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: 10262169
• 负责人: Victor Zhurkin
• 金额: $ 74.68万
• 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/10262169
• 关键词: 3-Dimensional; Address; Adhesions; Agreement; Binding; Bioinformatics; Biological; Biological Assay; Breathing; Cell Nucleus; Cell physiology; Chromatin; Chromatin Fiber; Chromatin Loop; Chromatin Structure; Chromosome Territory; DNA; DNA Binding; DNA Folding; DNA Sequence; DNA-Directed RNA Polymerase; Data; Disease; Electron Microscope; Equilibrium; Family; Fiber; Gel; Gene Expression Regulation; Genes; Genetic Polymorphism; Genetic Transcription; Genomic DNA; Geometry; Goals; Heterochromatin; Histone H2A; Histone H4; Histones; In Vitro; Length; Link; Linker DNA; Measurement; Modeling; Molecular Conformation; Nucleosomes; Oligonucleotides; Organism; Play; Positioning Attribute; Prevalence; Proteins; RNA; Resolution; Roentgen Rays; Role; Site; Spectrum Analysis; Structure; Structure-Activity Relationship; Superhelical DNA; TP53 gene; Tail; Techniques; Testing; Time; Transcription Process; Yeasts; base; cellular imaging; density; epigenetic regulation; experimental study; genome-wide analysis; in vivo; interest; novel; reconstitution; stem; tomography; transcription factor

Eukaryotic DNA is extremely tightly packed in the nucleus, nevertheless its sequence is effectively recognized by numerous protein factors. To elucidate structural mechanisms of this recognition one needs to have a detailed information about the second level of DNA organization, or 30-nm fibers. To tackle this problem, we computed all possible configurations of the two-start chromatin fibers with DNA linkers L = 10 - 70 bp (nucleosome repeat length, NRL = 157 - 217 bp). As a result, we observed two different families of conformations (i.e., topoisomers) 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. 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 yeast genes confirmed this assumption. We also found that the two fiber topoisomers (with L = 10n and 10n+5 bp) differ not only in their equilibrium configuration and average DNA linking number, but also in their dynamics. In particular, the novel 10n+5 topoisomer is characterized by an increased plasticity, which makes chromatin more accessible to transcription factors (TFs). 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 bioinformatic data indicating that the sequence-specific interaction between DNA and histone tails may be one of such factors. We have initiated in vitro experiments to test this assumption, analyzing positioning of nucleosomes reconstituted with histones H2A and H4 lacking the N-tails. 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. This, in turn, would open the prospects for revealing structural mechanisms of DNA recognition by TFs in the context of the three-dimensional organization of chromatin. In particular, wrapping DNA around the histone core can either facilitate the TF binding by exposing the cognate DNA site, or, by contrast, hinder the binding by placing the TF site inside the DNA loop. This consideration is directly related to the DNA recognition by p53 (see the second project).

真核DNA极其紧密地堆积在细胞核中，但其序列可以被许多蛋白质因子有效识别。为了阐明这种识别的结构机制，我们需要了解有关 DNA 组织的第二层（即 30 纳米纤维）的详细信息。为了解决这个问题，我们计算了带有 DNA 连接子 L = 10 - 70 bp（核小体重复长度，NRL = 157 - 217 bp）的双起始染色质纤维的所有可能配置。结果，我们观察到两个不同的构象家族（即拓扑异构体），它们具有不同的 DNA 拓扑结构。纤维的最佳几何形状取决于接头长度：接头 L = 10n 和 10n+5 bp 的纤维的每个核小体 delta(Lk) = -1.5 和 -1.0 的 DNA 连接数分别为 -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。因此，我们在解决长期存在的差异（称为链接数悖论）方面迈出了重要的一步。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，已知转录过程会在 RNA 聚合酶的上游和下游产生不同水平的 DNA 超螺旋。对酵母基因中 NRL 分布的全基因组分析证实了这一假设。我们还发现两种纤维拓扑异构体（L = 10n 和 10n+5 bp）不仅在平衡构型和平均 DNA 连接数方面不同，而且在动力学方面也不同。特别是，新型 10n+5 拓扑异构体的特点是可塑性增加，这使得染色质更容易接触转录因子 (TF)。此外，可以通过部分解开核小体来获取基因组 DNA。从力谱测量结果可以看出，染色质纤维的移动性非常大，以至于核小体在小外力 F = 2-3 pN 下会失去堆积，而在 F = 4-5 pN 时，核小体明显展开。 （这些力远低于 RNA 聚合酶产生的张力；因此，观察到的展开对应于“天然”条件。）重要的是，核小体呼吸是不对称发生的，一端打开比另一端强得多。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不平凡的效应。这一观察结果可能对转录和其他 DNA 相关细胞功能产生深远的影响。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，两端都暴露 20-30 bp）。因此，核小体的不对称呼吸增加了 DNA 与 TF 的可及性。核小体的定位并不完全由潜在的 DNA 序列决定，而是受到多种因素的调节。我们提供的生物信息学数据表明 DNA 和组蛋白尾部之间的序列特异性相互作用可能是此类因素之一。我们已启动体外实验来测试这一假设，分析用缺乏 N 尾的组蛋白 H2A 和 H4 重构的核小体的定位。这可能具有普遍的生物学意义，因为如果我们的假设得到证实，这将是第一次（据我们所知）核小体定位的表观遗传调控得到证实。反过来，这将为揭示转录因子在染色质三维​​组织背景下识别 DNA 的结构机制开辟前景。特别是，将 DNA 包裹在组蛋白核心周围可以通过暴露同源 DNA 位点来促进 TF 结合，或者相反，通过将 TF 位点置于 DNA 环内来阻碍结合。这种考虑与 p53 的 DNA 识别直接相关（参见第二个项目）。