BIOPHYSICAL CHARACTERIZATION OF THE SEQUSINGLE-STRANDED DNA-BINDING PROPERTIES OF MOUSE PUR : A REPRESSOR OF SMOOTH MUSCLE -ACTIN GENE EXPRESSION

Regulation of gene transcription by structural interconve rsions of genomic DNA is an emerging biochemical and genetic paradigm that adds to the already diverse repertoire of eukaryotic gene regulatory mechanisms. The appearance of paranemic structures coincident with changes in gene activity, as well as part icipation of transcription factors that recognize and bind single-stranded DNA at numerous gene promoters in vivo illustrates the authenticity of this concept and its impo rtance in cellular homeostasis. Despite its acceptance, this concept has been minimally described at the biochemical and biophysical levels, as the means by which sequence-specific single-stranded DNAbinding proteins exert transcriptional influence in double-strande d g nomes remains largely undefined. Purβ is a sequence-specific single-stranded DNA/RNA-binding prot ein that acts as a repressor of smooth muscle α-actin (SMαA) gene transcription, and mRNA translation. SMαA is an important cytoskeletal protein that contributes contractile, antimigratory, and nonproliferative functions in smooth muscle. In conce rt with Pur protein family member Purα, and Y-box protein MSY1, Pur β enacts repression of SM αA gene expression by interacting with a cryptic cis-regulatory element in the 5’ region of the SM αA promoter that has been shown to transiently adopt single-strande d conformations in vivo, and to confer transcriptional activation when trans-activator occupied while in a doublestranded conformation. Downregulation of SM αA gene expression has been identified to be a contributing factor to cardiovascular disease progre ssion; therefore a thorough understanding of SMαA repression mechanisms is critical for clinical mana gement of these conditions. Although highly homologous at the primary sequence level, P urβ and Pur α display significant conserved regions of sequence divergence that suggest these paralogs exert distinct cellular functions in various vertebrate class es. A goal of the studies presented herein was to delineate exhibited functional differences w ith respect to SMαA repression in pertinent mouse cell lines. Loss-of-function and chro matin immunoprecipitation studies verified that Pur β differs from Pur α in that Pur β is the dominant Pur protein repressor of SMαA expression in embryonic fibroblasts and vascular smoot h muscle cells, although by different, cell type-specific mechani sms. Biophysical assessment of Pur β single-stranded DNA binding properties showed that despite the ability of Pur β to self-dimerize in the absence of nucleic acid, Pur β binds to the cryptic SMαA enhancer by a sequential and cooperative mechanism, with remarkable affinity and a terminal stoichiometry of 2 to 1. Footprin ting and in vitro binding site characterization confirms two Pur β binding sites exist within this element and display slight degeneracy from a proposed Pur protein-binding consensus motif. These findings delineate binding mechanisms adopted by Pur β and provide a means to identify putative Purβ binding sites throughout the genome.