"""TSA-Seq"": a novel proximity mapping approach for studying three dimensional genome organization and function"
"The organization of chromatin and its localization in the eukaryotic cell nucleus plays a critical role in gene regulation (Agarwal and Rao, 1998; Brown, 2003; Ferrai et al., 2010) and is important for maintaining normal genome function. However, although it is known that the distribution of chromatin and genes in the eukaryotic cell nucleus is non-random and dynamic, the actual domain organization of chromatin, the mechanisms and dynamics which give rise to this domain organization, and the functional relationship between chromosome domain organization and transcriptional regulation are all unknown. The major technical limitations hindering research related to these problems include the difficulties in direct visualization of large scale chromatin structure, the lack of an efficient mapping approach to identify DNA position and distribution in the three dimensional (3D) nuclear space, and the inability to connect three dimensional microscopic information with the linear genome sequence. In this dissertation, I describe ""TSA-Seq"", a novel proximity mapping approach I have developed to study 3D genome organization. TSA-Seq transfers cytological distance information into a genomic plot, allowing us to detect chromosome spatial organization relative to any nuclear compartment. Using this method, I studied 3D genome organization by mapping the average distances of the entire genome relative to two major nuclear compartments- nuclear speckles and the nuclear lamina. We discovered that proximity to the nuclear lamina or to nuclear speckles correspond to the two extremes of the transcriptionally inactive versus active spectrum, with levels of gene expression and timing of replication distributed along this lamina-speckle-axis such that chromosome regions with the highest transcriptional activity associate closest to nuclear speckles and furthest away from the nuclear lamina. We demonstrated the ability of detecting chromatin loops and mean chromatin compaction rates from a single TSA genomic plot. Since it is a new method, I also included a step-by-step protocol of the TSA-Seq procedure. Further TSA-Seq mapping of multiple nuclear compartments may enable us to deconvolve the overall 3D genome nuclear organization. TSA-Seq mapping of cells in different differentiation stages or physiological states will allow us to investigate the functional impact of genome-wide changes in spatial organization during development and in health and disease. TSA-Seq should be a powerful experimental tool to bring our understanding of the genome organization and function to the next level."
TSA-Seq, 3D genome organization, nuclear speckles, nuclear Lamina
University of Illinois - Urbana-Champaign
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University of Illinois at Urbana-Champaign