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This Debye Lunch Lecture, Epigenetic modulation of nucleosome plasticity revealed by high-throughput AFM imaging, will be given by Willem Vanderlinden from Debye's Soft Condensed Matter & Biophysics research group.

Abstract: Nucleosomes, the fundamental units of genome compaction, wrap about 150 base pairs of DNA in 1.7 solenoidal turns around a protein core. Importantly, nucleosomes retain a certain level of plasticity, i.e. DNA wrapping is dynamic to ensure readout and processing of the genetic code. The energy landscape for spontaneous nucleosome wrapping thus governs nucleosome interactions with protein binding partners, but has not been experimentally determined in detail. In a first part of my talk I will discuss how we have exploited the exquisite signal-to-noise level of atomic force microscopy to tackle this issue. To this end, we developed a custom-written high-throughput image analysis platform that quantifies nucleosome conformations at the single molecule level and for thousands of molecules in an automated fashion (Konrad et al., 2021). From the large conformational ensembles, energy barriers to nucleosome unwrapping can be accurately recovered with a resolution <5 base pairs, and it is shown that nucleosome unwrapping occurs anti-cooperatively with approximately equal probability to unwrap via either end.

In the second part of the talk, I will present how we have used our high-throughput platform to investigate modulation of nucleosome plasticity by epigenetic factors. The epigenetic code consists of a set of chemical modifications to the DNA and histone proteins, that are recognized by specific epigenetic readers that govern gene regulation. We focus on the epigenetic modification H3K36Me3 (trimethylation of lysine 36 on histone 3) and the human epigenetic reader protein LEDGF/p75 that tethers cargo proteins such as HIV integrase to H3K36Me3 nucleosomes. We find that H3K36me3 nucleosomes unwrap significantly more than wild-type nucleosomes and additionally unwrap stochastically from both sides, in contrast to the highly anti-cooperative unwrapping seen for unmodified nucleosomes (Konrad et al., 2022). On addition of LEDGF/p75, nucleosome plasticity is further increased in a concentration-dependent manner (Vanderlinden et al., unpublished). Based on mutational analyses, we provide a model for the molecular mechanisms underlying the modulation of nucleosome plasticity by LEDGF/p75. Our results provide insights in the role of LEDGF/p75 as a nucleosome chaperone, and in the nuclear environment of HIV integration.

References

Konrad SF, Vanderlinden W, Frederickx W, Brouns T, Menze BH, De Feyter S, Lipfert J. High-throughput AFM analysis reveals unwrapping pathways of H3 and CENP-A nucleosomes. Nanoscale. 2021 Mar 18;13(10):5435-5447. doi: 10.1039/d0nr08564b.

Konrad SF, Vanderlinden W, Lipfert J. Quantifying epigenetic modulation of nucleosome breathing by high-throughput AFM imaging. Biophys J. 2022 Mar 1;121(5):841-851. doi: 10.1016/j.bpj.2022.01.014