title: p53 Specifically Binds Triplex DNA In Vitro and in Cells.
abstract: Triplex DNA is implicated in a wide range of biological activities, including regulation of gene expression and genomic instability leading to cancer. The tumor suppressor p53 is a central regulator of cell fate in response to different type of insults. Sequence and structure specific modes of DNA recognition are core attributes of the p53 protein. The focus of this work is the structure-specific binding of p53 to DNA containing triplex-forming sequences in vitro and in cells and the effect on p53-driven transcription. This is the first DNA binding study of full-length p53 and its deletion variants to both intermolecular and intramolecular T.A.T triplexes. We demonstrate that the interaction of p53 with intermolecular T.A.T triplex is comparable to the recognition of CTG-hairpin non-B DNA structure. Using deletion mutants we determined the C-terminal DNA binding domain of p53 to be crucial for triplex recognition. Furthermore, strong p53 recognition of intramolecular T.A.T triplexes (H-DNA), stabilized by negative superhelicity in plasmid DNA, was detected by competition and immunoprecipitation experiments, and visualized by AFM. Moreover, chromatin immunoprecipitation revealed p53 binding T.A.T forming sequence in vivo. Enhanced reporter transactivation by p53 on insertion of triplex forming sequence into plasmid with p53 consensus sequence was observed by luciferase reporter assays. In-silico scan of human regulatory regions for the simultaneous presence of both consensus sequence and T.A.T motifs identified a set of candidate p53 target genes and p53-dependent activation of several of them (ABCG5, ENOX1, INSR, MCC, NFAT5) was confirmed by RT-qPCR. Our results show that T.A.T triplex comprises a new class of p53 binding sites targeted by p53 in a DNA structure-dependent mode in vitro and in cells. The contribution of p53 DNA structure-dependent binding to the regulation of transcription is discussed.
authors.affiliation: Department of Biophysical Chemistry and Molecular Oncology, Institute of Biophysics, Academy of Sciences of the Czech Republic v.v.i., Brno, Czech Republic.
authors.forename: Marie
authors.initials: M
authors.lastname: Brázdová
authors.affiliation: Department of Biophysical Chemistry and Molecular Oncology, Institute of Biophysics, Academy of Sciences of the Czech Republic v.v.i., Brno, Czech Republic.
authors.forename: Vlastimil
authors.initials: V
authors.lastname: Tichý
authors.affiliation: Department of Biophysical Chemistry and Molecular Oncology, Institute of Biophysics, Academy of Sciences of the Czech Republic v.v.i., Brno, Czech Republic.
authors.forename: Robert
authors.initials: R
authors.lastname: Helma
authors.affiliation: Department of Biophysical Chemistry and Molecular Oncology, Institute of Biophysics, Academy of Sciences of the Czech Republic v.v.i., Brno, Czech Republic.
authors.forename: Pavla
authors.initials: P
authors.lastname: Bažantová
authors.affiliation: Department of Biophysical Chemistry and Molecular Oncology, Institute of Biophysics, Academy of Sciences of the Czech Republic v.v.i., Brno, Czech Republic.
authors.forename: Alena
authors.initials: A
authors.lastname: Polášková
authors.affiliation: Department of Molecular Biology and Pharmaceutical Biotechnology, Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences Brno, Brno, Czech Republic.
authors.forename: Aneta
authors.initials: A
authors.lastname: Krejčí
authors.affiliation: Department of Biophysical Chemistry and Molecular Oncology, Institute of Biophysics, Academy of Sciences of the Czech Republic v.v.i., Brno, Czech Republic.
authors.forename: Marek
authors.initials: M
authors.lastname: Petr
authors.affiliation: Department of Biophysical Chemistry and Molecular Oncology, Institute of Biophysics, Academy of Sciences of the Czech Republic v.v.i., Brno, Czech Republic.
authors.forename: Lucie
authors.initials: L
authors.lastname: Navrátilová
authors.affiliation: Department of Biophysical Chemistry and Molecular Oncology, Institute of Biophysics, Academy of Sciences of the Czech Republic v.v.i., Brno, Czech Republic.
authors.forename: Olga
authors.initials: O
authors.lastname: Tichá
authors.affiliation: Department of Biophysical Chemistry and Molecular Oncology, Institute of Biophysics, Academy of Sciences of the Czech Republic v.v.i., Brno, Czech Republic.
authors.forename: Karel
authors.initials: K
authors.lastname: Nejedlý
authors.affiliation: Biophysical Engineering Group, Faculty of Science and Technology, University of Twente, Enschede, The Netherlands.
authors.forename: Martin L
authors.initials: ML
authors.lastname: Bennink
authors.affiliation: Biophysical Engineering Group, Faculty of Science and Technology, University of Twente, Enschede, The Netherlands.
authors.forename: Vinod
authors.initials: V
authors.lastname: Subramaniam
authors.affiliation: Department of Molecular Biology and Pharmaceutical Biotechnology, Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences Brno, Brno, Czech Republic.
authors.forename: Zuzana
authors.initials: Z
authors.lastname: Bábková
authors.affiliation: Department of Computer Systems, Faculty of Information Technology, Brno University of Technology, Brno, Czech Republic.
authors.forename: Tomáš
authors.initials: T
authors.lastname: Martínek
authors.affiliation: Department of Information Technologies, Faculty of Informatics, Masaryk University, Brno, Czech Republic.
authors.forename: Matej
authors.initials: M
authors.lastname: Lexa
authors.affiliation: Department of Biophysical Chemistry and Molecular Oncology, Institute of Biophysics, Academy of Sciences of the Czech Republic v.v.i., Brno, Czech Republic.
authors.forename: Matej
authors.initials: M
authors.lastname: Adámik
mesh_terms: D001665:Binding Sites; D004247:DNA / Q000737:chemistry / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics*; D006801:Humans; D009690:Nucleic Acid Conformation; D059372:Nucleotide Motifs / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics; D017384:Sequence Deletion / Q000235:genetics; D015533:Transcriptional Activation / Q000235:genetics*; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry / Q000235:genetics*
chemical_list: D004268:DNA-Binding Proteins; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; C051802:triplex DNA; D004247:DNA