BPS2026 – Wielding DNA looping and supercoiling to understand nucleosome structure and dynamics

The goal of my PhD research project is to wield DNA looping and supercoiling to understand nucleosome structure and function. In cells, DNA is kept looped and slightly underwound (negatively supercoiled) relative to the well-known relaxed B-form DNA, and during transcription or replication, DNA becomes transiently extremely underwound or overwound. Chromatin is the structural dynamic framework that eukaryotic cells use to condense and protect DNA within the nucleus. Dysregulation of chromatin can promote many diseases, such as cancer, metabolic diseases, and neurological disorders. Nucleosomes are the building block of chromatin and are made up of DNA wrapped around a histone octamer. Most studies of nucleosomes use linear DNA and fail to capture the multitude of changes to DNA caused by looping and negative supercoiling. This failure means that there remains much we do not understand about nucleosome structure and function. Existing experimental data and structures of nucleosomes bound to DNA require special methods and sequences that bind DNA to histones. Although nucleosomes will only reconstitute on linear DNA containing nucleosome positioning sequences, I have reconstituted nucleosomes on negatively supercoiled minicircle DNA without such sequences. My results show that, compared to linear DNA, negatively supercoiled DNA allows nucleosomes to remain constituted at higher NaCl, indicating increased stability. I have imaged nucleosomes reconstituted on 336 bp negatively supercoiled DNA minicircles using cryo-electron microscopy and have seen both di-and mono-nucleosomes. In ongoing work, I have so far successfully produced an ∼8 Å structure. This work will advance the understanding of higher order chromatin dynamics and inform studies of chromatin dysregulation/dysfunction-related diseases.