SSeCKS (Src-Suppressed C kinase Substrate), 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 alpha and beta, separated by 68 Kb (a third isform, gamma, 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 alpha 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 alpha proximal promoter in v-Src/3T3 cells but not in untransformed NIH3T3 cells. SSeCKS alpha and beta 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 alpha, but not beta, proximal promoter in v-Src/3T3 cells. Recruitment of HDAC1 is necessary and sufficient to cause repression of alpha 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 alpha 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 beta transcription in v-Src/3T3 cells is likely coordinately regulated through distal changes in the chromatinization of the alpha promoter.... for 30 min, and then incubated with diluted anti-Sp1 (goat) and anti-Sp3 (rabbit ) antibodies (1:500 in 3% BSA) for 1 hour at room ... Total RNA from NIH3T3 cells was extracted using Trizol (Invitrogen) following the manufacturera#39;s instructions.
|Title||:||Dissection of the SSeCKS Metastasis Suppressor Gene Promoters: Identification of V-Src Responsive Elements|
|Publisher||:||ProQuest - 2007|