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。对于上面提到的CATG四聚体，我们的数据证明没有形成Hoogsteen碱基对（与三个x射线结构中的两个一致）。我们的研究结果还表明，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p53与核小体结合的四聚体一致）。DNA轨迹的详细可视化需要更多的放射性探测数据。在不久的将来，我们预计将获得一些DNA序列的数据，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）的数据。我们将特别关注DNA构象对p53 RE中心可变间隔S长度的依赖。比较CCA-和apo -基因中p53位点的分布，我们发现CCA-位点位于距靶基因转录起始位点（TSS） 2-3 kb的地方，而大多数apo -位点聚集在距TSS 1 kb的地方。值得注意的是，p53位点的这种分布是违反直觉的，因为p53与远端cca位点结合并诱导相应的cca基因似乎比p53与近端apo位点结合并激活apo基因更有效。我们进一步发现，具有中等或低GC含量（35- 55% GC）的cca位点的侧翼序列显示出与核小体DNA序列相似的富at和富GC簇的强周期性，表明稳定位置的核小体可能在这里形成。几个cca位点（p21、14-3-3sigma和GADD45）有限的实验数据与这一评估一致。预测这些核小体的旋转定位意味着p53 REs暴露在有利于p53识别的弯曲构象中。换句话说，cca位点附近的可弯曲DNA元件以这样一种方式组织，即核小体DNA是为p53四聚体结合而预先形成的。例如，体内最有效的p53 RE - p21 - 5-response元件与TSS相隔2.5 kb，并且弯曲成与结晶核小体相同的有利构象。我们认为p21和其他cca位点的暴露加速了p53在体内的结合过程。反过来，p53将p300/CBP和/或染色质重塑因子等共激活因子招募到启动子中，从而促进染色质的打开，提高转录水平。(这种远距离转移的详细分子机制尚不清楚。高阶染色质原纤维的增强子型环是可能的。在这种情况下，强cca位点和TSS之间的较长距离将是染色质刚性的自然结果：2-3 kb的原纤维的环状比0.5-1 kb的环状在能量上更有利。相比之下，载脂蛋白位点位于极富GC的区域（高达75- 80% GC）。这类序列的典型特征是核小体的多定位和相对容易重组，并且H1水平较低。我们假设这种动态环境干扰了p53对其同源结合位点的搜索，使其效率降低。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。我们的评估进一步证实了p53四聚体与核体DNA结合的合作实验研究（发表在J. Biol上）。化学)。根据我们的研究结果，p53与其同源位点的亲和力强烈依赖于该位点在核小体中的旋转定位。也就是说，当p53 RE的定位方式使四聚体CATG（如上所述）弯曲成主槽时，p53- dna结合更加有效，并且它们的小槽也相应地暴露出来。这正是我们在为p53结合而制备的cca位点的情况下所设想的情况。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。我们特别关注了假定的p53结合位点的全基因组分布及其与各种转座元件的关系，特别是Alu重复序列。首先，我们分析了迄今为止鉴定的160个功能性p53 REs，发现其中24个重复发生。这些重复相关的REs中有一半以上位于Alu元素中；它们位于内部RNA聚合酶III启动子框A和B附近。有趣的是，一些alu - p53 REs与凋亡基因（如CASP-10）有关。此外，使用位置权重矩阵方法，我们在全基因组的Alu元件中发现了大约40万个潜在的p53位点。这些位点与功能性p53 REs位于Alu重复序列的相同区域，因此根据它们与box A或box b的距离可以分为两组。先前对Alu元件进行的核小体定位实验表明，这两组p53位点具有不同的染色质环境，这对p53- dna结合至关重要。最后，我们将p53位点与相应的Alu共识序列进行了比较，得出结论：两组位点可能通过不同的机制进化：一组是由CG-to-CA:TG突变产生的；另一组明显早于几个Alu亚家族的祖先，如AluSp和AluSx。值得注意的是，这种评估适用于功能性p53 REs和推定的BSs。我们的观察结果对于理解p53调控网络在全基因组水平上的进化可能是重要的。

To elucidate DNA trajectory in the p53-DNA complex in solution, we used 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. As to the CATG tetramers mentioned above, our data testify against formation of the Hoogsteen base pairs (in agreement with two x-ray structures out of three). Our results also 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. In the near future, we anticipate obtaining such data for several DNA sequences, including those of the p53 REs activating cell cycle arrest and apoptotic genes (CCA and Apo-genes). Special attention will be paid to the dependence of DNA conformation on the length of a variable spacer S in the center of p53 RE. Comparing distribution of the p53 sites in the CCA- and Apo-genes, we found that the CCA-sites are located 2-3 kb away from the transcription start sites (TSS) of the target genes, whereas most of the Apo-sites are clustered within 1 kb from TSS. Note that such a distribution of the p53 sites is counter-intuitive because the p53 binding to a distal CCA-site and induction of the corresponding CCA-gene appears to be more efficient than the p53 binding to a close Apo-site and activation of the Apo-gene. We further showed that the flanking sequences of the CCA-sites, with moderate or low GC content (35-55 % GC), reveal strong periodicity of the AT-rich and the GC-rich clusters, similar to that observed in the nucleosomal DNA sequences, suggesting that stable positioned nucleosomes are likely to form here. The limited experimental data available for several CCA-sites (p21, 14-3-3sigma and GADD45) are consistent with this assessment. The predicted rotational positioning of these nucleosomes implies that the p53 REs are exposed in the bent conformation favorable for the p53 recognition. To put it differently, the bendable DNA elements in the vicinity of the CCA-sites are organized in such a way that the nucleosomal DNA is preformed for the p53 tetramer binding. For example, the p21 5-response element, the most effective p53 RE in vivo, is separated from TSS by 2.5 kb, and is bent in the same favorable conformation as observed in the crystallized nucleosomes. We suggest that exposure of the p21 and other CCA-sites accelerates the process of p53 binding in vivo. p53, in turn, recruits co-activators such as p300/CBP and/or chromatin remodeling factors to the promoters, thereby facilitating opening of chromatin and increasing the level of transcription. (The detailed molecular mechanisms of this long-distance transfer are not known. The enhancer-type looping of the higher-order chromatin fibril is a likely possibility. In such a case, the long distance between the strong CCA-sites and TSS would be a natural consequence of the chromatin rigidity: looping of 2-3 kb fibril is much more favorable energetically than looping of 0.5-1 kb.) By contrast, the Apo-sites are located in extremely GC-rich regions (up to 75-80 % GC). Such sequences are typically characterized by multiple positioning and relatively easy reorganization of nucleosomes, as well as low H1 level. We hypothesize that this dynamic environment interferes with the p53 search for its cognate binding site and makes it less effective. 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 assessment is further substantiated by a collaborative experimental study of the p53 tetramer binding to nucleosomal DNA (published in J. Biol. Chemistry). According to our results, the p53 affinity to its cognate site strongly depends on the rotational positioning of this site in nucleosome. Namely, the p53-DNA binding is much more effective when the p53 RE is positioned in such a way that the tetramers CATG (mentioned above) are bent into the major groove, and their minor groove is accordingly exposed. This is exactly the situation we envisioned in the case of the CCA-sites preformed for the p53 binding. 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. We paid special attention to genome-wide distribution of putative p53 binding sites and their relationship to various transposable elements, in particular Alu repeats. First, we analyzed 160 functional p53 REs identified so far and found that 24 of them occur in repeats. More than half of these repeat-associated REs reside in Alu elements; they are located in the vicinity of Boxes A and B of the internal RNA polymerase III promoter. Interestingly, several Alu-residing p53 REs are associated with apoptotic genes (e.g., CASP-10). In addition, using a position weight matrix approach, we found approximately 400,000 potential p53 sites in Alu elements genome-wide. These sites are located in the same regions of Alu repeats as the functional p53 REs and thus can be divided into two groups depending on their vicinity to the Boxes A or B. The nucleosome-mapping experiments made on Alu elements earlier, suggest that the p53 sites from these two groups have different chromatin environments which is critical for the p53-DNA binding. Finally, we compared the p53 sites with the corresponding Alu consensus sequences and concluded that the two groups of sites probably evolved through different mechanisms one group was generated by CG-to-CA:TG mutations; the other group apparently pre-existed in the progenitors of several Alu subfamilies, such as AluSp and AluSx. Remarkably, this assessments holds both for the functional p53 REs and for putative BSs. Our observations may be important for understanding the evolution of the p53 regulatory network at the genome-wide level.