Nucleosome Structure And Dynamics Investigated With Single Molecule Methods PDF Download

Nucleosomes, the basic building blocks of chromatin, condense linear DNA eventually into a chromosome, and serve as the platform of epigenetic information. DNA and histone modifications, histone variants, and nucleosome positioning form an epigenetic signal network, regulate various genome transactions such as transcription, replication, and DNA repair. It is crucial to learn how these epigenetic marks affect the structure and dynamic of nucleosomes and chromatin in order to understand their biophysical roles in epigenetic mechanisms for gene regulation.Single molecule fluorescence methods provide efficient means to study unsynchronized dynamics in a complex system. Fluorescence resonance energy transfer (FRET), serving as a molecular ruler, has helped solving many important biological problems. Variations of single molecule FRET (smFRET) are good methods to study the structure and dynamics of nucleosome, while there are still some technical barriers to overcome. I have developed two smFRET methods to study two problems in nucleosome biophysics. One method I developed was to study fast and unsynchronized nucleosomal DNA termini opening motion and how it is affected by salt conditions, DNA sequence, and histone H3 variation. My results indicate that nucleosome termini repeatedly open and close at 0.3~1 ms-1. The kinetics depends on salt and DNA-histone interactions but not much on DNA sequence, suggesting that this dynamics is universal, imposing the ultimate kinetic limit to gene access that can be modulated by histone variation or modification.The other method was to study transient interactions between nucleosomes and how they are affected by two histone acetylation marks and one SUMOylation mark. I found that internucleosomal interactions are inhibited by H4K16 acetylation (H4K16ac), H4K12 acetylation (H4K12ac), and H4K12 sumoylation (suH4). However, my single molecule observation suggests that the extents of inhibition in the long- and short-range internucleosomal interactions are different in each case. suH4 and H4K16ac inhibit long-range internucleosomal interactions more than the H4K12ac case. H4K16ac and H4K12ac inhibit short-range internucleosomal interactions more than the suH4 case. These observations led us to a conclusion that H4K16 acetylation inhibits nucleosome array folding by weakening both long- and short-range internucleosomal interactions, making this acetylation the most efficient way to inhibit nucleosome array folding, while H4K12ac and suH4 weaken mainly short-range and long-range interactions, respectively, making them less efficient in array folding inhibition than H4K16ac.My research demonstrates that single molecule methods can provide efficient means to study fast and unsynchronized dynamics of complex systems that are difficult to probe with conventional structural biology tools.