Online

Ferris, Lauren

Cellular, Molecular, and Biomedical Sciences Graduate Program

2018

Ph. D.

A number of physiologic processes require the expression of smooth muscle alpha actin (SM[alpha]A) to mediate cellular contraction. Stable expression of SM[alpha]A in differentiated vascular smooth muscle cells is associated with a contractile phenotype that is essential for regulation of blood flow and pressure. The transient expression of SM[alpha]A in myofibroblasts during wound repair facilitates wound closure. Hence, it is no surprise that dysregulation of SM[alpha]A gene expression in both cell types can have pathological consequences. Indeed, aberrant SM[alpha]A gene regulation has been implicated in diseases such as atherosclerosis and fibrosis. Therefore, a better understanding of the molecular mechanisms that regulate SM[alpha]A gene expression is necessary to uncover the factors involved in modulating the phenotype of vascular smooth muscle cells and fibroblasts in diseases affecting blood vessels and connective tissue. Previous studies have shown that the SM[alpha]A gene is regulated by a combination of transcriptional activators, repressors, and cofactors. Two members of the purine-rich element binding protein family known as Pur[alpha] and Pur[Beta] have been implicated in the repression of SM[alpha]A gene expression. Both proteins bind to single-stranded, purine-rich DNA sequences in the SM[alpha]A gene promoter with high affinity and specificity. However, published loss-of-function and gain-of-function analyses suggest that Pur[Beta] is the dominant repressor in vascular smooth muscle cells and fibroblasts. Thus, the principal objective of this dissertation project was to define the specific molecular mechanism(s) by which Pur[Beta] represses the SM[alpha]A gene. This undertaking was made possible by the prior identification of a functional core region in the center of the protein containing three regions of internal homology termed repeats I, II, and III. Amino terminal repeats I and II form an intramolecular DNA-binding domain, while two carboxy terminal repeats III form an intermolecular dimerization domain. Further analysis revealed that the dimerization domain is also capable of interacting with purine-rich single-stranded DNA and is absolutely necessary for the full SM[alpha]A gene repressor activity of Pur[Beta]. In this dissertation, experimental findings are presented indicating that Pur[Beta] binding to single-stranded DNA is mediated by both ionic and hydrophobic interactions. Site-directed mutation of specific positively-charged amino acid residues in each of the three repeats resulted in reduced repression of the SM[alpha]A gene promoter owing to diminished DNA binding affinity. Mutation of a positionally-conserved arginine residue in the third repeat had the most significant effect on the function of Pur[Beta]. In addition, biochemical characterization of rare single nucleotide polymorphism-encoded variants of Pur[Beta] revealed that other amino acid changes in the third repeat affect protein-protein interaction, but not DNA-binding activity. Lastly, evidence is shown indicating that Pur[Beta] inhibits the potent smooth muscle-restricted co-activator myocardin via a novel protein-protein interaction based mechanism. Interestingly, specific point mutations and variations in the third repeat impair the ability of Pur[Beta] to repress myocardin cofactor function. Collectively, these studies demonstrate that the third repeat/dimerization domain of Pur[Beta] is essential for full repression of the SM[alpha]A gene as specific amino acid residues in this region mediate both protein-DNA and protein-protein interactions.