Analysis of chromatin structure and dynamics with single nucleosome imaging

DNA is the media of hereditary information of living organisms on the earth. In eukaryotes, long genomic DNA is wrapped around core histones to form a nucleosome fiber. In addition, nucleosome fiber is organized three dimensionally as chromatin and folded within a tiny space such as a nucleus or chromosomes. In cellular functions based on DNA transaction including transcription and DNA replication, the cells must precisely execute the readout of information superimposed on genomic DNA. In this situation, a protein as molecular machinery needs to move around such complicated folded DNA and interrogate its target site, and the chromatin becomes a huge obstacle. Thus, chromatin structure and dynamics seem to be deeply related to diverse cellular functions but not merely packaging, and play a fundamental role in epigenetic regulation. Therefore, making the detailed observation and description of chromatin is essential to understand the various life phenomena in eukaryotes. The objective of this thesis is to contemplate the relationship between chromatin and epigenetics. To achieve this objective, multiple methods including genome-wide analysis of epigenetic regulation and live cell imaging of chromatin were performed. First, the epigenetic regulation and promoter types were analyzed based on genome-wide ChIP-Seq and DeepCAGE data. It was found that, in humans, broad promoters but not peak promoters had significant associations with histone modification and nucleosome position. Secondly, single nucleosome imaging was newly developed. To observe single nucleosomes in living mammalian cells, the histone was fused with photoactivatable (PA)-green fluorescent protein (GFP) and expressed in mammalian cells at a very low level. For single nucleosome imaging, an oblique illumination was used to illuminate a limited thin area within the cell. It was found that a small fraction of histone-PA-GFP was spontaneously activated without laser stimulation. Lastly, based on single nucleosome imaging, the chromatin structure and dynamics at single nucleosome level in a living mammalian cell and their variation related to different types of histone modification and chromatin associated proteins were successfully observed. Furthermore, numerous chromatin domains were identified throughout the cell cycle. These results will provide a basis of dynamic and flexible nature of chromatin toward understanding the function of chromatin in eukaryotic cell and allow new insight and perspective on the relationships between chromatin and epigenetics. Notes 慶應義塾大学湘南藤沢キャンパス先端生命科学研究会 2015年度学生論文集