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: 8937869
• 负责人: Victor Zhurkin
• 金额: $ 47.68万
• 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/8937869
• 关键词: Accounting; Agreement; Algorithms; Anisotropy; Base Pairing; Binding; Caliber; Cell physiology; Chromatin; Chromatin Fiber; Chromatin Loop; Code; Collaborations; Combined Modality Therapy; DNA; DNA Folding; DNA Sequence; DNA Sequence Rearrangement; Digestion; Eukaryota; Exonuclease; Fiber; Freedom; Genetic Crossing Over; Genetic Polymorphism; Genetic Transcription; Genomic DNA; Goals; Guanine + Cytosine Composition; Handedness; Histones; Human; In Situ; In Vitro; Lateral; Left; Link; Linker DNA; Maps; Measures; Methods; Micrococcal Nuclease; Minor Groove; Modeling; Modification; National Institute of Child Health and Human Development; Neurons; Nucleosomes; Pattern; Plasmids; Play; Positioning Attribute; Publishing; Relative (related person); Reporting; Resolution; Roentgen Rays; Role; Running; Sequence Analysis; Site; Slide; Solutions; Structure; Superhelical DNA; Technology; Testing; Transcriptional Activation; Yeasts; base; conformational conversion; cost; ds-DNA; genome-wide; in vivo; novel; preference; research study; retinal rods; stem; transcription factor; yeast genome

During the fiscal year 2013-2014, we 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 nucleosomes precisely mapped both in vitro and in vivo (in collaboration with F. Cui, Rochester Inst. of Technology, NY). We also ran all-atom energy minimization of numerous double-stranded DNA fragments undergoing conformational transitions similar to those observed in crystallized nucleosomes. This combined approach allowed us to make an important step forward, toward understanding the nucleosome code encripted in genomic DNA. 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. Recently, we developed a novel computational approach for calculation of topological polymorphism of the higher-order chromatin organization, the so-called 30-nm fibril. (This is important because the questions related to the structure of the 30-nm fibril still remain unanswered unambiguously. Most likely, the solenoid-type fibers proposed initially may be formed only for the inter-nucleosomal linkers L=50 bp or longer. When the linker L is 30 bp or shorter, which corresponds to chromatin in yeast and in human neurons, the two-start nucleosome fibers are formed.) We analyzed the two-start chromatin fibers for linkers L=13 to 37 bp. By optimizing the fiber energy with respect to the superhelical parameters we found two types of topological transition in fibers: one caused by an abrupt change in the linker DNA twisting, and another caused by over-crossing of the linkers. (The first transition is characterized by change in the DNA linking number, delta(Lk) = 1, and the second one by delta(Lk) = 2.) To the best of our knowledge, this topological polymorphism of the chromatin fibers was not reported in the computations published earlier. Importantly, the optimal configurations of the fibers with linkers L = 10n and 10n+5 bp are topologically different. Our results are consistent with experimental observations, such as the inclination 60-70 degrees (the angle between the nucleosomal disks and the fiber axis), helical rise, diameter and left-handedness of the fibers. In addition, we make several testable predictions, among them existence of different degree of DNA supercoiling in the fibers with L = 10n and 10n+5 bp, different stiffness of the two types of fibers, and a correlation between the local NRL and the level of transcription in different parts of the yeast genome. To test these predictions, we are planning two types of experiments. The first will be measuring the linking number difference in the plasmids containing arrays of '601' nucleosomes separated by linkers L = 20 and 25 bp (in collaboration with S. Grigoryev, Penn State). We will also analyze rearrangement of nucleosome positioning upon activation of transcription in yeast. Using the combined MNase/exoIII digestion described above will be essential for mapping nucleosomes with the highest possible resolution.

在2013-2014财政年度，我们扩大了我们的努力，以阐明指导核小体的旋转和平移定位的DNA序列模式。特别是，我们开发了一种新的DNA线程算法，正确预测核小体的定位，在体外和体内精确映射（与F。Cui，罗切斯特技术学院，纽约）。我们还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。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。核小体中DNA的折叠伴随着相邻碱基对的侧向位移，而这通常被忽视。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。最近，我们开发了一种新的计算方法，用于计算高级染色质组织的拓扑多态性，即所谓的30 nm原纤维。(This是重要的，因为与30纳米原纤维的结构有关的问题仍然没有明确的答案。最有可能的是，最初提出的螺线管型纤维可能仅形成于L=50 bp或更长的核小体间接头。当接头L为30 bp或更短时，其对应于酵母和人类神经元中的染色质，形成双起始核小体纤维。我们分析了双起始染色质纤维的接头L=13至37 bp。通过优化纤维能量相对于超螺旋参数，我们发现两种类型的拓扑转变的纤维：一个是由突然变化的接头DNA扭曲，另一个是由过度交叉的接头。(The第一个转变的特征在于DNA连接数的变化，δ（Lk）= 1，第二个转变的特征在于δ（Lk）= 2。据我们所知，这种染色质纤维的拓扑多态性在早期发表的计算中没有报道。重要的是，具有连接子L = 10 n和10 n +5 bp的纤维的最佳构型在拓扑上是不同的。我们的结果与实验观察一致，如60-70度（核小体盘和纤维轴之间的角度），螺旋上升，直径和左旋纤维之间的角度。此外，我们做了几个可检验的预测，其中存在不同程度的DNA超螺旋的纤维与L = 10 n和10 n +5 bp，不同的刚度的两种类型的纤维，和本地NRL和转录水平之间的相关性在不同的部分酵母基因组。为了验证这些预测，我们计划进行两种类型的实验。第一个将是测量含有由L = 20和25 bp的接头分开的“601”核小体阵列的质粒中的连接数差异（与S. Grigoryev，Penn State）.我们也将分析核小体在酵母中转录激活后的重排。使用上述组合的MNase/exoIII消化对于以最高可能分辨率绘制核小体是必不可少的。