p53-induced Regulation of Transcription in the Chromatin Context

为了阐明溶液中p53-DNA复合物中的DNA轨迹，我们使用碘-125放射探针（与美国国立卫生研究院临床中心的I. Panyutin和R. Neumann合作）。这种方法是基于对一种电子发射放射性同位素碘-125的衰变所产生的DNA链断裂的分析，碘-125结合在胞嘧啶的C5位置。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复合物。特别是，该方法可以直接比较结合p53核心结构域的DNA与野生型蛋白的构象，后者仍然超出了晶体学和核磁共振等传统方法的范围。我们的研究结果表明，在与wt p53的四聚体复合物中，一致的20 bp DNA片段（YYYRRR）的中心区域弯曲成小槽（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放射性探测数据。最近，一些DNA序列获得了这样的数据，包括p53 REs激活细胞周期阻滞和凋亡基因（CCA和载脂蛋白基因）的DNA序列。目前，我们正在分析放射性探测结果，将观察到的DNA链断裂强度与从p53-DNA共晶结构推断出的糖碘距离进行比较。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，我们分析了包含p53位点的人类基因组DNA的序列依赖弯曲各向异性。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定位一致。值得注意的是，p53位点和侧翼DNA的序列依赖的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载脂蛋白位点的旋转设置表明，许多载脂蛋白位点隐藏在核小体内部，从而阻止了p53的立即识别和延迟基因诱导。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对DNA的亲和力与其核小体中位置的旋转定位密切相关，这与上面描述的计算分析一致。核小体中暴露的p53位点（如cca位点）与隐藏的p53位点（如载脂蛋白位点）相比，具有更强的亲和力。因此，两组p53应答元件的核小体组织差异似乎是影响p53- dna结合强度和p53靶基因诱导动力学的关键因素。我们的模型不同于先前的概念，将CCA-和载脂蛋白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p53诱导的肿瘤抑制途径（细胞凋亡与细胞周期阻滞）的选择与染色质中相应p53结合位点的结构组织之间的直接关联。我们为现有的范式增加了新的维度，即p53 res的相对定位和染色质环境。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。

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.