Multiscale Chromatin Tracing of Domains and Compartments in Single Chromosomes

Abstract

How chromatin is folded and compacted in the cell nucleus has been a long-standing open question in cell biology. In particular, higher-order chromatin organization during interphase has been a very active area of research over the past two decades. The development of Hi-C has catapulted this latest wave of research and revealed structural features such as topologically associating domains (TADs), A/B compartments, as well as CTCF-anchored loop domains. At the beginning of my thesis research period, these intriguing results have led to an elevated interest in understanding whether these structural features are more than emergent properties of population averaging and if so, how they manifest and what their functional implications and dynamic properties are in single cells. My thesis research has focused on developing imaging-based tools address these questions in both fixed and live cells. In Chapter 2, my colleagues and I developed a multiplexed FISH-based chromatin tracing tools capable of mapping chromatin organization in single cells. In subsequent chapters, we systematically investigated the spatial organization of chromatin domains and compartments in single chromosomes across different length scales and how they correlate with nascent transcription. On the whole chromosome level, we observed spatial segregation of A/B compartments in several autosomes and revealed distinct compartment organization of the active and inactive copies of chromosome X. Using both super-resolution and conventional fluorescence microscopy, we revealed the existence of highly variable chromatin domains in single chromosomes that preferentially form boundaries at CTCF/cohesin-binding sites. Interestingly, the depletion of cohesin did not remove these chromatin domains, suggesting a potential cohesin-independent mechanism for the formation and maintenance of these structures. We further revealed that these chromatin domains preferentially interact with other domains containing genomic regions carrying similar ensemble A/B compartment identities, hence providing a physical mechanism on how single cell structures can give rise to ensemble structural features such as TADs and A/B compartments. Finally, we found that nascent transcription is influenced by the A/B microenvironment within the chromosome and A/B compartment segregation, but not relative positions within chromatin domains. Lastly, in Chapter 4, we report a two-color CRISPR labeling system that could be used for live chromatin imaging in human cells and validated it by simultaneously labeling telomeres and centromeres. Upon further modification, the system can potentially be used in conjunction with the chromatin tracing technology to gain further insights into the structure and dynamic of chromatin in cells.