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.
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.
Knapp
Anna M
AM
Department of Biochemistry, Cardiovascular Research Institute, University of Vermont College of Medicine, Burlington, Vermont 05405, USA.
Ramsey
Jon E
JE
Wang
Shu-Xia
SX
Godburn
Karolyn E
KE
Strauch
Arthur R
AR
Kelm
Robert J
RJ
D000199:Actins / Q000378:metabolism*; D000818:Animals; D001667:Binding, Competitive; D019921:Biotinylation; D015153:Blotting, Western; D004247:DNA / Q000737:chemistry; D004277:DNA, Single-Stranded / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000737:chemistry*; D004305:Dose-Response Relationship, Drug; D004742:Enhancer Elements, Genetic; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000737:chemistry; D005347:Fibroblasts / Q000378:metabolism; D017930:Genes, Reporter; D005822:Genetic Vectors; D051379:Mice; D008810:Mice, Inbred C57BL; D032389:Myocytes, Smooth Muscle / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000737:chemistry*; D009698:Nucleoproteins / Q000737:chemistry*; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012313:RNA / Q000737:chemistry; D034622:RNA Interference; D014157:Transcription Factors / Q000737:chemistry; D014158:Transcription, Genetic; D019076:Transgenes
D000199:Actins; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D000939:Epitopes; D009419:Nerve Tissue Proteins; D009698:Nucleoproteins; C121485:Pura protein, mouse; C108770:Purb protein, mouse; D014157:Transcription Factors; D012313:RNA; D004247:DNA