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Biomedical subjects

G E Muscat

Publications and source records attributed to G E Muscat.

At least 19 recordsLinked to original sources
PubMed2001

SOX18 directly interacts with MEF2C in endothelial cells.

Recently, we demonstrated that mutations in the Sry-related HMG box gene Sox18 underlie vascular and hair follicle defects in the mouse allelic mutants ragged (Ra) and RaJ. Ra mice display numerous anomalies in the homozygote including, oedema, peritoneal secretions, and are almost completely naked. Sox18 and the MADS box transcription factor, Mef2C, are expressed in developing endothelial cells. Null mutants in Sox18 and Mef2c display overlapping phenotypic abnormalities, hence, we investigated the relationship between these two DNA binding proteins. We report here the direct interaction between MEF2C and SOX18 proteins, and establish that these proteins are coexpressed in vivo in endothelial cell nuclei. MEF2C expression potentiates SOX18-mediated transcription in vivo and regulates the function of the SOX18 activation domain. Interestingly, MEF2C fails to interact or co-activate transcription with the Ra or RaJ mutant SOX18 proteins. These results suggest that MEF2C and SOX18 may be important partners directing the transcriptional regulation of vascular development.

Animals↗
PubMed2001

Sox18 expression in blood vessels and feather buds during chicken embryogenesis.

Sox18 encodes a transcription factor known to be important for the development of blood vessels and hair follicles in mice. In order to study the functional conservation of this gene through evolution, we have isolated and characterized Sox18 in chickens. cSox18 shows a high degree of sequence homology to both the mouse and human orthologues, particularly in the high mobility group DNA-binding domain and to a lesser extent in the transcriptional activation domain. A region of unusually high sequence conservation at the C-terminus may represent a further, previously unrecognized functional domain. Both the chicken and human proteins appear to be truncated at the N-terminus relative to mouse SOX18. In situ hybridization analyses showed expression in the developing vasculature and feather follicles, consistent with reported expression in the mouse embryo. In addition, cSox18 mRNA was observed in the retina and claw beds.

Amino Acid Sequence↗
PubMed2001

A dynamic role for HDAC7 in MEF2-mediated muscle differentiation.

The overlapping expression profile of MEF2 and the class-II histone deacetylase, HDAC7, led us to investigate the functional interaction and relationship between these regulatory proteins. HDAC7 expression inhibits the activity of MEF2 (-A, -C, and -D), and in contrast MyoD and Myogenin activities are not affected. Glutathione S-transferase pulldown and immunoprecipitation demonstrate that the repression mechanism involves direct interactions between MEF2 proteins and HDAC7 and is associated with the ability of MEF2 to interact with the N-terminal 121 amino acids of HDAC7 that encode repression domain 1. The MADS domain of MEF2 mediates the direct interaction of MEF2 with HDAC7. MEF2 inhibition by HDAC7 is dependent on the N-terminal repression domain and surprisingly does not involve the C-terminal deacetylase domain. HDAC7 interacts with CtBP and other class-I and -II HDACs suggesting that silencing of MEF2 activity involves corepressor recruitment. Furthermore, we show that induction of muscle differentiation by serum withdrawal leads to the translocation of HDAC7 from the nucleus into the cytoplasm. This work demonstrates that HDAC7 regulates the function of MEF2 proteins and suggests that this class-II HDAC regulates this important transcriptional (and pathophysiological) target in heart and muscle tissue. The nucleocytoplasmic trafficking of HDAC7 and other class-II HDACs during myogenesis provides an ideal mechanism for the regulation of HDAC targets during mammalian development and differentiation.

Animals↗
PubMed2001

Cloning and functional analysis of the Sry-related HMG box gene, Sox18.

The Sox gene family (Sry like HMG box gene) is characterised by a conserved DNA sequence encoding a domain of approximately 80 amino acids which is responsible for sequence specific DNA binding. We initially published the identification and partial cDNA sequence of murine Sox18, a new member of this gene family, isolated from a cardiac cDNA library. This sequence allowed us to classify Sox18 into the F sub-group of Sox proteins, along with Sox7 and Sox17. Recently, we demonstrated that mutations in the Sox18 activation domain underlie cardiovascular and hair follicle defects in the mouse mutation, ragged (Ra) (Pennisi et al., 2000. Mutations in Sox18 underlie cardiovascular and hair follicle defecs in ragged mice. Nat. Genet. 24, 434-437). Ra homozygotes lack vibrissae and coat hairs, have generalised oedema and an accumulation of chyle in the peritoneum. Here we have investigated the genomic sequences encoding Sox18. Screening of a mouse genomic phage library identified four overlapping clones, we sequenced a 3.25 kb XbaI fragment that defined the entire coding region and approximately 1.5 kb of 5' flanking sequences. This identified (i) an additional 91 amino acids upstream of the previously designated methionine start codon in the original cDNA, and (ii) an intron encoded within the HMG box/DNA binding domain in exactly the same position as that found in the Sox5, -13 and -17 genes. The Sox18 gene encodes a protein of 468 aa. We present evidence that suggests HAF-2, the human HMG-box activating factor -2 protein, is the orthologue of murine Sox18. HAF-2 has been implicated in the regulation of the Human IgH enhancer in a B cell context. Random mutagenesis coupled with GAL4 hybrid analysis in the activation domain between amino acids 252 and 346, of Sox18, implicated the phosphorylation motif, SARS, and the region between amino acid residues 313 and 346 as critical components of Sox18 mediated transactivation. Finally, we examined the expression of Sox18 in multiple adult mouse tissues using RT-PCR. Low-moderate expression was observed in spleen, stomach, kidney, intestine, skeletal muscle and heart. Very abundant expression was detected in lung tissue.

Amino Acid Sequence↗
PubMed2001

Class I histone deacetylases sequentially interact with MyoD and pRb during skeletal myogenesis.

We describe a functional and biochemical link between the myogenic activator MyoD, the deacetylase HDAC1, and the tumor suppressor pRb. Interaction of MyoD with HDAC1 in undifferentiated myoblasts mediates repression of muscle-specific gene expression. Prodifferentiation cues, mimicked by serum removal, induce both downregulation of HDAC1 protein and pRb hypophosphorylation. Dephosphorylation of pRb promotes the formation of pRb-HDAC1 complex in differentiated myotubes. pRb-HDAC1 association coincides with disassembling of MyoD-HDAC1 complex, transcriptional activation of muscle-restricted genes, and cellular differentiation of skeletal myoblasts. A single point mutation introduced in the HDAC1 binding domain of pRb compromises its ability to disrupt MyoD-HDAC1 interaction and to promote muscle gene expression. These results suggest that reduced expression of HDAC1 accompanied by its redistribution in alternative nuclear protein complexes is critical for terminal differentiation of skeletal muscle cells.

Animals↗
PubMed2001

Sox18 is transiently expressed during angiogenesis in granulation tissue of skin wounds with an identical expression pattern to Flk-1 mRNA.

Sox18 encodes a member of the Sry-related high mobility group box (SOX) family of developmental transcription factors. Examination of Sox18 expression during embryogenesis has shown that Sox18 is expressed transiently in endothelial cells of developing blood vessels, and mutations in Sox18 have been found to underlie the mouse vascular and hair follicle mutant ragged. In this study we have examined the expression of Sox18 in angiogenesis during wound healing. Full-thickness skin wounds were created in mice, and subsequent expression of vascular endothelial growth factor (VEGF), the VEGF receptor Flk-1, alpha1 (iv) collagen (Col4a1), and Sox18 were studied using in situ hybridization. As has been previously reported, VEGF was expressed predominantly in the keratinocytes at the wound margins. Sox18 expression was found five days after wounding during capillary sprouting in granulation tissue and persisted through the proliferative phase of healing, but was not detected in fully epithelialized wounds 21 days after wounding. Sox18 mRNA expression was detected in capillaries within the granulation tissue and showed an identical pattern of distribution to Flk-1 and Col4a1 mRNA expression in endothelial cells. Immunostaining with a polyclonal anti-Sox18 antibody showed SOX18 protein localized in capillary endothelial cells within the granulation tissue. Capillaries in the subcutaneous tissue of unwounded skin showed no Sox18 expression. Sox18 may therefore represent a transcription factor involved in the induction of angiogenesis during wound healing and tissue repair, but not in the maintenance of endothelial cells in undamaged tissue.

Animals↗
PubMed2001

Transcriptional modulation of mouse mu-opioid receptor distal promoter activity by Sox18.

Previously, we reported the presence of dual promoters, referred to as distal (DP) and proximal, with a negative regulatory element between them in the mouse mu-opioid receptor (mor) gene. Here we have identified a positive regulatory element influencing mor DP transcription, which contains multiple consensus binding motifs for Sox factors (sex-determining Sry-like high mobility group box-containing genes). In gel supershift assays, the Sox family member Sox18 bound directly to the multiple Sox consensus binding motifs of the mor DP enhancer. Overexpression of Sox18 cDNA increased luciferase activity regulated by the mor DP, and did so in a Sox18 concentration-dependent manner. In contrast, overexpression of another Sox member, Sox5, triggered no such trans-activation of mor DP-driven luciferase activity or DNA-protein binding activity. These results suggest that Sox18 directly and specifically stimulates mor gene expression, by trans-activating the mor DP enhancer.

5' Untranslated Regions↗
PubMed2001

Subcellular localization of the steroid receptor coactivators (SRCs) and MEF2 in muscle and rhabdomyosarcoma cells.

Skeletal muscle differentiation and the activation of muscle-specific gene expression are dependent on the concerted action of the MyoD family and the MADS protein, MEF2, which function in a cooperative manner. The steroid receptor coactivator SRC-2/GRIP-1/TIF-2, is necessary for skeletal muscle differentiation, and functions as a cofactor for the transcription factor, MEF2. SRC-2 belongs to the SRC family of transcriptional coactivators/cofactors that also includes SRC-1 and SRC-3/RAC-3/ACTR/AIB-1. In this study we demonstrate that SRC-2 is essentially localized in the nucleus of proliferating myoblasts; however, weak (but notable) expression is observed in the cytoplasm. Differentiation induces a predominant localization of SRC-2 to the nucleus; furthermore, the nuclear staining is progressively more localized to dot-like structures or nuclear bodies. MEF2 is primarily expressed in the nucleus, although we observed a mosaic or variegated expression pattern in myoblasts; however, in myotubes all nuclei express MEF2. GRIP-1 and MEF2 are coexpressed in the nucleus during skeletal muscle differentiation, consistent with the direct interaction of these proteins. Rhabdomyosarcoma (RMS) cells derived from malignant skeletal muscle tumors have been proposed to be deficient in cofactors. Alveolar RMS cells very weakly express the steroid receptor coactivator, SRC-2, in a diffuse nucleocytoplasmic staining pattern. MEF2 and the cofactors, SRC-1 and SRC-3 are abundantly expressed in alveolar and embryonal RMS cells; however, the staining is not localized to the nucleus. Furthermore, the subcellular localization and transcriptional activity of MEF2C and a MEF2-dependent reporter are compromised in alveolar RMS cells. In contrast, embryonal RMS cells express SRC-2 in the nucleus, and MEF2 shuttles from the cytoplasm to the nucleus after serum withdrawal. In conclusion, this study suggests that the steroid receptor coactivator SRC-2 and MEF2 are localized to the nucleus during the differentiation process. In contrast, RMS cells display aberrant transcription factor SRC localization and expression, which may underlie certain features of the RMS phenotype.

Acetyltransferases↗
PubMed2000

The steroid receptor coactivator, GRIP-1, is necessary for MEF-2C-dependent gene expression and skeletal muscle differentiation.

Nuclear receptor-mediated activation of transcription involves coactivation by cofactors collectively denoted the steroid receptor coactivators (SRCs). The process also involves the subsequent recruitment of p300/CBP and PCAF to a complex that synergistically regulates transcription and remodels the chromatin. PCAF and p300 have also been demonstrated to function as critical coactivators for the muscle-specific basic helix-loop-helix (bHLH) protein MyoD during myogenic commitment. Skeletal muscle differentiation and the activation of muscle-specific gene expression is dependent on the concerted action of another bHLH factor, myogenin, and the MADS protein, MEF-2, which function in a cooperative manner. We examined the functional role of one SRC, GRIP-1, in muscle differentiation, an ideal paradigm for the analysis of the determinative events that govern the cell's decision to divide or differentiate. We observed that the mRNA encoding GRIP-1 is expressed in proliferating myoblasts and post-mitotic differentiated myotubes, and that protein levels increase during differentiation. Exogenous/ectopic expression studies with GRIP-1 sense and antisense vectors in myogenic C2C12 cells demonstrated that this SRC is necessary for (1) induction/activation of myogenin, MEF-2, and the crucial cell cycle regulator, p21, and (2) contractile protein expression and myotube formation. Furthermore, we demonstrate that the SRC GRIP-1 coactivates MEF-2C-mediated transcription. GRIP-1 also coactivates the synergistic transactivation of E box-dependent transcription by myogenin and MEF-2C. GST-pulldowns, mammalian two-hybrid analysis, and immunoprecipitation demonstrate that the mechanism involves direct interactions between MEF-2C and GRIP-1 and is associated with the ability of the SRC to interact with the MADS domain of MEF-2C. The HLH region of myogenin mediates the direct interaction of myogenin and GRIP-1. Interestingly, interaction with myogenic factors is mediated by two regions of GRIP-1, an amino-terminal bHLH-PAS region and the carboxy-terminal region between amino acids 1158 and 1423 (which encodes an activation domain, has HAT activity, and interacts with the coactivator-associated arginine methyltransferase). This work demonstrates that GRIP-1 potentiates skeletal muscle differentiation by acting as a critical coactivator for MEF-2C-mediated transactivation and is the first study to ascribe a function to the amino-terminal bHLH-PAS region of SRCs.

Animals↗
PubMed2000

Expression and cytoplasmic localisation of deoxyuridine triphosphate pyrophosphatase encoded by a human endogenous retrovirus.

Many lentiviruses encode a dUTPase which may protect against toxic misincorporation of dUTP into cDNA during reverse transcription. However, the primate lentiviruses HIV and SIV do not express a dUTPase. Significantly, the host genomes of these lentiviruses contain a multicopy endogenous retrovirus which is absent in non-primate genomes. In humans, this endogenous retrovirus is known as HERV-K and encodes a potential dUTPase sequence. Previously, we have suggested that HIV infection is complemented by a cytosolic dUTPase derived from the dUTPase gene encoded by HERV-K. This study demonstrates expression of HERV-K dUTPase transcripts and protein in human cell lines using RT-PCR and western blot analysis. Immunocytochemistry showed that HERV-K dUTPase was predominantly located in cell cytoplasm when transiently expressed in COS-1 cells. These data provide substantiation and support for the hypothesis above and is the first documentation of expression of an enzyme of nucleotide metabolism expressed by an endogenous retrovirus.

Blotting, Western↗
PubMed2000

Domains of Brn-2 that mediate homodimerization and interaction with general and melanocytic transcription factors.

The class III POU gene brn-2, encoding the Brn-2/N-Oct-3 transcription factor, is widely expressed in the developing mammalian central nervous system. Brn-2 has also been found to regulate the melanocytic phenotype with N-Oct-3 DNA binding activity elevated in malignant melanoma, however, its mode of action is yet to be defined. The functional role of the Brn-2 transcription factor has been investigated through the analysis of protein-protein interactions it forms with a number of basal and melanocytic transcriptional regulatory proteins. In vivo interactions were tested by gene-cotransfection using the mammalian GAL4-Herpes Simplex viral protein 16 (VP16) two hybrid formation and direct protein binding by in vitro glutathione S-transferase (GST)-pull down assay. The Brn-2 protein was found to homodimerize in vivo with high affinity, using Brn-2 deletion constructs dimer complex formation was found to be dependent on the presence of both the homeodomain and linker regions of the POU-domain. However, the POU-homoedomain was dispensable for the formation of the dimerization interface in one of the partner molecules but not both, when the POU-linker region was removed the ability to interact was lost irrespective of the presence of the homeodomain. Dimerization of Brn-2/N-Oct-3 was also found to occur in DNA binding assays using melanoma cell line nuclear extracts and a recently reported dimer target sequence probe, which may have significant consequences for gene regulation in melanocytic tumours. Low affinity Brn-2 protein contacts have also been found with the basal transcription complex, including TATA binding protein (TBP) and the transcriptional coactivator p300, and with the Sox-10 and Pax-3 transcription factors that are known to play an important role in melanocyte cell formation.

Binding Sites↗
PubMed2000

Structure-function analysis of the Rev-erbA and RVR ligand-binding domains reveals a large hydrophobic surface that mediates corepressor binding and a ligand cavity occupied by side chains.

Rev-erbA/RVR are closely related orphan nuclear receptors (NRs) functioning as dominant transcriptional silencers through an association with the nuclear receptor corepressor N-CoR. In contrast with ligand-regulated NRs, Rev-erbA/RVR lack the ligand-binding domain (LBD) C-terminal activation helix, H12. In the case of retinoid acid receptor and thyroid hormone receptor, ligand binding is thought to reposition H12, causing corepressor dissociation and coactivator recruitment, thus leading to transcriptional activation. Here we present homology models of the Rev-erbA/RVR LBDs, which show that the putative ligand cavity is occupied by side chains, suggesting the absence of endogenous ligands. Modeling also revealed a very hydrophobic surface due to the absence of H12, exposing residues from H3, loop 3-4, H4, and H11. Mutation of specific residues from this surface severely impaired the in vitro and in vivo interaction of the Rev-erbA/RVR LBD with the receptor-interacting domain of the corepressors N-CoR or its splice variant RIP13delta1, reinforcing the view of the physical association of N-CoR with a LBD surface encompassing H3-H4 and H11. Furthermore, mutations in the LBD surface significantly reduced the ability of Rev-erbA and RVR to function as repressors of transcription. Interestingly, a hydrophobic surface comprised of H3-H4 and H12 in liganded NRs mediates the interaction with coactivators. Hence, it appears that corepressors and coactivators bind to overlapping surfaces of NR LBDs, the conformational change associated with H12 upon ligand binding resulting in a switch from a corepressor- to a coactivator-binding surface.

Amino Acid Sequence↗
PubMed1999

Structure/function analysis of a dUTPase: catalytic mechanism of a potential chemotherapeutic target.

dUTP pyrophosphatase catalyses hydrolysis of deoxyuridine triphosphate (dUTP) to deoxyuridine monophosphate (dUMP) and inorganic pyrophosphate (PPi). Elimination of dUTP is vital since its misincorporation into DNA by DNA polymerases can initiate a damaging iterative repair and misincorporation cycle, resulting in DNA fragmentation and cell death. The anti-tumour activity of folate agonists and thymidylate synthase inhibitors is thought to rely on dUTP misincorporation. Furthermore, retroviral cDNA production may be particularly susceptible to the effects of dUTP misincorporation by virtue of the error-prone nature of reverse trans criptase. Consequently, dUTPase activity is an ideal point of intervention in both chemotherapy and anti-retroviral therapy. In particular, the dUTPase encoded by a human endogenous retrovirus (HERV-K) has been suggested to complement HIV infection and so is an attractive target for specific inhibition. Hence, we used site photoaffinity labelling, site-directed mutagenesis and molecular modelling to assign catalytic roles to the conserved amino acid residues in the active site of the HERV-K dUTPase and to identify structural differences with other dUTPase enzymes. We found that dUTP photoaffinity labelling was specific for a beta-hairpin motif in HERV-K dUTPase. Mutagenesis of aspartate residues Asp84 and 86 to asparagine within this beta-hairpin showed the carboxylate moiety of both residues was required for catalysis but not for dUTP binding. An increase in the pKa of both aspartate residues brought about by substitution of a serine residue with a glutamate residue adjacent to the aspartate residues increased activity by a factor of 1.67 at pH 8.0, implicating general base catalysis as the enzyme's catalytic mechanism. Conservative mutagenesis of Tyr87 to Phe resulted in a sevenfold reduction of dUTPase activity and a 3.3-fold reduction in binding activity, whilst substitution with an isoleucine residue totally abolished both catalytic activity and dUTP binding, suggesting that binding/activity is dependent on an aromatic side-chain at the base of the hairpin. Comparison of a homology-based three-dimensional model structure of HERV-K dUTPase with a crystallographic structure of the human dUTPase revealed displacement of a conserved alpha-helix in the HERV-K enzyme causing expansion of the HERV-K active site. This expansion may be responsible for the ability of the HERV-K enzyme to hydrolyse dTTP and bind the bulkier dNTPs in contrast to the majority of dUTPases which are highly specific for dUTP. Knowledge of the dUTPase catalytic mechanism and the distinctive topography of the HERV-K active site provides a molecular basis for the design of HERV-K dUTPase-specific inhibitors.

Amino Acid Sequence↗
PubMed1999

Exogenous expression of a dominant negative RORalpha1 vector in muscle cells impairs differentiation: RORalpha1 directly interacts with p300 and myoD.

ROR/RZR is an orphan nuclear receptor that has no known ligand in the 'classical sense'. In the present study we demonstrate that RORalpha is constitutively expressed during the differentiation of proliferating myoblasts to post-mitotic multinucleated myotubes, that have acquired a contractile phenotype. Exogenous expression of dominant negative RORalpha1DeltaE mRNA in myogenic cells significantly reduces the endogenous expression of RORalpha1 mRNA, represses the accumu-lation and delays the activation of mRNAs encoding MyoD and myogenin [the muscle-specific basic helix-loop-helix (bHLH) proteins] and p21(Waf-1/Cip-1) (a cdk inhibitor). Immunohistochemistry demonstrates that morpho-logical differentiation is delayed in cells expressing the RORDeltaE transcript. Furthermore, the size and development of mutlinucleated myotubes is impaired. The E region of RORalpha1 interacts with p300, a cofactor that functions as a coactivator in nuclear receptor and MyoD-mediated transactivation. Consistent with the functional role of RORalpha1 in myogenesis, we observed that RORalpha1 directly interacts with the bHLH protein MyoD. This interaction was mediated by the N-terminal activation domain of the bHLH protein, MyoD, and the RORalpha1 DNA binding domain/C region. Furthermore, we demonstrated that p300, RORalpha1 and MyoD interact in a non-competitive manner. In conclusion, this study provides evidence for a biological role and positive influence of RORalpha1 in the cascade of events involved in the activation of myogenic-specific markers and cell cycle regulators and suggests that crosstalk between theretinoid-relatedorphan (ROR) nuclear receptors and the myogenic bHLH proteins has functional consequences for differentiation.

Antigens, Differentiation↗
PubMed1999

The nuclear receptor corepressor N-CoR regulates differentiation: N-CoR directly interacts with MyoD.

Classical ligand-activated nuclear receptors (e.g. thyroid hormone receptor, retinoic acid receptor), orphan nuclear receptors (e.g. Rev-erbAalpha/beta), Mad/Max bHLH (basic helix loop helix)-LZ proteins, and oncoproteins, PLZF and LAZ3/BCL6, bind DNA and silence transcription by recruiting a repressor complex that contains N-CoR (nuclear receptor corepressor)/SMRT (silencing mediator of retinoic acid and thyroid hormone receptor), Sin3A/B, and HDAc-1/-2 proteins. The function of the corepressor, N-CoR, in the process of cellular differentiation and coupled phenotypic acquisition, has not been investigated. We examined the functional role of N-CoR in myogenesis (muscle differentiation), an ideal paradigm for the analysis of the determinative events that govern the cell's decision to divide or differentiate. We observed that the mRNA encoding N-CoR was suppressed as proliferating myoblasts exited the cell cycle, and formed morphologically and biochemically differentiated myotubes. Exogenous expression of N-CoR (but not RIP13) in myogenic cells ablated 1) myogenic differentiation, 2) the expression of the myoD gene family that encode the myogenic specific bHLH proteins, and 3) the crucial cell cycle regulator, p21Waf-1/Cip-1 mRNA. Furthermore, N-CoR expression efficiently inhibits the myoD-mediated myogenic conversion of pluripotential C3H10T1/2 cells. We demonstrate that MyoD-mediated transactivation and activity are repressed by N-CoR. The mechanism involves direct interactions between MyoD and N-CoR; moreover, the interaction was dependent on the amino-terminal repression domain (RD1) of N-CoR and the bHLH region of MyoD. Trichostatin A treatment significantly stimulated the activity of MyoD by approximately 10-fold and inhibited the ability of N-CoR to repress MyoD-mediated transactivation, consistent with the involvement of the corepressor and the recruitment of a histone deacteylase activity in the process. This work demonstrates that the corepressor N-CoR is a key regulator of MyoD activity and mammalian differentiation, and that N-CoR has a multifaceted role in myogenesis.

Animals↗
PubMed1998

The orphan nuclear receptor, COUP-TF II, inhibits myogenesis by post-transcriptional regulation of MyoD function: COUP-TF II directly interacts with p300 and myoD.

COUP-TF II is an orphan nuclear receptor that has no known ligand in the 'classical sense'. COUP-TF interacts with the corepressors N-CoR, SMRT and RIP13, and silences transcription by active repression and trans-repression. Forced expression of the orphan nuclear receptor COUP-TF II in mouse C2 myogenic cells has been demonstrated to inhibit morphological differentiation, and to repress the expression of: (i) the myoD gene family which encodes myogenic basic helix-loop-helix (bHLH) proteins; and (ii) the cell cycle regulator, p21(Waf-1/Cip-1). In the present study, we show that COUP-TF II efficiently inhibits the myoD -mediated myogenic conversion of pluripotential C3H10T1/2 cells by post-transcriptional mechanisms. Furthermore, repression of MyoD-dependent transcription by COUP-TF II occurs in the absence of the nuclear receptor cognate binding motif. The inhibition of MyoD-mediated trans-activation involves the direct binding of the DNA binding domain/C-region and hinge/D-regions [i.e. amino acid (aa) residues 78-213] of COUP-TF II to the N-terminal activation domain of MyoD. Over-expression of the cofactor p300, which functions as a coactivator of myoD-mediated transcription, alleviated repression by COUP-TF II. Further binding analysis demonstrated that COUP-TF II interacted with the N-terminal 149 aa residues of p300 which encoded the receptor interaction domain of the coactivator. Finally we observed that COUP-TF II, MyoD and p300 interact in a competitive manner, and that increasing amounts of COUP-TF II have the ability to reduce the interaction between myoD and p300 invitro. The experiments presented herein suggest thatCOUP-TF II post-transcriptionally regulates myoD activity/function, and that crosstalk between orphan nuclear receptors and the myogenic bHLH proteins has functional consequences for differentiation.

3T3 Cells↗