p53-induced Regulation of Transcription in the Chromatin Context

p53调节广泛的基因。p53诱导的反应的绝对水平变化很大，这对生物功能很重要。特别是，p53诱导的细胞周期阻滞基因（CCA-基因）的激活比凋亡基因（Apo-基因）的激活更强且发生得更快。由于p53可以结合核小体DNA，我们试图了解两组p53位点在嵌入核小体时的可及性是否不同。为此，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核小体在CCA位点附近的定位一致。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。我们还在体外测定了p53与其嵌入强定位的“601”核小体中的同源位点的结合。我们的数据表明，与我们的计算分析一致，p53与DNA的亲和力与其RE在核小体中的旋转定位密切相关。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。最近，我们对已发表的p53顺反序列进行了全面分析，并在正常细胞和癌细胞中鉴定了数千个结合位点。我们的分析揭示了两个不同的表观遗传特征潜在的p53-DNA相互作用在体内。首先，我们发现p53结合位点与正常细胞染色质中的转录活性组蛋白标记（H3 K4 me 3和H3 K36 me 3）相关，但与癌细胞染色质中的抑制性组蛋白标记（H3 K27 me 3）相关。第二，癌细胞中的p53结合位点的特征在于DNA甲基化水平低于正常细胞中的对应物，这可能与癌症中的整体低甲基化有关。此外，无论细胞类型如何，p53位点高度富集在ERV 1家族的内源性逆转录病毒元件中，突出了该重复序列家族在形成p53转录网络中的重要性。此外，p53位点表现出一种不寻常的染色质模式组合：高核小体占有率，同时对DNA酶I具有高敏感性。我们的研究结果表明，p53可以访问它的靶位点在染色质环境中，是不允许的大多数DNA结合因子，这可能允许p53作为一个先锋转录因子的背景下，染色质。此外，我们还分析了Alu重复序列附近的核小体组织和这些重复序列中发生的p53 RE。我们的观察表明，乳腺癌（BRC）的核小体间距显着缩短，特别是在5 '端的Alu元素纳入基因。正如我们先前所证明的，核小体间距缩短5 bp与转录水平增加有关。我们预计，专注于这些观察，我们将能够揭示转录重编程的重要决定因素在不同阶段的BRC转换。最近，我们研究了p53与修饰的组蛋白结合的核小体，模拟组蛋白N-尾的表观遗传乙酰化。我们开发了一个体外模型的表观遗传乙酰化的赖氨酸K5和K7的组蛋白H2 A。为此，我们使用非洲爪蟾组蛋白八聚体与野生型（WT）和H2 A无尾组蛋白（H2 A-del）重组定位良好的核小体。分析p53与WT和H2 A-del核小体的结合，我们观察到两者之间的以下差异。如上所述，p53对核小体DNA的亲和力与其RE在WT核小体中的旋转设置强烈相关。这种相关性也适用于H2 A-del核小体。此外，对于H2 A-del核小体，p53对RE的亲和力与其与核小体末端的接近度之间的关系是单调的（这在直觉上是显而易见的，因为RE越接近核小体末端，它就越灵活，因此，p53结合就越容易。然而，对于WT核小体，上述关系是非单调的。这是一种新的效应，显然与组蛋白尾的存在相结合。基于这些结果，我们得出结论，组蛋白H2 A尾巴产生的“屏蔽”效应调节p53-DNA结合亲和力。重要的是，这种“屏蔽”效应是局部的，并且非常有选择性，因为它改变了p53对X射线结构中H2 A尾与DNA相互作用的位置附近的RE的亲和力，而不是对整个核小体的亲和力。H4 N尾是否产生类似的效果还有待观察，这将是我们未来研究的主题。潜在地，这一观察结果可能与其他TF相关，因此，可能对TF与染色质结合的表观遗传调控具有深远的影响。

p53 regulates a wide spectrum of genes. The absolute level of the p53-induced response varies drastically, which is important for biological functioning. In particular, the p53-induced activation of the cell cycle arrest genes (CCA-genes) is stronger and occurs much faster than activation of the apoptotic genes (Apo-genes). Since p53 can bind nucleosomal DNA, we sought to understand if the two groups of p53 sites differ in their accessibility when embedded in nucleosomes. To this aim, 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 nucleosome surface. This is consistent with experimentally observed positioning of human nucleosomes in the vicinity of the CCA-sites. 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 in vitro. Our data suggest that the p53 affinity to DNA strongly correlates with the rotational positioning of its RE in nucleosome, in agreement with our computational analysis. 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. Recently, we performed a comprehensive analysis of the published p53 cistromes and identified thousands binding sites in normal and cancer cells. Our analysis revealed two distinct epigenetic features underlying p53-DNA interactions in vivo. First, we found that p53 binding sites are associated with transcriptionally active histone marks (H3K4me3 and H3K36me3) in normal-cell chromatin, but with repressive histone marks (H3K27me3) in cancer-cell chromatin. Second, p53 binding sites in cancer cells are characterized by a lower level of DNA methylation than their counterparts in normal cells, probably related to global hypomethylation in cancers. In addition, regardless of the cell type, p53 sites are highly enriched in the endogenous retroviral elements of the ERV1 family, highlighting the importance of this repeat family in shaping the transcriptional network of p53. Moreover, the p53 sites exhibit an unusual combination of chromatin patterns: high nucleosome occupancy and, at the same time, high sensitivity to DNase I. Our results suggest that p53 can access its target sites in a chromatin environment that is non-permissive to most DNA-binding factors, which may allow p53 to act as a pioneer transcription factor in the context of chromatin. In addition, we analyzed the nucleosome organization in the vicinity of Alu repeats and the p53 REs occurring in these repeats. Our observations indicate dramatic shortening of nucleosome spacing in breast cancer (BRC), in particular, at the 5'-ends of Alu elements incorporated in genes. As we demonstrated earlier, the shortening of inter-nucleosome distances by 5 bp is associated with the increased level of transcription. We anticipate that focusing on these observations, we will be able to reveal important determinants of transcription reprogramming at various stages of the BRC transformation. Recently, we studied p53 binding to nucleosomes with modified histones, mimicking epigenetic acetylation of the histone N-tails. We developed an in vitro model of the epigenetic acetylation of the lysines K5 and K7 of histone H2A. To this aim, we reconstituted well-positioned nucleosomes using Xenopus histone octamers with wild type (WT) and H2A tail-less histones (H2A-del). Analyzing p53 binding to the WT and H2A-del nucleosomes, we observed the following difference between the two. As mentioned above, the p53 affinity to nucleosomal DNA strongly correlates with the rotational setting of its RE in WT nucleosome. This correlation holds for H2A-del nucleosomes as well. In addition, for the H2A-del nucleosomes, the relationship between the p53 affinity to RE and its proximity to the nucleosome end is monotonic (which is intuitively obvious because the closer is RE to the nucleosome end, the more flexible it is, and hence, the more accessible for p53 binding.) However, for the WT nucleosomes the above relationship is non-monotonic. This is a novel effect, apparently coupled with the presence of histone tails. Based on these results, we conclude that the histone H2A tails produce a 'shielding' effect modulating the p53-DNA binding affinity. Importantly, this 'shielding' effect is local and very selective, because it changes the p53 affinity to the REs in the vicinity of the position where the H2A tail interacts with DNA in x-ray structure, rather than to the whole nucleosome. It remains to be seen whether the H4 N-tail produces a similar effect, and it will be the subject of our future studies. Potentially, this observation may be relevant to other TFs as well and thus, may have far-reaching implications for epigenetic regulation of TF binding to chromatin.