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 α-actin (SMαA) is a biomarker of myofibroblast differentiation. Expression of the SMαA gene, ACTA2, is regulated by cis-acting elements and transcription factors that activate or repress the ACTA2 promoter.
Purine-rich element binding proteins A (Purα) and B (Purβ) 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 Purα, Purβ 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 Purβ reversibly self-associates in solution to form a homodimer. Quantitative DNA-binding assays revealed that Purβ interacts with the purine-rich strand of the ACTA2 MCAT motif via a cooperative, multisite binding mechanism to form a high-affinity 2:1 Purβ-ssDNA complex.
In this dissertation, a combination of computational, biochemical, and cell-based approaches were employed to elucidate the molecular basis of Purβ repressor interaction with the ACTA2 gene. Limited proteolysis of recombinant mouse Purβ 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 Purβ in mouse embryonic fibroblasts via RNA interference induced SMαA expression and conversion to a myofibroblast-like phenotype. To map the specific structural domains in the core region of Purβ that account for its unique ACTA2 repressor and ssDNA-binding functions, computational homology models of the Purβ monomer and dimer were generated based on the x-ray crystal structure of an intramolecular subdomain of Drosophila melanogaster Purα. Empirical biochemical and cell-based analyses of rationally-designed Purβ truncation proteins revealed that the assembled Purβ 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 Purβ 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 Purβ. Collectively, the findings described herein establish the structural and chemical basis for the cooperative interaction of Purβ with the ACTA2 MCAT enhancer and for Purβ-dependent suppression of myofibroblast differentiation.
| Identifer | oai:union.ndltd.org:uvm.edu/oai:scholarworks.uvm.edu:graddis-1276 |
| Date | 01 January 2014 |
| Creators | Rumora, Amy |
| Publisher | ScholarWorks @ UVM |
| Source Sets | University of Vermont |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Graduate College Dissertations and Theses |
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