Spatial genome organization: contrasting views from chromosome conformation capture and fluorescence in situ hybridization

Abstract

Although important for gene regulation, most studies of genome organization use either fluorescence in situ hybridization (FISH) or chromosome conformation capture (3C) methods. FISH directly visualizes the spatial relationship of sequences but is usually applied to a few loci at a time. The frequency at which sequences are ligated together by formaldehyde cross-linking can be measured genome-wide by 3C methods, with higher frequencies thought to reflect shorter distances. FISH and 3C should therefore give the same views of genome organization, but this has not been tested extensively. We investigated the murine HoxD locus with 3C carbon copy (5C) and FISH in different developmental and activity states and in the presence or absence of epigenetic regulators. We identified situations in which the two data sets are concordant but found other conditions under which chromatin topographies extrapolated from 5C or FISH data are not compatible. We suggest that products captured by 3C do not always reflect spatial proximity, with ligation occurring between sequences located hundreds of nanometers apart, influenced by nuclear environment and chromatin composition. We conclude that results obtained at high resolution with either 3C methods or FISH alone must be interpreted with caution and that views about genome organization should be validated by independent methods.

Label-free imaging of the native, living cellular nanoarchitecture using partial-wave spectroscopic microscopy

“…Live-cell PWS is a natural supplement to superresolution fluorescence techniques, providing quantifiable information about unstained cellular organization to examine the role of the nanoarchitecture on molecular interactions in live cells. In the future, we envision that live-cell PWS can be applied to a broad range of critical studies of structure-function in live cells, leveraging the multimodal potential in conjunction with existing SRM to study: (i) the interaction between chromatin structure and mRNA transport; (ii) the accessibility of euchromatin and heterochromatin to transcription factors (45)(46)(47); (iii) the relationship between chromatin looping, as measured by techniques such as Hi-C, to the physical chromatin structure (6,16,48); (iv) why and how higher-order chromatin structure changes in cancer (14); (v) the role of nuclear architecture as an epigenetic regulator of gene expression (6,16,48); (vi) the effect of metabolism on chromatin structure (40,49); and (vii) the role of chromatin dynamics in stem cell development (50,51).…”

Nanoscale spatial organization of the HoxD gene cluster in distinct transcriptional states

“…20), and a chromatin structure in the nuclear space of single cells, on the other hand, is of great interest and has recently come under discussion (see ref. 21). In this study, we used DNA FISH to show that the two TADs splitting the HoxD locus are distinct chromatin units, which rarely overlap despite their close association in space.…”

FISH Is in the Limelight Again As More Than a Cytogenetical Technique for Metaphase Chromosomes

“…The distribution of TADs is also supported by 3D-FISH (Nora et al 2012). However, some genome organization revealed by chromosome conformation capture techniques could not be confirmed by 3D-FISH (Williamson et al 2014). The inconsistency would be derived from the technical difference between FISH and Hi-C.…”