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ABSTRACT: Previous studies from our laboratories have implicated two members of the Pur family of single-stranded DNA/RNA-binding proteins, Pur alpha and Pur beta, in transcriptional repression of the smooth muscle alpha-actin gene in vascular cell types. Although Pur alpha and Pur beta share substantial sequence homology and nucleic acid binding properties, genomic promoter and cis-element occupancy studies reported herein suggest that Pur beta is the dominant factor in gene regulation. To dissect the molecular basis of Pur beta repressor activity, site-directed mutagenesis was used to map amino acids critical to the physical and functional interaction of Pur beta with the smooth muscle alpha-actin promoter. Of all the various acidic, basic, and aromatic residues studied, mutation of positionally conserved arginines in the class I or class II repeat modules significantly attenuated Pur beta repressor activity in transfected vascular smooth muscle cells and fibroblasts. DNA binding and protein-protein interaction assays were conducted with purified recombinant Pur beta and selected mutants to reveal the physical basis for loss-of-function. Mutants R57E, R57E/R96E, and R57A/R96A each exhibited reduced single-stranded DNA binding affinity for an essential promoter element and diminished interaction with corepressor YB-1/MSY1. Structural analyses of the R57A/R96A and R57E/R96E double mutants in comparison to the wild-type Pur beta homodimer revealed aberrant self-association into higher order oligomeric complexes, which correlated with decreased alpha-helical content and defective DNA and protein binding in vitro. These findings point to a previously unrecognized structural role for certain core arginine residues in forming a conformationally stable Pur beta protein capable of physical interactions necessary for smooth muscle alpha-actin gene repression.
Journal of Biological Chemistry 01/2008; 282(49):35899-909. · 4.77 Impact Factor
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ABSTRACT: Previous studies from our laboratories have implicated two members of the Pur family of single-stranded DNA/RNA-binding proteins,
Purα and Purβ, in transcriptional repression of the smooth muscle α-actin gene in vascular cell types. Although Purα and Purβ
share substantial sequence homology and nucleic acid binding properties, genomic promoter and cis-element occupancy studies reported herein suggest that Purβ is the dominant factor in gene regulation. To dissect the molecular
basis of Purβ repressor activity, site-directed mutagenesis was used to map amino acids critical to the physical and functional
interaction of Purβ with the smooth muscle α-actin promoter. Of all the various acidic, basic, and aromatic residues studied,
mutation of positionally conserved arginines in the class I or class II repeat modules significantly attenuated Purβ repressor
activity in transfected vascular smooth muscle cells and fibroblasts. DNA binding and protein-protein interaction assays were
conducted with purified recombinant Purβ and selected mutants to reveal the physical basis for loss-of-function. Mutants R57E,
R57E/R96E, and R57A/R96A each exhibited reduced single-stranded DNA binding affinity for an essential promoter element and
diminished interaction with corepressor YB-1/MSY1. Structural analyses of the R57A/R96A and R57E/R96E double mutants in comparison
to the wild-type Purβ homodimer revealed aberrant self-association into higher order oligomeric complexes, which correlated
with decreased α-helical content and defective DNA and protein binding in vitro. These findings point to a previously unrecognized structural role for certain core arginine residues in forming a conformationally
stable Purβ protein capable of physical interactions necessary for smooth muscle α-actin gene repression.
Journal of Biological Chemistry 12/2007; 282(49):35899-35909. · 4.77 Impact Factor
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ABSTRACT: Pur alpha and Pur beta are structurally related single-stranded DNA/RNA-binding proteins implicated in the control of cell growth and differentiation. The goal of this study was to determine whether Pur alpha and Pur beta function in a redundant, distinct, or collaborative manner to suppress smooth muscle alpha-actin gene expression in cell types relevant to wound repair and vascular remodeling. RNA interference-mediated loss-of-function analyses revealed that, although Pur beta was the dominant repressor, the combined action of endogenous Pur alpha and Pur beta was necessary to fully repress the full-length smooth muscle alpha-actin promoter in cultured fibroblasts but to a lesser extent in vascular smooth muscle cells. The activity of a minimal core enhancer containing a truncated 5' Pur repressor binding site was unaffected by knockdown of Pur alpha and/or Pur beta in fibroblasts. Conversely, gain-of-function studies indicated that Pur alpha or Pur beta could each independently repress core smooth muscle alpha-actin enhancer activity albeit in a cell type-dependent fashion. Biochemical analyses indicated that purified recombinant Pur alpha and Pur beta were essentially identical in terms of their binding affinity and specificity for GGN repeat-containing strands of several cis-elements comprising the core enhancer. However, Pur alpha and Pur beta exhibited more distinctive protein interaction profiles when evaluated for binding to enhancer-associated transcription factors in extracts from fibroblasts and vascular smooth muscle cells. These findings support the hypothesis that Pur alpha and Pur beta repress smooth muscle alpha-actin gene transcription by means of DNA strand-selective cis-element binding and cell type-dependent protein-protein interactions.
Journal of Biological Chemistry 04/2006; 281(12):7907-18. · 4.77 Impact Factor
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[show abstract]
[hide abstract]
ABSTRACT: Purα and Purβ are structurally related single-stranded DNA/RNA-binding proteins implicated in the control of cell growth and
differentiation. The goal of this study was to determine whether Purα and Purβ function in a redundant, distinct, or collaborative
manner to suppress smooth muscleα-actin gene expression in cell types relevant to wound repair and vascular remodeling. RNA
interference-mediated loss-of-function analyses revealed that, although Purβ was the dominant repressor, the combined action
of endogenous Purα and Purβ was necessary to fully repress the full-length smooth muscle α-actin promoter in cultured fibroblasts
but to a lesser extent in vascular smooth muscle cells. The activity of a minimal core enhancer containing a truncated 5′
Pur repressor binding site was unaffected by knockdown of Purα and/or Purβ in fibroblasts. Conversely, gain-of-function studies
indicated that Purα or Purβ could each independently repress core smooth muscle α-actin enhancer activity albeit in a cell
type-dependent fashion. Biochemical analyses indicated that purified recombinant Purα and Purβ were essentially identical
in terms of their binding affinity and specificity for GGN repeat-containing strands of several cis-elements comprising the core enhancer. However, Purα and Purβ exhibited more distinctive protein interaction profiles when
evaluated for binding to enhancer-associated transcription factors in extracts from fibroblasts and vascular smooth muscle
cells. These findings support the hypothesis that Purα and Purβ repress smooth muscle α-actin gene transcription by means
of DNA strand-selective cis-element binding and cell type-dependent protein-protein interactions.
Journal of Biological Chemistry 03/2006; 281(12):7907-7918. · 4.77 Impact Factor
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ABSTRACT: Expression of smooth muscle alpha-actin in growth factor-induced myofibroblasts and in differentiated vascular smooth muscle cells is transcriptionally controlled by multiple positive or negative trans-acting factors interacting with distinct cis-elements in the 5'-flanking region of the gene. Because none of the transcriptional regulators reported to date is smooth muscle cell- or myofibroblast-specific per se, the dynamic interplay among many factors interacting at specific sites along the promoter appears to be a signature feature of smooth muscle alpha-actin gene regulation in these cell types. Herein, the ability of the adenovirus E1A 12 S protein to bind and functionally inactivate specific cell regulatory factors has been exploited to identify several previously unknown coactivators of the mouse smooth muscle alpha-actin promoter in rodent fibroblasts and vascular smooth muscle cells. In transient cotransfection assays, ectopic expression of wild type E1A suppressed promoter activity in a dose- and cis-element-dependent manner. In asynchronous cells, N-terminal E1A mutants defective in CREB-binding protein (CBP) and p300 binding capacity exhibited markedly reduced inhibitory activity toward a smooth muscle alpha-actin promoter driven by a composite TEF-1-, SRF-, and Sp1/3-regulated enhancer. In synchronized cells, however, a more complex mutant E1A inhibitory pattern indicated that collaboration between CBP/p300 and the retinoblastoma family of pocket proteins was required to produce a fully functional enhancer. Cotransfection experiments conducted with Rb(-/-) fibroblasts demonstrated the necessity of pRB in augmenting smooth muscle alpha-actin enhancer/promoter activity. Physical interaction studies with the use of purified wild type and mutant E1A proteins confirmed that CBP, p300, and pRB were targets of E1A binding in nuclear extracts of vascular smooth muscle cells and/or fibroblasts. Collectively, these results suggest that a repertoire of E1A-interacting proteins, namely CBP/p300 and pRB, serve to integrate the activities of multiple trans-acting factors to control smooth muscle alpha-actin gene transcription in a cell type- and cell cycle-dependent manner.
Journal of Biological Chemistry 03/2005; 280(7):6204-14. · 4.77 Impact Factor
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ABSTRACT: Plasticity of smooth muscle alpha-actin gene expression in fibroblasts and vascular smooth muscle cells is mediated by opposing effects of transcriptional activators and repressors. Among these factors, three single-stranded DNA-binding proteins, Puralpha, Purbeta, and MSY1, have been implicated as coregulators of a cryptic 5'-enhancer module. In this study, a molecular analysis of Purbeta, the least well characterized member of this group, was conducted. Southwestern and Northwestern blotting of purified Purbeta deletion mutants using smooth muscle alpha-actin-derived probes mapped the minimal single-stranded DNA/RNA-binding domain to a conserved region spanning amino acids 37-263. Quantitative binding assays indicated that the relative affinity and specificity of Purbeta for single-stranded DNA were influenced by purine/pyrimidine content; by non-conserved regions outside amino acids 37-263; and by cell-derived proteins, specifically MSY1. When overexpressed in A7r5 vascular smooth muscle cells, Purbeta (but not Puralpha) inhibited transcription of a smooth muscle-specific mouse alpha-actin promoter transgene. Structural domains required for Purbeta repressor activity included the minimal DNA-binding region and a C-terminal domain required for stabilizing high affinity protein and nucleic acid interactions. Purbeta inhibitory activity in transfected A7r5 cells was potentiated by MSY1, but antagonized by serum response factor, reinforcing the idea that interplay among activators and repressors may account for phenotypic changes in smooth muscle alpha-actin-expressing cell types.
Journal of Biological Chemistry 11/2003; 278(40):38749-57. · 4.77 Impact Factor
