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DNA Folding in Chromatin at the Supra-nucleosome Level

核小体上水平的染色质 DNA 折叠

基本信息

批准号：
10926081
负责人：
Victor Zhurkin
金额：
$ 51.39万
依托单位：
DIVISION OF BASIC SCIENCES - NCI
依托单位国家：
美国
项目类别：
财政年份：
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/10926081
关键词：
Address Agreement Biological Assay Breast Cancer Cell Breast Cancer Patient Cancer Diagnostics Cell Nucleus Cell physiology Cells Chromatin Chromatin Fiber Chromatin Structure Chromophobe Renal Cell Carcinoma Clear cell renal cell carcinoma Clinic Collaborations Coupled DNA DNA Binding DNA Folding DNA Methylation DNA Sequence DNA-Directed RNA Polymerase Data Diagnostic Disease Electron Microscope Equilibrium Family Fiber Foundations Future Gel Gene Expression Regulation Genes Genetic Polymorphism Genetic Transcription Genome Genomic DNA Geometry Goals Heterochromatin Histones Human Knowledge Length Link Linker DNA Malignant Neoplasms Maps Medical Methodology Molecular Conformation Monitor Normal tissue morphology Nucleic Acid Regulatory Sequences Nucleosomal Repeat Length Nucleosomes Nucleotides Organism Paper Patients Positioning Attribute Prevalence Printing Proteins RNA Regulation Renal Cell Carcinoma Research Personnel Resolution Roentgen Rays Side Solid Stratification Structure Structure-Activity Relationship Superhelical DNA Techniques Time Tissues Transcription Process Tumor Tissue Universities Urologic Oncology Variant Yeasts anticancer research cancer type cell free DNA cellular imaging cohort density genome-wide genome-wide analysis in vivo liquid biopsy malignant breast neoplasm neoplastic cell novel stem tomography transcription factor tumor ultra high resolution

项目摘要

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. This allowed us to resolve 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 characterized 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 develop solid theoretical foundations for cfDNA-based nucleosomics analysis for patient diagnostics, monitoring and stratification. The paper describing these results has been submitted to Cancer Research; see also bioRxiv preprint: https://doi.org/10.1101/2023.04.17.537031. Future studies are needed to apply this methodology to larger patient cohorts and more cancer types. Therefore, we established a collaboration with W.M. Linehan (Urologic Oncology Branch, NCI) to compare nucleosome landscapes in several types of renal cell carcinoma (clear cell, sarcomatoid and chromophobe renal cell carcinoma). The initial results suggest that there are significant rearrangements of nucleosomes in the tumor tissues (compared to the healthy ones). Importantly, we observe a strong correlation between the level of aggressiveness of the tumor cells and the changes in nucleosome positioning, characterized numerically as a decrease in nucleosome repeat length (in progress).

基因组功能的变化与染色质空间组织的变化相结合。在过去的一年中，我们获得了一个详细的全基因组信息的染色质重组在核小体水平上的乳腺癌。真核DNA紧密地堆积在细胞核中，然而它的序列被许多调节所必需的蛋白质因子有效地识别。为了阐明这种识别的结构机制，人们需要有关于DNA组织的第二级或30纳米纤维的详细信息。为了解决这个问题，我们之前计算了具有DNA接头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构型。结果，我们观察到两种不同的构象家族（即，拓扑异构体），其特征在于不同的DNA拓扑结构。纤维的最佳几何形状取决于接头长度：接头L = 10 n和10 n +5 bp的纤维的每个核小体的DNA连接数δ（Lk）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。这使我们能够解决长期存在的被称为联系数悖论的差异。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RNA聚合酶上游和下游产生不同水平的DNA超螺旋。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 = 10 n和10 n +5 bp）不仅在平衡构型和平均DNA连接数上不同，而且在动力学上也不同。特别是，新的10 n +5拓扑异构体的特征在于增加的可塑性，这使得染色质更容易接近转录因子（TF）。此外，基因组DNA可以通过部分解开核小体来获得。有趣的是，看看染色质结构，拓扑结构和基因调控之间的这种结构-功能关系是否也存在于人类中。为此，我们彻底研究了乳腺癌（BRC）细胞中的核小体重新定位（与英国埃塞克斯大学的V. Teif合作）。我们在来自相同BRC患者的配对肿瘤和正常组织中生成高分辨率核小体图，并将其与相应的无细胞DNA（cfDNA）进行比较。肿瘤组织的特征在于：（1）单个核小体在关键调控区重新定位，（2）部分核小体解包裹的全基因组增加，以及（3）NRL减少5-10 bp。这些影响受到核苷酸含量和DNA序列重复的存在的调节，并与差异DNA甲基化和稳定染色体的接头组蛋白变体H1.4和H1 X的结合有关。肿瘤组织中NRL缩短的观察开启了许多关于癌症中核小体到细胞水平重组的重要问题，这些问题需要在未来进行额外的分析。在实践方面，我们首次分析了来自相同患者的配对正常组织和肿瘤组织中的核小体定位，并为先前在cfDNA中观察到的并在临床中经验性使用的效果提供了可能的解释。此外，我们的发现为液体活检的NRL相关分析开辟了新的途径。总之，这项研究使我们能够为基于cfDNA的核组学分析开发坚实的理论基础，用于患者诊断，监测和分层。描述这些结果的论文已提交给Cancer Research;另见bioRxiv预印本：https://doi.org/10.1101/2023.04.17.537031。未来的研究需要将这种方法应用于更大的患者队列和更多的癌症类型。因此，我们与W.M.建立了合作关系。Linehan（泌尿肿瘤学分支，NCI）比较几种类型肾细胞癌（透明细胞、肉瘤样和嫌色肾细胞癌）的核小体景观。初步结果表明，在肿瘤组织中存在显著的核小体重排（与健康组织相比）。重要的是，我们观察到肿瘤细胞的侵袭性水平与核小体定位的变化之间存在很强的相关性，其在数字上表征为核小体重复长度的减少（进行中）。

项目成果

期刊论文数量（18）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Topological polymorphism of the two-start chromatin fiber.

DOI：
10.1016/j.bpj.2015.04.015
发表时间：
2015-05
期刊：
Biophysical journal
影响因子：
3.4
作者：
D. Norouzi;V. Zhurkin
通讯作者：
D. Norouzi;V. Zhurkin

Topological polymorphism of nucleosome fibers and folding of chromatin.

DOI：
10.1016/j.bpj.2021.01.008
发表时间：
2021-02-16
期刊：
Biophysical journal
影响因子：
3.4
作者：
Zhurkin VB;Norouzi D
通讯作者：
Norouzi D

Trajectories of microsecond molecular dynamics simulations of nucleosomes and nucleosome core particles.

核小体和核小体核心颗粒的微秒分子动力学模拟轨迹。

DOI：
10.1016/j.dib.2016.04.073
发表时间：
2016
期刊：
Data in brief
影响因子：
1.2
作者：
Shaytan,AlexeyK;Armeev,GrigoriyA;Goncearenco,Alexander;Zhurkin,VictorB;Landsman,David;Panchenko,AnnaR
通讯作者：
Panchenko,AnnaR

Regulation of chromatin folding by conformational variations of nucleosome linker DNA.