Brázdová Marie, Tichý Vlastimil, Helma Robert, Bažantová Pavla, Polášková Alena, Krejčí Aneta, Petr Marek, Navrátilová Lucie, Tichá Olga, Nejedlý Karel, Bennink Martin L, Subramaniam Vinod, Bábková Zuzana, Martínek Tomáš, Lexa Matej, Adámik Matej
Department of Biophysical Chemistry and Molecular Oncology, Institute of Biophysics, Academy of Sciences of the Czech Republic v.v.i., Brno, Czech Republic.
Department of Molecular Biology and Pharmaceutical Biotechnology, Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences Brno, Brno, Czech Republic.
PLoS One. 2016 Dec 1;11(12):e0167439. doi: 10.1371/journal.pone.0167439. eCollection 2016.
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.
三链DNA参与多种生物学活动，包括基因表达调控以及导致癌症的基因组不稳定性。肿瘤抑制因子p53是细胞命运对不同类型损伤作出反应的核心调节因子。DNA识别的序列和结构特异性模式是p53蛋白的核心属性。这项工作的重点是p53在体外和细胞中与含有三链形成序列的DNA的结构特异性结合以及对p53驱动转录的影响。这是全长p53及其缺失变体对分子间和分子内T.A.T三链体的首次DNA结合研究。我们证明p53与分子间T.A.T三链体的相互作用与对CTG发夹非B型DNA结构的识别相当。使用缺失突变体，我们确定p53的C端DNA结合结构域对三链体识别至关重要。此外，通过竞争和免疫沉淀实验检测到p53对分子内T.A.T三链体（H-DNA）的强烈识别，该三链体在质粒DNA中由负超螺旋稳定，并通过原子力显微镜可视化。此外，染色质免疫沉淀揭示了p53在体内与T.A.T形成序列的结合。通过荧光素酶报告基因检测观察到，在将三链体形成序列插入具有p53共有序列的质粒后，p53增强了报告基因的反式激活。通过计算机扫描人类调控区域中共有序列和T.A.T基序的同时存在，确定了一组候选p53靶基因，并通过RT-qPCR证实了其中几个基因（ABCG5、ENOX1、INSR、MCC、NFAT5）的p53依赖性激活。我们的结果表明，T.A.T三链体构成了一类新的p53结合位点，p53在体外和细胞中以DNA结构依赖性模式靶向这些位点。讨论了p53 DNA结构依赖性结合对转录调控的贡献。
