v-Src-mediated down-regulation of SSeCKS metastasis suppressor gene promoter by the recruitment of HDAC1 into a USF1-Sp1-Sp3 complex

SSeCKS ( (S) under bar -rc (s) under bar uppr (e) under bar ssed (C) under bar (k) under bar inase (s) under bar 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 alpha and beta, separated by 68 kb. 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 identified the sequence between -106 and -49 in the alpha proximal promoter as the minimal v-Src-responsive element, which contains E- and GC-boxes bound by USF1 and Sp1/Sp3, respectively. Both E- and GC-boxes are crucial for v-Src-responsive 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. SSeCKS alpha and beta transcript levels in v-Src/3T3 cells can be restored by treatment with the histone deacetylase inhibitor, trichostatin A, but not with the DNA demethylation agent, 5-azacytidine. Chromatin changes are found only on the alpha promoter even though the beta proximal promoter contains a similar E- and GC-box arrangement. 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. Our data suggest that suppression of the beta promoter is facilitated by Src- induced changes in the alpha promoter chromatinization mediated by a USF1-Sp1-Sp3 complex.