INTRODUCTION
HIGHLIGHT OF TN5-FISH
1 A The upper panel demonstrated the BAC FISH probe (green rectangle box) compared with the Tn5-FISH probe (red rectangle box) marked using triple color. The lower panel demonstrated Tn5-FISH (red) and BAC probe (green) in GM19705 and Platr22. B The upper panel demonstrated Tn5-FISH probes across the beta-Globin loop (90 kb apart) along with its major components (represented in blue and purple rectangle boxes). The lower panel demonstrated Tn5-FISH 3’HS1 (red) probe and the LCR5 probe (green). C The full width at half maximum (FWHM) signal comparison between Tn5-FISH and traditional BAC-FISH. The result suggests that the Tn5-FISH probe is better to resolve smaller genomic components. D The schematic demonstration of Tn5-FISH from identification of the chromatin interaction loci to in situ hybridization (Distributed under the terms of the Creative Commons CC BY license, Niu et al. 2020) |
Six applications for TN5-FISH or FISH
(Tn5-)FISH application in mESCs
2 A Gse1 and Bco1, Uhrf1 and Msh6 are paired genes in CTCF-enriched loci from different Compartment A. Through the employment of 3D-FISH, the change of distance of the paired gene can be imaged and examined. B Welch’s t-test on Gse1 and Bco1, Uhrf1 and Msh6. Gse1 and Bco1 obtained p-value of 5.12 × 10-7 and 5.782 × 10-8 (from left to right) suggesting distance increased upon the absence of Rybp. C A schematic demonstration of inter-compartment interactions under the presence and absence of Rybp along with a demonstration of induced CTCF phase separation in Rybp knocked out cell line (Distributed under the terms of the Creative Commons CC BY license, Wei et al. 2022) |
lncRNA Platr22 regulation on nearby super-enhancers
3 Platr22 imaged using Tn5-based RNA-FISH on mESCs. A Upper panel: the Antisense. Bottom panel: sense control functioned as a control. In the antisense, one to two foci could be identified. The upper panel demonstrated how Platr22 would interact solely within its own genomic region. B A statistical summary of embryonic stem cells contains either none, one, or two spots of Platr22. C A contact ability map based on Hi-C showing the interaction between genomic regions, and the results revealed that Platr22’s tendency to interact within its genomic loci (Distributed under the terms of the Creative Commons CC BY license, Yan et al. 2020) |
Tn5-based FISH in differentiated cells
Validating MyoD as a genome structure organizer in muscle cells
4 A Tn5-based DNA FISH utilized to examine the spatial distancing of the wild-type group compared with E-box deletion (upper panel) and MKO (lower panel). B Statistical analysis confirming the increase in spatial distancing between MyoG (red) and Mybph (green). C The schematic demonstration of how E-box and MyoD assist the folding of DNA (Distributed under the terms of the Creative Commons CC BY license, Wang et al. 2022) |
Validating spatial distance between different genomic loci in subcompartments
5 A The upper panel on the left demonstrated genomic loci L1 and L2 through DNA-FISH. L1 and L2 belong to Subcompartment B1. The bottom panel on the left demonstrated genomic L1 and L3 through DNA-FISH. L3 belongs to Subcompartment B2. B The cumulative distribution function (CDF) on the right shows the spatial distance of L1–L2 (blue line) is closer than L1–L3 (orange line) since L1 and L2 are in the same subcompartment (Abbas et al. 2019). C The blue region marks Subcompartment B, and the red region marks Subcompartment A. L1 and L2 located in Subcompartment B1 (the red hollow circle), and L3 located in the yellow hollow circle of Subcompartment B. L1 and L2 demonstrated to be spatially closer compared with L3. D Chi-seq data showing the distance of different histone marks of L1, L2, and L3 (Distributed under the terms of the Creative Commons CC BY license, Abbas et al. 2019) |
Tn5-FISH application in leukemia cells
6 A The Tn5-based FISH showed that distances between GATA2 promoter (green) and enhancer (red) in the ATRA-treated group demonstrated to be longer than the control group. B Mann-Whitney U-test demonstrated the control group has more interactions between GATA2 promoters and enhancers than the ATRA-treated group. The visual illustration of GATA2 promoter and enhancers under ATRA-induction. C A demonstration of induced-ATRA would increase distances between GATA2 and other enhancers (Distributed under the terms of the Creative Commons CC BY license, Li et al. 2018) |