DNA Folding in Chromatin at the Supra-nucleosome Level

真核生物的DNA紧密堆积在细胞核中，但其序列却能被多种蛋白质因子有效识别。为了阐明这种识别的结构机制，我们需要有关于第二级DNA组织或30纳米纤维的详细信息。为了解决这个问题，我们计算了两个起始染色质纤维的所有可能的构型，其中DNA连接子L=10-70bp(核小体重复长度，nr1=157-217bp)。结果，我们观察到了两类不同的构象(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+5BP的纤维每核小体增量的DNA连接数(LK)分别为-1.5和-1.0。换句话说，DNA超卷曲的程度与染色质中的核小体间距直接相关。因此，我们朝着解决长期存在的被称为链接数悖论的矛盾迈出了重要的一步。我们假设上述染色质纤维的拓扑多态可能在转录过程中发挥作用，众所周知，转录过程会产生不同水平的DNA超卷曲，从RNA聚合酶的上游和下游。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，L=10n和10n+5bp的两个纤维拓扑异构体不仅在平衡构型和平均DNA连接数上存在差异，而且在动力学上也存在差异。特别是，新的10n+5拓扑异构体的特征是可塑性增加，这使得染色质更容易被转录因子(TF)访问。此外，基因组DNA是通过部分解开核小体来获得的。力谱测量表明，染色质纤维是如此可移动，以至于在小的外力F=2-3Pn下，核小体失去了堆积，而当F=4-5Pn时，核小体显著解缠。(这些力远远低于RNA聚合酶产生的张力；因此，观察到的解缠对应于“自然”条件。)重要的是，核小体的呼吸是不对称的，一端张开的强度比另一端大得多。我们能够从理论上解释这种非平凡的影响，考虑到描述DNA和组蛋白核心之间相互作用的非线性附着能函数。这一观察结果可能对转录和其他与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的不对称解缠在一端暴露了50-60个碱基(相比之下，对称解缠的情况下两端暴露了20-30个碱基)。因此，核小体的不对称呼吸增加了DNA对转录因子的可及性。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其中一个因素。我们已经开始了体外实验来验证这一假设，分析了由缺少N-尾部的组蛋白H2A和H4重组的核小体的定位。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，将是第一次(据我们所知)证明核小体定位的表观遗传调节。这反过来又为在染色质的三维组织背景下揭示转录因子识别DNA的结构机制开辟了前景。特别是，将DNA包裹在组蛋白核心上可以通过暴露同源DNA位点来促进Tf结合，或者相反，可以通过将Tf位点放置在DNA环内来阻碍结合。这一考虑与p53对DNA的识别直接相关(见第二个项目)。

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).