Walking along chromosomes with super-resolution imaging, contact maps, and integrative modeling

Nir, Guy; Farabella, Irene; Estrada, Cynthia Pérez; Ebeling, Carl G.; Beliveau, Brian J.; Sasaki, Hiroshi; Lee, S. Dean; Nguyen, Son C.; McCole, Ruth B.; Chattoraj, Shyamtanu; Erceg, Jelena; Abed, Jumana AlHaj; Martins, Nuno M. C.; Nguyen, Huy; Hannan, Mohammed A.; Russell, Sheikh; Durand, Neva C.; Rao, Suhas S.P.; Kishi, Jocelyn Y.; Soler-Vila, Paula; Pierro, Michele Di; Onuchic, José N.; Callahan, Steven P.; Schreiner, John; Stuckey, Jeff; Yin, Peng; Aiden, E; Martí‐Renom, Marc A.; Wu, C-ting

doi:10.1371/journal.pgen.1007872

Cited by 215 publications

(199 citation statements)

References 110 publications

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“…In the meantime, we emphasize that the framework is general and additional experimental data other than Hi-C 21,63 can be further incorporated to improve model accuracy. It would be particularly interesting to integrate the Hi-C based model with results obtained from super-resolution imaging 55,[64][65][66][67] to characterize single-cell genome structures.…”

Section: Discussionmentioning

confidence: 99%

Data-driven polymer model for mechanistic exploration of diploid genome organization

Reyes

Johnstone

et al. 2020

Preprint

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Chromosomes are positioned non-randomly inside the nucleus to coordinate with their transcriptional activity. The molecular mechanisms that dictate the global genome organization and the nuclear localization of individual chromosomes are not fully understood. We introduce a polymer model to study the organization of the diploid human genome: it is data-driven as all parameters can be derived from Hi-C data; it is also a mechanistic model since the energy function is explicitly written out based on a few biologically motivated hypotheses. These two features distinguish the model from existing approaches and make it useful both for reconstructing genome structures and for exploring the principles of genome organization. We carried out extensive validations to show that simulated genome structures reproduce a wide variety of experimental measurements, including chromosome radial positions and spatial distances between homologous pairs. Detailed mechanistic investigations support the importance of both specific inter-chromosomal interactions and centromere clustering for chromosome positioning. We anticipate the polymer model, when combined with Hi-C experiments, to be a powerful tool for investigating large scale rearrangements in genome structure upon cell differentiation and tumor progression.

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Section: Discussionmentioning

confidence: 99%

Data-driven polymer model for mechanistic exploration of diploid genome organization

Reyes

Johnstone

et al. 2020

Preprint

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“…A remarkable complexity of chromatin organization, involving several levels of order of DNA coiling around the nucleosome cores and beyond the 30-nm "beads on a string" nucleosomal order, characterizes interphase chromatin and mitotic chromosomes (Mateo et al, 2019;Tan, Xing, Chang, Li, & Xie, 2018;Wang & Hayes, 2006;Bintu et al, 2018). Furthermore, considerable variability of chromatin organization characterizes different parts of genomes, various cell types, sequential developmental or differentiation stages of cells, and the respective cell cycle phases (Bannister & Kouzarides, 2011;Finn et al, 2019;Maleszewska, Wojtas, & Kamińska, 2018;Nir et al, 2018;Tolsma & Hansen, 2019). This variability comprises altered degrees of chromatin compaction (condensation) that represent both divergence in chromatin protein constituents and local epigenetic changes involving DNA and protein modifications such as phosphorylation, acetylation, methylation, among others.…”

Section: Of 14mentioning

confidence: 99%

Assessment of DNA Susceptibility to Denaturation as a Marker of Chromatin Structure

et al. 2019

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The susceptibility of DNA in situ to denaturation is modulated by its interactions with histone and nonhistone proteins, as well as with other chromatin components related to the maintenance of the 3D nuclear structure. Measurement of DNA proclivity to denature by cytometry provides insight into chromatin structure and thus can be used to recognize cells in different phases of the cell cycle, including mitosis, quiescence (G 0 ), and apoptosis, as well as to identify the effects of drugs that modify chromatin structure. Particularly useful is the method's ability to detect chromatin changes in sperm cells related to DNA fragmentation and infertility. This article presents a flow cytometric procedure for assessing DNA denaturation based on application of the metachromatic property of acridine orange (AO) to differentially stain singleversus double-stranded DNA. This approach circumvents limitations of biochemical methods of examining DNA denaturation, in particular the fact that the latter destroy higher orders of chromatin structure and that, being applied to bulk cell populations, they cannot detect heterogeneity of individual cells. Because the metachromatic properties of AO have also found application in other cytometric procedures, such as differential staining of RNA versus DNA and assessment of lysosomal proton pump including autophagy, to avert confusion between these approaches and the use of this dye in the DNA denaturation assay, these AO applications are briefly outlined in this unit as well. C 2019 by John Wiley & Sons, Inc. Basic Protocol: Differential staining of single-versus double-stranded DNA with acridine orange Keywords: acridine orange r apoptosis r autophagy r cell cycle r lysosomes r mitosis r sperm chromatin fertility assay How to cite this article: Darzynkiewicz, Z., Halicka, D. H., Zhao, H., & Li, J. (2019). Assessment of DNA susceptibility to denaturation as a marker of chromatin structure. BASIC PROTOCOL DNA DenaturationThe process of splitting double-stranded DNA (dsDNA) into single strands, which involves breaking hydrogen bonds between the bases in the DNA double-helical structure, is defined as DNA denaturation. DNA in solution undergoes denaturation when heated, and the temperature at which half remains in double-stranded conformation while the other half is single stranded is called the melting temperature (T m ). Because the G-C base pair, maintained by three hydrogen bonds, is stronger that the two-base pairing of AO has unique metachromatic properties: under the correct conditions (AO concentration, ionic strength, temperature), it can differentially stain ssDNA and dsDNA. Specifically, when AO intercalates between the base pairs of dsDNA, its fluorescence is maximally excited at 480 nm, its emission is at 530 nm, and its excitation lifetime is ß2 ns (Kapuscinski & Darzynkiewicz, 1984Kapuscinski, Darzynkiewicz, & Melamed, 1982. These values reflect singlet excitation of AO. The interaction of AO with ssDNA is more complex: it involves the insertion of the AO cation between adj...

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“…Multiplexed super-resolution FISH 12,13 and different orthogonal CRISPR–dCas9 systems 14,15 can be employed to label DNA at large scale and to identify cooperative higher-order chromatin interactions. With super-resolution 16–21 and even electron microscopy with higher resolution 22 , more and more detailed genomic features about regulatory architecture such as chromatin loops, TADs and contact domains could be investigated.…”

Section: Introductionmentioning

confidence: 99%

Imaging chromatin interactions at sub-kilobase resolution Via Tn5-FISH

Zhang

Niu

et al. 2019

Preprint

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There is increasing interest in understanding how the three-dimensional organization of the genome is regulated. Different strategies have been employed to identify chromatin interactions genome wide. However, due to the current limitations in resolving genomic contacts, visualization and validation of these genomic loci with sub-kilobase resolution remain the bottleneck for many years. Here, we describe Tn5 transposase-based Fluorescence in situ Hybridization (Tn5-FISH), a Polymerase Chain Reaction (PCR)-based, cost-effective imaging method, which achieved the co-localization of genomic loci with sub-kilobase resolution, to fine dissect genome architecture at sub-kilobase resolution and to verify chromatin interactions detected by Chromatin Configuration Capture (3C)-derivative methods. Especially, Tn5-FISH is very useful to verify short-range chromatin interactions inside of contact domain and Topologically Associated Domain (TAD). It also offers one powerful molecular diagnosis tool for clinical detection of cytogenetic changes in cancers.the genomic resolution of traditional FISH based on BAC clones is too low to precisely label and to image the interactions within the genomic distance less than 500 Kb, such as inside of a TAD or contact domain, whereas most of the chromatin interactions identified by 3C-based techniques fall into this range [25][26][27] .There are advanced FISH techniques with higher genomic resolution, such as oligopaint 28 , HD-FISH 29 , CasFISH 30 and MB-FISH 31 , etc. But they are either very expensive 28,31 or require complex preparation 29,30 , which is difficult for laboratories that do not routinely use various FISH techniques to adopt and to study genomic loci. Molecular beacon-based FISH 31 can offer a resolution of 2.5 kb, but is also technically complex and lacks cost-effectiveness.Here we report a novel and cost-effective method named Tn5-FISH (Tn5 transposase based fluorescence in situ hybridization) that offers more than one order of magnitude higher resolution than traditional FISH. Tn5-FISH utilized the hyperactive Tn5 transposase for probe library construction, and PCR for probe library amplification and labeling. Foreign DNA sequences can be inserted into host genome with the help of Tn5 transposase through "cut and paste" mechanism 32 , which granted Tn5 transposase the ability to induce the DNA double breaks at insertion site. Efficient segmentation of targeted DNA sequence is favored in probe library construction 33,34 . With the aid of bioinformatic tools, we can obtain the repeat-free DNA sequence suitable for probing via genomic PCR.Then Tn5-FISH was employed to verify the chromatin interactions, for example, the ones inside of type II KRT locus in chromosome 12 of K562 cells measured by Hi-C contact map from published data. Triple-color Tn5-FISH can verify these interactions well, with triple-color traditional BAC FISH as positive control, indicating that Tn5-FISH is suitable to visualize specific genomic loci and chromatin interactions inside a typical TAD, in ei...

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Walking along chromosomes with super-resolution imaging, contact maps, and integrative modeling

Cited by 215 publications

References 110 publications

Data-driven polymer model for mechanistic exploration of diploid genome organization

Data-driven polymer model for mechanistic exploration of diploid genome organization

Assessment of DNA Susceptibility to Denaturation as a Marker of Chromatin Structure

Imaging chromatin interactions at sub-kilobase resolution Via Tn5-FISH

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