Nucleoprotein Interactions Governing Cell Type-dependent Repression of the Mouse Smooth Muscle Alpha-actin Promoter by Single-stranded Dna-binding Proteins Pur Alpha and Pur Beta.
From: Department of Biochemistry, Cardiovascular Research Institute, University of Vermont College of Medicine, Burlington, Vermont 05405, USA.
The Journal of biological chemistry
- Publish Date: Mar 2006
- ISSN: 0021-9258
- Volume: 281
- Issue: 12
- Pages: 7907-18
- Medium: Print
- Language: English
- Citation (JAMA): Knapp Anna M, Ramsey Jon E, Wang Shu-Xia, et al. Nucleoprotein Interactions Governing Cell Type-dependent Repression of the Mouse Smooth Muscle Alpha-actin Promoter by Single-stranded Dna-binding Proteins Pur Alpha and Pur Beta.. J. Biol. Chem. Mar 2006;281:7907-18
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.
Mesh Headings (Keywords): Actins, Animals, Binding, Competitive, Biotinylation, Blotting, Western, DNA, DNA, Single-Stranded, DNA-Binding Proteins, Dose-Response Relationship, Drug, Enhancer Elements (Genetics), Enzyme-Linked Immunosorbent Assay, Epitopes, Fibroblasts, Genes, Reporter, Genetic Vectors, Mice, Mice, Inbred C57BL, Myocytes, Smooth Muscle, Nerve Tissue Proteins, Nucleoproteins, Plasmids, Promoter Regions (Genetics), Protein Binding, RNA, RNA Interference, Transcription Factors, Transcription, Genetic, Transgenes
Check for Full Text / PubMed Unique Identifier (PMID): 16436378
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