Transforming Growth Factor-β/SMAD Target Gene SKIL Is Negatively Regulated by the Transcriptional Cofactor Complex SNON-SMAD4

Angeles C Tecalco-Cruz,Marcela Sosa-Garrocho,Genaro Vázquez-Victorio,Layla Ortiz-García,Elisa Domínguez-Hüttinger,Marina Macías-Silva

doi:10.1074/jbc.m112.386599

Angeles C Tecalco-Cruz, Marcela Sosa-Garrocho + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m112.386599

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Abstract

The human SKI-like (SKIL) gene encodes the SMAD transcriptional corepressor SNON that antagonizes TGF-β signaling. SNON protein levels are tightly regulated by the TGF-β pathway: whereas a short stimulation with TGF-β decreases SNON levels by its degradation via the proteasome, longer TGF-β treatment increases SNON levels by inducing SKIL gene expression. Here, we investigated the molecular mechanisms involved in the self-regulation of SKIL gene expression by SNON. Bioinformatics analysis showed that the human SKIL gene proximal promoter contains a TGF-β response element (TRE) bearing four groups of SMAD-binding elements that are also conserved in mouse. Two regions of 408 and 648 bp of the human SKIL gene (∼2.4 kb upstream of the ATG initiation codon) containing the core promoter, transcription start site, and the TRE were cloned for functional analysis. Binding of SMAD and SNON proteins to the TRE region of the SKIL gene promoter after TGF-β treatment was demonstrated by ChIP and sequential ChIP assays. Interestingly, the SNON-SMAD4 complex negatively regulated basal SKIL gene expression through binding the promoter and recruiting histone deacetylases. In response to TGF-β signal, SNON is removed from the SKIL gene promoter, and then the activated SMAD complexes bind the promoter to induce SKIL gene expression. Subsequently, the up-regulated SNON protein in complex with SMAD4 represses its own expression as part of the negative feedback loop regulating the TGF-β pathway. Accordingly, when the SNON-SMAD4 complex is absent as in some cancer cells lacking SMAD4 the regulation of some TGF-β target genes is modified.

Highlights

Human SKIL gene encodes for SNON, a negative regulator of the TGF-␤/SMAD pathway
S4 was bound to the SKIL promoter in basal conditions, and the binding was further increased by TGF-␤ stimulation (Fig. 2G). These results demonstrate that SNON and SMAD proteins bind differentially to the human SKIL gene promoter and indicate that the repression and activation of the SKIL gene promoter occur at different time points during TGF-␤ signaling
Because the response of cells to TGF-␤ depends on the cell context, we evaluated the effect of TGF-␤ on SKIL gene expression in two different cell types: A549 cells that undergo epithelial-mesenchymal transition in response to TGF-␤ and exhibit a high level of SNON protein and a low level of SKI protein and the colon cancer cell line SW480 that lacks S4, has a mesenchymal phenotype, and contains very high levels of both SNON and SKI proteins that are not down-regulated in response to TGF-␤

Summary

Introduction

Human SKIL gene encodes for SNON, a negative regulator of the TGF-␤/SMAD pathway. Significance: The formation and function of complex SNON-SMAD4 are impaired in cancer cells lacking SMAD4, which affects TGF ␤-target gene regulation. The human SKI-like (SKIL) gene encodes the SMAD transcriptional corepressor SNON that antagonizes TGF-␤ signaling. Bioinformatics analysis showed that the human SKIL gene proximal promoter contains a TGF-␤ response element (TRE) bearing four groups of SMAD-binding elements that are conserved in mouse. Binding of SMAD and SNON proteins to the TRE region of the SKIL gene promoter after TGF-␤ treatment was demonstrated by ChIP and sequential ChIP assays. The SNON-SMAD4 complex negatively regulated basal SKIL gene expression through binding the promoter and recruiting histone deacetylases. When the SNON-SMAD4 complex is absent as in some cancer cells lacking SMAD4 the regulation of some TGF-␤ target genes is modified

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