Imaging the Genome in 3D at Super Resolution
Lauren Gagnon, Ph.D. Bruker Vutara Applications Scientist, Bruker Nano Surfaces Division, 630 Komas Dr., Ste. 230, Salt Lake City, UT 84108
Lauren.Gagnon@
bruker.com
Abstract: The spatial organization of chromatin has become recog- nized as a major factor in the regulation of gene expression. While chromosome conformation capture
techniques, such as
high-
throughput chromosome conformation capture (Hi-C), have advanced our understanding of the role of 3D organization of chromatin on chro- mosomal function, Hi-C data are typically ensemble in nature and relies on intense computational methods to derive genomic structural information. The field is beginning to shift in recognizing the potential value of imaging methods to study spatial organization of chroma- tin. Imaging allows acquisition and analysis at the individual cell level while providing direct visualization and analysis of 3D chromatin orga- nization. This feature will highlight how single-molecule localization microscopy (SMLM) using the Bruker Vutara SMLM system in con- junction with sequential labeling via microfluidics is revolutionizing our understanding of nuclear spatial organization and its role in regulation of gene function.
Keywords: single-molecule localization, chromosomes, chromatin, oligopaints, OligoSTORM, genomics
Introduction DNA organization is complex. Within every human cell,
roughly two meters of DNA is packed by many means into a 5 μm space—the nucleus—in the form of 23 pairs of chro- mosomes. Tere are nucleosomes that are packed and wound into primary, secondary, and tertiary chromatin structures, which are further organized into distinct territories within the nucleus. Te estimated 20–25 thousand genes that make up these chromosomes are responsible for the production of all proteins in the human body. But not every cell in our body expresses every gene. Te expression of the different genes across tissue and across time is regulated by the structure of the genome within the nucleus. Understanding the structure, function, evolution, and mapping of the genome is essential to discovering the genetic basis for health and disease and is the foundation of the field of genomics.
Traditional Approaches to Understanding Genomic Organization How DNA is packaged into the nucleus is one of the fun-
damental questions of genomics because the structure of DNA has a direct impact on gene expression and regulation. Te past two decades since the first chromosome conformation cap- ture (3C) assay was described have seen great advancement in biochemical techniques for mapping genomic interaction [1]. 3C assays began as a means of determining the interactions between two genomic loci by estimating contact frequencies. Since its initial inception, 3C has inspired an abundance of derivative techniques that improve upon the limitations of the original method. Tese include the broader categories of 4C (circularized chromosome conformation capture), 5C (chromosome conformation capture carbon copy), and Hi-C
18 doi:10.1017/S1551929520001546
(high-throughput chromosome conformation capture) assays. While 3C probes one-to-one interaction of two loci, 4C tech- niques expand upon this to look at genome-wide interactions of a single locus (one-to-all). On the other hand, 5C is a many- to-many approach that does not rely on the selection of a sin- gle locus to study, thereby providing more general views on chromosome organization than 3C and 4C. Arguably, though, the greatest advancements in the technology to date have come from the development of Hi-C assays (Figure 1), which use an all-to-all approach to capturing interactions within the genome and have resulted in observations of finer details of genome organization [2]. Researchers have used 3C techniques to discover that
chromosomes are packaged into territories and observe that individual chromosomes mostly interact with themselves, though they do have small regions of interaction with each other. Within a Hi-C map, it is possible to look at individ- ual chromosomes and observe sub-features like compart- ments, topologically associated domains, and individual loops (Figure 1). This organization plays an important role in gene expression. For example, a loop may enable one gene to come in proximity with an enhancer that will then drive expression of that gene. On a linear scale, the enhancer is far away from the gene itself, but interaction is enabled within the loop [3].
Visualizing Genomic Organization with Microscopy Knowing the genetic composition of the genome is essen-
tial, but establishing how genes are organized with one another and their environment is equally important. Being able to visu- alize organization and structure on a sub-chromosomal level is necessary for understanding these relationships and how genes function. However, visualizing genomic structure at this level can prove difficult because of such a large and densely packed number of targets within the genome. Tere simply are not enough chromatically separable probes available to image them using conventional fluorescence microscopy methodolo- gies. Fortunately, new approaches are beginning to allow for sequential labeling of targets using the same fluorescent probe for imaging.
Chromosome Conformation Capture Versus Imaging Chromosome conformation capture techniques such as
Hi-C are ensemble-sequencing techniques, providing an aver- age structure of the genome from millions of cells. Tis pro- vides researchers the ability to look at the entire genome at high resolution. While these techniques provide a good over- view of the average cell, 3C data lose cellular context because
www.microscopy-today.com • 2020 November
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