Dissection of the SSeCKS metastasis suppressor gene promoters: Identification of v-Src responsive elements
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SSeCKS ( S rc- S uppressed C k inase S ubstrate), also called gravin/AKAP12, is a large scaffolding protein with metastasis suppressor activity. Two major isoforms of SSeCKS are expressed in most cell and tissue types under the control of two independent promoters, designated α and β, separated by 68 Kb (a third isform, γ, is driven by a testes-specific promoter). SSeCKS transcript and protein levels are severely decreased in Src- and Ras-transformed fibroblasts and in many epithelial tumors. By dissecting its promoters with progressive deletion analysis we identify the sequence between -106 and -43 in the α proximal promoter as the minimal v-Src-responsive element (VSRE), which contains E- and GC-boxes bound by the USF1 and Sp1/Sp3 transcription factors, respectively. Mutation analyses reveal that both E- and GC-boxes are crucial for v-Src-responsive (VSR) and basal promoter activities. v-Src does not alter USF1 binding levels at the E-box, but it increases Sp1/Sp3 binding to the GC-box despite no change in their cellular protein abundance and nuclear translocation. Overexpression of Sp3 together with Sp1 can antagonize the Sp1 mediated transactivation of the α proximal promoter in v-Src/3T3 cells but not in untransformed NIH3T3 cells. SSeCKS α and β transcript levels in v-Src/3T3 cells can be restored by treatment with the histone deacetylase inhibitor, Trichostatin A (TSA), but not with the DNA demethylation agent, 5-aza-cytidine. Histone hypoacetylations are found only on the α, but not β, proximal promoter in v-Src/3T3 cells. Recruitment of HDAC1 is necessary and sufficient to cause repression of α proximal promoter activity, and the addition of Sp1 and/or Sp3 potentiates the repression. Taken together, our data indicate that the v-Src mediated repression of SSeCKS α transcription is controlled by dynamic changes in USF1/Sp1/Sp3-associated complexes and by increased histone deacetylation at the proximal promoter, and suggest that the repression of the β transcription in v-Src/3T3 cells is likely coordinately regulated through distal changes in the chromatinization of the α promoter.