Higher-Order RNA and DNA Hubs Shape Genome Organization in the Nucleus

Citation

Quinodoz, Sofia Agustina (2020) Higher-Order RNA and DNA Hubs Shape Genome Organization in the Nucleus. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/k33x-9k36. http://resolver.caltech.edu/CaltechTHESIS:03132019-143409731

Abstract

Although the entire genome is present within the nucleus of every cell, distinct genes need to be accessed and expressed in different cellular conditions. Accordingly, the nucleus of each cell is a highly organized arrangement of DNA, RNA, and protein that is dynamically assembled and regulated in different cellular states. These dynamic nuclear structures are largely arranged around functionally related roles and often occur across multiple chromosomes. These include large nuclear bodies (i.e., nucleolus, nuclear speckle), smaller nuclear bodies (i.e., Cajal bodies and histone locus bodies), and gene-gene interactions (i.e., transcription compartments and loops). Yet, what molecular components are involved in establishing this dynamic organization have been largely unknown due to a lack of methods to measure the RNA and DNA components of nuclear bodies and their spatial arrangements in the nucleus. Here, we present Split-Pool Recognition of Interactions by Tag Extension (SPRITE), which enables genome-wide detection of higher-order interactions within the nucleus. In the second chapter, we introduce SPRITE and recapitulate known structures identified by proximity ligation and identify additional interactions occurring across larger distances, including two hubs of inter-chromosomal interactions that are arranged around the nucleolus and nuclear speckles. We show that a substantial fraction of the genome exhibits preferential organization relative to these nuclear bodies. Our results generate a global model whereby nuclear bodies act as inter-chromosomal hubs that shape the overall packaging of DNA in the nucleus. In the third chapter, we provide a detailed experimental protocol for performing SPRITE and an automated computational pipeline for analyzing SPRITE data. Finally, in the fourth chapter, we present a dramatically improved implementation of the SPRITE method that enables comprehensive mapping of all classes of RNA in the nucleus, from abundant RNAs encoded from DNA repeats to low abundance RNAs such as nascent pre-mRNAs and lncRNAs. We find that RNAs localize broadly across the nucleus, with individual RNAs localizing within discrete territories ranging from nuclear bodies to individual topologically associated domains. We uncover that nascent mRNAs interact in structures corresponding to nascent mRNA chromosome territories and compartments. Together, these results uncover a central and widespread role for non-coding RNA in demarcating 3D nuclear structures within the nucleus.

Thesis Files