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J. Biol. Chem., Vol. 281, Issue 12, 7907-7918, March 24, 2006
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Nucleoprotein Interactions Governing Cell Type-dependent Repression of the Mouse Smooth Muscle 
-Actin Promoter by Single-stranded DNA-binding Proteins Pur and Pur
*
12
3
From the
Departments of Biochemistry and Medicine, Cardiovascular Research Institute, University of Vermont College of Medicine, Burlington, Vermont 05405 and the ¶Department of Physiology and Cell Biology, Dorothy M. Davis Heart and Lung Research Institute, Ohio State University College of Medicine and Public Health, Columbus, Ohio 43210
Pur
and Pur
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 and Pur function in a redundant, distinct, or collaborative manner to suppress smooth muscle-actin gene expression in cell types relevant to wound repair and vascular remodeling. RNA interference-mediated loss-of-function analyses revealed that, although Pur was the dominant repressor, the combined action of endogenous Pur and Pur was necessary to fully repress the full-length smooth muscle -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 and/or Pur in fibroblasts. Conversely, gain-of-function studies indicated that Pur or Pur could each independently repress core smooth muscle -actin enhancer activity albeit in a cell type-dependent fashion. Biochemical analyses indicated that purified recombinant Pur and Pur 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 and Pur 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 and Pur repress smooth muscle -actin gene transcription by means of DNA strand-selective cis-element binding and cell type-dependent protein-protein interactions.
Received for publication, September 1, 2005 , and in revised form, January 5, 2006.
* This work was supported in part by Grant HL 54281(to R. J. K.) from NHLBI, National Institutes of Health (NIH). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The on-line version of this article (available at http://www.jbc.org) contains supplemental "Methods," "Results and Discussion," references, and Figs. S1–S5.
1 Supported by an NIH Training Grant HL07594.
2 Supported by American Heart Association predoctoral fellowship Grant 0515620T.
3 To whom correspondence should be addressed: UVM Colchester Research Facility, 208 South Park Dr., Colchester, VT 05446. Tel.: 802-656-0329; Fax: 802-656-8969; E-mail: Robert.Kelm{at}uvm.edu.
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