p53-induced Regulation of Transcription in the Chromatin Context

p53 诱导的染色质转录调节

• 批准号: 7733278
• 负责人: Victor Zhurkin
• 金额: $ 20.71万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7733278
• 关键词: Affect; Affinity; Apoptosis; Apoptotic; Binding; Binding Sites; Bioinformatics; Biological Process; CDKN1A gene; Cell Cycle Arrest; Cellular Stress; Chromatin; Chromatin Fibril; Chromatin Loop; Chromatin Remodeling Factor; Code; Complex; DNA; DNA Binding; DNA Damage; DNA Sequence; Data; Decompression Sickness; Dimensions; Distal; EP300 gene; Enhancers; Environment; Event; GC Rich Sequence; Gene Activation; Gene Expression Regulation; Gene Targeting; Genes; Genetic Transcription; Genome; Genomics; Goals; Guanine + Cytosine Composition; Histones; Human; Human Genome; In Vitro; Indium; Kinetics; Length; Major Groove; Manuscripts; Measures; Minor Groove; Modeling; Molecular; Molecular Conformation; Motivation; Muscle Rigidity; Nucleosomes; Periodicity; Play; Pliability; Positioning Attribute; Preparation; Process; Promoter Regions; Property; Protein p53; Range; Recruitment Activity; Regulation; Relative (related person); Response Elements; Role; Scheme; Side; Site; TP53 gene; Transactivation; Transcription Initiation Site; Transcriptional Regulation; Tumor Suppression; Tumor Suppressor Proteins; concept; expectation; gel electrophoresis; in vivo; insight; oncoprotein p21; promoter; research study; response; tool

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.

出乎意料的是，我们发现CCA位点距离目标基因的转录起始位点（TSS）2-3 kb，而大多数Apo位点聚集在距离TSS 1 kb以内。请注意，p53 位点的这种分布与我们天真的预期相反，是反直觉的，p53 结合到远端 CCA 位点并诱导相应的 CCA 基因似乎比 p53 结合到附近的 Apo 位点和激活 Apo 基因更有效。我们进一步表明，具有中度或低GC含量（35-55％GC）的CCA位点的侧翼序列揭示了富含AT和富含GC的簇的强周期性，与在核小体DNA序列中观察到的相似，表明稳定定位的核小体可能在这里形成。 （几个 CCA 位点 p21、14-3-3σ 和 GADD45 的有限实验数据与此评估一致。）这些核小体的预测旋转定位意味着 p53 RE 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 元件的组织方式使得核小体 DNA 预先形成用于 p53 四聚体结合。例如，p21 5-响应元件（体内最有效的 p53 RE）与 TSS 相距 2.5 kb，并以与结晶核小体中观察到的相同的有利构象弯曲。我们认为 p21 和其他 CCA 位点的暴露加速了 p53 体内结合的过程。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并提高转录水平。 （这种长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之间的长距离将是2-3 kb原纤维的染色质刚性环比0.5-1 kb原纤维的染色质刚性环更有利的自然结果。）相比之下， Apo 位点位于 GC 极其丰富的区域（高达 75-80% GC）。此类序列的典型特征是核小体的多重定位和相对容易的重组，以及低 H1 水平。我们假设这种动态环境会干扰 p53 寻找其同源结合位点并使其效率降低。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。 p53 四聚体与核小体 DNA 结合的合作实验研究（正在准备中）进一步证实了我们的评估。根据我们的结果，p53 与其同源位点的亲和力很大程度上取决于该位点在核小体中的旋转定位。即，当p53 RE以四聚体CWWG（如上所述）弯曲到大沟内且其小沟相应地暴露的方式定位时，p53-DNA结合更加有效。这正是我们在为 p53 结合预先形成的 CCA 位点的情况下设想的情况。我们的模型不同于早期将 CCA 和 Apo 基因的选择性激活与其 RE 与 p53 的结合亲和力联系起来的概念。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停滞）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相关性。我们向现有范式添加了新的维度：p53 RE 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，而且还为 p53 激活数百个基因的细胞机制提供了新的见解。例如，有趣的是我们的简单模型是否具有超越 CCA 和 Apo 基因限制的更普遍的意义。为此，我们比较了数百个人类基因，揭示了同一类型实验中 p53 诱导激活的各种动力学。具体来说，我们询问 p53 诱导的早期反应基因与晚期反应基因在其 RE 相对于 TSS 的定位方面是否有所不同。对于大多数这些基因，功能性 p53 RE 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位点的分布与上述类似：早期反应的基因的p53位点距离启动子区域2-3kb，而对于晚期反应的基因，p53位点大多在距离TSS 1kb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中的重要元素。另一项正在进行的研究涉及更一般的进化方面，例如人类基因组中的 p53 RE 与散布的重复序列之间的相互关系。首先，我们分析了间隔区长度 S 的全基因组分布，发现这是高度非随机的。 （间隔区插入两个 p53 半位点之间，RRRCWWGYYY。）特别是，S=0 和 S=3 bp 的 p53 位点的频率几乎是 S=2、4 或 5 bp 的位点的两倍。其次，我们证明这些差异是由转座子引起的，特别是由 Alu 重复引起的。此外，我们还比较了 2000 个已知受 p53 调节的人类基因的间隔区谱。 研究发现，p53 激活基因大部分被 S=0 的 RE 包围，而 p53 抑制基因的启动子区域则富含 S=3 bp 的 p53 位点。 重要的是，当仅选择具有最强 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）。 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.