Category: 3D Visualization and Analysis of Genome Organization

Description: Coupling fluorescence in situ hybridization (FISH) with 3D microscopy and image reconstruction to study spatial organization of the nuclear genome, chromatin topology and interactions between chromosomes/chromatins, which play important roles in transcriptional and epigenetic regulation of the genome function.

The images from the gallery are cited below. Click on a selection in orange to go to the publication.
Images 1, 2

Kubalová, I. et al. (2021) Helical metaphase chromatid coiling is conserved. preprint. Cell Biology. doi:10.1101/2021.09.16.460607.

Boyle, S. et al. (2020) ‘A central role for canonical PRC1 in shaping the 3D nuclear landscape’, Genes & Development, 34(13–14), pp. 931–949. doi:10.1101/gad.336487.120.

Development involves a cascade of temporal and spatial controls. The fidelity of this cascade is under the control of transcription factors acting together with the epigenome to regulate gene expression. A group of epigenetic regulators known as polycomb repressive complexes (PRCs) work to place the chromatin in a transcriptionally silent but poised state. Complementing Hi-C data DNA FISH was used in mouse embryonic stem cells to demonstrate how PCR1 specifically contributes to nuclear organization and its role in the 3D topology of the mammalian genome.

Category: Chromosome Structure and Arrangement

Description: Using FISH-based assay to reveal chromosome structures and rearrangements including translocations, segmental duplications/deletions, and presence-absence variations, which is essential for studying recombination between chromosomes, chromosome synteny and evolution, massive gene duplication and amplification events, etc.

Images 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13

do Vale Martins, L. et al. (2019) ‘Meiotic crossovers characterized by haplotype-specific chromosome painting in maize’, Nature Communications, 10(1), p. 4604. doi:10.1038/s41467-019-12646-z.

The generation of hybrids is crucial in the plant and animal industries. During meiotic crossover events DNA is exchanged between the homologous parental chromosomes. This exchange of DNA is pivotal in the generation of genetic diversity. Utilizing myTags fluorescent probes generated for haplotype analysis do Vale Martins e al. were able to effectively identify parental and recombinant regions of chromosome 10 in hybrids and F2 progeny of Maize B73 and Mo70 hybrids.

Nozawa, R.-S. et al. (2017) ‘SAF-A Regulates Interphase Chromosome Structure through Oligomerization with Chromatin-Associated RNAs’, Cell, 169(7), pp. 1214-1227.e18. doi:10.1016/j.cell.2017.05.029.

Beliveau, B.J. et al. (2012) ‘Versatile design and synthesis platform for visualizing genomes with Oligopaint FISH probes’, Proceedings of the National Academy of Sciences, 109(52), pp. 21301–21306. doi:10.1073/pnas.1213818110.

Applications of synthetic oligonucleotides in FISH is a powerful tool that can be used in any organism whose genome has been sequenced. The probes are renewable, reliable, and able to label both DNA and RNA sequences in cell culture, fixed tissue and metaphase chromosome spreads. Using a bioinformatic platform for design the probes precisely target desired sequences and allow for single and multicolor imaging of regions that range from kilobases to megabases in length.

Albert, P.S. et al. (2019) ‘Whole-chromosome paints in maize reveal rearrangements, nuclear domains, and chromosomal relationships’, Proceedings of the National Academy of Sciences, p. 201813957. doi:10.1073/pnas.1813957116.

Piperidis, N. and D’Hont, A. (2020) ‘Sugarcane genome architecture decrypted with chromosome‐specific oligo probes’, The Plant Journal, 103(6), pp. 2039–2051. doi:10.1111/tpj.14881.

Šimoníková, D. et al. (2019) ‘Chromosome Painting Facilitates Anchoring Reference Genome Sequence to Chromosomes In Situ and Integrated Karyotyping in Banana (Musa Spp.)’, Frontiers in Plant Science, 10, p. 1503. doi:10.3389/fpls.2019.01503.

Boyle, S. et al. (2020) ‘A central role for canonical PRC1 in shaping the 3D nuclear landscape’, Genes & Development, 34(13–14), pp. 931–949. doi:10.1101/gad.336487.120.

Boyle et al., Genes and Development, 2022. A central role for canonical PRC1 in shaping the 3D nuclear landscape.

Category: Chromosome Identification, Karyotyping and Chromosome Painting

Description: Using oligo-FISH to develop chromosome identification/barcode system, allowing karyotyping based on individually identified chromosomes and studies of chromosome-scale genetic adaptation and evolution; Chromosome/haplotype-specific painting using chromosome or haplotype-specific oligo probes can be applied to research in chromosomal evolution, monitoring chromosome paring and transmission and assessing genomic sequence assembly.

Images 14, 15, 16, 17, 18, 19, 20, 21, 22

Beliveau, B.J. et al. (2012) ‘Versatile design and synthesis platform for visualizing genomes with Oligopaint FISH probes’, Proceedings of the National Academy of Sciences, 109(52), pp. 21301–21306. doi:10.1073/pnas.1213818110.

Albert, P.S. et al. (2019) ‘Whole-chromosome paints in maize reveal rearrangements, nuclear domains, and chromosomal relationships’, Proceedings of the National Academy of Sciences, p. 201813957. doi:10.1073/pnas.1813957116.

do Vale Martins, L. et al. (2019) ‘Meiotic crossovers characterized by haplotype-specific chromosome painting in maize’, Nature Communications, 10(1), p. 4604. doi:10.1038/s41467-019-12646-z.

Hou, L. et al. (2018) ‘Chromosome painting and its applications in cultivated and wild rice’, BMC Plant Biology, 18(1). doi:10.1186/s12870-018-1325-2.

Šimoníková, D. et al. (2019) ‘Chromosome Painting Facilitates Anchoring Reference Genome Sequence to Chromosomes In Situ and Integrated Karyotyping in Banana (Musa Spp.)’, Frontiers in Plant Science, 10, p. 1503. doi:10.3389/fpls.2019.01503.

Piperidis, N. and D’Hont, A. (2020) ‘Sugarcane genome architecture decrypted with chromosome‐specific oligo probes’, The Plant Journal, 103(6), pp. 2039–2051. doi:10.1111/tpj.14881.

Beliveau, B.J. et al. (2012) ‘Versatile design and synthesis platform for visualizing genomes with Oligopaint FISH probes’, Proceedings of the National Academy of Sciences, 109(52), pp. 21301–21306. doi:10.1073/pnas.1213818110.

Category: Oligonucleotides Based Fluorescent in situ Hybridization (Oligo-FISH)

Description: Oligo-FISH, which utilizes custom-designed synthetic single stranded oligonucleotides probes, is the fundamental technique required for chromosome identification, chromosome painting, chromosome rearrangement and study of three-dimensional chromatin organization, etc.

Images: 23, 24, 25, 26, 27, 28

Beliveau, B.J. et al. (2012) ‘Versatile design and synthesis platform for visualizing genomes with Oligopaint FISH probes’, Proceedings of the National Academy of Sciences, 109(52), pp. 21301–21306. doi:10.1073/pnas.1213818110.

Albert, P.S. et al. (2019) ‘Whole-chromosome paints in maize reveal rearrangements, nuclear domains, and chromosomal relationships’, Proceedings of the National Academy of Sciences, p. 201813957. doi:10.1073/pnas.1813957116.

do Vale Martins, L. et al. (2019) ‘Meiotic crossovers characterized by haplotype-specific chromosome painting in maize’, Nature Communications, 10(1), p. 4604. doi:10.1038/s41467-019-12646-z.

Hou, L. et al. (2018) ‘Chromosome painting and its applications in cultivated and wild rice’, BMC Plant Biology, 18(1). doi:10.1186/s12870-018-1325-2.

Category: Evaluation of Gene Expression and Regulation

Description: Visualization of chromatin interactions and spatial arrangement/localization of target genes or mRNA transcripts using oligo-FISH allows for investigation of transcriptional and epigenetic regulation activities.

Images: 29, 30, 31, 32

Mirabelli, C. et al. (2021) ‘Morphological cell profiling of SARS-CoV-2 infection identifies drug repurposing candidates for COVID-19’, Proceedings of the National Academy of Sciences, 118(36), p. e2105815118. doi:10.1073/pnas.2105815118.

The global pandemic of SARS CoV2 initiated the search for therapeutic interventions that could be rapidly identified and moved into therapeutic use. Drug repurposing can quickly move therapeutics into use. Utilizing bioinformatic design synthetic oligos were generated to recognize the SARS-CoV2 (+) strand in both human and African green monkey cells. High-throughput screening assays for 1425 FDA approved drugs were done on cells growing in 385 well plates. Analysis of infection rate and morphological compartmentalization of the virus was determined utilizing a combination of viral genomic RNA in situ hybridization and immunocytochemistry for viral specific proteins.

Carmen Mirabelli, et al. Proceedings of the National Academy of Sciences Sep 2021, 118 (36) e2105815118; DOI: 10.1073/pnas.2105815118

Boyle, S. et al. (2020) ‘A central role for canonical PRC1 in shaping the 3D nuclear landscape’, Genes & Development, 34(13–14), pp. 931–949. doi:10.1101/gad.336487.120.

Boyle et al., (2022) Genes and Development, A central role for canonical PRC1 in shaping the 3D nuclear landscape.

Beliveau, B.J. et al. (2012) ‘Versatile design and synthesis platform for visualizing genomes with Oligopaint FISH probes’, Proceedings of the National Academy of Sciences, 109(52), pp. 21301–21306. doi:10.1073/pnas.1213818110.

Category: Super-resolution Imaging of Nuclear Organizations and Structures

Description: Oligo-FISH in combination with super-resolution imaging techniques allows for analyzing the subcellular localization and spatial arrangement of targeted DNA sequences and RNA transcripts at the single cell level.

Images: 33

Kubalová, I. et al. (2021) Helical metaphase chromatid coiling is conserved. Preprint. Cell Biology. doi:10.1101/2021.09.16.460607.

Multiple applications were used to elucidate the chromosome organization in the large chromosomes of barley. Super resolution microscopy was used to detect the localization of fluorescent oligo nucleotides designed specifically to analyze the morphological structure of the helical organization. The data determined the presence of a 400 nm chromatin fiber in a helical arrangement. The number of turns was correlated with the length of the chromatid arm. Reviewing data from non-plant published data it was determined the helical turning of metaphase chromatid arms is conserved across large chromosomes.