Online

Rumora, Amy

Biochemistry

2014

PhD

The trans-differentiation of injury-activated fibroblasts to myofibroblasts is a process that provides contractile strength for wound closure. Persistent myofibroblast differentiation, however, is associated with fibrotic pathologies such as organ fibrosis, vascular remodeling, and atherosclerotic plaque formation. Myofibroblasts acquire a contractile phenotype with biochemical properties characteristic of both smooth muscle cells and stromal fibroblasts. The cyto-contractile protein, smooth muscle alpha-actin (SM[alpha]A) is a biomarker of myofibroblast differentiation. Expression of the SM[alpha]A gene, ACTA2, is regulated by cis-acting elements and transcription factors that activate or repress the ACTA2 promoter. Purine-rich element binding proteins A (Pur[alpha]) and B (Pur[beta]) are sequence-specific, single-stranded DNA (ssDNA)/RNA-binding proteins that act as transcriptional repressors of ACTA2 expression. Both Pur proteins interact with the purine-rich strand of a cryptic muscle-CAT (MCAT) enhancer motif in 5'-flanking region of the ACTA2 promoter. Despite significant sequence homology with Pur[alpha], Pur[beta] was identified as the dominant repressor of ACTA2 expression in mouse embryonic fibroblasts and vascular smooth muscle cells by virtue of gain-of function and loss-of-function analyses in cultured cells. Biophysical studies indicated that Pur[beta] reversibly self-associates in solution to form a homodimer. Quantitative DNA-binding assays revealed that Pur[beta] interacts with the purine-rich strand of the ACTA2 MCAT motif via a cooperative, multisite binding mechanism to form a high-affinity 2:1 Pur[beta]-ssDNA complex. In this dissertation, a combination of computational, biochemical, and cell-based approaches were employed to elucidate the molecular basis of Pur[beta] repressor interaction with the ACTA2 gene. Limited proteolysis of recombinant mouse Pur[beta] in the presence and absence of the purine-rich strand of the ACTA2 MCAT element led to the identification of a core ssDNA-binding region that retains the ability to dimerize in solution. Knockdown of endogenous Pur[beta] in mouse embryonic fibroblasts via RNA interference induced SM[alpha]A expression and conversion to a myofibroblast-like phenotype. To map the specific structural domains in the core region of Pur[beta] that account for its unique ACTA2 repressor and ssDNA-binding functions, computational homology models of the Pur[beta] monomer and dimer were generated based on the x-ray crystal structure of an intramolecular subdomain of Drosophila melanogaster Pur[alpha]. Empirical biochemical and cell-based analyses of rationally-designed Pur[beta] truncation proteins revealed that the assembled Pur[beta] homodimer is composed of three separate purine-rich ssDNA-binding subdomains. Evaluation of the effects of anionic detergent and high-salt on the binding of Pur[beta] to ssDNA implicated the involvement of hydrophobic and electrostatic interactions in mediating high-affinity nucleoprotein complex formation. This inference was validated by site-directed mutagenesis experiments, which identified several basic amino acid residues required for the ACTA2 repressor activity of Pur[beta]. Collectively, the findings described herein establish the structural and chemical basis for the cooperative interaction of Pur[beta] with the ACTA2 MCAT enhancer and for Pur[beta]-dependent suppression of myofibroblast differentiation.