Transcription factor YB-1 mediates DNA polymerase alpha gene expression.
Y-box protein-1 involvement in cyclin A and B1 gene regulation has recently been demonstrated. A more generalized role of this protein for cell replication is hypothesized as numerous regulatory sequences of cell cycle-related genes contain putative binding sites. In the present study the DNA polymerase alpha (DPA) gene is identified as another YB-1-responsive gene with a Y-box and 3' inverted repeat sequence, designated DPA RE-1, in the serum-responsive promoter region. Overexpressed YB-1 concentration-dependently trans-activated DPA gene expression in reporter assays and Southwestern blotting as well as DNA binding analyses revealed binding of distinct endogenous proteins to the RE-1 with molecular sizes of 26, 32 and 52 kDa. Among these, YB-1 binding was confirmed using recombinant as well as endogenous proteins, with preferential single-stranded DNA binding. Early serum growth response in mesangial cells was accompanied by a nuclear YB-1 shift and nucleocomplex formation at the RE-1. Fine mapping of the DPA RE-1 sequence unraveled a dependence on co-factors for trans-regulation with gene activation in the context of a heterologous SV40 promoter but suppression in the context of the abbreviated homologous promoter sequence. A YB-1 knock down resulted in decreased DPA transcription rates and abrogated the serum-dependent induction of DPA transcription. These results link YB-1 with serum responsiveness of DPA gene expression and provide insight into the required sequence and protein binding context.
En-Nia
Abdelaziz
A
Department of Nephrology and Clinical Immunology, University Hospital of Aachen, 52057 Aachen, Germany.
Yilmaz
Emek
E
Klinge
Uwe
U
Lovett
David H
DH
Stefanidis
Ioannis
I
Mertens
Peter R
PR
D000111:Acetylcysteine / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D015139:Blotting, Southern; D015153:Blotting, Western; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D049109:Cell Proliferation; D047369:Chromatin Immunoprecipitation; D003593:Cytoplasm / Q000378:metabolism; D004247:DNA / Q000737:chemistry; D004256:DNA Polymerase I / Q000096:biosynthesis* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism / Q000502:physiology*; D004305:Dose-Response Relationship, Drug; D005786:Gene Expression Regulation; D015971:Gene Expression Regulation, Enzymologic; D017930:Genes, Reporter; D006801:Humans; D047468:Immunoprecipitation; D008156:Luciferases / Q000378:metabolism; D008969:Molecular Sequence Data; D009687:Nuclear Proteins; D016376:Oligonucleotides, Antisense / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D034741:RNA, Small Interfering / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry; D027602:Serum Response Element; D013539:Simian virus 40 / Q000378:metabolism; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000378:metabolism; D013997:Time Factors; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection; D051840:Y-Box-Binding Protein 1
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D016376:Oligonucleotides, Antisense; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; D051840:Y-Box-Binding Protein 1; C070856:YBX1 protein, human; C486169:Ybx1 protein, rat; D004247:DNA; D008156:Luciferases; D004256:DNA Polymerase I; D015719:Single-Strand Specific DNA and RNA Endonucleases; D000111:Acetylcysteine