Abstract
Single-strand DNA-binding proteins, Puralpha and Purbeta, play a role in cell growth and differentiation by modulating both transcriptional and translational controls of gene expression. We have previously characterized binding of Puralpha and Purbeta proteins to a purine-rich negative regulatory (PNR) element of the rat cardiac alpha-myosin heavy chain (MHC) gene that controls cardiac muscle specificity. In this study we investigated the role of upstream sequences of the alpha-MHC promoter in Purbeta-mediated gene repression. In the transient transfection analysis overexpression of Purbeta revealed a negative regulatory effect on serum response factor (SRF)-dependent alpha-MHC and alpha-skeletal actin expression in muscle cell background. Contrary, in nonmuscle cells, Purbeta showed no repressive effect. The results obtained from gel-shift assays demonstrated a sequence specific competitive binding of Purbeta to the minus strand of the SRF-binding, CArG box sequences of different muscle genes, but not to the SRF-binding, SRE sequences of the c-fos gene. These element-specific associations of Purbeta with muscle CArG boxes may, in part, explain why muscle gene expression is downregulated in disease states in which Purbeta levels are elevated. This data also provide a mechanistic distinction between muscle CArG boxes and nonmuscle serum response element (SRE) sequences in terms of their affinity to bind to SRF and their ability to regulate cell-specific gene expression.
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