DNA Folding in Chromatin at the Supra-nucleosome Level

Division Of Basic Sciences - Nci

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Abstract

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).