p53-induced Regulation of Transcription in the Chromatin Context

p53 诱导的染色质转录调节

• 批准号: 9153693
• 负责人: Victor Zhurkin
• 金额: $ 27.76万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9153693
• 关键词: Affect; Affinity; Agreement; Anisotropy; Apoptosis; Apoptotic; Binding; Binding Sites; Biological Process; Cell Cycle Arrest; Chromatin; Chromatin Loop; Clinical; Collaborations; Complex; Computer Analysis; Consensus; Crystallography; Cytosine; DNA; DNA Binding; DNA Sequence; DNA strand break; Data; Dimensions; Electrons; Environment; GADD45; Gene Activation; Gene Targeting; Genes; Genetic Transcription; Genomic DNA; Genomics; Goals; Histones; Human; I125 isotope; Imagery; In Vitro; Iodine; Iodine Radioisotopes; Kinetics; Major Groove; Measures; Methods; Minor Groove; Modeling; Molecular Conformation; Motivation; Nucleosomes; Pattern; Positioning Attribute; Proteins; Radioisotopes; Relative (related person); Response Elements; Scheme; Sequence Analysis; Side; Signal Transduction; Site; Solutions; Structure; Surface; TP53 gene; Transactivation; Transcriptional Regulation; Tumor Suppression; United States National Institutes of Health; Work; base; gel electrophoresis; gene induction; in vivo; insight; prevent; research study; response; sugar

To elucidate DNA trajectory in the p53-DNA complex in solution, we are using Iodine-125 radioprobing (in collaboration with I. Panyutin and R. Neumann, Clinical Center, NIH). This method is based on analysis of the DNA strand breaks produced by the decay of an electron-emitting radioisotope, Iodine-125, incorporated in the C5 position of cytosine. The weaker the DNA strand break, the larger the distance from the radioisotope to the cleavage site. The major advantage of radioprobing is its applicability for very large protein-DNA complexes. In particular, this method allows direct comparison of the conformations of DNA bound to the p53 core domain and to the wild type protein, the latter still being beyond the scope of conventional methods such as crystallography and NMR. Our results indicate that in a tetrameric complex with wt p53, the central region of the consensus 20-bp DNA fragment (YYYRRR) is bent into the minor groove (that is, consistent with our model and with the p53 tetramer binding to nucleosome). The detailed visualization of the DNA trajectory requires more radioprobing data. Recently, such data were obtained for several DNA sequences, including those of the p53 REs activating cell cycle arrest and apoptotic genes (CCA and Apo-genes). Currently, we are analyzing the radioprobing results, comparing the observed DNA strand breaks intensities with the sugar-iodine distances deduced from the p53-DNA co-crystal structures. To compare the chromatin context of the p53 sites associated with the CCA- and Apo-genes, we analyzed the sequence-dependent bending anisotropy of human genomic DNA containing p53 sites. We calculated rotational positioning patterns predicting that most of the CCA-sites are exposed on the nucleosomal surface. This is consistent with experimentally observed positioning of human nucleosomes. Remarkably, the sequence-dependent DNA anisotropy of both the p53 sites and flanking DNA work in concert producing strong positioning signals. By contrast, both the predicted and observed rotational settings of the Apo-sites in nucleosomes suggest that many of these sites are buried inside, thus preventing immediate p53 recognition and delaying gene induction. We also measured the p53 binding to its cognate sites embedded in the in strongly positioned '601' nucleosome. Our data suggest that the p53 affinity to DNA strongly correlates with the rotational positioning of its site in nucleosome, in agreement with the computational analysis described above. The exposed configurations of the p53 sites in nucleosome (like CCA-sites) demonstrate significantly stronger affinity to p53 compared to the buried configurations (similar to the Apo-sites). Thus, the difference in nucleosomal organization of the two sets of p53 response elements appears to be a key factor affecting the strength of p53-DNA binding and kinetics of induction of the p53 target genes. Our model differs from the earlier concept connecting the selective activation of the CCA- and Apo-genes to the binding affinities of their REs to p53. Instead, we emphasize a direct correlation between the selection of p53-induced tumor suppression pathway (apoptosis versus cell cycle arrest) and structural organization of the corresponding p53-binding sites in chromatin. We add new dimensions to the existing paradigm, the relative positioning and chromatin environment of the p53 REs. Our scheme not only explains the above cases but also provides a new insight into the cellular mechanisms of activation of hundreds of genes by p53.

为了阐明溶液中p53-DNA复合体中的DNA轨迹，我们使用了碘-125放射性探针(与NIH临床中心的I.Panyutin和R.Neumann合作)。这种方法是基于对胞嘧啶的C5位结合的电子放射性同位素碘-125的衰变所产生的DNA链断裂的分析。DNA链断裂越弱，放射性同位素到裂解位点的距离就越大。放射性探测的主要优点是它适用于非常大的蛋白质-DNA复合体。特别是，这种方法允许直接比较与P53核心区结合的DNA的构象和与野生型蛋白的构象，后者仍然超出了传统方法如结晶学和核磁共振的范围。我们的结果表明，在与wt p53的四聚体复合体中，共识20-bp DNA片段(YYYRRR)的中心区域被弯曲到小沟中(即与我们的模型和与核小体结合的P53四聚体一致)。DNA轨迹的详细可视化需要更多的无线电探测数据。最近，一些DNA序列获得了这样的数据，包括激活细胞周期停滞的P53 RES和凋亡基因(CCA和Apo-基因)。目前，我们正在分析放射性探测结果，将观察到的DNA链断裂强度与由p53-DNA共晶结构推导出的糖碘距离进行比较。为了比较与CCA基因和Apo基因相关的P53位点的染色质背景，我们分析了包含P53位点的人类基因组DNA的序列依赖的弯曲各向异性。我们计算了旋转定位模式，预测大多数CCA位点暴露在核小体表面。这与实验观察到的人类核小体的定位是一致的。值得注意的是，P53位点和侧翼DNA序列依赖的DNA各向异性协同工作，产生强烈的定位信号。相比之下，预测和观察到的核小体中载脂蛋白位点的旋转设置都表明，其中许多位点被埋在里面，从而阻止了对p53的即时识别和延迟了基因诱导。我们还测量了P53与其同源位点的结合，嵌入在In强定位的‘601’核小体中。我们的数据表明，P53与DNA的亲和力与其在核小体中的旋转位置密切相关，这与上述计算分析一致。与埋藏构型(类似于载脂蛋白位点)相比，核小体中暴露的P53位点(如CCA位点)对P53的亲和力显著增强。因此，这两组P53反应元件在核小体组织上的差异似乎是影响P53-DNA结合强度和P53靶基因诱导动力学的关键因素。我们的模型不同于早期的概念，将CCA基因和Apo基因的选择性激活与其RES与P53的结合亲和力联系起来。相反，我们强调选择P53诱导的肿瘤抑制途径(细胞凋亡与细胞周期停滞)与染色质中相应的P53结合部位的结构组织之间存在直接关联。我们在现有的范例、P53 RES的相对定位和染色质环境中增加了新的维度。我们的方案不仅解释了上述情况，而且为P53激活数百个基因的细胞机制提供了新的见解。