Proximity Ligation Assay to Study Oncogene-Derived Transcription-Replication Conflicts

Summary

Here, we present a sensitive and specific method to detect oncogene-induced transcription-replication conflicts (TRCs) using the proximity ligation assay (PLA). This approach leverages specific antibodies targeting PCNA and phospho-CTD RNAPII, enabling the assessment of TRC prevalence between RNA polymerase II transcription and DNA replication machinery.

Abstract

Both DNA replication and RNA transcription utilize genomic DNA as their template, necessitating spatial and temporal separation of these processes. Conflicts between the replication and transcription machinery, termed transcription-replication conflicts (TRCs), pose a considerable risk to genome stability, a critical factor in cancer development. While several factors regulating these collisions have been identified, pinpointing primary causes remains difficult due to limited tools for direct visualization and clear interpretation. In this study, we directly visualize TRCs using a proximity ligation assay (PLA), leveraging antibodies specific to PCNA and phosphorylated CTD of RNA polymerase II. This approach allows precise measurement of TRCs between replication and transcription processes mediated by RNA polymerase II. The method is further enhanced through DNA primers conjugated covalently to these antibodies, coupled with PCR amplification using fluorescent probes, providing a highly sensitive and specific means of detecting endogenous TRCs. Fluorescence microscopy enables the visualization of these conflicts, offering a powerful tool to study genome instability mechanisms associated with cancer. This technique addresses the gap in direct TRC visualization, allowing for a more comprehensive analysis and understanding of the underlying processes driving genome instability in cells.

Introduction

The genome serves as a template for essential biological processes, including replication and transcription. In healthy cells, RNA transcription and DNA replication machinery typically cooperate and are spatially and temporally segregated^1,2,3,4. However, under pathological conditions, such as oncogene overexpression, this harmonious cooperation can be disrupted.

Oncogenes often drive elevated transcription levels, increasing the likelihood of collisions between the transcription and replication machinery⁵. Some oncogenes alter chromatin architecture, making it more accessible for transcription but potentially hindering replication fork progression⁵. Additionally, oncogene activation can induce replication stress by accelerating the cell cycle⁶. RNA polymerase II (RNAPII) activity is significantly elevated during the G1/S phase transition due to cyclin-dependent kinase (CDK) phosphorylation of Rb, which releases E2F transcription factors that promote the transcription of essential proteins, including cyclins, CDKs, and housekeeping genes⁷. Histone gene expression peaks during the S phase, coinciding with DNA replication⁸. Moreover, transcribing the full-length transcripts of some very long genes requires more than one cell cycle⁹. With each entry into the S phase, the simultaneous activity of transcription and replication machinery at the same genomic regions may result in detrimental encounters, leading to conflicts. These conflicts are a primary intrinsic source of genome instability, which is closely associated with tumorigenesis. How the replication machinery coordinates with RNAPII transcription to prevent harmful clashes and maintain genomic stability remains an important question. Therefore, direct visualization of RNAPII-associated transcription-replication conflicts (TRCs) has become essential for understanding the molecular mechanisms that resolve these conflicts and may eventually lay the foundation for harnessing these conflicts for therapeutic purposes.

Transcription-replication conflicts (TRCs) can emerge in two orientations: head-on, where transcription and replication progress towards each other, and co-directional, where they move in the same direction. Head-on collisions are far more destructive than co-directional ones^10,11,12,13. The formation of DNA-RNA hybrids (R-loops) has been identified as a major driver of TRCs; thus, a considerable amount of research has inferred the presence of TRCs by assessing the occurrence of R-loops and their regions in response to dysregulated replication and/or transcription^10,11. However, whether the accumulation of transcription-derived R-loops serves as a proportional obstacle to replication and directly correlates with TRCs remains an unresolved question. Researchers have also investigated TRCs by analyzing replication fork dynamics. For instance, two-dimensional gel electrophoresis of reporter genes can uncover locus-specific replisome pausing¹², while DNA fiber assays allow researchers to examine alterations in replication fork speed, origin density, and fork asymmetry¹³. The advancement of next-generation sequencing technology has led to the development of several innovative methods, such as Okazaki fragment sequencing (Ok-seq)^14,15, initiation site sequencing (ini-seq)^16,17, short nascent strand sequencing (SNS-seq)¹⁸, Repli-seq¹⁹, and polymerase usage sequencing (Pu-seq)²⁰. These techniques have provided genome-wide profiles of replication origin usage, fork directionality, and replication termination, revealing key factors involved in the coordination of replication and transcription. TRIPn-seq is one of the few methods capable of directly detecting transcription and replication co-occupancy, significantly expanding our understanding of the dynamic organization of DNA replication and transcription under physiological and pathological conditions²¹. Despite the valuable insights provided by these sequencing-based methods, their high cost and time-intensive nature limit their broader application. Therefore, it is crucial to establish a more definitive, highly sensitive, convenient, and rapid detection method to visualize and quantify TRCs at the single-cell level.

The in situ proximity ligation assay (PLA), also known as Duolink PLA technology, is a powerful method for evaluating the physical proximity of target proteins in tissue sections or cell cultures. This technique generates a signal only when two proteins or protein complexes are within 40 nm of each other. It requires two primary antibodies against the target proteins, each from a different species (e.g., mouse/rabbit, rabbit/goat, or mouse/goat). After incubating the sample with these primary antibodies, secondary antibodies known as PLA probes (one PLUS and one MINUS) are applied. Unlike regular secondary antibodies that bind to the constant regions of primary antibodies, PLA probes are covalently linked to specific DNA primers. When the target proteins are in close proximity (less than 40 nm), a connector oligonucleotide can hybridize with both PLA probes, facilitating the enzymatic ligation of their attached oligonucleotides into a full-length DNA molecule. This newly formed DNA serves as a surrogate marker for the detected protein interaction and acts as a template for amplification. The amplified product is then visualized using fluorescently labeled oligonucleotide probes. If the proteins are more than 40 nm apart, the PLA probes cannot form a DNA template, resulting in no detectable signal. The schematic diagram of PLA is depicted in Figure 1. The proximity and/or interaction are visualized by fluorescence microscopy as fluorescent dots, and the number and intensity of these dots can be quantified. Since its invention in 2002, PLA has become popular for monitoring protein interactions due to its high sensitivity and specificity. Unlike sequencing-based assays, PLA requires only small quantities of samples, making it favorable for analyzing scarce samples.

Studies have demonstrated that the expression of the oncogenic KRAS G12D mutation leads to transcription-replication conflicts (TRCs)^22,23. For instance, research presented by Meng et al.²³ indicates that ectopic expression of KRAS G12D in human pancreatic ductal epithelial (HPNE) cells enhances TRCs and TRC-related R-loops. To enable the detection of collisions between RNAPII transcription and replication machinery in cell lines, we provide a protocol specifically developed for this purpose. The KRAS (G12D) plasmid and vector control are transfected into human lung epithelial BEAS-2B cells, and KRAS (G12D) expression, along with DNA damage (γH2AX), is verified by Western blot. R-loop accumulation is confirmed by S9.6 dot blot analysis, which together indicates the accumulation of replication roadblocks and genome instability in cells expressing KRAS (G12D). Finally, the cells are fixed onto slides for the PLA assay. For localized detection of collisions, cells are first stained with RNAPII and PCNA primary antibodies, followed by staining with secondary antibodies conjugated with PLA probes. A circular DNA molecule forms through successful ligation, serving as a template for subsequent rolling circle amplification (RCA). The slides are then mounted with appropriate mounting media and visualized under a fluorescence microscope. The images can be analyzed using ImageJ.

Protocol

1. Lentivirus production and transduction into target cell line

1. Lentivirus production²⁴​
   1. Seed 293T packaging cells at 3 × 10⁶ cells per 10 cm-plate in high glucose Dulbecco's Modified Eagle Medium (DMEM; complete medium). Grow cells at 37 °C in a humidified 5% CO₂ incubator for an additional 18-20 h.
   2. In two 1.5 mL tubes, prepare a DNA mixture of a total of 24 µg plasmid DNA in 500 µL of Opti-MEM (reduced serum medium) that contains 9.2 µg of psPAX2, 2.8 µg of pMD2.G, along with 12 µg of pLVX-KRAS plasmid or empty vector respectively.
      NOTE: Because endotoxins significantly decrease transfection efficiency, it is recommended to remove endotoxin from the plasmid during purification.
   3. Dilute 144 µL of polyethylenimine (PEI; 1 mg/mL) in 1 mL of reduced serum medium, mix, and incubate for 5 min at room temperature (RT).
   4. Add 500 µL of diluted PEI dropwise to the diluted DNA mixture while gently flicking the tube. Incubate the PEI/DNA mixtures for 15 min at RT.
   5. During the incubation, gently aspirate the media from the previously seeded 10 cm plates. Replace with 10 mL of fresh complete DMEM.
   6. After the incubation, slowly pipette the DNA: PEI transfection mix onto the 10 cm plates, making sure not to detach the cells.
      NOTE: PEI is a commonly used transfection reagent, but its concentration must be carefully optimized to balance transfection efficiency and cytotoxicity. Despite the higher cost of Lipofectamine 3000 (transfection reagent), it has demonstrated higher transfection efficiency and lower cytotoxicity compared to PEI in certain cell lines, leading to improved viral production. Electroporation is another alternative that uses electrical pulses to introduce nucleic acids into cells. While effective, it requires specialized equipment and can be more labor-intensive.
   7. Grow the cells for 18 h, then carefully replace the transfection media with fresh 10 mL of DMEM Complete medium containing 25 μM of chloroquine.
   8. After 72 h of transfection, collect the culture medium containing the virus particle. Remove the packaging 293T cells by centrifuging at 500 x g for 5 min.
   9. Collect the supernatant and filter it through a 0.45 µm polyethersulfone(PES) filter. Use the viral supernatant directly.
      NOTE: The viral supernatant should be aliquoted and snap-frozen in liquid nitrogen and kept at -80 °C to avoid loss of titer.
2. Lentiviral Infection of target cells
   1. Seed 2 × 10⁶ target cells BEAS-2B in two 6 cm plates and grow at 37 °C, 5% CO₂ overnight. Upon transduction, ensure that the BEAS-2B cells are approximately 70% confluent.
   2. Carefully remove the old medium and add the appropriate 0.5 mL of KRAS or vector lentiviral particles together with 3 mL of fresh complete RPMI-1640 medium into two plates, respectively.
   3. Grow the cells at 37 °C for 18-20 h in a 5% CO₂ incubator. Replace the old medium containing lentiviral particles with 3.5 mL of fresh pre-warmed complete culture medium. After 72 h transduction, collect the cells for the following assay.
      NOTE: If overnight incubation with the virus causes toxicity, the incubation time can be shortened to 4 h. To achieve the balance of transduction efficiency and cellular health, the following strategies need to be considered: (1) Optimize multiplicity of infection ; (2) Use high-quality viral preparations; (3) Utilize polycations (such as polybrene) to improve efficiency, allowing for the use of lower viral titers and reducing potential toxicity; (4) For long-term studies, consider using inducible expression systems to control transgene expression temporally, reducing the burden on cells and minimizing potential toxicity.

2. Verification of KRAS (G12D) derived DNA damage and R-loop formation

1. Confirm gene expression and DNA damage by WB assay
   1. Aspirate the culture medium from the plates.
   2. Gently wash the cells with 1x phosphate-buffered saline (PBS) to remove any residual medium or debris.
   3. Add a small volume of 1x PBS to keep the cells moist during the scraping process.
   4. Use a cell scraper, holding it at an angle, and gently scrape the cells from the surface in a consistent direction.
      NOTE: Be careful not to apply too much pressure, which could damage the cells.
   5. Tilt the plate and use a pipette to collect the cell suspension into a tube and spin the cells at 500 x g for 5 min. Discard the supernatant.
   6. Prepare the 1x RIPA buffer by diluting the 2x RIPA buffer (see Table 1), and add the protease inhibitor cocktail and phosphatase inhibitor cocktail immediately before use.
   7. Lyse the cell pellet with pre-chilled RIPA buffer (1x) and incubate for 30 min on ice. Remove the debris by centrifuging at 15,000 x g for 10 min. Collect the supernatant and measure the protein concentration using the Bradford assay.
   8. Add 4 x sodium dodecyl sulfate (SDS) sample loading buffer to the supernatant and denature the proteins for 5 min at 95 ˚C. First, separate 40 µg of protein from each sample in a 12.5% SDS-polyacrylamide gel electrophoresis (PAGE) gel and then transfer it to 0.2 µm nitrocellulose membranes (see Table of Materials).
   9. After finishing the transfer, block the membranes with Blocking Buffer (5% nonfat milk in Tris-buffered saline containing 0.1% Tween 20 [TBST; Table 1]) for 1 h.
   10. Briefly rinse the membrane with TBST, and then incubate the membrane with primary antibodies against FLAG-tag (1: 2500 dilution in PBST) and γH2AX (1:1000 dilution in PBST) (see Table of Materials) overnight at 4 ˚C.
   11. Remove the primary antibodies, and wash the membrane with TBST 3 times, each time for 10 min.
   12. Immerse the membranes in diluted horseradish peroxidase (HRP)-conjugated secondary antibodies (1:5,000 in TBST buffer containing 5% nonfat milk) for 1 h at room temperature (RT).
   13. Wash the membrane with TBST 3 times, each time for 10 min.
   14. Visualize the protein levels via the enhanced chemiluminescence (ECL) reagents (see Table of Materials).
2. Detection of R-loop by Dot blot²⁵​
   1. Aspirate the culture medium from the plates.
   2. Gently wash the cells with 1x PBS to remove any residual medium or debris.
   3. Add a small volume of 1x PBS to keep the cells moist during the scraping process.
   4. Use a cell scraper, holding it at an angle, and gently scrape the cells from the surface in a consistent direction.
      NOTE: Be careful not to apply too much pressure, which could damage the cells.
   5. Tilt the plate and use a pipette to collect the cell suspension into a tube and spin the cells at 500 x g for 5 min. Discard the supernatant.
   6. Lyse 2 × 10⁶ cells with 300 µL of ice-cold Cell Lysis Buffer (see Table 1). Incubate on ice for 10 min. Spin at 500 x g for 5 min at 4 ˚C and discard the supernatant. The pellet contains nuclei.
   7. Use a wide-orifice tip to re-suspend each pellet with 300 µL of pre-chilled Nuclear Lysis Buffer (see Table 1).
   8. Add 3 µL of 20 mg/mL proteinase K (see Table of Materials) to each sample and digest for 3-5 h at 55 °C.
   9. Add 100 µL of Elution Buffer (see Table 1) to each tube and mix thoroughly.
   10. Add 400 µL of phenol: chloroform: isoamyl alcohol (25:24:1 pH 8.0) to each tube. After vigorous vortex, centrifuge at 12,000 x g for 5 min at 4 °C to separate DNA from other cell content.
   11. Carefully transfer the upper aqueous phase to the new tubes.
   12. Precipitate the DNA by adding 1/10 volume of 3 M sodium acetate (pH 5.2) and 2.5 volume of pre-chilled 100% ethanol to the sample. Mix thoroughly and incubate for 4 h or overnight at -20 ˚C.
       ​NOTE: The addition of glycogen (See Table of Materials) to the sample can increase DNA recovery from ethanol precipitation. The final glycogen concentration is about 0.05-1 µg/µL.
   13. Pellet the DNA by centrifuging at 12,000 x g for 30 min at 4 °C. Discard the supernatant without disturbing the pellet.
   14. Rinse the pellet with 1 mL of ice-cold 70% ethanol and centrifuge again for 5-15 min at 12,000 x g at 4 °C.
   15. Discard the supernatant carefully to avoid disturbing the DNA pellet.
   16. Air dry the DNA pellet for 5-10 min.
   17. Dissolve the DNA pellet by adding 50 µL of pre-warmed TE buffer (pH8.0) and incubate for 30 min at 37 °C. Measure the DNA concentration of each sample.
   18. Prepare dilutions of DNA to desired concentrations in TE buffer (5 ng/ µL to 200 ng/µL). Evenly spot 20 µL of every sample onto a positively charged nylon membrane by a dot-blot apparatus.
       NOTE: To detect R-loops using a dot blot assay, it is recommended to apply approximately 500 ng of genomic DNA per dot. This amount has been shown to provide sufficient sensitivity for detecting RNA-DNA hybrids when using the S9.6 monoclonal antibody, which specifically binds to these structures.
   19. Immobilize the DNA by crosslinking the DNA to the nylon membrane (see Table of Materials) via UV crosslinker "Auto Crosslink" setting (1,200 µJ x 100) (see Table of Materials).
   20. Immerse the membranes in Blocking Buffer (see Table 1) for 1 h at room temperature. Then briefly rinse the membranes with TBST.
   21. Overnight incubate the membrane with primary antibody (See Table of Materials) at 4°C. The dilution of S9.6 is 1:1,000 in TBST containing 5% BSA.
   22. Remove primary antibody and wash 3 times with TBST, and each time for 10 min.
   23. Incubate the membrane with HRP-conjugated secondary antibody in 5% BSA in TBST at room temperature for 1 h.
   24. Remove secondary antibody and wash 3 times with TBST, and each time for 10 min.
   25. Visualize the R-loop levels via the ECL (Enhanced Chemiluminescence) reagents See Table of Materials).

3. PLA assay

1. Cell preparation
   NOTE: Transduction of cells with the KRAS G12D oncogene leads to an increase in TRCs, as evidenced by a positive PLA signal. This method effectively distinguishes oncogene-driven TRCs from background levels without the need for antibiotic selection. However, implementing antibiotic selection post-transduction can enhance the overall signal by enriching the population of successfully transduced cells, thereby improving the assay's sensitivity and specificity.
   1. Place 12-mm-diameter cover glasses in each well of a 12-well plate.
   2. Seed the lentivirus-infected cells into these wells 24 h post-infection, and maintain the cells on cover glasses in 500 µL of complete RPMI-1640 medium at 37 °C with 5% CO₂.
   3. Optimal) If the experimental design includes antibiotic selection to enrich for successfully transduced cells, add the appropriate antibiotic to the culture medium at this stage.
   4. Monitor the cells to ensure they reach approximately 60% confluence 48-72 h post-infection.
   5. Carefully remove RPMI-1640 and pre-extract cells with cold 0.5% NP-40 for 4 min on ice.
   6. Add 500 µL of Fixation Buffer (see Table 1) directly to fix the cells for 15 min at RT.
      NOTE: It is advised not to wash cells before the fixation step as this can cause them to detach from the surface they are growing on, especially if the cell line is sensitive to detachment; therefore, directly adding the fixative solution is often preferred.
   7. Stop the reaction by removing the fixation buffer and carefully washing the cells with 1x PBS 3 times.
   8. Aspirate off PBS and add 500 µL of pre-chilled 100% methanol to cells.
   9. Permeabilize the cells by incubating at -20 °C for 15-30 min.
   10. Block cells with Duolink block solution for 1 h at RT.
2. Antibody staining
   1. Dilute mouse anti-RNAPII 8WG16 (1:500) and rabbit anti-PCNA(D3H8P) (1:500) in Duolink antibody dilution buffer. Incubate the cells with the primary antibodies mixture overnight at 4 °C in a humidity chamber.
      NOTE: Perform the PLA protocol using only one of the primary antibodies (either anti-RNAPII or anti-PCNA) while omitting the other. This helps identify any background signal resulting from nonspecific interactions of the individual antibodies or PLA probes.
   2. Gently remove the primary antibodies from the slides using a lint-free waste paper sheet without touching the cells. Then, wash the cells 3 times with 1x PBS.
   3. Prepare PLA probes (or secondary antibody) according to the following procedure (see Table 2), then mix and let stand for 20 min at room temperature.
   4. Add the secondary antibody mix to the slides and incubate in a humidity chamber for 2 h at RT.
3. Ligation and amplification
   1. Discard the secondary antibody mix and wash slides twice with 1x Buffer A each for 5 min.
   2. Prepare the ligation mix according to the following procedure (see Table 2).
   3. Apply 15 µL of ligation mix to each cover slide and incubate at 37 °C for 30 min in a humidity chamber.
   4. Wash slides twice with 1x Buffer A each for 2 min.
   5. Prepare the Amplification Mix according to the following procedure (see Table 2).
   6. Apply 15 µL of Amplification Mix to each slide and incubate for 100 min at 37 °C in a humidity chamber. Discard Amplification Mix and wash twice with 1x Buffer B each for 10 min.
   7. Wash slides once with 0.01x buffer B for 1 min.
   8. Aspirate off buffer B and mount the slides in Duolink In Situ mounting medium with DAPI.
4. Image analysis and threshold determination:
   1. Quantify the number of PLA foci by ImageJ. Automated image analysis software can assist in quantifying PLA signals objectively. Choose a threshold of 3 or more PLA foci per nucleus as a threshold because less than 1% of control cells acquire 3 PLA foci under unperturbed conditions.
      NOTE: Since the threshold is determined experimentally by analyzing multiple samples and controls to account for variability, consistent imaging settings (e.g., exposure time, gain) have to be maintained across all samples to ensure comparability.

Results

γH2AX serves as a biomarker for DNA damage. Overexpression of KRAS (G12D) impairs genomic stability in these cells, as evidenced by the increased γH2AX signal in Western blot analysis (Figure 2A). Additionally, the intensified S9.6 dot blot signal in KRAS (G12D)-expressing cells compared to vector controls (Figure 2B) indicates that oncogenic KRAS expression leads to the accumulation of aberrant R-loops, which may contribute to DNA break formation.

...

Discussion

The RNAPII transcription machinery can create a hindrance to DNA replication fork progression^26,27, promoting TRCs and DNA damage, especially in cancer cells^28,29. Deciphering the proteins that regulate TRCs and understanding the detailed mechanisms can help us comprehend how these harmful events occur and guide the development of new therapeutic approaches in the future. Therefore, to fully understand TR...

Materials

Name	Company	Catalog Number	Comments
Clarity Western ECL Substrate	Bio-Rad	1705061
Duolink In Situ Detection Reagents Green	Sigma	DUO92014
FLAG antibody	Millipore	F7425
gamma H2AX antibody	Cell Signaling	25955
Glycogen, molecular biology grade	ThermoFisher	R0561
Image J	NIH	https://imagej.net/ij/
nitrocellulose membranes	Amersham	10600004
PCNA antibody	Cell Signaling	13110
pLVX-Kras G12D	N/A	N/A
pMD2.G	Addgene	12259
Proteinase K, recombinant, PCR grade	ThermoFisher	EO0491
psPAX2	Addgene	12260
RNAPII antibody	SCBT	sc-56767
S9.6 antibody	Active motif	65683
UV crosslinkers	Fisher Scientific	FB-UVXL-1000