Innovative Tools for DNA Topology Probing in Human Cells Reveal a Build-Up of Positive Supercoils Following Replication Stress at Telomeres and at the FRA3B Fragile Site
<p>In vitro topological properties of Topotools. (<b>a</b>) Schematics of Topotools proteins. For purification purposes, recombinant Topotools were fused to an N-terminal (His)6 tag. (<b>b</b>) 2D Gels of Topo assays where 1 µM Topotools were incubated with a relaxed circular plasmid in the presence of wheat germ Topoisomerase I. Note the appearance of topoisomers and their inverse orientation. Chloroquine, which creates (+) supercoils, increases migration for RFP-GyrA CTD topoisomers, indicating that RFP-GyrA CTD also creates (+) supercoils. In contrast, chloroquine slows down migration for RFP-HMfB topoisomers, indicating that RFP-HMfB creates (−) supercoils.</p> "> Figure 2
<p>Topotools bind chromatin all along mitosis. Representative images of fixed Topotools-expressing HT1080 ST cells synchronized by treatment with RO-3306 and then released. Images were taken at different time points, allowing observation of different stages of mitosis. Cells were fixed and no labeling or IF were performed. DAPI and Topotools RFP signals were monitored.</p> "> Figure 3
<p>Topoisomerase 2 inhibition and replication stress cause a build-up of positive supercoils on telomeres. (<b>a</b>) Representative images of Hela-38 cells transduced by Topotools-expressing lentiviruses and treated with ICRF-193 at 35 µM for 5 h. Topotools were detected by IF using a rat anti-HA antibody and telomeres by PNA FISH using a telomeric probe. Circles indicate co-localizations. (<b>b</b>) Quantification of the co-localizations between Topotools and telomeres observed in the experiment shown in (<b>a</b>). Statistics were performed compared to control condition using Kruskal–Wallis followed by Dunn tests. **** <span class="html-italic">p</span> < 0.0001. (<b>c</b>) Representative Slot blot of ChIP experiments performed with HT1080 ST transduced by Topotools-expressing or Empty lentiviruses. Cells were treated using 150 nM of Aphidicolin and 10 µM of ICRF-193 for 24 h. Topotools-bound chromatin was immuno-precipitated using an anti-HA antibody. (<b>d</b>) Quantification of the ChIP experiment. Error bars represent maximal values obtained between two biological replicates.</p> "> Figure 4
<p>Inhibition of HDAC I and II by Trichostatin-A (TSA) causes telomere relaxation. Quantification of the co-localizations between Topotools and telomeres observed in Hela-38 cells transduced by Topotools-expressing lentiviruses and treated with 2.5 µM of TSA for 5 h. Topotools were detected by IF using a rat anti-HA antibody and telomeres by PNA FISH using a telomeric probe. Statistics were performed compared to control condition using Kruskal–Wallis tests followed by Dunn tests. *** <span class="html-italic">p</span> < 0.001.</p> "> Figure 5
<p>Knockdown of TRF2 causes supercoil build-up on telomeres. (<b>a</b>) Representative images of HCT116 cells expressing the corresponding Topotools and transfected with vectors expressing either a siRNA targeting <span class="html-italic">TERF2</span> mRNA or a siRNA of identical composition but with a random sequence (siCtrl). Topotools were detected by IF using a rat anti-HA antibody and telomeres by PNA FISH using a telomeric probe (Telo). Circles indicate co-localizations. (<b>b</b>) Quantification of the co-localizations between Topotools and telomeres observed in the experiment shown in (<b>a</b>). Statistics were performed compared to control conditions using Kruskal–Wallis tests followed by Dunn tests. **** <span class="html-italic">p</span> < 0.0001. (<b>c</b>) QPCR analysis of TRF2 expression in the HCT116 cells used in (<b>a</b>). (<b>d</b>) Western blot showing Topotools and Actin expression in the experiment shown in Figure (<b>a</b>). Anti-tRFP and anti-Actin antibodies were used.</p> "> Figure 6
<p>Mild replication stress causes topological changes in the <span class="html-italic">FHIT</span> gene. Top: graphical representation of the <span class="html-italic">FHIT</span> gene and position of primers pairs used to analyze ChIP samples. Bottom: quantitative analysis of ChIP samples by qPCR using primers pairs A to F and performed on HCT116 cells expressing the Topotools and the RFP control. Three conditions were analyzed: DMSO (− DOX), Doxycyclin induction of Topotool expression (+ DOX), and Doxycyclin induction and treatment with Aphidicolin (150 nM for 24 h, + DOX + APH). Antibodies used were rat anti-HA and rat IgG.</p> ">
Abstract
:1. Introduction
2. Materials and Methods
2.1. Vectors
2.2. Topotool Purification
2.3. In Vitro Topology Assay and 2D Gel
2.4. Cell Culture, Transfections, Lentiviral Production, and Infections
2.5. Direct Observation of Topotools in Cells
2.6. PNA-FISH IF
2.7. ChIP
2.8. Slot Blot
2.9. qPCR
A for | CAGCCACCCTTCCTTACTGG |
A rev | GCCACTAGAGTCAGCCAAGG |
B for | CCTCTTTGCCACACACTTGC |
B rev | TAATTGGCACAGGGCCTGAC |
C for | GAGTCAACAGAGTGGACCTACC |
C rev | TAGATGACCGGAAGGTGTGTT |
D for | CCAGCAGTTAATGGCTTGCT |
D rev | GTTGGGCCATGACCAGTTAC |
E for | GTAAACAATGCAGGATCACCGTGTA |
E rev | TTCCACCTACTTTGGGCCTGAG |
F for | TGTGGTCATCACCAACCCAG |
F rev | CAGGTTAGCAGGTCTCGGTG |
Sub-telo for | CCCAAACCCTAACCCTAAAA |
Sub-telo rev | CTTCCTGTTTGCAGCACTGA |
2.10. Immunoblots
2.11. qRT–PCR for Measuring TRF2 Expression
2.12. siRNA KD of TERF2 mRNA
3. Results
3.1. GyrA CTD and HMfB-Based Topotools to Investigate DNA Topology Inside the Nucleus
3.2. Topotools Are Nuclear and Bind Chromatin
3.3. Topoisomerase 2 Inhibition Causes a Build-Up of Positive Supercoils at Telomeres
3.4. Replicative Stress Causes a Build-Up of Positive Supercoils at Telomeres
3.5. Trichostatin-A Treatment Causes an Increase in HMfB Binding
3.6. TRF2 Depletion Has a Dual Effect on Telomere Topology
3.7. Replicative Stress Causes Topological Changes in the Fragile FRA3B Site
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
T-loop | Telomeric loop a lasso-like structure ending telomeres of most species |
R-loop | RNA loop, a loop created by the retention of an RNA transcript on a transcribed region |
HA tag | tag derived from the human influenza hemagglutinin (HA) protein |
NLS | Nuclear Localization Signal |
RFP | Red Fluorescent Protein |
IPTG | Isopropyl β- d-1-thiogalactopyranoside |
PMSF | phenylmethylsulfonyl fluoride |
PNA-FISH | Peptide nucleic Acid—Fluorescence in situ Hybridization |
IF | Immuno-Fluorescence assay |
ChIP | Chromatin Immuno-Precipitation |
qPCR | quantitative Polymerase Chain Reaction |
qRT-PCR | Reverse transcription followed by qPCR |
KD | knock-down |
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Ghilain, C.; Vidal-Cruchez, O.; Joly, A.; Debatisse, M.; Gilson, E.; Giraud-Panis, M.-J. Innovative Tools for DNA Topology Probing in Human Cells Reveal a Build-Up of Positive Supercoils Following Replication Stress at Telomeres and at the FRA3B Fragile Site. Cells 2024, 13, 1361. https://doi.org/10.3390/cells13161361
Ghilain C, Vidal-Cruchez O, Joly A, Debatisse M, Gilson E, Giraud-Panis M-J. Innovative Tools for DNA Topology Probing in Human Cells Reveal a Build-Up of Positive Supercoils Following Replication Stress at Telomeres and at the FRA3B Fragile Site. Cells. 2024; 13(16):1361. https://doi.org/10.3390/cells13161361
Chicago/Turabian StyleGhilain, Claire, Olivia Vidal-Cruchez, Aurélia Joly, Michelle Debatisse, Eric Gilson, and Marie-Josèphe Giraud-Panis. 2024. "Innovative Tools for DNA Topology Probing in Human Cells Reveal a Build-Up of Positive Supercoils Following Replication Stress at Telomeres and at the FRA3B Fragile Site" Cells 13, no. 16: 1361. https://doi.org/10.3390/cells13161361