DNA Folding in Chromatin and Interaction with Transcription Factors

在2013-2014财年，我们继续努力阐明指导核小体旋转和平移定位的DNA序列模式。特别是，我们开发了一种新的DNA线程算法，正确地预测了在体外和体内精确定位的核小体的定位(与F·崔，Rochester Inst合作)。纽约理工大学)。我们还对许多双链DNA片段进行了全原子能量最小化处理，这些片段经历了类似于在结晶核小体中观察到的构象转变。这种结合的方法使我们向前迈出了重要的一步，朝着理解基因组DNA中加密的核小体密码迈进了一步。核小体中DNA的折叠伴随着相邻碱基对的侧向位移，这一点通常被忽略。然而，我们发现，剪切变形，称为滑动，在DNA折叠中发挥着比之前想象的更重要的作用。首先，在DNA的局部各向异性弯曲部位观察到的横向滑动变形定义了它在染色质中的超螺旋轨迹。其次，在核小体上变形DNA的计算成本是特定于序列的：在最优定位的序列中，最容易变形的碱基对步骤(CA：TG和TA)发生在大的正向滑动和负向滚动的位置(DNA在这些位置强烈弯曲或扭结到小槽中)。在这里，我们纳入了“真实”DNA的所有自由度，从而超越了传统模型的限制，忽略了碱基对的横向滑动位移。注意，我们的结果与体外序列选择(SELEX)实验非常一致。对不同GC含量序列的核小体定位的成功预测表明了我们基于DNA变形能计算的结构分析的潜在优势。最近，我们开发了一种新的计算方法来计算高阶染色质组织的拓扑多态，即所谓的30 nm纤维。(这一点很重要，因为与30 nm原纤维结构相关的问题仍然没有明确的答案。最有可能的是，最初提出的螺线管类型的纤维可能只针对核小体间连接子L=50bp或更长而形成。当连接子L为30bp或更短时，对应于酵母和人类神经元中的染色质，就形成了两个起始的核小体纤维。)我们分析了连接子L=13到37个碱基的两个起始染色质纤维。通过对超螺旋参数对纤维能量的优化，我们发现了纤维中两种类型的拓扑转变：一种是由于连接子DNA扭曲的突变引起的，另一种是由连接子的过度交叉引起的。(第一个转变的特征是DNA连接数的变化，增量(LK)=1，第二个转变的特征是增量(LK)=2。)就我们所知，染色质纤维的这种拓扑多态在之前发表的计算中没有报道。重要的是，具有连接基L=10N和10N+5BP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，如倾斜60-70度(核小体圆盘与纤维轴的夹角)、螺旋上升、直径和纤维的左旋。此外，我们还做了几个可检验的预测，其中包括在L=10N和10N+5BP的纤维中存在不同程度的DNA超卷曲，这两种类型的纤维的硬度不同，以及局部NRL与酵母基因组不同部分的转录水平之间的相关性。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连接物L=20和25个碱基对分隔的核小体阵列的质粒中的连接数差异(与宾夕法尼亚州立大学S.Grigoryev合作)。我们还将分析酵母中转录激活时核小体位置的重排。使用上述结合的MNase/exoIII消化对于以尽可能高的分辨率绘制核小体图谱将是必不可少的。

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.