title: Superresolution imaging of nanoscale chromosome contacts.
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.
authors.affiliation: Mechanobiology Institute and Department of Biological Sciences, National University of Singapore 117411, Singapore.
authors.forename: Yejun
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Mechanobiology Institute and Department of Biological Sciences, National University of Singapore 117411, Singapore.|FIRC Institute for Molecular Oncology (IFOM), Milan 20139, Italy.
authors.forename: Prasuna
authors.initials: P
authors.lastname: Ratna
authors.affiliation: Mechanobiology Institute and Department of Biological Sciences, National University of Singapore 117411, Singapore.|FIRC Institute for Molecular Oncology (IFOM), Milan 20139, Italy.
authors.forename: G V
authors.initials: GV
authors.lastname: Shivashankar
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D018797:Cell Cycle Proteins; D002460:Cell Line; D002467:Cell Nucleus; D002843:Chromatin / Q000235:genetics / Q000378:metabolism; D047369:Chromatin Immunoprecipitation; D002875:Chromosomes / Q000235:genetics* / Q000378:metabolism*; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods; D051379:Mice; D016328:NF-kappa B / Q000378:metabolism; D010750:Phosphoproteins; D011485:Protein Binding; D026362:Serum Response Factor / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D000091102:YAP-Signaling Proteins
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D018797:Cell Cycle Proteins; D002843:Chromatin; D016328:NF-kappa B; D010750:Phosphoproteins; D026362:Serum Response Factor; D014157:Transcription Factors; D000091102:YAP-Signaling Proteins; C486223:Yap1 protein, mouse