p53-induced Regulation of Transcription in the Chromatin Context

作为一种转录因子，p53在调节广泛的基因方面是独一无二的。p53诱导反应的绝对水平也有很大差异，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，发生得更快。由于p53可以结合核小体DNA，我们试图了解当嵌入核小体时，两组p53位点的可及性是否不同。为此，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。我们还在体外测量了p53与其嵌入在强定位“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。最近，我们对已发表的p53细胞进行了全面分析，并在正常细胞和癌细胞中发现了数千个结合位点。我们的分析揭示了体内p53-DNA相互作用的两个不同的表观遗传特征。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（H3K4me3和H3K36me3）相关，但与癌细胞染色质中的抑制性组蛋白标记（H3K27me3）相关。其次，癌细胞中的p53结合位点的特征是DNA甲基化水平低于正常细胞中的对应位点，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位点在ERV1家族的内源性逆转录病毒元素中高度富集，这突出了该重复家族在塑造p53转录网络中的重要性。此外，p53位点表现出不同寻常的染色质模式组合：高核小体占用，同时对dna酶i高度敏感。我们的研究结果表明，p53可以在大多数dna结合因子不允许的染色质环境中进入其靶位点，这可能使p53成为染色质背景下的先驱转录因子。此外，我们启动了多形性胶质母细胞瘤（GBM）细胞核小体重定位的全基因组分析（与英国埃塞克斯大学的V. Teif合作）。我们的目的是比较GBM组织和GBM患者的健康脑组织中的核小体占用（从而DNA对tf的可及性）。特别是，我们对CTCF结合位点附近的核小体重排（其中许多参与染色质环的稳定），CTCF位点周围丰富的Alu重复序列以及这些重复序列中经常出现的p53 REs感兴趣。我们预计，专注于这三个要素，我们将能够揭示在GBM转化的各个阶段转录重编程的重要决定因素。不幸的是，由于实验室空间的限制，我们的研究被推迟了。希望这项研究能在2020年底前完成。

As a transcription factor, p53 is unique in regulating a wide spectrum of genes. The absolute level of the p53-induced response also 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 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. 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 initiated genome-wide analysis of the nucleosome repositioning in glioblastoma multiforme (GBM) cells (in collaboration with V. Teif, Essex University, UK). Our aim is to compare the nucleosome occupancy (and thus, the DNA accessibility to TFs) in the GBM tissues and in healthy brain tissues from GBM patients. In particular, we are interested in the nucleosome rearrangement in the vicinity of CTCF binding sites (many of which are involved in stabilization of chromatin loops), in Alu repeats that are abundant around the CTCF sites, and the p53 REs frequently occurring in these repeats. We anticipate that focusing on these three elements, we will be able to reveal important determinants of transcription reprogramming at various stages of GBM transformation. Unfortunately, there was a delay in our research due to restricted access to the lab space. Hopefully, this study will be completed by the end of 2020.