p53-induced Regulation of Transcription in the Chromatin Context

出乎意料的是，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，而大多数载脂蛋白位点聚集在距TSS 1 kb的地方。值得注意的是，这种p53位点的分布与我们最初的预期相反，p53结合到远端cca位点并诱导相应的cca基因似乎比p53结合到近端apo位点并激活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站点的有限实验数据p21, 14-3-3&#963；和GADD45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位点与TSS之间的较长距离将是染色质刚性的自然结果，2-3 kb原纤维的环在能量上比0.5-1 kb的环更有利。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p53四聚体结合核小体DNA（准备）的合作实验研究。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的位置使四聚体CWWG（如上所述）弯曲成主槽时，p53- dna结合更加有效，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。例如，除了CCA-和载脂蛋白基因的限制外，我们的简单模型是否具有更普遍的意义，这很有趣。为此，我们比较了数百个人类基因，揭示了在同一类型的实验中p53诱导激活的各种动力学。具体来说，我们想知道在相对于TSS的REs定位方面，早期反应的p53诱导基因与晚期反应的p53诱导基因是否存在差异。对于这些基因中的大多数，功能p53 REs是未知的，因此，我们分析了我们之前开发的生物信息学工具预测的假定p53结合位点的定位。假设的p53位点分布与上述相似：早期应答基因的p53位点位于距离启动子区域2-3 kb处，而晚期应答基因的p53位点大多位于距离TSS 1 kb以内。因此，我们认为p53结合位点的基因组环境（特别是侧翼序列的染色质组织）是p53靶基因有序反式激活的一般机制的重要因素。另一项正在进行的研究与更一般的进化方面有关，例如人类基因组中p53 REs与穿插重复序列之间的相互关系。首先，我们分析了间隔长度S的全基因组分布，发现这是高度非随机的。（间隔插入在两个p53半位点RRRCWWGYYY之间。）特别是，S=0和S=3 bp的p53位点几乎是S=2、4或5 bp位点的两倍。其次，我们发现这些差异是由转座子引起的，特别是由Alu重复序列引起的。此外，我们比较了2000个已知受p53调控的人类基因的间隔序列。结果发现，p53活化基因的周围多为S=0的REs，而p53抑制基因的启动子区域则富含S= 3bp的p53位点。重要的是，当只选择具有最强p53诱导效应的基因时，这种区别变得更加明显（手稿正在准备中）。这一结果将使我们更好地理解基因调控机制与参与调控的TF结合位点的基因组环境之间的关系。

Unexpectedly, 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 contrary to our naive expectations, 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, 14-3-3σ 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 (in preparation). 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 CWWG (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. For example, its intriguing to see whether our simple model has a more general significance, beyond the limit of the CCA- and Apo-genes. To this aim, we compared several hundred human genes revealing various kinetics of the p53-induced activation in the same type of experiments. Specifically, we asked whether the p53-induced genes with early response differ from those with late response in terms of positioning of their REs relative to TSS. For most of these genes, the functional p53 REs are unknown, therefore, we analyzed positioning of the putative p53 binding sites predicted by the bioinformatic tool developed by us earlier. The distribution of hypothetical p53 sites is similar to that described above: the genes with early response have p53 sites located 2-3 kb away from the promoter region, while for the genes with late response, p53 sites are mostly within 1 kb from TSS. Thus, we suggest that the genomic environment of the p53 binding sites (in particular, the chromatin organization of the flanking sequences) is an important element in a general mechanism of orderly trans-activation of the p53 target genes. Another study in progress is related to more general evolutionary aspects, such as the inter-relationship between the p53 REs in human genome and interspersed repeat sequences. First, we analyzed the genome-wide distribution of the spacer length, S, and found this to be highly non-random. (The spacer is inserted between two p53 half-sites, RRRCWWGYYY.) In particular, the p53 sites with S=0 and S=3 bp are nearly twice as frequent as the sites with S=2, 4 or 5 bp. Second, we showed that these differences are caused by transposons, in particular, by Alu repeats. In addition, we compared the spacer profiles for 2000 human genes known to be regulated by p53. It was found that the p53-activated genes are surrounded mostly by REs with S=0, whereas the promoter regions of p53-repressed genes are enriched with the p53 sites having S=3 bp. Importantly, this distinction becomes even more pronounced when only the genes with the strongest p53-induced effect are selected (manuscript in preparation). The results will give us a better understanding of the relationship(s) between the mechanism(s) of gene regulation and the genomic environment of the TF binding sites operative in this regulation.