliest known event of terminal differentiation and marks irreversible commitment to this, while MyoD-negative cells did not differentiate and became the reserve cells. We also demonstrated that ectopic expression of MyoD converted the reserve cells to differentiating cells, indicating that down-regulation of MyoD is a causal event in the formation of reserve cells.

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Regulation of the twist target gene tinman by modular cis-regulatory elements during early mesoderm development

Development ◽

10.1242/dev.124.24.4971 ◽

1997 ◽

Vol 124 (24) ◽

pp. 4971-4982 ◽

Cited By ~ 12

Author(s):

Z. Yin ◽

X.L. Xu ◽

M. Frasch

Keyword(s):

Homeobox Gene ◽

Regulatory Elements ◽

Bhlh Protein ◽

Expression Domain ◽

Enhancer Activity ◽

Enhancer Element ◽

Mesoderm Development ◽

Visceral Mesoderm

The Drosophila tinman homeobox gene has a major role in early mesoderm patterning and determines the formation of visceral mesoderm, heart progenitors, specific somatic muscle precursors and glia-like mesodermal cells. These functions of tinman are reflected in its dynamic pattern of expression, which is characterized by initial widespread expression in the trunk mesoderm, then refinement to a broad dorsal mesodermal domain, and finally restricted expression in heart progenitors. Here we show that each of these phases of expression is driven by a discrete enhancer element, the first being active in the early mesoderm, the second in the dorsal mesoderm and the third in cardioblasts. We provide evidence that the early-active enhancer element is a direct target of twist, a gene encoding a basic helix-loop-helix (bHLH) protein, which is necessary for tinman activation. This 180 bp enhancer includes three E-box sequences which bind Twist protein in vitro and are essential for enhancer activity in vivo. Ectodermal misexpression of twist causes ectopic activation of this enhancer in ectodermal cells, indicating that twist is the only mesoderm-specific activator of early tinman expression. We further show that the 180 bp enhancer also includes negatively acting sequences. Binding of Even-skipped to these sequences appears to reduce twist-dependent activation in a periodic fashion, thus producing a striped tinman pattern in the early mesoderm. In addition, these sequences prevent activation of tinman by twist in a defined portion of the head mesoderm that gives rise to hemocytes. We find that this repression requires the function of buttonhead, a head-patterning gene, and that buttonhead is necessary for normal activation of the hematopoietic differentiation gene serpent in the same area. Together, our results show that tinman is controlled by an array of discrete enhancer elements that are activated successively by differential genetic inputs, as well as by closely linked activator and repressor binding sites within an early-acting enhancer, which restrict twist activity to specific areas within the twist expression domain.

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The Oncogenic Potential of the Homeobox Gene Cdx2 Is Depending on the MAPK Pathway and Can Be Antagonized by the MEK1/2 Inhibitor PD98059 in a Mouse Model of t(12;13)(p13;q12) Positive AML.

Blood ◽

10.1182/blood.v104.11.1975.1975 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1975-1975

Author(s):

Vijay P.S. Rawat ◽

Vegi M. Naidu ◽

Christina Schessl ◽

Monica Cusan ◽

R.Keith Humphries ◽

...

Keyword(s):

Bone Marrow Cells ◽

Fusion Gene ◽

Mapk Pathway ◽

Phosphorylation Site ◽

Ectopic Expression ◽

Homeobox Gene ◽

Mapk Signaling ◽

Transactivation Domain ◽

Oncogenic Potential

Abstract In AML the translocation t(12;13)(p13;q12) results in the ectopic expression of the homeobox gene Cdx2 and the expression of the ETV6-CDX2 fusion. We have recently shown that myeloid leukemogenesis is induced by the ectopic expression of the proto-oncogene Cdx2 and not by the ETV6-CDX2 fusion gene in a murine model of t(12;13) AML. To characterize the contribution of different Cdx2 motifs to the transforming capacity of the gene we generated different mutants, inactivating the DNA binding homeodomain (N51S-Cdx2), or the PBX1-interacting motif (W167A-Cdx2), or deleting the N-terminal portion of Cdx2 (N-Cdx2). Expression of Cdx2 and the different mutants were induced in primary murine bone marrow cells by retroviral gene transfer, using an MSCV based retroviral construct with an IRES-YFP cassette. Expression of Cdx2 and the W167A-Cdx2 mutant significantly increased primary colony formation (3-fold) (n=3;p<0.001) with a higher number of CFU-G/GM colonies (p<0.015). Furthermore, both constructs enhanced the replating capacity of clonogenic progenitors with an 80-100fold increase in secondary colonies (p<0.005). In addition, both constructs induced the outgrowth of blast colonies (2700fold; p<0.02). In contrast, cells transduced with N51S-Cdx2 and N-Cdx2 lost their clonogenic potential after replating. In vivo all mice transplanted with cells expressing Cdx2 or the W167A-Cdx2 mutant developed transplantable AML. However, in Cdx2 leukemic mice > 90% of the cells co-expressed Gr-1+ and Mac1+, whereas in W167A mice 40% of the leukemic population were Gr-1+ only. The N51S mutant induced a distinct leukemia phenotype with 90 % Gr-1+/c-Kit+. We extended structure-function analyses, inactivating the phosphorylation site (S60) in the Cdx2 transactivation domain, previously shown to be regulated by the MAPK family. We confirmed that oncogenic Cdx2 is phophorylated at the N-terminal in primary BM cells by Western blotting using a P-Cdx2-S60 specific antibody. S60 position mutation slightly reduced the hematopoietic activity of wild-type Cdx2. Incubation with the MEK1 inhibitor PD98059 inhibited phosphorylation, decreased the frequency of CFU-S 8fold (n=7; p<0.001) and blocked growth of leukemic Cdx2 transfected blasts in vitro. In contrast, the p38 inhibitor SB2059 did not prevent phosphorylation and was unable to antagonize Cdx2 induced transformation. These data demonstrate that the transforming activity of Cdx2 and the phenotype of Cdx2 induced leukemias is depending on the functional integrity of distinct Cdx2 domains. Furthermore, our data link the oncogenic potential of Cdx2 directly to the MAPK signaling, opening the possibility to counteract Cdx2 associated leukemogenesis by kinase inhibitors.

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Hes6 regulates myogenic differentiation

Development ◽

10.1242/dev.129.9.2195 ◽

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.

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Hoxa5 overexpression correlates with IGFBP1 upregulation and postnatal dwarfism: evidence for an interaction between Hoxa5 and Forkhead box transcription factors

Development ◽

10.1242/dev.129.17.4065 ◽

2002 ◽

Vol 129 (17) ◽

pp. 4065-4074

Author(s):

Isabelle Foucher ◽

Michel Volovitch ◽

Monique Frain ◽

J. Julie Kim ◽

Jean-Claude Souberbielle ◽

...

Keyword(s):

Transcription Factors ◽

Growth Factor ◽

Promoter Activity ◽

Homeobox Gene ◽

Direct Interaction ◽

Fold Increase ◽

Regulatory Elements ◽

Insulin Like Growth Factor ◽

Forkhead Box

Transgenic mice expressing the homeobox gene Hoxa5 under the control of Hoxb2 regulatory elements present a growth arrest during weeks two and three of postnatal development, resulting in proportionate dwarfism. These mice present a liver phenotype illustrated by a 12-fold increase in liver insulin-like growth factor binding protein 1 (IGFBP1) mRNA and a 50% decrease in liver insulin-like growth factor 1 (IGF1) mRNA correlated with a 50% decrease in circulating IGF1. We show that the Hoxa5 transgene is expressed in the liver of these mice, leading to an overexpression of total (endogenous plus transgene) Hoxa5 mRNA in this tissue. We have used several cell lines to investigate a possible physiological interaction of Hoxa5 with the main regulator of IGFBP1 promoter activity, the Forkhead box transcription factor FKHR. In HepG2 cells, Hoxa5 has little effect by itself but inhibits the FKHR-dependent activation of the IGFBP1 promoter. In HuF cells, Hoxa5 cooperates with FKHR to dramatically enhance IGFBP1 promoter activity. This context-dependent physiological interaction probably corresponds to the existence of a direct interaction between Hoxa5 and FKHR and FoxA2/HNF3β, as demonstrated by pull-down experiments achieved either in vitro or after cellular co-expression. In conclusion, we propose that the impaired growth observed in this transgenic line relates to a liver phenotype best explained by a direct interaction between Hoxa5 and liver-specific Forkhead box transcription factors, in particular FKHR but also Foxa2/HNF3β. Because Hoxa5 and homeogenes of the same paralog group are normally expressed in the liver, the present results raise the possibility that homeoproteins, in addition to their established role during early development, regulate systemic physiological functions.

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Epigenetic Manipulation Facilitates the Generation of Skeletal Muscle Cells from Pluripotent Stem Cells

Stem Cells International ◽

10.1155/2017/7215010 ◽

2017 ◽

Vol 2017 ◽

pp. 1-8 ◽

Cited By ~ 2

Author(s):

Tomohiko Akiyama ◽

Shunichi Wakabayashi ◽

Atsumi Soma ◽

Saeko Sato ◽

Yuhki Nakatake ◽

...

Keyword(s):

Stem Cells ◽

Pluripotent Stem Cells ◽

Myogenic Differentiation ◽

Ectopic Expression ◽

Cell Types ◽

Culture Conditions ◽

The Body ◽

Specific Cell ◽

Modifying Factors

Human pluripotent stem cells (hPSCs) have the capacity to differentiate into essentially all cell types in the body. Such differentiation can be directed to specific cell types by appropriate cell culture conditions or overexpressing lineage-defining transcription factors (TFs). Especially, for the activation of myogenic program, early studies have shown the effectiveness of enforced expression of TFs associated with myogenic differentiation, such as PAX7 and MYOD1. However, the efficiency of direct differentiation was rather low, most likely due to chromatin features unique to hPSCs, which hinder the access of TFs to genes involved in muscle differentiation. Indeed, recent studies have demonstrated that ectopic expression of epigenetic-modifying factors such as a histone demethylase and an ATP-dependent remodeling factor significantly enhances myogenic differentiation from hPSCs. In this article, we review the recent progress for in vitro generation of skeletal muscles from hPSCs through forced epigenetic and transcriptional manipulation.

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The giant gene of Drosophila encodes a b-ZIP DNA-binding protein that regulates the expression of other segmentation gap genes

Development ◽

10.1242/dev.114.1.99 ◽

1992 ◽

Vol 114 (1) ◽

pp. 99-112 ◽

Cited By ~ 11

Author(s):

M. Capovilla ◽

E.D. Eldon ◽

V. Pirrotta

Keyword(s):

Dna Binding ◽

Leucine Zipper ◽

Ectopic Expression ◽

Regulatory Elements ◽

In Vitro Translation ◽

Gap Genes ◽

Zip Family ◽

Negative Effect ◽

Drosophila Embryos

The sequence of a cDNA from the giant gene of Drosophila shows that its product has a basic domain followed by a leucine zipper motif. Both features contain characteristic conserved elements of the b-ZIP family of DNA-binding proteins. Expression of the gene in bacteria or by in vitro translation yields a protein that migrates considerably faster than the protein extracted from Drosophila embryos. Treatment with phosphatase shows that this difference is due to multiple phosphorylation of the giant protein in the embryo. Ectopic expression of the protein in precellular blastoderm embryos produces abnormal phenotypes with a pattern of segment loss closely resembling that of Kruppel mutant embryos. Immunological staining shows that giant, ectopically expressed from the hsp70 promoter, represses the expression of both the Kruppel and knirps segmentation gap genes. The analysis of the interactions between Kruppel, knirps and giant reveals a network of negative regulation. We show that the apparent positive regulation of knirps by Kruppel is in fact mediated by a negative effect of Kruppel on giant and a negative effect of giant on knirps. giant protein made in bacteria or in embryos binds in vitro to the Kruppel regulatory elements CD1 and CD2 and recognizes a sequence resembling the binding sites of other b-ZIP proteins.

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Ehrlichia chaffeensis TRP120 Moonlights as a HECT E3 Ligase Involved in Self- and Host Ubiquitination To Influence Protein Interactions and Stability for Intracellular Survival

Infection and Immunity ◽

10.1128/iai.00290-17 ◽

2017 ◽

Vol 85 (9) ◽

Cited By ~ 7

Author(s):

Bing Zhu ◽

Seema Das ◽

Shubhajit Mitra ◽

Tierra R. Farris ◽

Jere W. McBride

Keyword(s):

Ubiquitin Ligase ◽

Protein Interactions ◽

Ectopic Expression ◽

E3 Ligase ◽

Intracellular Survival ◽

Host Protein ◽

Ehrlichia Chaffeensis ◽

Content Type ◽

Host Interactions

ABSTRACT Ehrlichia chaffeensis secretes tandem repeat protein (TRP) effectors that are involved in a diverse array of host cell interactions, some of which directly activate cell signaling pathways and reprogram host gene transcription to promote survival in the mononuclear phagocyte. However, the molecular details of these effector-host interactions and roles in pathobiology are incompletely understood. In this study, we determined that the E. chaffeensis effector TRP120 is posttranslationally modified by ubiquitin (Ub) and that ubiquitination occurs through intrinsic and host-mediated HECT ligase activity. A functional HECT E3 ligase domain with a conserved catalytic site was identified in the C-terminal region of TRP120, and TRP120 autoubiquitination occurred in vitro in the presence of host UbcH5b/c E2 enzymes. TRP120 ubiquitination sites were mapped using a high-density microfluidic peptide array and confirmed by ectopic expression of TRP120 lysine mutants in cells. Moreover, we determined that the HECT E3 ubiquitin ligase, Nedd4L, interacts with TRP120 during infection and also mediates TRP120 ubiquitination. Nedd4L knockdown resulted in the reduction of TRP120-Ub, decreased ehrlichial infection, and reduced recruitment of a known TRP120-interacting host protein, PCGF5, to ehrlichial inclusions. TRP120-mediated PCGF5 polyubiquitination was associated with a reduction in PCGF5 levels. Inhibition of ubiquitination with small molecules also significantly decreased ehrlichial infection, indicating that the Ub pathway is critical for ehrlichial intracellular replication and survival. The current study identified a novel E. chaffeensis ubiquitin ligase and revealed an important role for the ubiquitin pathway in effector-host interactions and pathogen-mediated host protein stability in order to promote intracellular survival.

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The homeobox gene Msx1 is expressed in a subset of somites, and in muscle progenitor cells migrating into the forelimb

Development ◽

10.1242/dev.126.12.2689 ◽

1999 ◽

Vol 126 (12) ◽

pp. 2689-2701 ◽

Cited By ~ 5

Author(s):

D. Houzelstein ◽

G. Auda-Boucher ◽

Y. Cheraud ◽

T. Rouaud ◽

I. Blanc ◽

...

Keyword(s):

Progenitor Cells ◽

Myogenic Differentiation ◽

Homeobox Gene ◽

Limb Bud ◽

Axial Position ◽

Limb Muscle ◽

Pax3 Gene ◽

Muscle Progenitor Cells ◽

Myoblast Cell

In myoblast cell cultures, the Msx1 protein is able to repress myogenesis and maintain cells in an undifferentiated and proliferative state. However, there has been no evidence that Msx1 is expressed in muscle or its precursors in vivo. Using mice with the nlacZ gene integrated into the Msx1 locus, we show that the reporter gene is expressed in the lateral dermomyotome of brachial and thoracic somites. Cells from this region will subsequently contribute to forelimb and intercostal muscles. Using Pax3 gene transcripts as a marker of limb muscle progenitor cells as they migrate from the somites, we have defined precisely the somitic origin and timing of cell migration from somites to limb buds in the mouse. Differences in the timing of migration between chick and mouse are discussed. Somites that label for Msx1(nlacZ)transgene expression in the forelimb region partially overlap with those that contribute Pax3-expressing cells to the forelimb. In order to see whether Msx1 is expressed in this migrating population, we have grafted somites from the forelimb level of Msx1(nlacZ)mouse embryos into a chick host embryo. We show that most cells migrating into the wing field express the Msx1(nlacZ)transgene, together with Pax3. In these experiments, Msx1 expression in the somite depends on the axial position of the graft. Wing mesenchyme is capable of inducing Msx1 transcription in somites that normally would not express the gene; chick hindlimb mesenchyme, while permissive for this expression, does not induce it. In the mouse limb bud, the Msx1(nlacZ)transgene is downregulated prior to the activation of the Myf5 gene, an early marker of myogenic differentiation. These observations are consistent with the proposal that Msx1 is involved in the repression of muscle differentiation in the lateral half of the somite and in limb muscle progenitor cells during their migration.

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