Transcriptional Repression by v-Ski and c-Ski Mediated by a Specific DNA Binding Site

Abstract

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.

Transcriptional Control by the SMADs

“…SKI and SKIL bind SMAD4 and SMAD2/3 (Luo 2004), and, strikingly, both SKI and SKIL are rapidly degraded on TGF-b stimulation (Stroschein et al 1999;Sun et al 1999). SKI and SKIL were shown by DNA-binding site selection to bind to GTCTAGAC elements (Nicol and Stavnezer 1998), which were subsequently identified as the SBEs described above. The relevance of this became clear when SMAD4 and SKIL were shown to form a complex on repeated SBEs in the absence of signal, which functions to keep target genes repressed (Stroschein et al 1999), as a result of recruitment of corepressors such as NCOR (formerly N-CoR) or SIN3A (Luo 2004).…”

Cloning and functional characterization of a new Ski homolog, Fussel-18, specifically expressed in neuronal tissues

“…[9][10][11][12] A 8-bp palindromic sequence (GTCTAGAC) was initially identified to be a high-affinity binding site for Ski. 13 However, Ski cannot bind to this sequence directly. Instead, Ski binds to this site along with Smad3 and Smad4 13 leading to the hypothesis that Ski may bind to the GTCTAGAC sequence indirectly through interaction with Smad4 or Smad3.…”

“…13 However, Ski cannot bind to this sequence directly. Instead, Ski binds to this site along with Smad3 and Smad4 13 leading to the hypothesis that Ski may bind to the GTCTAGAC sequence indirectly through interaction with Smad4 or Smad3. Ski and SnoN interaction with Smad4 is constitutive, whereas Ski and SnoN interact with Smad2 and Smad3 in a TGF-b-dependent manner.…”

Ski acts as a co-repressor with Smad2 and Smad3 to regulate the response to type β transforming growth factor

“…c-Ski and v-Ski do not bind DNA on their own, but we have shown that they participate in a multiprotein complex that binds specifically to the consensus element, GTCTAGAC (5). Binding to this element mediates transcriptional repression by Ski, and these activities are tightly linked to Ski's ability to transform cells and induce muscle differentiation (6).…”

“…T he products of the cellular and retroviral ski genes are nuclear proteins that either activate or repress transcription, depending on the cellular and promoter context (1)(2)(3)(4)(5)(6). The v-Ski oncoprotein of the SKV retrovirus (7) is truncated by 312 amino acids relative to its cellular homolog, c-Ski (8), but this truncation per se is not oncogenic.…”