Tools to Study the Role of Architectural Protein HMGB1 in the Processing of Helix Distorting, Site-specific DNA Interstrand Crosslinks

Podsumowanie

Targeted DNA damage can be achieved by tethering a DNA damaging agent to a triplex-forming oligonucleotide (TFO). Using modified TFOs, DNA damage-specific protein association, and DNA topology modification can be studied in human cells by the utilization of modified chromatin immunoprecipitation assays and DNA supercoiling assays described herein.

Streszczenie

High mobility group box 1 (HMGB1) protein is a non-histone architectural protein that is involved in regulating many important functions in the genome, such as transcription, DNA replication, and DNA repair. HMGB1 binds to structurally distorted DNA with higher affinity than to canonical B-DNA. For example, we found that HMGB1 binds to DNA interstrand crosslinks (ICLs), which covalently link the two strands of the DNA, cause distortion of the helix, and if left unrepaired can cause cell death. Due to their cytotoxic potential, several ICL-inducing agents are currently used as chemotherapeutic agents in the clinic. While ICL-forming agents show preferences for certain base sequences (e.g., 5'-TA-3' is the preferred crosslinking site for psoralen), they largely induce DNA damage in an indiscriminate fashion. However, by covalently coupling the ICL-inducing agent to a triplex-forming oligonucleotide (TFO), which binds to DNA in a sequence-specific manner, targeted DNA damage can be achieved. Here, we use a TFO covalently conjugated on the 5' end to a 4'-hydroxymethyl-4,5',8-trimethylpsoralen (HMT) psoralen to generate a site-specific ICL on a mutation-reporter plasmid to use as a tool to study the architectural modification, processing, and repair of complex DNA lesions by HMGB1 in human cells. We describe experimental techniques to prepare TFO-directed ICLs on reporter plasmids, and to interrogate the association of HMGB1 with the TFO-directed ICLs in a cellular context using chromatin immunoprecipitation assays. In addition, we describe DNA supercoiling assays to assess specific architectural modification of the damaged DNA by measuring the amount of superhelical turns introduced on the psoralen-crosslinked plasmid by HMGB1. These techniques can be used to study the roles of other proteins involved in the processing and repair of TFO-directed ICLs or other targeted DNA damage in any cell line of interest.

Wprowadzenie

Triplex-forming oligonucleotides (TFOs) bind duplex DNA in a sequence-specific fashion via Hoogsteen-hydrogen bonding to form triple-helical structures^1-5. Triplex technology has been used to interrogate a variety of biomolecular mechanisms, such as transcription, DNA damage repair, and gene targeting (reviewed in references^6-8). TFOs have been used extensively to induce site-specific damage on reporter plasmids^9,10. Our lab and others have previously used a TFO, AG30, tethered to a psoralen molecule to induce site-specific DNA interstrand crosslinks (ICLs) in the supF gene on the plasmid pSupFG1^5,10-12. ICLs are highly cytotoxic as these lesions covalently crosslink the two DNA strands, and if left unrepaired, can block gene transcription and impede the DNA replication machinery^13,14. Because of their cytotoxic potential, ICL-inducing agents have been used as chemotherapeutic drugs in the treatment of cancer and other diseases¹⁵. However, the processing and repair of ICLs in human cells is not well understood. Thus, a better understanding of the mechanisms involved in the processing of ICLs in human cells may help to improve the efficacy of ICL-based chemotherapeutic regimens. TFO-induced ICLs and their repair intermediates have the potential to cause significant structural distortions to the DNA helix. Such distortions are probable targets for architectural proteins, which bind to distorted DNA with higher affinity than to canonical B-form duplex DNA^16-20. Here, we studied the association of a highly abundant architectural protein, HMGB1 with ICLs in human cells via chromatin immunoprecipitation (ChIP) assays on psoralen-crosslinked plasmids and identified a role for HMGB1 in modulating the topology of the psoralen-crosslinked plasmid DNA in human cancer cell lysates.

HMGB1 is a highly abundant and ubiquitously expressed non-histone architectural protein that binds to damaged DNA and alternatively structured DNA substrates with higher affinity than canonical B-form DNA^17-20. HMGB1 is involved in several DNA metabolic processes, such as transcription, DNA replication, and DNA repair^16,21-23. We have previously demonstrated that HMGB1 binds to TFO-directed ICLs in vitro with high affinity²⁰. Further, we have demonstrated that lack of HMGB1 increased the mutagenic processing of TFO-directed ICLs and identified HMGB1 as a nucleotide excision repair (NER) co-factor^23,24. Recently, we have found that HMGB1 is associated with TFO-directed ICLs in human cells and its recruitment to such lesions is dependent upon the NER protein, XPA¹⁶. Negative supercoiling of DNA has been shown to promote the efficient removal of DNA lesions by NER²⁵, and we have found that HMGB1 induces negative supercoiling preferentially on TFO-directed ICL-containing plasmid substrates (relative to non-damaged plasmids substrates)¹⁶, providing a better understanding of the potential role(s) of HMGB1 as an NER co-factor. The processing of ICLs is not fully understood in human cells; thus, the techniques and assays developed based on the molecular tools described herein could lead to the identification of additional proteins involved in ICL repair, which in turn may serve as pharmacological targets that can be exploited to improve the efficacy of cancer chemotherapy regimens.

Here, an effective approach to assess the efficiency of TFO-directed ICL formation in plasmid DNA by denaturing agarose gel electrophoresis has been discussed. Further, using the plasmids containing the TFO-directed ICLs, techniques to determine the association of HMGB1 with ICL-damaged plasmids in a cellular context using modified ChIP assays have been described. Additionally, a facile method to study topological modifications introduced by the architectural protein HMGB1, specifically on ICL-damaged plasmid substrates in human cell lysates has been determined by performing supercoiling assays via two-dimensional agarose gel electrophoresis. The techniques described can be used to further the understanding of the involvement of DNA repair and architectural proteins in the processing of targeted DNA damage on plasmids in human cells.

We describe detailed protocols for the formation of TFO-directed site-specific psoralen ICLs on plasmid DNA, and subsequent plasmid ChIP and supercoiling assays to identify proteins that associate with the lesions, and proteins that alter the DNA topology, respectively. These assays can be modified to perform with other DNA damaging agents, TFOs, plasmid substrates, and mammalian cell lines of interest. In fact, we have shown that there is at least one potential unique and high affinity TFO-binding site within every annotated gene in the human genome²⁶. However, for clarity, we described these techniques for the use of a specific psoralen-conjugated TFO (pAG30) on a specific mutation-reporter plasmid (pSupFG1) in human U2OS cells as we have utilized in Mukherjee & Vasquez, 2016¹⁶.

Protokół

1. Preparation of TFO-directed ICLs on Plasmid Substrates

1. Incubate equimolar amounts of plasmid pSupFG1^10,11 DNA (5 µg plasmid DNA is a good starting point) with psoralen-conjugated TFO AG30 in 8 µl of triplex binding buffer [50% glycerol, 10 mM Tris (pH 7.6), 10 mM MgCl₂] and dH₂O to a final volume of 40 µl in an amber tube. Mix thoroughly by pipetting up and down. Incubate the reaction at 37 °C water bath for 12 hr.
2. Warm up the UVA lamp to ensure full power output. Place the triplex reaction on paraffin film, and place the paraffin film on ice under the UVA (365 nM) lamp. Place a Mylar filter between the lamp and the reaction.
3. UVA irradiate for a total dose of 1.8 J/cm². Store the samples at 4 °C for further use.
   Caution: Wear proper protective eyewear and clothing with UVA irradiation.
4. Linearize 200 ng of the above prepared plasmid with the restriction enzyme EcoR1 in a 20 µl total volume using the manufacturer supplied 10x buffer. Heat denature the enzyme for 20 min at 65 °C. Spin the samples for 10 min at 10,000 x g and store at 4 °C.
5. Prepare 50x alkaline buffer [1.5 M NaOH, 50 mM Ethylenediaminetetraacetic acid (EDTA)]. Add 0.5 mg of agarose to 50 ml dH₂O. Boil to dissolve the agarose and place it in a 50 °C water bath.
6. After the temperature of the dissolved agarose is down to 50 °C, add 1 ml of the 50x alkaline buffer, mix it and then pour the gel into the gel tray. Allow time for the gel to solidify at room temperature prior to use.
7. Add 4 µl of 0.5 M EDTA to the 20 µl of linearized DNA sample and incubate for 10 min at room temperature.
8. Add 6x alkaline gel loading buffer (300 mM NaOH, 6 mM EDTA, 18% (w/v) high molecular weight polysaccharide, 0.06% Bromocresol Green in dH₂O) to the samples and to a 1 kb DNA ladder.
   NOTE: It is important to use 6x alkaline gel loading buffer, please see discussion.
9. Load samples onto the gel in the cold room and run overnight in 1x alkaline buffer (12-16 hr) at 3.2 volts per cm. Once the samples have entered the gel, place a glass plate on top of the gel to prevent it from floating.
10. Neutralize the samples by soaking the gel in neutralization buffer [1 M Tris-Cl (pH 7.6), 1.5 M NaCl, 3 M sodium acetate (pH 5.2)] for 45 min at room temperature.
11. Stain the gel for DNA with 4 µl of 1% ethidium bromide (EtBr) in 50 ml water for 1 hr at room temperature. Destain with water for 15 min. Visualize the DNA by using an imaging system.
    Caution: EtBr is a potent mutagen and can be absorbed through the skin. Avoid direct contact with EtBr.

2. Transfection and Immunoprecipitation of TFO-directed ICL-containing Plasmids in Human Cells

1. Plate 400,000 mammalian cells (e.g., U2OS osteosarcoma cells) per 60 mm dish in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) without antibiotics 24 hr before transfection. Incubate cells overnight at 37 °C with 5% CO₂.
2. Prior to transfection, warm growth media and phosphate buffered saline (PBS) to 37 °C in a water bath. Warm the transfection reagent to room temperature.
3. Incubate 30 µl of transfection reagent with 500 µl of 1x growth media (mix 1) and 2 µg of TFO-directed ICL-containing plasmids in 500 µl of 1x growth media (mix 2) in separate 14 ml round bottom tubes for 10 min at room temperature.
4. Add mix 1 to mix 2, mix well by pipetting up and down. Incubate for 25-30 min at room temperature.
5. Wash cells twice with warm PBS. Add the transfection mix in a drop-wise fashion distributing evenly throughout the plate of cells. Add 1 ml of growth media to the plate and bring the final volume to 2 ml. Mix well. Place the culture plates in the 37 °C incubator with 5% CO₂ for 4 hr.
6. Replace the media with 3 ml growth media supplemented with 10% FBS, and incubate further for 16 hr. Do not use antibiotics.
7. Treat cells with 80 µl of 37% fresh formaldehyde per plate to a final concentration of approximately 1% and incubate for 10 min at room temperature in the absence of light.
   Caution: Formaldehyde is toxic and should be handled according to its safety instructions. Formaldehyde exposure can cause harm to skin, eye and respiratory tract.
8. Quench formaldehyde crosslinking by adding 300 µl of chilled glycine (supplied in commercially available kits) per plate and incubating for 5 min. Remove the media and wash twice with chilled PBS, and then collect cells by scraping in 1 ml chilled (4 °C) PBS into a microcentrifuge tube. Keep the samples on ice at all times.
9. Prepare cell pellets by centrifuging at 4 °C for 5 min at 13,400 x g using a tabletop refrigerated centrifuge. Pipet out the supernatant and place the cell pellet on ice.
10. Homogeneously resuspend the pellet in 1 ml chilled (4 °C) Buffer A (supplied in commercially available kits) and incubate on ice for 10 min. Mix occasionally by inverting the tube. Pellet cells as before (see step 2.9 above).
11. Homogeneously resuspend the pellet in 1 ml chilled (4 °C) Buffer B (supplied in commercially available kits) and incubate on ice for 10 min with occasional mixing by inverting. Pellet cells by centrifugation as before (see step 2.9 above).
12. Resuspend the cells again in 200 µl chilled Buffer B. Add 2 µl of Micrococcal nuclease (MNase) and incubate at room temperature for 10 min. Mix reaction occasionally by flicking the tube. Stop the MNase reaction by placing the tube on ice and adding 40 mM EDTA. Pellet cells as before (see step 2.9 above).
13. Resuspend cells in 100 µl 1x chromatin immunoprecipitation buffer (ChIP) buffer (supplied in commercially available kits) supplemented with protease inhibitor cocktail.
14. Place the resuspended cells in a thin-walled microfuge tube. Sonicate the samples by floating them on water bath sonicator for 20 sec followed by 20 sec incubation on ice. Repeat sonication steps 9 times to generate ~800-1,200 bp fragments.
    1. To 20 µl of the sonicated samples add 3 µl 5 M NaCl and 1 µl RNase A. Mix well by vortexing and incubate at 37 °C for 30 min. Subsequently, add 2 µl Proteinase K and incubate for 2 hr at 65 °C. Purify the samples using a PCR purification kit. Run samples on 1% agarose gel and visualize with EtBr.
       NOTE: Maximum intensity of the resulting DNA should be at or under 1,000 bp.
15. In three separate tubes, add 30 µl of lysate in a pre-chilled siliconized tube and then add 70 µl of chilled 1x ChIP buffer with added protease inhibitors. Add 1 µg of anti-immunoglobulin G (IgG), anti-histone H3 (as a positive control), or anti-HMGB1 antibodies to the respective tubes. Incubate overnight in a cold room with continuous rotation.
16. Resuspend protein G beads homogeneously in the cold room. Add 4 µl of protein G beads per tube and incubate for another 2-4 hr in the cold room with rotation.
17. Using a magnetic rack, pellet the beads and remove the supernatant. Add 200 µl 1x ChIP buffer mixed with protease inhibitor cocktail to the pellet and wash for 5 min in the cold room with rotation. Repeat wash twice.
18. Perform one additional wash with high salt 1x ChIP buffer (containing 70 mM NaCl).
19. Resuspend the pellets with 160 µl elution buffer (supplied in commercially available kits) and incubate at 37 °C with rotation for 30 min.
20. Pellet the beads and collect the supernatant in a fresh tube. Add 6 µl of 5 M NaCl and 2 µl of proteinase K, and incubate overnight at 65 °C.
21. Purify the DNA using a PCR product purification kit and elute the DNA using 50 µl dH₂O.
22. Perform PCR¹⁶ reactions using primer sets to the region(s) of interest, and resolve the products on a 1% agarose gel stained with EtBr.

3. Supercoiling Assay and 2-dimensional Agarose Gel Electrophoresis

1. Prepare DNA repair synthesis buffer (45 mM Hepes-KOH, pH 7.8; 70 mM KCl; 7.4 mM MgCl₂; 0.9 mM DTT; 0.4 mM EDTA; 2 mM ATP, 20 µM dNTPs, 3.4% glycerol and 18 µg bovine serum albumin). Store at -80 °C. Thaw on ice and prior to use add 40 mM phosphocreatine and 2.5 µg creatine phosphokinase.
2. Prepare 10x supercoiling buffer [500 mM Tris (pH 8.0), 500 mM NaCl, 25 mM MgCl₂, 1 mM EDTA], and store at room temperature.
3. Linearize 300 ng of supercoiled plasmid pSupFG1 DNA completely using 1 µl Vaccinia Topoisomerase I (5-15 U/µl) with 1x supercoiling buffer in a 10 µl reaction. Incubate the mixture at 37 °C for 1 hr.
4. Thaw HeLa cell-free extract²⁴ on ice. To the completely relaxed plasmids, add 25 µl HeLa cell-free extract and DNA repair synthesis buffer. Mix well. Add additional 1 µl of Vaccinia Topoisomerase I to the mix, and incubate for 1 hr at 37 °C.
5. Terminate the reactions by adding sodium dodecyl sulfate (SDS) (1% final concentration) and proteinase K (0.25 mg/ml final concentration). Incubate at 65 °C for 2 hr.
6. Cast a 1% agarose gel with 1x Tris Borate EDTA (TBE) buffer. Add DNA loading dye and load the reactions directly onto the gel at least 4 lanes apart. Run the gel for 8-10 hr at 2 V/cm in 1x TBE (dimension 1) at room temperature to resolve the topoisomers.
   NOTE: Prepare a 5x TBE stock solution in 1 L of dH₂O by adding 54 g of Tris base. 27.5 g of boric acid and 20 ml of 0.5 M EDTA (pH 8.0).
7. Prepare 2 L of 1x TBE with 3 µg/ml chloroquine-phosphate. Soak the gel in 200 ml of this solution for 20 min. Cover the gel tray with aluminum foil.
8. To electrophorese the gel in the second dimension, turn the gel 90° in a clockwise fashion and run it for 4-6 hr at 2 V/cm in chloroquine containing 1x TBE to resolve the positive and negative supercoils.
9. Stain the gel with EtBr for 1 hr on a rocker. Destain the gel with dH₂O for 15 min and subsequently visualize the thermal distribution of the topoisomers using an imager.

Wyniki

Formation of the TFO-directed ICL is critical for the plasmid based assays, which are used to interrogate the roles of architectural proteins in ICL processing in human cells. Denaturing agarose gel electrophoresis is a facile way to determine the efficiency of TFO-directed ICL formation. The plasmids harboring TFO-directed ICLs migrate with a slower mobility through the agarose gel matrix (Figure 1A, lane 3) when compared to the un-crosslinked control plasmids (F...

Dyskusje

The efficient formation of TFO-directed ICLs is dependent upon two crucial factors: First, proper buffer components (e.g., MgCl₂) and time of incubation of the TFO with its target DNA substrate (dependent upon the binding affinity of the TFO to its target duplex, and the concentrations used); and second, the proper dose of UVA (365 nm) irradiation to form psoralen crosslinks efficiently. Optimal triplex formation can be achieved by using HPLC or gel purified TFOs. Impurities contaminating the TFO may ...

Materiały

Name	Company	Catalog Number	Comments
5'HMT Psoralen-AG30	Midland Certified Reagent Co., Midland, TX	Custom order	HPLC (or gel) purified
Simple ChIP Enzymatic Chromatin IP Kit	Cell signaling Inc.	9003	Manufacturer suggested protocol is optimized for chromatin IP and not for plasmid IP. The control IgG and H3 antibodies as well as the Protein G beads and micrococcal nuclease are supplied by the manufacturer.
GoTaq Green	Promega	M7122	PCR master mix
HMGB1 antibody	Abcam	Ab18256
Wizard Gel and PCR cleanup kit	Promega	A9281
Chloroquine-diphosphate salt	Sigma	C6628-50G	For two-dimensional agarose gel electrophoresis
EcoRI	New England BioLabs	R0101S
GenePORTER transfection reagent	Genlantis	T201015
Vaccinia Topoisomerase I	Invitrogen	38042-024
Blak-Ray long wave ultraviolet lamp	Upland, CA	B 100 AP	UVA lamp
EpiSonic Multi-Functional Bioprocessor 1100	Epigentek	EQC-1100	Water bath sonicator
Mylar filter	GE Healthcare Life Sciences	80112939
Agarose	Sigma-Aldrich	A6111
BIO-RAD Chemidoc	BIO-RAD	XRS+ system	DNA imaging system
Glycine	Fisher Scientific	BP381-500
37% Formaldehyde	Sigma-Aldrich	F8775-25ML	Used for crosslinking
Proteinase K	New England Biolabs	P8107S
DMEM	ThermoFisher	11965092	Cell culture media
PBS	ThermoFisher	70013073	Cell culture
Trypsin-EDTA	ThermoFisher	25200056	Cell culture
Bromocresol green	Sigma-Aldrich	114-359	DNA loading dye
Siliconized tubes	Fisher Scientific	02-681-320	Low retention microfuge tube
Tris base	Fisher Scientific	BP152-1
Boric Acid	Fisher Scientific	A74-1
EDTA	Fisher Scientific	BP24731
Photometer	InternationalLight Technologies	ILT1400-A	UVA dose measurement
Cell lines
Human U2OS osteosarcoma	ATCC	ATCC HTB-96	Cultured according to supplier’s recommendation
Human cervical adenocarcinoma HeLa	ATCC	ATCC-CCL-2	Cultured according to supplier’s recommendation
Proximal forward primer	5′-gcc ccc ctg acg agc atc ac
Proximal reverse primer	5′-tag tta ccg gat aag gcg cag cgg
Distal forward primer	5′-aat acc gcg cca cat agc ag
Distal reverse primer	5′-agt att caa cat ttc cgt gtc gcc