Id3 Is a Direct Transcriptional Target of Pax7 in Quiescent Satellite Cells

Pax7 is a key regulator of skeletal muscle stem cells and is required along with Pax3 to generate skeletal muscle precursors. We have identified a collection of genes induced by either Pax3 or Pax7 in C2C12 muscle cells. Two notable Pax3/7 targets are the inhibitory helix-loop-helix (HLH) proteins inhibitor of DNA binding (Id) 2 and Id3, both of which are coordinately expressed with Pax7 in quiescent satellite cells and are induced in quiescent C2C12 myogenic cells after ectopic expression of either Pax3 or Pax7. Ectopic Pax7 activates expression of a luciferase reporter driven by the Id3 promoter, and maximal induction of this reporter requires a conserved Pax7 binding site located upstream of the Id3 gene. Chromatin immunoprecipitation indicated that Pax7 is bound upstream of the Id3 promoter in quiescent satellite cells. In addition, short hairpin RNA-mediated knockdown of Pax7 expression in cultured satellite cells coordinately decreased both Id2 and Id3 expression. Together, these findings indicate that Id3 is a direct transcriptional target for Pax7 in quiescent satellite cells, and they suggest that Pax7 acts to block premature differentiation of quiescent satellite cells by inducing the expression of Id2 and Id3, which in turn may act to block either the precocious induction of myogenic basic (b)HLH proteins, the activity of myogenic bHLH proteins, or both.

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RUNX1/core binding factor A2 regulates platelet 12-lipoxygenase gene (ALOX12): studies in human RUNX1 haplodeficiency

Blood ◽

10.1182/blood-2009-04-214601 ◽

2010 ◽

Vol 115 (15) ◽

pp. 3128-3135 ◽

Cited By ~ 46

Author(s):

Gurpreet Kaur ◽

Gauthami Jalagadugula ◽

Guangfen Mao ◽

A. Koneti Rao

Keyword(s):

Expression Profiling ◽

Region Of Interest ◽

Luciferase Reporter ◽

Direct Transcriptional Target ◽

Erythroleukemia Cells ◽

12 Lipoxygenase ◽

Sirna Knockdown ◽

Induced Aggregation ◽

Transcriptional Target

Abstract Haploinsufficiency of RUNX1 (also known as CBFA2/AML1) is associated with familial thrombocytopenia, platelet dysfunction, and predisposition to acute leukemia. We have reported on a patient with thrombocytopenia and impaired agonist-induced aggregation, secretion, and protein phosphorylation associated with a RUNX1 mutation. Expression profiling of platelets revealed approximately 5-fold decreased expression of 12-lipoxygenase (12-LO, gene ALOX12), which catalyzes 12-hydroxyeicosatetraenoic acid production from arachidonic acid. We hypothesized that ALOX12 is a direct transcriptional target gene of RUNX1. In present studies, agonist-induced platelet 12-HETE production was decreased in the patient. Four RUNX1 consensus sites were identified in the 2-kb promoter region of ALOX12 (at −1498, −1491, −708, −526 from ATG). In luciferase reporter studies in human erythroleukemia cells, mutation of each site decreased activity; overexpression of RUNX1 up-regulated promoter activity, which was abolished by mutation of RUNX1 sites. Gel shift studies, including with recombinant protein, revealed RUNX1 binding to each site. Chromatin immunoprecipitation revealed in vivo RUNX1 binding in the region of interest. siRNA knockdown of RUNX1 decreased RUNX1 and 12-LO proteins. ALOX12 is a direct transcriptional target of RUNX1. Our studies provide further proof of principle that platelet expression profiling can elucidate novel alterations in platelets with inherited dysfunction.

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Differential regulation of granulopoiesis by the basic helix-loop-helix transcriptional inhibitors Id1 and Id2

Blood ◽

10.1182/blood-2004-12-4883 ◽

2005 ◽

Vol 105 (11) ◽

pp. 4272-4281 ◽

Cited By ~ 45

Author(s):

Miranda Buitenhuis ◽

Hanneke W. M. van Deutekom ◽

Liesbeth P. Verhagen ◽

Anders Castor ◽

Sten Eirik W. Jacobsen ◽

...

Keyword(s):

Critical Role ◽

Ectopic Expression ◽

Constitutive Expression ◽

Retroviral Transduction ◽

Basic Helix Loop Helix ◽

Helix Loop Helix ◽

Cd34 Cells ◽

Final Maturation ◽

Inhibitor Of Dna Binding

Abstract Inhibitor of DNA binding (Id) proteins function as inhibitors of members of the basic helix-loop-helix family of transcription factors and have been demonstrated to play an important role in regulating lymphopoiesis. However, the role of these proteins in regulation of myelopoiesis is currently unclear. In this study, we have investigated the role of Id1 and Id2 in the regulation of granulopoiesis. Id1 expression was initially up-regulated during early granulopoiesis, which was then followed by a decrease in expression during final maturation. In contrast, Id2 expression was up-regulated in terminally differentiated granulocytes. In order to determine whether Id expression plays a critical role in regulating granulopoiesis, Id1 and Id2 were ectopically expressed in CD34+ cells by retroviral transduction. Our experiments demonstrate that constitutive expression of Id1 inhibits eosinophil development, whereas in contrast neutrophil differentiation was modestly enhanced. Constitutive Id2 expression accelerates final maturation of both eosinophils and neutrophils, whereas inhibition of Id2 expression blocks differentiation of both lineages. Transplantation of β2-microglobulin-/- nonobese diabetic severe combined immunodeficient (NOD/SCID) mice with CD34+ cells ectopically expressing Id1 resulted in enhanced neutrophil development, whereas ectopic expression of Id2 induced both eosinophil and neutrophil development. These data demonstrate that both Id1 and Id2 play a critical, although differential role in granulopoiesis.

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An Examination of the Role of Transcriptional and Posttranscriptional Regulation in Rhabdomyosarcoma

Stem Cells International ◽

10.1155/2017/2480375 ◽

2017 ◽

Vol 2017 ◽

pp. 1-10 ◽

Cited By ~ 1

Author(s):

Alexander J. Hron ◽

Atsushi Asakura

Keyword(s):

Skeletal Muscle ◽

Soft Tissue ◽

Posttranscriptional Regulation ◽

Progenitor Cell ◽

Treatment Options ◽

Soft Tissue Tumors ◽

Basic Helix Loop Helix ◽

Helix Loop Helix ◽

Bhlh Proteins

Rhabdomyosarcoma (RMS) is an aggressive family of soft tissue tumors that most commonly manifests in children. RMS variants express several skeletal muscle markers, suggesting myogenic stem or progenitor cell origin of RMS. In this review, the roles of both recently identified and well-established microRNAs in RMS are discussed and summarized in a succinct, tabulated format. Additionally, the subtypes of RMS are reviewed along with the involvement of basic helix-loop-helix (bHLH) proteins, Pax proteins, and microRNAs in normal and pathologic myogenesis. Finally, the current and potential future treatment options for RMS are outlined.

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Inhibition of Tcf3 Binding by I-mfa Domain Proteins

Molecular and Cellular Biology ◽

10.1128/mcb.21.5.1866-1873.2001 ◽

2001 ◽

Vol 21 (5) ◽

pp. 1866-1873 ◽

Cited By ~ 36

Author(s):

Lauren Snider ◽

Hilary Thirlwell ◽

Jeffrey R. Miller ◽

Randall T. Moon ◽

Mark Groudine ◽

...

Keyword(s):

Transcription Factor ◽

Wnt Signaling ◽

Binding Sites ◽

Ectopic Expression ◽

Wnt Signaling Pathway ◽

Developmental Pathways ◽

Basic Helix Loop Helix ◽

Helix Loop Helix ◽

Dorsal Axis ◽

Bhlh Proteins

ABSTRACT We have determined that I-mfa, an inhibitor of several basic helix-loop-helix (bHLH) proteins, and XIC, a Xenopusortholog of human I-mf domain-containing protein that shares a highly conserved cysteine-rich C-terminal domain with I-mfa, inhibit the activity and DNA binding of the HMG box transcription factor XTcf3. Ectopic expression of I-mfa or XIC in early Xenopus embryos inhibited dorsal axis specification, the expression of the Tcf3/β-catenin-regulated genessiamois and Xnr3, and the ability of β-catenin to activate reporter constructs driven by Lef/Tcf binding sites. I-mfa domain proteins can regulate both the Wnt signaling pathway and a subset of bHLH proteins, possibly coordinating the activities of these two critical developmental pathways.

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miR-194-5p negatively regulates the proliferation and differentiation of rabbit skeletal muscle satellite cells

Molecular and Cellular Biochemistry ◽

10.1007/s11010-020-03918-0 ◽

2020 ◽

Author(s):

Yu Shi ◽

Xudong Mao ◽

Mingcheng Cai ◽

Shenqiang Hu ◽

Xiulan Lai ◽

...

Keyword(s):

Skeletal Muscle ◽

Satellite Cells ◽

Mammalian Species ◽

Muscle Growth ◽

Luciferase Reporter ◽

Stem Cell Population ◽

Muscle Satellite Cells ◽

Skeletal Muscle Satellite Cells ◽

Proliferation And Differentiation

Abstract Skeletal muscle satellite cells (SMSCs), also known as a multipotential stem cell population, play a crucial role during muscle growth and regeneration. In recent years, numerous miRNAs have been associated with the proliferation and differentiation of SMSCs in a number of mammalian species; however, the regulatory mechanisms of miR-194-5p in rabbit SMSCs still remain scarce. In this study, miR-194-5p was first observed to be highly expressed in the rabbit leg muscle. Furthermore, both the mimics and inhibitor of miR-194-5p were used to explore its role in the proliferation and differentiation of rabbit SMSCs cultured in vitro. Results from both EdU and CCK8 assays showed that miR-194-5p inhibited the proliferation of SMSCs. Meanwhile, Mef2c was identified as a target gene of miR-194-5p based on the dual-luciferase reporter assay results. In addition, upregulation of miR-194-5p decreased the expression levels of Mef2c and MyoG during rabbit SMSCs differentiation on Days 3 and 7 of in vitro culture. Taken together, these data demonstrated that miR-194-5p negatively regulates the proliferation and differentiation of rabbit SMSCs by targeting Mef2c.

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Molecular Distinction between Specification and Differentiation in the Myogenic Basic Helix-Loop-Helix Transcription Factor Family

Molecular and Cellular Biology ◽

10.1128/mcb.21.7.2404-2412.2001 ◽

2001 ◽

Vol 21 (7) ◽

pp. 2404-2412 ◽

Cited By ~ 86

Author(s):

Donald A. Bergstrom ◽

Stephen J. Tapscott

Keyword(s):

Skeletal Muscle ◽

Transcriptional Activation ◽

Molecular Basis ◽

Alpha Helix ◽

Endogenous Gene ◽

Transcriptional Activation Domain ◽

Basic Helix Loop Helix ◽

Helix Loop Helix ◽

Muscle Genes ◽

Bhlh Proteins

ABSTRACT The myogenic basic helix-loop-helix (bHLH) proteins regulate both skeletal muscle specification and differentiation: MyoD and Myf5 establish the muscle lineage, whereas myogenin mediates differentiation. Previously, we demonstrated that MyoD was more efficient than myogenin at initiating the expression of skeletal muscle genes, and in this study we present the molecular basis for this difference. A conserved amphipathic alpha-helix in the carboxy terminus of the myogenic bHLH proteins has distinct activities in MyoD and myogenin: the MyoD helix facilitates the initiation of endogenous gene expression, whereas the myogenin helix functions as a general transcriptional activation domain. Thus, the alternate use of a similar motif for gene initiation and activation provides a molecular basis for the distinction between specification and differentiation within the myogenic bHLH gene family.

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Basic Helix-Loop-Helix Proteins Bind to TrkB and p21Cip1 Promoters Linking Differentiation and Cell Cycle Arrest in Neuroblastoma Cells

Molecular and Cellular Biology ◽

10.1128/mcb.24.7.2662-2672.2004 ◽

2004 ◽

Vol 24 (7) ◽

pp. 2662-2672 ◽

Cited By ~ 60

Author(s):

Yuhui Liu ◽

Mario Encinas ◽

Joan X. Comella ◽

Martí Aldea ◽

Carme Gallego

Keyword(s):

Cell Cycle ◽

Cell Cycle Arrest ◽

Neuronal Differentiation ◽

Ectopic Expression ◽

Neurotrophin Receptor ◽

Basic Helix Loop Helix ◽

Helix Loop Helix ◽

Cycle Arrest ◽

E Box ◽

Bhlh Proteins

ABSTRACT Differentiation of precursor into specialized cells involves an increasing restriction in proliferative capacity, culminating in cell cycle exit. In this report we used a human neuroblastoma cell line to study the molecular mechanisms that coordinate cell cycle arrest and neuronal differentiation. Exposure to retinoic acid (RA), a differentiation agent in many cell types, causes an upregulation of neurotrophin receptor TrkB and the cyclin kinase inhibitor p21Cip1 at a transcriptional level. Full transcriptional activation of these two genes requires canonical E-box sequences found in the TrkB and p21Cip1 promoters. As reported for other E-box-regulated promoters, ectopic expression of E47 and E12 basic helix-loop-helix (bHLH) proteins enhances RA-dependent expression of TrkB and p21Cip1 , whereas the inhibitory HLH Id2 exerts opposite effects. In addition, ectopic expression of E47 and NeuroD, a neuronal bHLH protein, is able to activate TrkB transcription in the absence of RA. More importantly, we show that E47 and NeuroD proteins bind the TrkB and p21Cip1 promoter sequences in vivo. Since they establish a direct transcriptional link between a cell cycle inhibitor, p21Cip1 , and a neurotrophic receptor, TrkB, bHLH proteins would play an important role in coordinating key events of cell cycle arrest and neuronal differentiation.

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ACTA1 is inhibited by PAX3-FOXO1 through RhoA-MKL1-SRF signaling pathway and impairs cell proliferation, migration and tumor growth in Alveolar Rhabdomyosarcoma

Cell & Bioscience ◽

10.1186/s13578-021-00534-3 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Qiande Hu ◽

Liang Zhu ◽

Yuan Li ◽

Jianjun Zhou ◽

Jun Xu

Keyword(s):

Skeletal Muscle ◽

Cell Proliferation ◽

Tumor Growth ◽

Signaling Pathway ◽

Fusion Gene ◽

Ectopic Expression ◽

Alveolar Rhabdomyosarcoma ◽

Luciferase Reporter ◽

Protein Levels ◽

Malignant Soft Tissue Tumor

Abstract Background Alveolar Rhabdomyosarcoma (ARMS) is a pediatric malignant soft tissue tumor with skeletal muscle phenotype. Little work about skeletal muscle proteins in ARMS was reported. PAX3-FOXO1 is a specific fusion gene generated from the chromosomal translocation t (2;13) (q35; q14) in most ARMS. ACTA1 is the skeletal muscle alpha actin gene whose transcript was detected in ARMS. However, ACTA1 expression and regulation in ARMS have not been well investigated. This work aims to explore the expression, regulation and potential role of ACTA1 in ARMS. Results ACTA1 protein was detected in the studied RH30, RH4 and RH41 ARMS cells. ACTA1 was found to be inhibited by PAX3-FOXO1 at transcription and protein levels by employing western blot, luciferase reporter, qRT-PCR and immunofluorescence assays. The activities of ACTA1 gene reporter induced by RhoA, MKL1, SRF, STARS or Cytochalasin D molecule were reduced in the presence of overexpressed PAX3-FOXO1 protein. CCG-1423 is an inhibitor of RhoA-MKL1-SRF signaling, we observed there was a synergistic effect between this inhibitor and PAX3-FOXO1 to suppress ACTA1 reporter activity. Furthermore, PAX3-FOXO1 overexpression decreased ACTA1 protein level and knockdown of PAX3-FOXO1 by siRNA enhanced ACTA1 expression. In addition, both MKL1 and SRF, but not RhoA were also found to be inhibited by PAX3-FOXO1 gene at protein levels and increased once knockdown of PAX3-FOXO1 expression. The association between MKL1 and SRF in cells was decreased accordingly with ectopic expression of PAX3-FOXO1. However, the distribution of MKL1 and SRF in nuclear or cytoplasm fraction was not changed by PAX3-FOXO1 expression. Finally, we showed that ACTA1 overexpression in RH30 cells could inhibit cell proliferation and migration in vitro and impair tumor growth in vivo compared with the control groups. Conclusions ACTA1 is inhibited by PAX3-FOXO1 at transcription and protein levels through RhoA-MKL1-SRF signaling pathway and this inhibition may partially contribute to the tumorigenesis and development of ARMS. Our findings improved the understanding of PAX3-FOXO1 in ARMS and provided a potential strategy for the treatment of ARMS in future.

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