DNA Folding in Chromatin at the Supra-nucleosome Level

核小体上水平的染​​色质 DNA 折叠

• 批准号: 10014465
• 负责人: Victor Zhurkin
• 金额: $ 57.41万
• 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/10014465
• 关键词: Adhesions; Agreement; Algorithms; Analytical Centrifugation; Anisotropy; Binding; Bioinformatics; Biological; Biological Assay; Breathing; Cell physiology; Chromatin; Chromatin Fiber; Chromatin Loop; Collaborations; Combined Modality Therapy; DNA; DNA Folding; DNA Repair; DNA Sequence; DNA-Directed RNA Polymerase; Data; Digestion; Dimensions; Electron Microscopy; Epigenetic Process; Eukaryota; Exonuclease; Family; Fiber; Gel; Gene Expression; Genes; Genetic Polymorphism; Genetic Transcription; Genomic DNA; Geometry; Goals; Higher Order Chromatin Structure; Histone H1; Histones; In Situ; In Vitro; Length; Link; Linker DNA; Literature; Location; Measurement; Methods; Micrococcal Nuclease; Modeling; Modification; Molecular Conformation; Monte Carlo Method; Mus; National Institute of Child Health and Human Development; Nucleosomes; Play; Positioning Attribute; Resolution; Roentgen Rays; Role; Sequence Analysis; Site; Spectrum Analysis; Structure; Superhelical DNA; TP53 gene; Tail; Testing; Time; Transcription Process; Transcriptional Regulation; Yeasts; base; design; epigenetic regulation; experimental study; flexibility; genetic information; genome-wide; genome-wide analysis; histone modification; in vivo; interest; nuclease; preference; retinal rods; stem; transcription factor

Eukaryotic DNA is spatially organized in a hierarchical manner starting from single nucleosomes folded into the higher-order chromatin structures. The DNA trajectory at the second level of organization, in 30-nm fibers, still remains the subject of heated discussion in the literature. To tackle this problem, we computed all possible configurations of chromatin fibers with DNA linkers L = 10 - 70 bp (nucleosome repeat length, NRL = 157 - 217 bp). For the first time, we observed two different families of conformations characterized by different DNA topologies. The optimal geometry of a fiber depends on the linker length: the fibers with linkers L = 10n and 10n+5 bp have DNA linking numbers per nucleosome delta(Lk) = -1.5 and -1.0, respectively. In other words, the level of DNA supercoiling is directly related to the nucleosome spacing in chromatin. This theoretical prediction was corroborated by topological gel assays indicating that the DNA supercoiling differs by as much as 50% in nucleosomal arrays with linkers L = 20 and 25 bp, in excellent agreement with our computations. Thus, we made an important step toward resolving the long-standing discrepancy known as the linking-number paradox. We hypothesize that topological polymorphism of chromatin fibers described above may play a role in the process of transcription, which is known to generate different levels of DNA supercoiling upstream and downstream from RNA polymerase. A genome-wide analysis of the NRL distribution in active and silent yeast genes confirmed this assumption. Our results may reflect a general tendency of chromosomal domains containing active or repressed genes to retain topologically distinct higher-order structures. We also analyzed nucleosomal arrays with linkers containing curved DNA fragments, A-tracts. This is important for elucidating contribution of linker DNA conformation and flexibility to nucleosome chain folding and accessibility of DNA in chromatin, because nucleosome linkers are enriched with the A-tracts. Linker DNA conformational variability has been proposed to direct nucleosome array folding into more or less compact chromatin fibers but direct experimental evidence for such models are lacking. We tested this hypothesis by designing nucleosome arrays with A-tracts at specific locations in the nucleosome linkers to induce inward (AT-IN) and outward (AT-OUT) bending of the linker DNA. Using electron microscopy and analytical centrifugation, we observed spontaneous folding of AT-IN nucleosome arrays into highly compact structures, comparable to those induced by linker histone H1. In contrast, AT-OUT nucleosome arrays formed less compact structures with decreased nucleosome interactions similar to wild-type nucleosome arrays. Electron microscopy analysis and Monte Carlo simulations are consistent with a profound zigzag linker DNA configuration and closer nucleosome proximity in the AT-IN arrays due to inward linker DNA bending. We propose that the evolutionary preferred positioning of A-tracts in DNA linkers may control chromatin higher-order folding and thus influence cellular processes such as gene expression, transcription and DNA repair. Indeed, the chromatin folding regulates accessibility of the DNA-encrypted genetic information for transcription factors (TFs), as indicated by a strong difference between the restriction nuclease digestion of nucleosome linkers in AT-IN and AT-OUT arrays. In addition, genomic DNA is made accessible by partial unwrapping of nucleosomes. As follows from the force spectroscopy measurements, the chromatin fiber is so mobile that nucleosomes lose their stacking under small external forces F = 2-3 pN, whereas at F = 4-5 pN, the nucleosomes are significantly unwrapped. (These forces are well below the tension produced by RNA polymerase; therefore, the observed unwrapping corresponds to 'native' conditions.) Importantly, the nucleosome breathing occurs asymmetrically, with one end opened much stronger than the other one. We were able to explain this non-trivial effect theoretically, taking into account a non-linear adhesion energy function describing interactions between DNA and histone core. This observation may have profound implications for transcription and other DNA-related cellular functions. According to our data, asymmetric unwrapping of nucleosomal DNA exposes 50-60 bp at one end (compared to 20-30 bp at both ends in the case of symmetric unwrapping). Therefore, asymmetric breathing of nucleosomes increases accessibility of DNA to TFs. The nucleosome positioning is not entirely determined by underlying DNA sequence, but rather is modulated by a plethora of various factors. We provide the data indicating that the sequence-specific interaction between DNA and histone tails may be one of such factors. We are suggesting specific ways to test this assumption using bioinformatic approaches. This may be of general biological interest because, if our hypothesis is confirmed, it would be for the first time (to the best of our knowledge) when the epigenetic regulation of nucleosome positioning is demonstrated. These considerations are critical for designing the experiments on p53 binding to chromatin.

真核DNA在空间上以分级方式组织，从单个核小体开始折叠成更高级的染色质结构。在第二层次的组织，在30纳米的纤维中的DNA轨迹，仍然是在文献中热烈讨论的主题。为了解决这个问题，我们计算了所有可能的构型的染色质纤维与DNA接头L = 10 - 70 bp（核小体重复长度，NRL = 157 - 217 bp）。这是我们第一次观察到两个不同的家庭的构象特征不同的DNA拓扑结构。纤维的最佳几何形状取决于接头长度：接头L = 10 n和10 n +5 bp的纤维的每个核小体的DNA连接数δ（Lk）分别为-1.5和-1.0。换句话说，DNA超螺旋的水平与染色质中的核小体间距直接相关。拓扑凝胶分析证实了这一理论预测，表明DNA超螺旋的差异高达50%，在核小体阵列与接头L = 20和25 bp，与我们的计算非常一致。因此，我们朝着解决长期存在的被称为联系数悖论的差异迈出了重要的一步。我们假设，上述染色质纤维的拓扑多态性可能在转录过程中发挥作用，这是已知的RNA聚合酶上游和下游产生不同水平的DNA超螺旋。对活性和沉默酵母基因中NRL分布的全基因组分析证实了这一假设。我们的研究结果可能反映了一个普遍的趋势，染色体域含有活性或抑制基因保留拓扑结构不同的高阶结构。我们还分析了含有弯曲DNA片段的接头的核小体阵列。这对于阐明连接体DNA构象和柔性对核小体链折叠和染色质中DNA可及性的贡献是重要的，因为核小体连接体富含A段。已经提出了连接DNA构象变异性来指导核小体阵列折叠成或多或少紧凑的染色质纤维，但缺乏这种模型的直接实验证据。我们通过设计核小体阵列来测试这一假设，其中在核小体连接体的特定位置处具有A束，以诱导连接体DNA的向内（AT-IN）和向外（AT-OUT）弯曲。使用电子显微镜和分析离心，我们观察到AT-IN核小体阵列自发折叠成高度紧凑的结构，与连接体组蛋白H1诱导的结构相当。相比之下，AT-OUT核小体阵列形成的结构不太紧凑，核小体相互作用减少，类似于野生型核小体阵列。电子显微镜分析和Monte Carlo模拟是一致的，深刻的锯齿形接头DNA的配置和更紧密的核小体接近的AT-IN阵列由于向内的接头DNA弯曲。我们提出，在DNA接头的A-tracts的进化优选定位可能控制染色质高阶折叠，从而影响细胞过程，如基因表达，转录和DNA修复。事实上，染色质折叠调节转录因子（TF）的DNA加密的遗传信息的可及性，如AT-IN和AT-OUT阵列中的核小体接头的限制性核酸酶消化之间的强烈差异所示。此外，基因组DNA可以通过部分解开核小体来获得。从力谱测量结果可以看出，染色质纤维是移动的，使得核小体在小的外力F = 2-3 pN下失去它们的堆积，而在F = 4-5 pN时，核小体被显著地解开。（这些力远低于RNA聚合酶产生的张力;因此，观察到的解缠绕对应于“天然”条件。重要的是，核小体呼吸是不对称的，一端的开放比另一端强得多。考虑到描述DNA和组蛋白核心之间相互作用的非线性粘附能函数，我们能够从理论上解释这种非平凡效应。这一观察结果可能对转录和其他DNA相关的细胞功能有深远的影响。根据我们的数据，核小体DNA的不对称解包在一端暴露50-60 bp（相比之下，在对称解包的情况下在两端暴露20-30 bp）。因此，核小体的不对称呼吸增加了DNA对TF的可及性。核小体的定位并不完全由潜在的DNA序列决定，而是由多种因素调节。我们提供的数据表明，DNA和组蛋白尾部之间的序列特异性相互作用可能是这样的因素之一。我们建议使用生物信息学方法来测试这一假设的具体方法。这可能是普遍的生物学兴趣，因为，如果我们的假设得到证实，这将是第一次（尽我们所知），当核小体定位的表观遗传调控被证明。这些考虑对于设计p53与染色质结合的实验至关重要。