Date of Award


Document Type


Degree Name

Doctor of Philosophy (PhD)



First Advisor

Robert J. Kelm

Second Advisor

Sylvie Doublie


A number of physiologic processes require the expression of smooth muscle alpha actin (SMαA) to mediate cellular contraction. Stable expression of SMαA 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 SMαA in myofibroblasts during wound repair facilitates wound closure. Hence, it is no surprise that dysregulation of SMαA gene expression in both cell types can have pathological consequences. Indeed, aberrant SMαA gene regulation has been implicated in diseases such as atherosclerosis and fibrosis. Therefore, a better understanding of the molecular mechanisms that regulate SMαA 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 SMαA gene is regulated by a combination of transcriptional activators, repressors, and cofactors. Two members of the purine-rich element binding protein family known as Purα and Purβ have been implicated in the repression of SMαA gene expression. Both proteins bind to single-stranded, purine-rich DNA sequences in the SMαA gene promoter with high affinity and specificity. However, published loss-of-function and gain-of-function analyses suggest that Purβ 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 Purβ represses the SMαA 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 SMαA gene repressor activity of Purβ.

In this dissertation, experimental findings are presented indicating that Purβ 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 SMαA 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 Purβ. In addition, biochemical characterization of rare single nucleotide polymorphism-encoded variants of Purβ 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 Purβ 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 Purβ to repress myocardin cofactor function. Collectively, these studies demonstrate that the third repeat/dimerization domain of Purβ is essential for full repression of the SMαA gene as specific amino acid residues in this region mediate both protein-DNA and protein-protein interactions.



Number of Pages

291 p.

Included in

Biochemistry Commons