Methods for visualizing a whole human chromosome in super resolution and distinguishing maternal and paternal homologs genome-wide

Abstract

Chromatin is organized into discrete units called chromosomes, which form chromosome territories (CTs) in interphase cells. Chromatin and chromosome organization play a critical role in genome regulation and inheritance, with misfolding leading to diseases such as developmental disorders and cancers. Thus, these fundamental features of chromatin organization and their consequences are being intensely studied across genomic scales. The process, however, is complex and demanding. For example, at the single-cell level, the genome exhibits extensive structural variability, making it challenging to establish structure-function relationship. Furthermore, using allele-specific techniques, variability has been observed between homologs at known imprinted regions and between the two X chromosomes during X-chromosome inactivation. Yet, how sub-chromosomal regions fold across an entire chromosomal fiber to form CTs in single cells and how the paternal and maternal homologs differ remain challenging to address. In my dissertation, I describe the development of two in situ imaging technologies. The first method enables imaging of an entire chromosome in super-resolution using OligoSTORM single-molecule localization microscopy (SMLM). We applied this technology to capture human chromosome 19, revealing its 3D volumetric structure from a 1-Mb genomic resolution to the scale of an entire 56-Mb chromosome with nanometer precision. My goal was not only to reveal chromosome 19 in 3D super resolution, but also to capture its 3D volumetric features, including volume and ellipticity, among others. The second method extends the application of Homolog-specific Oligopaints (HOPs), a FISH-based technology, to identify the parent-of-origin (PO) of homologous chromosomes across the entire human genome. We demonstrated the scalability of HOPs by identifying the PO for all 22 pairs of homologous chromosomes, plus the sex chromosomes. Finally, we integrated chromosome-19 HOPs with whole chromosome-19 OligoSTORM imaging, offering a first look at the 3D structures of both the paternal and maternal chromosome 19 with super resolution in human cells. We envision that our OligoSTORM imaging approach could scale to whole-genome studies. Furthermore, these methods together could enhance studies of allelic regulation, such as X-chromosome inactivation and genomic imprinting, and how allelic regulation is linked to allelic chromatin organization at the single-cell level.