Nucleoprotein interactions governing cell type-dependent repression of the mouse smooth muscle α-actin promoter by single-stranded DNA-binding proteins Purα and Purβ

Purα and Purβ are structurally related single-stranded DNA/RNA-binding proteins implicated in the control of cell growth and differentiation. The goal of this study was to determine whether Purα and Purβ function in a redundant, distinct, or collaborative manner to suppress smooth muscle α-actin gene expression in cell types relevant to wound repair and vascular remodeling. RNA interference-mediated loss-of-function analyses revealed that, although Purβ was the dominant repressor, the combined action of endogenous Purα and Purβ was necessary to fully repress the full-length smooth muscle α-actin promoter in cultured fibroblasts but to a lesser extent in vascular smooth muscle cells. The activity of a minimal core enhancer containing a truncated 5′ Pur repressor binding site was unaffected by knockdown of Purα and/or Purβ in fibroblasts. Conversely, gain-of-function studies indicated that Purα or Purβ could each independently repress core smooth muscle α-actin enhancer activity albeit in a cell type-dependent fashion. Biochemical analyses indicated that purified recombinant Purα and Purβ were essentially identical in terms of their binding affinity and specificity for GGN repeat-containing strands of several cis-elements comprising the core enhancer. However, Purα and Purβ exhibited more distinctive protein interaction profiles when evaluated for binding to enhancer-associated transcription factors in extracts from fibroblasts and vascular smooth muscle cells. These findings support the hypothesis that Purα and Purβ repress smooth muscle α-actin gene transcription by means of DNA strand-selective cis-element binding and cell type-dependent protein-protein interactions.