Spectral imaging to visualize higher-order genomic organization

Abstract

A concern in the field of genomics is the proper interpretation of large, high-throughput sequencing datasets. The use of DNA FISH followed by high-content microscopy is a valuable tool for validation and contextualization of frequently occurring gene pairing events at the single-cell level identified by deep sequencing. However, these techniques possess certain limitations. Firstly, they do not permit the study of colocalization of many gene loci simultaneously. Secondly, the direct assessment of the relative position of many clustered gene loci within their respective chromosome territories is impossible. Thus, methods are required to advance the study of higher-order nuclear and cellular organization. Here, we describe a multiplexed DNA FISH technique combined with indirect immunofluorescence to study the relative position of 6 distinct genomic or cellular structures. This can be achieved in a single hybridization step using spectral imaging during image acquisition and linear unmixing. Here, we detail the use of this method to quantify gene pairing between highly expressed spliceosomal genes and compare these data to randomly positioned in silico simulated gene clusters. This is a potentially universally applicable approach for the validation of 3C-based technologies, deep imaging of spatial organization within the nucleus and global cellular organization.

Cajal body function in genome organization and transcriptome diversity

“…We have recently provided new evidence to support a genome-wide organizational role for the CB [30, 40]. Using a combination of advanced multicolor microscopic mapping tools and high-throughput sequencing techniques, we explored the specific clustering network, consisting of distant chromosomal targeting regions proximal to CBs [30].…”

“…However, NBs are multifunctional structures that influence the DNA damage response, protein and RNA modifications, as well as gene activity [1]. Importantly, CBs are known to assemble at specific gene loci, including both spliceosomal snRNA genes and histone gene loci (through its physical association with the HLB) [22–26, 30, 40]. Thus, CBs concentrate essential but frequently limited factors for expedited snRNA transcription, extended 3’end RNA processing and snRNP maturation processes at target snRNA gene loci.…”

Specific genomic cues regulate Cajal body assembly

“…In particular, the CB has long been known to be frequently associated with specific genomic loci enriched with U snRNA and intron-encoded snoRNA, scaRNA and histone gene loci. 15,20,[40][41][42][43][44][45] However, it has not been fully explored and genomically mapped whether the initiation of CB formation is restricted to a limited number of prominent primary nucleation sites (such as the highly expressed and prominent RNU1 and RNU2 gene arrays 46 or histone gene clusters) or other highly expressed individual gene loci or repeats. It is unknown whether these must be positioned either in cis configurations on the same chromosome or in trans on different chromosomes to trigger CB formation.…”

“…Finally, the growing CB attracts other target genes in cis and trans configurations to its physical proximity. 20,44,71 It is unclear whether these target gene loci may possess their own smaller CBs (that are sub-microscopic) prior to amalgamation into a larger structure, 94 or if there are only moderate local concentrations of coilin and the various sn/snoRNA transcriptional and processing complexes. Nevertheless, the CB induces a topological rearrangement of the genome and is seen to frequently associate with several target gene loci simultaneously, while pushing away nearby non-target genomic regions.…”

“…Nevertheless, the CB induces a topological rearrangement of the genome and is seen to frequently associate with several target gene loci simultaneously, while pushing away nearby non-target genomic regions. 20,44 The biological consequences of this cluster formation around the CB is not apparent and further work is required to describe the stability and benefit of these higher-order gene association events. Several studies have indicated that CBs are capable of both fusion and fission, but it is unknown with which genomic loci these CBs are associated.…”