Superresolution imaging of nanoscale chromosome contacts

Yejun Wang,Prasuna Ratna,G V Shivashankar

doi:10.1038/srep42422

Yejun Wang, Prasuna Ratna + Show 1 more

Open Access

https://doi.org/10.1038/srep42422

Copy DOI

Abstract

Co-expression of a specific group of genes requires physical associations among these genes, which form functional chromosomal contacts. While DNA fluorescence in situ hybridization (FISH) pinpoints the localization of genes within the 3D nuclear architecture, direct evidence of physical chromosomal contacts is still lacking. Here, we report a method for the direct visualization of transcription-dependent chromosomal contacts formed in two distinct mechanical states of cells. We prepared open chromatin spreads from isolated nuclei, ensuring 2D rendering of chromosome organization. Superresolution imaging of these chromatin spreads resolved the nanoscale organization of genome contacts. We optimized our imaging method using chromatin spreads from serum+/− cells. We then showed direct visualization of functional gene clusters targeted by YAP (Yes-associated protein) and SRF (Serum response factor) transcription factors. In addition, we showed the association of NF-κB bound gene clusters induced by TNF-α addition. Furthermore, EpiTect ChIP qPCR results showed that these nanoscale clusters were enriched with corresponding transcription factors. Taken together, our method provides a robust platform to directly visualize and study specific genome-wide chromosomal contacts.

Highlights

In this paper, we describe an open chromatin spread system that was developed by modifying our previously described method[22], and implementing the optimized 2D superresolution imaging technique to directly visualize the functional genomic contacts at a nanometer scale resolution
To visualize chromosomal contacts associated with specific transcription factors, we advanced our open chromatin method[22] for chromosomal contact preparation, by incorporating one major modification
We have described a method to systematically visualize functional chromosomal contacts by combining modified open chromatin spreads and superresolution microscopy

Summary

Introduction

We describe an open chromatin spread system that was developed by modifying our previously described method[22], and implementing the optimized 2D superresolution imaging technique to directly visualize the functional genomic contacts at a nanometer scale resolution. Superresolution imaging resolved the short chromatin fragments as structures with more than one DNA fiber associated with 5 S RNA pol[2] and specific transcription factors. In serum-starved cells, we observed few contacts, whereas the amount of contacts increased significantly upon serum stimulation, indicating the functionality of the observed chromosomal contacts Using this method, we directly visualized specific chromosomal contacts, in particular those targeted by transcription factors/cofactors such as YAP, SRF, and NF-κB. It is worth noting that, by seeding cells sparsely onto glass slides, we were able to image chromosomal contacts from one cell without mixing contacts from other cells This method allows us to reveal heterogeneity in the level of specific contacts between cells. Our system could serve as a robust platform to study the molecular mechanisms of contact formation

Methods

Results

Conclusion