Structure/Function Analysis of Mouse Pur , a Single-stranded DNA-binding Repressor of Vascular Smooth Muscle -Actin Gene Transcription*

Plasticity of smooth muscle -actin gene expression in fibroblasts and vascular smooth muscle cells is mediated by opposing effects of transcriptional activators and repressors. Among these factors, three singlestranded DNA-binding proteins, Pur , Pur , and MSY1, have been implicated as coregulators of a cryptic 5 enhancer module. In this study, a molecular analysis of Pur , the least well characterized member of this group, was conducted. Southwestern and Northwestern blotting of purified Pur deletion mutants using smooth muscle -actin-derived probes mapped the minimal single-stranded DNA/RNA-binding domain to a conserved region spanning amino acids 37–263. Quantitative binding assays indicated that the relative affinity and specificity of Pur for single-stranded DNA were influenced by purine/pyrimidine content; by non-conserved regions outside amino acids 37–263; and by cell-derived proteins, specifically MSY1. When overexpressed in A7r5 vascular smooth muscle cells, Pur (but not Pur ) inhibited transcription of a smooth muscle-specific mouse -actin promoter transgene. Structural domains required for Pur repressor activity included the minimal DNA-binding region and a C-terminal domain required for stabilizing high affinity protein and nucleic acid interactions. Pur inhibitory activity in transfected A7r5 cells was potentiated by MSY1, but antagonized by serum response factor, reinforcing the idea that interplay among activators and repressors may account for phenotypic changes in smooth muscle -actin-expressing cell types.