Puralpha activates PDGF-A gene transcription via interactions with a G-rich, single-stranded region of the promoter.
Transcription of the PDGF-A chain gene is regulated by multiple promoter and silencer elements that are GC-rich and exhibit considerable single-stranded character. In this study, the 42 kDa single-stranded DNA and RNA binding protein, Puralpha, was investigated with respect to its ability to bind and interact functionally with single-stranded DNA elements in the PDGF-A gene. Recombinant GST-Puralpha bound with high affinity and sequence-specificity to the G-rich strands of two such transcriptional control elements, the 5'-S1 nuclease-hypersensitive silencer (5'SHS; -1418 to -1388) and the nuclease-hypersensitive element (NHE; -92 to -48). Ethylation interference footprinting localized binding of Puralpha to a region between nucleotides -91 and -77 within the NHE element, which contains binding sites for the double-stranded DNA-binding transcription factors Sp1, EGR-1 and WT1. Forced expression of Puralpha upregulated transcriptional activity of the PDGF-A promoter but not the 5'SHS silencer in HepG2 cells, demonstrating Puralpha has the potential to activate PDGF-A gene expression. Targeted disruption of the Puralpha gene reduced NHE activity and PDGF-A mRNA expression in mouse embryo fibroblasts, consistent with a physiological role for Puralpha in maintaining optimal transcription of the PDGF-A gene. These results indicate Puralpha enhances transcription of the PDGF-A gene through its interactions with single-stranded, G-rich strands in the promoter, perhaps by stabilizing non-B-form DNA conformations.
Zhang
Qingbei
Q
Department of Molecular and Biomedical Pharmacology, University of Kentucky Medical Center, Lexington, KY 40536, USA.
Pedigo
Nancy
N
Shenoy
Satyendra
S
Khalili
Kamel
K
Kaetzel
David M
DM
D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D045744:Cell Line, Tumor; D017362:Cyclic AMP Response Element-Binding Protein / Q000235:genetics / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation; D005838:Genotype; D006147:Guanine / Q000378:metabolism; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D008954:Models, Biological; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D010982:Platelet-Derived Growth Factor / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014157:Transcription Factors; D014158:Transcription, Genetic / Q000235:genetics; D015854:Up-Regulation / Q000235:genetics
D017362:Cyclic AMP Response Element-Binding Protein; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D009419:Nerve Tissue Proteins; C501130:PURA protein, human; D010982:Platelet-Derived Growth Factor; C121485:Pura protein, mouse; D012333:RNA, Messenger; D014157:Transcription Factors; C075278:platelet-derived growth factor A; D006147:Guanine