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Structure and Dynamics of Replication Domains in Single Chromosome Territories of Interphase Nuclei

Xiang, Wanqing

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Abstract

Knowing the three-dimensional organization of chromatin sets the framework for understanding genome regulation. Our picture of higher order chromatin structure insitu however remains fragmentary at many scales, since it is not directly accessible by imaging technologies available today. The recently revealed domain organization of chromatin subunits into sub-megabasepair sized topologically associating domains (TADs), enabled by chromosome conformation capture based techniques, marks a significant advancement in understanding chromatin architecture. Similarly quantitative methods for the analysis of global structure and dynamics of chromatin in single living cells are currently lacking, leaving it unclear how TADs are manifested within a single nucleus and how dynamic topological chromatin interactions are in living cells. To start to address this gap in our knowledge, I set out to systematically probe the basic polymer features of chromatin at the level of replication domains (RDs) in single cells as a basis for a model of higher order chromatin organization. I have addressed both structural and dynamic aspects of RD organization during interphase. Using super-resolution microscopy, I was able to investigate RD organization at unprecedented resolution. I found that the median RD diameter is ~150 nm, significantly smaller than the ~270 nm distance to the nearest neighbor, which leaves sufficient physical space for extended linker regions between RDs. By quantifying correlated motion of neighboring RDs, I could reveal the typical elastic coupling range between RDs to be ~500 nm. Combining super-resolution microscopy with a perturbation experiment I could further obtain evidence for the model that chromatin compaction upon ATP depletion is predominantly mediated by preferential compaction of linker regions between RDs, rather than by compaction of RDs themselves. In addition to these structural parameters of RD organization, I also characterized the diffusional behavior of interphase RDs of single chromosome territories. Tracking 1,372 RDs of 141 chromosome territories allowed me to obtain a global and statistically robust view of interphase chromatin dynamics across the entire nucleus. My data confirms that heterochromatin chromatin is immobile within a few hundred nanometers of the nuclear membrane and nucleolar surface over the time scale of several minutes and that nucleoplasmic dynamics is characterized by anomalous diffusion. I did not observe reproducible directed motion of RDs on the timescale of seconds to a minute. I observed a systematic reduction in chromatin motion as the cell cycle progressed from G1 to late S-phase and an increase in mobility if I artificially increased nuclear volume by allowing cells to grow when DNA replication was inhibited. My observations on native and perturbed chromatin structure and dynamics in nuclei of living cells allow me to propose a comprehensive model of higher order chromatin organization in single cells, that consists of stable structuring units of RDs, which are connected by extended flexible linker domains, whose dynamics are limited by attachment to the nuclear periphery and nucleoli and the available free volume inside the nucleus.

Item Type: Dissertation
Supervisor: Ellenberg, PD Dr. Jan
Date of thesis defense: 9 November 2015
Date Deposited: 03 Nov 2017 12:00
Date: 2017
Faculties / Institutes: The Faculty of Bio Sciences > Dean's Office of the Faculty of Bio Sciences
Subjects: 570 Life sciences
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