p53-induced Regulation of Transcription in the Chromatin Context

为了阐明p53-DNA复合物在溶液中的DNA轨迹，我们正在使用碘-125放射性探针（与I。Panyeland和R. Neumann，临床中心，NIH）。这种方法是基于分析的DNA链断裂所产生的电子发射放射性同位素，碘-125，掺入胞嘧啶的C5位置的衰变。DNA链断裂越弱，从放射性同位素到切割位点的距离越大。放射探测的主要优点是它适用于非常大的蛋白质-DNA复合物。特别地，该方法允许直接比较与p53核心结构域和野生型蛋白结合的DNA的构象，后者仍然超出常规方法如晶体学和NMR的范围。我们的研究结果表明，在与野生型p53的四聚体复合物中，共有的20-bp DNA片段（YYYRRR）的中心区域被弯曲成小沟（即，与我们的模型一致，并与p53四聚体结合到核小体）。DNA轨迹的详细可视化需要更多的放射探测数据。最近，这样的数据获得了几个DNA序列，包括那些p53 RE激活细胞周期阻滞和凋亡基因（CCA和载脂蛋白基因）。目前，我们正在分析放射性探测结果，比较观察到的DNA链断裂强度与从p53-DNA共晶体结构推导出的糖碘距离。为了比较与CCA和载脂蛋白基因相关的p53位点的染色质背景，我们分析了含有p53位点的人类基因组DNA的序列依赖性弯曲各向异性。我们计算了旋转定位模式预测，大部分的CCA-网站暴露在核小体表面上。这与实验观察到的人类核小体的定位一致。值得注意的是，p53位点和侧翼DNA的序列依赖性DNA各向异性协同工作，产生强烈的定位信号。相比之下，核小体中Apo位点的预测和观察到的旋转设置都表明这些位点中的许多位点被埋在内部，从而阻止了p53的立即识别并延迟了基因诱导。我们还测量了p53与其嵌入在强定位的"601"核小体中的同源位点的结合。我们的数据表明，p53与DNA的亲和力与其在核小体中的位点的旋转定位密切相关，与上述计算分析一致。核小体中p53位点的暴露构型（如CCA-位点）与埋藏构型（类似于Apo-位点）相比，对p53表现出显著更强的亲和力。因此，两组p53反应元件的核小体组织的差异似乎是影响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连接CCA和载脂蛋白基因的选择性激活的结合亲和力的RE的p53。相反，我们强调p53诱导的肿瘤抑制途径（细胞凋亡与细胞周期阻滞）的选择和相应的p53结合位点在染色质中的结构组织之间的直接相关性。我们增加了新的维度，现有的范例，相对定位和染色质环境的p53 REs。我们的计划不仅解释了上述情况下，但也提供了一个新的见解，数百个基因的激活的细胞机制的p53。

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.