DNA Folding in Chromatin at the Supra-nucleosome Level

基因组功能的变化与基因组空间组织的变化相结合。 染色质在过去的一年中，我们获得了关于染色质的详细的全基因组信息 乳腺癌在核小体水平的重组。真核生物的DNA紧密地包裹在 细胞核，然而它的序列被许多蛋白质因子有效地识别 对于监管至关重要。为了阐明这种识别的结构机制，需要 有关于第二层次的DNA组织，或30纳米纤维的详细信息。到 为了解决这个问题，之前我们计算了双头的所有可能配置 染色质纤维与DNA接头L = 10 - 70 bp（核小体重复长度，NRL = 157 - 217 bp）。结果，我们观察到两种不同的构象家族（即，拓扑异构体） 以不同的DNA拓扑结构为特征。光纤的最佳几何形状取决于 接头长度：接头L = 10 n和10 n +5 bp的纤维具有每个接头的DNA连接数。 核小体δ（Lk）分别为-1.5和-1.0。换句话说，DNA的水平 超螺旋与染色质中的核小体间距直接相关。因此，我们做出了一个 这是解决长期存在的联系号码不一致问题的重要一步 悖论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，这是已知的产生不同水平的 RNA聚合酶上游和下游的DNA超螺旋。一个全基因组的分析， NRL在酵母基因中的分布证实了这一假设。我们还发现这两种纤维 拓扑异构体（L = 10 n和10 n +5 bp）不仅在平衡构型上不同， 平均DNA连接数，以及它们的动力学。特别是，小说10 n +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近 转录因子（TF）。此外，基因组DNA可以通过部分 核小体的展开有趣的是，这种结构-功能关系 染色质结构、拓扑学和基因调控之间的关系也存在于人类中。为此， 我们彻底研究了乳腺癌（BRC）细胞中核小体的重新定位（在 与V. Teif，埃塞克斯大学，英国合作）。我们制作了高分辨率的核小体 图在配对的肿瘤和正常组织从相同的BRC患者，并比较这些与 相应的无细胞DNA（cfDNA）。肿瘤组织的特征是：（1）单个核小体 在关键调控区域的重新定位，（2）部分核小体在全基因组范围内的增加 解缠绕和（3）NRL减少5-10 bp。这些作用受到核苷酸的调节 含量和DNA序列重复的存在，并与差异DNA甲基化有关 以及稳定染色体的接头组蛋白变体H1.4和H1 X的结合。的 肿瘤组织中NRL缩短的观察揭示了许多关于 癌症中核小体到细胞水平的重组，需要在 未来在实践方面，我们第一次分析了核小体定位在配对 正常组织和肿瘤组织，并提供了一个可能的解释， 先前在cfDNA中观察到并在临床上经验性使用的效果。另外我们 这一发现为液体活检的NRL相关分析开辟了新的途径。总的来说，这 这项研究使我们能够为基于cfDNA的核组学建立坚实的理论基础 用于患者诊断、监测和分层的分析。未来的研究需要 将这种方法应用于更大的患者群体和更多的癌症类型。

Changes in genome functioning are coupled with the changes in spatial organization of chromatin. In the past year, we obtained a detailed genome-wide information about chromatin reorganization in breast cancer at the nucleosome level. Eukaryotic DNA is tightly packed in the nucleus, nevertheless its sequence is effectively recognized by numerous protein factors essential for regulation. 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, earlier 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. It is interesting to see if this structure-function relationship between chromatin structure, topology and gene regulation also exists in humans. To this aim, we thoroughly investigated nucleosome repositioning in breast cancer (BRC) cells (in collaboration with V. Teif, Essex University, UK). We generated high-resolution nucleosome maps in paired tumor and normal tissues from the same BRC patients and compared these with the corresponding cell-free DNA (cfDNA). Tumor tissues were characterised by (1) single-nucleosome repositioning at key regulatory regions, (2) genome-wide increase in partial nucleosome unwrapping and (3) decrease in NRL by 5-10 bp. These effects were modulated by the nucleotide content and the presence of DNA sequence repeats and linked to differential DNA methylation and binding of linker histone variants H1.4 and H1X which stabilize chromatosomes. The observation of NRL shortening in tumor tissues opens a number of important questions about the nucleosome-to-cell level reorganization in cancer that require additional analyses in the future. On the practical side, we analyzed for the first time nucleosome positioning in paired normal and tumor tissues from the same patients and provided a possible explanation for the effects previously observed in cfDNA and used empirically in the clinic. In addition, our findings opened a new venue for NRL-related analyses of liquid biopsies. Taken together, this study allows us to establish solid theoretical foundations for cfDNA-based nucleosomics analysis for patient diagnostics, monitoring and stratification. Future studies are needed to apply this methodology to larger patient cohorts and more cancer types.