DNA Folding in Chromatin and Interaction with Transcription Factors

染色质中的 DNA 折叠及其与转录因子的相互作用

• 批准号: 8157498
• 负责人: Victor Zhurkin
• 金额: $ 60.94万
• 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/8157498
• 关键词: Chromatin; DNA Folding; transcription factor

During the fiscal year 2009-2010, we further extended our efforts to elucidate the DNA sequence patterns guiding rotational and translational positioning of nucleosomes. In particular, we developed a novel DNA threading algorithm correctly predicting positioning of 70 % nucleosomes precisely mapped in vitro. This is based on our earlier analyses of the DNA deformability in crystal structures (the so-called knowledge-based approach) as well as on new unpublished results obtained in the course of theoretical conformational analysis of DNA. To this aim, we ran all-atom energy minimization of numerous double-stranded DNA fragments undergoing conformational transitions similar to those observed in crystallized nucleosomes. In other words, combining together the crystal structures with the results of computer simulations of DNA, we have significantly extended the list of flexible DNA sequences which are ready to accommodate the distortions imposed on nucleosomal DNA by histones. This combined approach allowed us to make an important step forward, toward understanding the nucleosome code encripted in genomic DNA. The results of these studies (two papers) have been published in the special issue of the Journal of Biomolecular Structure and Dynamics, dedicated to Nucleosome Positioning (June 2010). The folding of DNA in nucleosomes is accompanied by the lateral displacements of adjacent base pairs, which are usually ignored. We have found, however, that the shear deformation, called Slide, plays a much more important role in DNA folding than was previously imagined. First, the lateral Slide deformations observed at sites of local anisotropic bending of DNA define its superhelical trajectory in chromatin. Second, the computed cost of deforming DNA on the nucleosome is sequence-specific: in optimally positioned sequences the most easily deformed base-pair steps (CA:TG and TA) occur at the sites of large positive Slide and negative Roll (where the DNA strongly bends, or kinks, into the minor groove). Here, we incorporate all the degrees of freedom of 'real' DNA, thereby going beyond the limits of the conventional model ignoring the lateral Slide displacements of base pairs. Note that our results are in remarkable agreement with the in vitro sequence selection (SELEX) experiments. The successful prediction of nucleosome positioning for sequences of various GC-content demonstrates the potential advantage of our structural analysis, based on calculations of the DNA deformation energy. Next, we intend to apply our method to the analysis of GC-rich mammalian promoters. In this regard, it is important that our knowledge-based model of nucleosome positioning takes into account the sequence-specific effects caused by linker histones (LH). LHs demonstrate a higher affinity for the AT-rich sequences at the entry-exit points of nucleosomes, which is consistent with a general tendency of AT-rich DNA for a tight compactization in chromatin. On the other hand, the GC-rich promoters are often depleted of nucleosomes and thus are easily accessible for transcription machinery. The situation is quite different, however, when DNA is methylated. In this case, the stability of nucleosomes in particular, and of chromatin in general, is increased, the promoters become less accessible, and the level of transcription is significantly decreased. The methylation-induced silencing of tumor suppressor genes is frequently related to human cancer. We link this epigenetic effect with the sequence-specific properties of LHs, known to have a higher affinity not only for the AT-rich sequences, but also for the methylated DNA. According to our model, the LHs bind to thymines and methylated cytosines through hydrophobic interactions in the major groove (published in Nucleic Acids Research). Our new experimental data confirm the model postulating hydrophobic interactions between the linker histone H1-0 and the AT-rich fragments embedded in the linker DNA. Mutating the DNA sequence at the entry-exit point in nucleosome, we demonstrated that indeed, the presence of thymine cluster in the predicted position does increase the affinity of histone H1-0 to nucleosome. Our next step is to study interactions of the linker histone H1-0 with methylated DNA. In this regard, it is important that the H1-0 variant of linker histone is involved in terminal differentiation. Therefore, we anticipate that our efforts may help understanding the molecular mechanisms responsible for the epigenetic effects caused by DNA methylation in particular, the roles played by different H1 variants. In addition to DNA folding in nucleosomes, the shearing deformations described above, are implicated in the sequence-specific recognition of DNA by transcription factors, such as the tumor suppressor protein p53. The DNA bending, twisting and sliding (first, predicted by us and then observed in solution upon p53 binding) are entirely consistent with the 'Kink-and-Slide' conformation described above. Therefore, structural organization of a p53 binding site in chromatin can regulate its affinity to p53 - for example, exposure of the DNA site on nucleosomal surface would facilitate the p53 binding to the response elements regulating cell cycle arrest genes (p21, GADD45, etc.). Our results indicate that there is a complex interplay between the structural codes encrypted in eukaryotic genomes - one code for DNA packaging in chromatin, and the other code for DNA recognition by regulatory proteins. Rather than being mutually exclusive (as was assumed earlier), the two codes appear to be consistent with each other. At least in some cases, such as p53 and NF-kappaB, the DNA wrapping in nucleosomes can facilitate binding of the transcription factor to its cognate sequence, provided that the latter is properly exposed in chromatin.

在2009-2010财政年度，我们进一步扩大了我们的努力，以阐明DNA序列模式指导旋转和平移定位的核小体。特别是，我们开发了一种新的DNA线程算法，正确预测70%的核小体在体外精确映射的定位。这是基于我们早期对晶体结构中DNA变形性的分析（所谓的基于知识的方法）以及在DNA理论构象分析过程中获得的新的未发表的结果。为了这个目的，我们运行了所有原子能量最小化的许多双链DNA片段进行构象转变类似于那些在结晶核小体中观察到的。换句话说，将晶体结构与DNA的计算机模拟结果结合在一起，我们已经大大扩展了灵活的DNA序列的列表，这些序列可以适应组蛋白对核小体DNA施加的扭曲。这种结合的方法使我们朝着理解基因组DNA中的核小体密码迈出了重要的一步。这些研究的结果（两篇论文）已发表在《生物分子结构与动力学杂志》的特刊上，致力于核小体定位（2010年6月）。 DNA在核小体中的折叠伴随着相邻碱基对的侧向位移，这通常被忽略。然而，我们已经发现，剪切变形，称为滑动，在DNA折叠中起着比以前想象的更重要的作用。首先，在DNA局部各向异性弯曲的位点处观察到的横向滑动变形定义了其在染色质中的超螺旋轨迹。其次，计算出的核小体上DNA变形的代价是序列特异性的：在最佳定位的序列中，最容易变形的碱基对步骤（CA：TG和TA）发生在大的正滑动和负滚动的位点（DNA强烈弯曲或扭结到小沟中）。在这里，我们将所有的自由度的“真实的”DNA，从而超越了传统的模型的限制，忽略了横向滑动位移的碱基对。请注意，我们的结果与体外序列选择（SELEX）实验非常一致。成功预测的核小体定位的序列的各种GC含量证明了我们的结构分析的潜在优势，基于计算的DNA变形能。接下来，我们打算将我们的方法应用于富含GC的哺乳动物启动子的分析。 在这方面，重要的是，我们的核小体定位的知识为基础的模型考虑到连接组蛋白（LH）引起的序列特异性的影响。LH在核小体的入口-出口点处对富含AT的序列表现出更高的亲和力，这与富含AT的DNA在染色质中紧密致密化的一般趋势一致。另一方面，富含GC的启动子通常耗尽核小体，因此易于被转录机器接近。然而，当DNA被甲基化时，情况就完全不同了。在这种情况下，特别是核小体的稳定性，以及一般染色质的稳定性增加，启动子变得更难接近，并且转录水平显著降低。甲基化诱导的肿瘤抑制基因沉默通常与人类癌症有关。我们将这种表观遗传效应与LH的序列特异性特性联系起来，已知LH不仅对富含AT的序列具有更高的亲和力，而且对甲基化DNA也具有更高的亲和力。根据我们的模型，LH通过大沟中的疏水相互作用与胸腺嘧啶和甲基化胞嘧啶结合（发表在《核酸研究》上）。 我们新的实验数据证实了该模型假定的连接体组蛋白H1-0和AT-丰富的片段嵌入在连接体DNA之间的疏水相互作用。在核小体的入口-出口点突变DNA序列，我们证明了确实，在预测位置的胸腺嘧啶簇的存在确实增加了组蛋白H1-0对核小体的亲和力。我们的下一步是研究连接体组蛋白H1-0与甲基化DNA的相互作用。在这方面，重要的是接头组蛋白的H1-0变体参与终末分化。因此，我们预计，我们的努力可能有助于理解DNA甲基化引起的表观遗传效应的分子机制，特别是不同H1变体所起的作用。 除了核小体中的DNA折叠之外，上述剪切变形还涉及转录因子（如肿瘤抑制蛋白p53）对DNA的序列特异性识别。DNA的弯曲、扭曲和滑动（首先，由我们预测，然后在p53结合后在溶液中观察到）与上述“扭结和滑动”构象完全一致。因此，染色质中p53结合位点的结构组织可以调节其与p53的亲和力-例如，核小体表面上DNA位点的暴露将促进p53与调节细胞周期阻滞基因（p21，GADD 45等）的反应元件结合。我们的研究结果表明，在真核生物基因组中加密的结构代码之间存在复杂的相互作用-一个代码用于染色质中的DNA包装，另一个代码用于调节蛋白的DNA识别。这两个守则并不相互排斥（如前面所假设的那样），而是似乎相互一致。至少在某些情况下，如p53和NF-κ B，包裹在核小体中的DNA可以促进转录因子与其同源序列的结合，只要后者适当地暴露在染色质中。