Overexpression of the Ski pro-oncogene has been shown to induce myogenesis in non-muscle cells, to promote muscle hypertrophy in postnatal mice, and to activate transcription of muscle-specific ...genes. However, the precise role of Ski in muscle cell differentiation and its underlying molecular mechanism are not fully understood. To elucidate the involvement of Ski in muscle terminal differentiation, two retroviral systems were used to achieve conditional overexpression or knockdown of Ski in satellite cell-derived C2C12 myoblasts. We found that enforced expression of Ski promoted differentiation, whereas loss of Ski severely impaired it. Compromised terminal differentiation in the absence of Ski was likely because of the failure to induce myogenin (Myog) and p21 despite normal expression of MyoD. Chromatin immunoprecipitation and transcriptional reporter experiments showed that Ski occupied the endogenous Myog regulatory region and activated transcription from the Myog regulatory region upon differentiation. Transactivation of Myog was largely dependent on a MEF3 site bound by Six1, not on the binding site of MyoD or MEF2. Activation of the MEF3 site required direct interaction of Ski with Six1 and Eya3 mediated by the evolutionarily conserved Dachshund homology domain of Ski. Our results indicate that Ski is necessary for muscle terminal differentiation and that it exerts this role, at least in part, through its association with Six1 and Eya3 to regulate the Myog transcription.
The c-ski protooncogene encodes a transcription factor that binds DNA only in association with other proteins. To identify co-binding proteins, we performed a yeast two-hybrid screen. The results of ...the screen and subsequent co-immunoprecipitation studies identified Smad2 and Smad3, two transcriptional activators that mediate the type β transforming growth factor (TGF-β ) response, as Ski-interacting proteins. In Ski-transformed cells, all of the Ski protein was found in Smad3-containing complexes that accumulated in the nucleus in the absence of added TGF-β . DNA binding assays showed that Ski, Smad2, Smad3, and Smad4 form a complex with the Smad/Ski binding element GTCTAGAC (SBE). Ski repressed TGF-β -induced expression of 3TP-Lux, the natural plasminogen activator inhibitor 1 promoter and of reporter genes driven by the SBE and the related CAGA element. In addition, Ski repressed a TGF-β -inducible promoter containing AP-1 (TRE) elements activated by a combination of Smads, Fos, and/or Jun proteins. Ski also repressed synergistic activation of promoters by combinations of Smad proteins but failed to repress in the absence of Smad4. Thus, Ski acts in opposition to TGF-β -induced transcriptional activation by functioning as a Smad-dependent co-repressor. The biological relevance of this transcriptional repression was established by showing that overexpression of Ski abolished TGF-β -mediated growth inhibition in a prostate-derived epithelial cell line.
Summary
The SKI protein represses the TGF‐β tumor suppressor pathway by associating with the Smad transcription factors. SKI is upregulated in human malignant melanoma tumors in a disease‐progression ...manner and its overexpression promotes proliferation and migration of melanoma cells in vitro. The mechanisms by which SKI antagonizes TGF‐β signaling in vivo have not been fully elucidated. Here we show that human melanoma cells in which endogenous SKI expression was knocked down by RNAi produced minimal orthotopic tumor xenograft nodules that displayed low mitotic rate and prominent apoptosis. These minute tumors exhibited critical signatures of active TGF‐β signaling including high levels of nuclear Smad3 and p21Waf1, which are not found in the parental melanomas. To understand how SKI promotes tumor growth we used gain‐ and loss‐of‐function approaches and found that simultaneously to blocking the TGF‐β‐growth inhibitory pathway, SKI promotes the switch of Smad3 from tumor suppression to oncogenesis by favoring phosphorylations of the Smad3 linker region in melanoma cells but not in normal human melanocytes. In this context, SKI is required for preventing TGF‐β‐mediated downregulation of the oncogenic protein c‐MYC, and for inducing the plasminogen activator inhibitor‐1, a mediator of tumor growth and angiogenesis. Together, the results indicate that SKI exploits multiple regulatory levels of the TGF‐β pathway and its deficiency restores TGF‐β tumor suppressor and apoptotic activities in spite of the likely presence of oncogenic mutations in melanoma tumors.
Schwann cell proliferation and subsequent differentiation to nonmyelinating and myelinating cells are closely linked processes. Elucidating the molecular mechanisms that control these events is key ...to the understanding of nerve development, regeneration, nerve-sheath tumors, and neuropathies. We define the protooncogene Ski, an inhibitor of TGF-β signaling, as an essential component of the machinery that controls Schwann cell proliferation and myelination. Functional Ski overexpression inhibits TGF-β-mediated proliferation and prevents growth-arrested Schwann cells from reentering the cell cycle. Consistent with these findings, myelinating Schwann cells upregulate Ski during development and remyelination after injury. Myelination is blocked in myelin-competent cultures derived from
Ski-deficient animals, and genes encoding myelin components are downregulated in
Ski-deficient nerves. Conversely, overexpression of Ski in Schwann cells causes an upregulation of myelin-related genes. The myelination-regulating transcription factor Oct6 is involved in a complex modulatory relationship with Ski. We conclude that Ski is a crucial signal in Schwann cell development and myelination.
The Ski oncoprotein has been shown to bind DNA and activate transcription in conjunction with other cellular factors. Because tumor cells or myogenic cells were used for those studies, it is not ...clear that those activities of Ski are related to its transforming ability. In this study, we use a nuclear extract of c-ski-transformed cells to identify a specific DNA binding site for Ski with the consensus sequence GTCTAGAC. We demonstrate that both c-Ski and v-Ski in nuclear extracts are components of complexes that bind specifically to this site. By evaluating the features of the sequence that are critical for binding, we show that binding is cooperative. Although Ski cannot bind to this sequence on its own, we use cross-linking with ultraviolet light to show that Ski binds to this site along with several unidentified cellular proteins. Furthermore, we find that Ski represses transcription either through upstream copies of this element or when brought to the promoter by a heterologous DNA binding domain. This is the first demonstration that Ski acts as a repressor rather than an activator and could provide new insights into regulation of gene expression by Ski.
Overexpression of the
Ski
pro-oncogene has been shown to induce
myogenesis in non-muscle cells, to promote muscle hypertrophy in postnatal
mice, and to activate transcription of muscle-specific ...genes. However, the
precise role of
Ski
in muscle cell differentiation and its underlying
molecular mechanism are not fully understood. To elucidate the involvement of
Ski
in muscle terminal differentiation, two retroviral systems were
used to achieve conditional overexpression or knockdown of Ski in satellite
cell-derived C2C12 myoblasts. We found that enforced expression of Ski
promoted differentiation, whereas loss of Ski severely impaired it.
Compromised terminal differentiation in the absence of Ski was likely because
of the failure to induce myogenin (Myog) and p21 despite normal expression of
MyoD. Chromatin immunoprecipitation and transcriptional reporter experiments
showed that Ski occupied the endogenous
Myog
regulatory region and
activated transcription from the
Myog
regulatory region upon
differentiation. Transactivation of
Myog
was largely dependent on a
MEF3 site bound by Six1, not on the binding site of MyoD or MEF2. Activation
of the MEF3 site required direct interaction of Ski with Six1 and Eya3
mediated by the evolutionarily conserved Dachshund homology domain of Ski. Our
results indicate that Ski is necessary for muscle terminal differentiation and
that it exerts this role, at least in part, through its association with Six1
and Eya3 to regulate the
Myog
transcription.
The 5' end of the Rous sarcoma virus (RSV) RNA around the primer-binding site forms a series of RNA secondary stem/loop structures (U5-IR stem, TpsiC interaction region, U5-leader stem) that are ...required for efficient initiation of reverse transcription. The U5-IR stem and loop also encode the U5 integrase (IN) recognition sequence at the level of DNA such that this region has overlapping biological functions in reverse transcription and integration.
We have investigated the ability of RSV to tolerate mutations in and around the U5 IR stem and loop. Through the use of viral libraries with blocks of random sequence, we have screened for functional mutants in vivo, growing the virus libraries in turkey embryo fibroblasts. The library representing the U5-IR stem rapidly selects for clones that maintain the structure of the stem, and is subsequently overtaken by wild type sequence. In contrast, in the library representing the U5-IR loop, wild type sequence is found after five rounds of infection but it does not dominate the virus pool, indicating that the mutant sequences identified are able to replicate at or near wild type levels.
These results indicate that the region of the RNA genome in U5 adjacent to the PBS tolerates much sequence variation even though it is required for multiple biological functions in replication. The in vivo selection method utilized in this study was capable of detecting complex patterns of selection as well as identifying biologically relevant viral mutants.
Overexpression of the oncoprotein SKI correlates with the progression of human melanoma in vivo. SKI is known to curtail the growth inhibitory activity of tumor growth factor beta through the ...formation of repressive transcriptional complexes with Smad2 and Smad3 at the p21(Waf-1) promoter. Here, we show that SKI also stimulates growth by activating the Wnt signaling pathway. From a yeast two-hybrid screen and immunoprecipitation studies, we identified the protein FHL2/DRAL as a novel SKI binding partner. FHL2, a LIM-only protein, binds beta-catenin and can function as either a transcriptional repressor or activator of the Wnt signaling pathway. SKI enhanced the activation of FHL2 and/or beta-catenin- regulated gene promoters in melanoma cells. Among the SKI targets were microphthalmia-associated transcription factor and Nr-CAM, two proteins associated with melanoma cell survival, growth, motility, and transformation. Transient overexpression of SKI and FHL2 in ski(-/-) melanocytes synergistically enhanced cell growth, and stable overexpression of SKI in a poorly clonogenic human melanoma cell line was sufficient to stimulate rapid proliferation, decreasing the number of cells in the G(1) phase of the cell cycle, and dramatically increasing clonogenicity, colony size and motility. Taken together, these results suggest that by targeting members of the tumor growth factor beta and beta-catenin pathways, SKI regulates crucial events required for melanoma growth, survival, and invasion.
Experiments involving overexpression of Ski have suggested that this gene is involved in neural tube development and muscle differentiation. In agreement with these findings, Ski−/− mice display a ...cranial neural tube defect that results in exencephaly and a marked reduction in skeletal muscle mass. Here we show that the penetrance and expressivity of the phenotype changes when the null mutation is backcrossed into the C57BL6/J background, with the principal change involving a switch from a neural tube defect to midline facial clefting. Other defects, including depressed nasal bridge, eye abnormalities, skeletal muscle defects and digit abnormalities, show increased penetrance in the C57BL6/J background. These phenotypes are interesting because they resemble some of the features observed in individuals diagnosed with 1p36 deletion syndrome, a disorder caused by monosomy of the short arm of human chromosome 1p (refs. 6-9). These similarities prompted us to re-examine the chromosomal location of human SKI and to determine whether SKI is included in the deletions of 1p36. We found that human SKI is located at distal 1p36.3 and is deleted in all of the individuals tested so far who have this syndrome. Thus, SKI may contribute to some of the phenotypes common in 1p36 deletion syndrome, and particularly to facial clefting.
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