Nucleoprotein interactions governing cell type-dependent repression of the mouse smooth muscle alpha-actin promoter by single-stranded DNA-binding proteins Pur alpha and Pur beta

Pur alpha and Pur beta 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 Pur alpha and Pur beta function in a redundant, distinct, or collaborative manner to suppress smooth muscle alpha-actin gene expression in cell types relevant to wound repair and vascular remodeling. RNA interference-mediated loss-of-function analyses revealed that, although Pur beta was the dominant repressor, the combined action of endogenous Pur alpha and Pur beta was necessary to fully repress the full-length smooth muscle alpha-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 Pur alpha and/or Pur beta in fibroblasts. Conversely, gain-of-function studies indicated that Pur alpha or Pur beta could each independently repress core smooth muscle alpha-actin enhancer activity albeit in a cell type-dependent fashion. Biochemical analyses indicated that purified recombinant Pur alpha and Pur beta 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, Pur alpha and Pur beta 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 Pur alpha and Pur beta repress smooth muscle alpha-actin gene transcription by means of DNA strand-selective cis-element binding and cell type-dependent protein-protein interactions.