Electrostatic and Hydrophobic Interactions Mediate Single-Stranded DNA Recognition and <i>Acta2</i> Repression by Purine-Rich Element-Binding Protein B

Myofibroblast differentiation is characterized by an increased level of expression of cytoskeletal smooth muscle α-actin. In human and murine fibroblasts, the gene encoding smooth muscle α-actin (<i>Acta2</i>) is tightly regulated by a network of transcription factors that either activate or repress the 5′ promoter–enhancer in response to environmental cues signaling tissue repair and remodeling. Purine-rich element-binding protein B (Purβ) suppresses the expression of <i>Acta2</i> by cooperatively interacting with the sense strand of a 5′ polypurine sequence containing an inverted MCAT <i>cis</i> element required for gene activation. In this study, we evaluated the chemical basis of nucleoprotein complex formation between the Purβ repressor and the purine-rich strand of the MCAT element in the mouse <i>Acta2</i> promoter. Quantitative single-stranded DNA (ssDNA) binding assays conducted in the presence of increasing concentrations of monovalent salt or anionic detergent suggested that the assembly of a high-affinity nucleoprotein complex is driven by a combination of electrostatic and hydrophobic interactions. Consistent with the results of pH titration analysis, site-directed mutagenesis revealed several basic amino acid residues in the intermolecular (R267) and intramolecular (K82 and R159) subdomains that are essential for Purβ transcriptional repressor function in <i>Acta2</i> promoter–reporter assays. In keeping with their diminished <i>Acta2</i> repressor activity in fibroblasts, purified Purβ variants containing an R267A mutation exhibited reduced binding affinity for purine-rich ssDNA. Moreover, certain double and triple-point mutants were also defective in binding to the <i>Acta2</i> corepressor protein, Y-box-binding protein 1. Collectively, these findings establish the repertoire of noncovalent interactions that account for the unique structural and functional properties of Purβ