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Quantifying Symmetrically Methylated H4R3 on the Kaposi’s Sarcoma-associated Herpesvirus (KSHV) Genome by ChIP-Seq
采用ChIP-Seq定量卡波西肉瘤相关疱疹病毒(KSHV)基因组上的对称甲基化H4R3    

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PLOS Pathogens
Jul 2017

Abstract

Post-translational modifications to histone tails contribute to the three-dimensional structure of chromatin and play an important role in determining the relative expression of nearby genes. One such modification is symmetric di-methylation of arginine residues, which may exhibit different effects on gene expression including blocking the binding of transcriptional activators, or recruiting repressive effector molecules. Recent ChIP-Seq studies have demonstrated the importance of cross-talk between different histone modifications in gene regulation. Thus, to acquire a comprehensive understanding of the combined efforts of these epigenetic marks, ChIP-Seq must be utilized for identifying specific enrichment on the chromatin. Tumorigenic herpesvirus KSHV, employs epigenetic mechanisms for gene regulation, and by evaluating relative abundance of multiple histone modifications in a thorough, unbiased way, using ChIP-Seq, we can get a superior insight concerning the complex mechanisms of viral replication and pathogenesis.

Keywords: KSHV (KSHV), Viral chromatin (病毒染色质), ChIP-Seq (ChIP-Seq), Arginine methylation (精氨酸甲基化)

Background

Kaposi’s sarcoma-associated herpesvirus (KSHV) is an oncogenic human virus with two distinct phases during its lifecycle. After initial infection, KSHV establishes a persistent life-long infection in the host that is particularly problematic to the immune-compromised individuals. KSHV can cause various tumors in HIV/AIDS patients including Kaposi’s sarcoma, and multiple B-cell lymphomas (Chang et al., 1994; Cesarman et al., 1995; Russo et al., 1996; Soulier et al., 1995). With a large genome of about 165,000 bp, which encodes nearly 90 different open reading frames, KSHV has ample tools to evade the host immune surveillance system, alter host-cell growth pathways and produce infectious progeny virions.

During the latent phase only a fraction of the viral genes is expressed, which are oncogenic in nature and also help in replication and passaging of the viral episomes into the divided tumor cells (Uppal et al., 2014; Purushothaman et al., 2016). However, many factors including viral co-infection (HIV) and other stimulus such as, hypoxia, oxidative stress, or immune-suppressant medications can trigger the virus to shift into the active, lytic phase of the lifecycle leading to the production of infectious progeny virions, which egress from host cells surface to infect surrounding tissues (Purushothaman et al., 2015). These complex processes are carried out in a coordinated fashion with specific genes expressed in a sequential manner during the switch to a lytic (virus-producing) phase of the viral life cycle (Purushothaman et al., 2015; Aneja and Yuan, 2017).

KSHV employs epigenetic mechanisms to carefully regulate differential gene expression needed to maintain the virus in a specific phase of the lifecycle. Upon infection and entry into the human cells, viral genome acquires cellular histones, similar to the host genomes, and persists in euchromatin (transcription-permissive) or heterochromatin (transcription-repressive) states (Toth et al., 2013; Uppal et al., 2015). By these means, KSHV is able to restrict gene expression during latency to only a minimal subset of genes and is yet poised to rapidly shift into lytic replication phase. The development of KSHV-induced malignancies involves both phases of the lifecycle, thus it is essential for researchers to have a clear understanding of the mechanisms of how these epigenetic changes regulate viral genes expression.

Epigenetic regulation of gene expression relies on conformational changes to chromatin that alter the availability of certain proteins for transcription (Bernstein et al., 2007). One of the best-studied ways organisms induce conformational changes to the chromatin is through histone-tail modifications. Histone residues may be ‘modified’ by the addition of different molecules to specific residues on the histone proteins (Bannister and Kouzarides, 2011). While several modifications have been identified, two of the most-commonly studied types are lysine residue acetylation or lysine residue methylation and they can dictate the transcriptional state of the genes occupied by those modified histones (Zhang et al., 2015). For example, lysine acetylation is generally considered an ‘activating’ mark that upregulates gene expression. Another activating mark is histone 3, lysine 4 trimethylation (H3K4me3), yet another modification on histone H3 at lysine 27 in the same fashion (H3K27me3) is a ‘repressive’ mark, leads to transcriptional silencing (Bannister and Kouzarides, 2011).

Histone lysine acetylation (H3ac), H3K4me3, and H3K27me3 levels have been assessed on the KSHV genome but the landscape of other histone modifications during lytic reactivation had not yet been ascertained (Gunther and Grundhoff, 2010; Toth et al., 2010 and 2013). So, when proteomic interaction studies conducted in our lab suggested that a lytic viral protein, ORF59, could be involved in chromatin remodeling, particularly in regards to arginine methylation, we set out to study the histone arginine methylation. Our recent study, ‘KSHV encoded ORF59 modulates histone arginine methylation of the viral genome to promote viral reactivation’ examined the enrichment of a specific histone modification H4R3me2s (histone 4 arginine 3 symmetric di-methylation) across the viral genome (Strahan et al., 2017). H4R3me involves first the mono-methylation of the arginine followed by the addition of another methyl group in either a symmetric, or asymmetric fashion (H4R3me2s or H4R3me2a, respectively) (Di Lorenzo and Bedford, 2011). Arginine methylation is important for various several cellular processes including RNA processing, DNA repair, transcription, signal transduction, and chromatin remodeling (Pahlich et al., 2006). While the addition of methyl groups on arginine residues increases hydrophobicity that blocks hydrogen bonding but the overall charge is not altered so the binding between nucleic acids or other proteins remains undisturbed (Pahlich et al., 2006). Various protein arginine methyltransferases (PRMTs) are expressed in multiple subcellular locales to modify the protein arginine residues of nuclear as well as cytoplasmic proteins (Bedford and Clarke, 2009).

Interestingly, the conformational difference between asymmetrically modified H4R3me2 and symmetrically H4R3me2 affects transcriptional activation/repression very distinctly. Symmetrically modified H4R3me2 favors the repression of gene expression and transcriptional silencing, while in contrast asymmetrically modified H4R3me2 favors upregulation of gene transcription (Di Lorenzo and Bedford, 2011).

In order to specifically capture the symmetrically modified, H4R3me2s chromatin, it was first absolutely essential to verify that the antibody used for ChIP-Seq purposes would not cross-react with H4R3me2a. Anti-H4R3me2s antibody was obtained and the specificity to the symmetrically modified H4R3me2 was tested before using for immunoprecipitating chromatin from KSHV infected cells. Following the confirmation of its specificity, we proceeded to perform ChIP-Seq from latent and lytically reactivating KSHV positive TRExBCBL1-RTA cells. The advantage of using TRExBCBL1-RTA cells (KSHV infected cell line) was to induce the expression of Replication and Transcription Activator (RTA) by tetracycline/doxycycline, which is both necessary and sufficient to trigger lytic reactivation (Nakamura et al., 2003). The ChIP assay was performed similarly to previously done ChIP-Seq assays that include the following basic steps: harvest the cells and cross-link with formaldehyde, isolate and shear chromatin, immunoprecipitate DNA-protein complexes of interest, purify the DNA, and prepare sequencing libraries from the immunoprecipitated and respective inputs DNA (Figure 1). To obtain a more thorough understanding of the chromatin remodeling role of viral protein ORF59, we needed to test the relative enrichment of several different factors at the KSHV genome including H4R3me2s, ORF59, PRMT5, COPR5. Each of these ChIPs were done in triplicate and samples were combined for library preparation. ChIP-Seq experiments traditionally use approximately 20 million cells per sample; however, to quantify H4R3me2s (and enrichment of other factors as well) on the KSHV genome, we chose to use a modified Low-Cell ChIP protocol with 5 million cells per sample instead (Park, 2009). To accomplish this, the chromatin-shearing step of the ChIP assay was optimized using the Diagenode Bioruptor® Pico to improve the efficiency of the DNA immunoprecipitation.


Figure 1. Flow-chart depiction of H4R3me2s ChIP. Cells from latent and lytic KSHV-positive cells were cross-linked to preserve DNA-protein interactions and the DNA was sheared into small fragments. DNA fragments were then immunoprecipitated, purified, and subjected to next-generation sequencing.

Another unique challenge faced in assessing chromatin structure of viral genomes is that H4R3me2s ChIP assay isolates both viral and cellular host DNA bound to H4R3me2s; and furthermore, upon lytic reactivation viral genomes are multiplied resulting in drastically different levels of viral DNA between two samples with an approximately identical number of cells (Figure 2). For this reason, we assessed H4R3me2s levels at a very early time point during lytic reactivation (12 h), before viral genome copies have had a chance to accumulate and possibly bias the downstream ChIP-Seq. As a result of these careful adjustments, we were able to successfully quantify relative enrichment of a repressive chromatin mark on the viral genome during two different phases of the lifecycle and demonstrate a novel chromatin-remodeling role for the early viral protein, ORF59.


Figure 2. Schematic depiction of H4R3me2s role in KSHV lytic reactivation. H4R3me2s is an abundant hallmark of transcriptionally silent, heterochromatin and must be removed to favor active gene transcription.

Materials and Reagents

  1. Materials
    1. Pipette tips
    2. 1.5 ml Bioruptor® Pico Microtubes with Caps (Diagenode, catalog number: C30010016 )
    3. Illumina NextSeq 500 Mid Output KT v2 (150 cycles) (Illumina, catalog number: FC-404-2001 )
      Note: Researchers should select the most appropriate flow cell for their experimental specifications. To sequence arginine methylation on the KSHV genome, this specific flow cell was sufficient.

  2. Chemicals/stock solutions
    1. Protease inhibitors (leupeptin, aprotinin, sodium fluoride, pepstatin, and phenylmethylsulfonyl fluoride) (Sigma-Aldrich, catalog number: S8830 )
    2. Pierce 16% formaldehyde (w/v), Methanol-Free (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 28906 )
    3. 1 M glycine
    4. Bioruptor® Pico sonication beads (Diagenode, catalog number: C01020031 )
    5. RNase A, 20 mg/ml
    6. 1.5% agarose gel
    7. Specific ChIP grade antibodies
      Note: To quantify H4R3me2s on the KSHV genome, following ChIP grade antibodies were used, rabbit anti-H4R3me2s (Active Motif, catalog number: 61187 ), rabbit anti-Histone H4 (Active Motif, catalog number: 61299 ), rabbit anti-control IgG (ChIP grade—Cell Signaling Technology, catalog number: 2729 ).
    8. 1x TE
    9. 5 M NaCl
    10. 7.5 M ammonium acetate
    11. 100% ethanol
    12. GlycoBlue Coprecipitant (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM9516 )
    13. Proteinase K
    14. QIAGEN MinElute PCR purification Kit (QIAGEN, catalog number: 28004 )
    15. Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific, InvitrogenTM, catalog number: Q32851 )
    16. NEXTflexTM ChIP Seq Kit (Illumina compatible) (Bioo Scientific, catalog number: NOVA-5143-02 )
    17. NEXTflexTM ChIP Seq Barcodes - 12 (Illumina compatible) (Bioo Scientific, catalog number: NOVA-514120 )
    18. KAPA Library Quantification Complete Kit (Universal), kit code KK4824 (Kapa Biosystems, catalog number: 07960140001 )
    19. Agilent Bioanalyzer High Sensitivity DNA chip Kit (Agilent Technologies, catalog number: 5067-4626 )
    20. 0.5 M PIPES, pH 8.0
    21. 1.7 M KCl
    22. 10% Nonidet-P40 (NP-40)
    23. 0.5 M EDTA, pH 8.0
    24. 1 M Tris-HCl pH 8.1
    25. Magnetic Protein A, and G beads
      1. Protein A Mag Sepharose (GE Healthcare, catalog number: 28944006 )
      2. Protein G Mag Sepharose (GE Healthcare, catalog number: 28944008 )
    26. Salmon sperm DNA, sheared 10 mg/ml (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM9680 )
    27. 10% Triton X-100
    28. 10% SDS
    29. 1 M NaHCO3

  3. Buffers
    1. 1x PBS (137 mM NaCl, 10 mM phosphate, 2.7 mM KCl, and a pH of 7.4)
    2. Buffer D Chromatin Shearing Buffer (Diagenode, catalog number: C01020030 )
    3. Cell lysis buffer (see Recipes)
    4. ChIP dilution buffer (see Recipes)
    5. ChIP Magnetic A+G beads (see Recipes)
    6. ChIP Low Salt Wash (see Recipes)
    7. ChIP elution buffer (see Recipes)

Equipment

  1. Pipettes
  2. Tabletop Eppendorf centrifuge, refrigerated and non-refrigerated
  3. Bioruptor® Pico Sonication device (Diagenode, catalog number: B01060001 )
  4. Magnetic stand
  5. Water bath
  6. Tube rotators
  7. Thermocycler
  8. Qubit Fluorometer (Thermo Fisher Scientific)
  9. Agilent 2100 Bioanalyzer (Agilent Technologies, model: Agilent 2100 , catalog number: G2939BA)
  10. Quantitative PCR machine
  11. Vortexer
  12. Illumina NextSeq 500 (Illumina, model: NextSeq 500 )
    Note: The Illumina NextSeq machine used for these studies is the property of the Nevada Genomics Center, who performed all sequencing runs for this study.

Software

  1. CLC workbench 10.0.1 (Licensed from QIAGEN, Germantown, MD)

Procedure

  1. Chromatin immunoprecipitation assay
    1. Harvest approximately 20 million cells and centrifuge into a pellet. Aspirate culture medium, then resuspend in 5 ml ice-cold PBS containing protease inhibitors (1 μg/ml leupeptin, 1 μg/ml aprotinin, 1 μg/ml sodium fluoride, 1 μg/ml pepstatin, and 1 mM phenylmethylsulfonyl fluoride) and centrifuge again to wash cells.
    2. Resuspend cell pellet in 10 ml of 1% formaldehyde in PBS and incubate samples at room temperature for 10 min with gentle rocking.
      Note: If using 16% methanol-free formaldehyde solution, mix 1 ml + 15 ml PBS to obtain 1% formaldehyde solution to cross-link cells in.
    3. Immediately add glycine to samples at a final concentration of 125 mM to quench the cross-linking reaction and rock samples 5 min at room temperature.
      Note: For 10 ml of cross-linking cell suspension, add 1.25 ml 1 M glycine to obtain a final concentration of 125 mM.
    4. Centrifuge cells at 70 x g for 5 min, wash once more in 5 ml ice-cold PBS with protease inhibitors.
    5. Aspirate PBS from cell pellet and resuspend cells in 1 ml cell lysis buffer [5 mM piperazine-N,N’-bis (2-ethanesulfonic acid) (PIPES)-KOH (pH 8.0)-85 mM KCl-0.5% NP-40; Recipe 1] supplemented with protease inhibitors and incubate on ice for 10 min.
    6. Centrifuge at 800 x g for 5 min at 4 °C to collect the nuclei, aspirate supernatant from pellet.
    7. Meanwhile, prepare 1.5 ml sonication tubes by adding approximately 100 mg of Bioruptor® sonication beads and labeling tubes appropriately.
    8. Resuspend pellet in Buffer D Chromatin Shearing Buffer (Diagenode Inc.) in preparation for chromatin shearing using the Bioruptor® Pico instrument (Diagenode Inc.). For 20 million cells, resuspend nuclei in 1.8 ml Buffer D, and distribute 300 μl to 6 prepared sonication tubes.
    9. Sonicate using the Bioruptor® Pico sonication device using On/Off time for each cycle of 30 sec/30 sec for 60 cycles.
      Note: Sonication parameters may take further optimization of conditions to obtain ideal chromatin shearing where fragments about 300 bp long are obtained. See Diagenode manuals for further suggestions on optimizing sonication efficiency.
    10. Centrifuge sonication tubes in a tabletop centrifuge at 4,000 x g for 1 min to remove cell debris and transfer supernatant to clean tubes. Supernatant should be fairly clear after sonication. If desired, a small 15-20 μl aliquot may be taken from this step and treated with 1 μl RNase (20 mg/ml stock) for 30 min, then cross-links reversed by incubation at 65 °C for at least 4 h or overnight and resolved on a 1.5% agarose gel to check shearing efficiency (see Figure 3 for an example of shearing results).


      Figure 3. Chromatin shearing of KSHV positive cell lines. Lane 1, 100 bp marker. Lane 2-5 are shearing controls from TRExBCBL1-RTA cells, lanes 2 and 4 latent uninduced samples, lanes 3 and 5 are lytically reactivated cells. Lanes 2 and 3 demonstrate efficient shearing in preparation for ChIP assay, while lanes 4 and 5 show some residual DNA fragments (smearing) that are too large for ChIP-Seq assay. Lanes 2 and 3 were sheared using the Bioruptor® Pico for 30 sec on/off cycles for 60 min; lanes 4-5 were sheared using the identical parameters but the shorter time (30 min).

    11. Add 1-3x volume of ChIP dilution buffer (1.2 mM EDTA, 16.7 mM Tris, pH 8.1, 167 mM NaCl, and protease inhibitors; Recipe 2) to sheared chromatin samples and divide volume in two, one set for IgG control ChIP, the other for specific antibody.
      Note: For H4R3me2s ChIP from KSHV cells, we used 3x dilution of ChIP dilution buffer and 2 μg of specific antibody. Depending on antibody efficiency, a lesser dilution (1x or 2x volume of ChIP dilution buffer) of sheared chromatin can be used for ChIP assay.
    12. Pre-clear samples with addition of 15 μl/ml of ChIP Magnetic A + G beads (Recipe 3) and rotate at 4 °C 30 min.
      Note: If antibody specificity is excellent, the pre-clearing step may be omitted, however, to reduce background signal in the IgG samples when performing ChIP-Seq we recommend keeping this step.
    13. Retain 10% of ChIP lysate for input fraction, store samples at 4 °C.
    14. Collect beads against a magnetic stand and transfer ChIP lysate to fresh tubes. Add IgG or specific antibody (2 μg or according to supplier’s instructions) and rotate ChIP samples overnight 4 °C.
    15. Next day, collect DNA-protein complexes by adding 25 μl/ml of ChIP Magnetic A + G beads and rotating 4 °C for 2 h.
    16. Wash ChIP samples 3 times each with 1 ml of ChIP Low Salt Wash buffer (0.1% SDS-1.0% Triton X-100-2 mM EDTA-20 mM Tris [pH 8.1]-150 mM NaCl; Recipe 4), and twice with 1x TE.
      Note: Refer to Figure 4 for visual representation of magnetic bead washing procedure.


      Figure 4. Magnetic bead washing procedure. A. Gently invert magnetic bead slurry with wash buffer to evenly mix and wash beads. B. Place the sample in magnetic rack and let stand until slurry appears clear because all magnetic beads have settled at the edge of the tube near the magnet. Carefully remove supernatant from beads and proceed with further washes or DNA elution step. This method removes loosely bound non-specific DNA fragments that contribute to background noise during sequencing.

    17. Elute chromatin complexes from magnetic beads by adding 150 μl of freshly-prepared elution buffer (1% SDS-0.1 M NaHCO3; Recipe 5) and vortex very mildly for 15 min. Collect eluates by applying magnetic stand and transferring supernatant to clean tubes.
      Note: To elute, set vortexer to a lowest speed and secure samples for continual vortexing for 15 min. Alternately, samples may be rotated for 15 min to achieve the same mild agitating effect. The elution step must be carried out at room temperature.
    18. Repeat Step A17 for a total of 2 elution steps, and a total of 300 μl of eluted chromatin. Also, bring ChIP Input samples to a total volume of 300 μl, adding elution buffer if need be.
    19. Reverse cross-links by adding 18 μl of 5 M NaCl (0.3 M NaCl final concentration) and 1 μl RNase A (10 mg/ml), and incubating samples at 65 °C for at least 4 h or overnight.
      Note: If the shorter reverse-crosslinking time is preferred (4 h), we recommend incubating samples in 65 °C water-bath to ensure efficient sample heating. If reversing crosslinks overnight, a hybridization oven set to 65 °C is sufficient.
    20. After reverse-crosslinking is complete, precipitate samples by adding 1/15th volume 7.5 M ammonium acetate, and 2 volumes of 100% ethanol and incubating at -80 °C for at least 30 min.
      Note: We recommend also adding a co-precipitant like GlycoBlue, 1 μl, to the samples at this stage to make visualizing the pellet easier.
    21. Centrifuge at max speed in a 4 °C benchtop centrifuge for 15 min to pellet DNA. Semi-dry the pellet for approximately 5 min by leaving the tube open.
    22. Resuspend pellet in 100 μl 1x TE, and add 2 μl Proteinase K (10 mg/ml), and incubate samples at 45 °C for 2 h.
    23. Purify DNA using QIAGEN MinElute PCR Purification Kit, according to manufacturer’s instructions.
      Note: Alternately, phenol-chloroform extraction may be performed at this step, and resulting DNA can be precipitated as in Step A20. Then add 1 ml 70% ethanol to each sample, centrifuging at max speed, 4 °C for 15 min, discarding ethanol, and repeating for a total of 3 washes. Let DNA pellets air dry briefly, then resuspend in 20-30 μl nuclease free water to proceed library preparation and sequencing.
    24. Quantify DNA concentration using the Qubit High-Sensitivity DNA fluorometer, following manufacturer’s instructions. Briefly, Qubit dsDNA HS Reagent is mixed with an appropriate volume of Qubit dsDNA HS Buffer to make the Qubit dsDNA HS working solution. 1 μl of sample is mixed with 199 μl Qubit dsDNA HS working solution and the tube containing the sample is put into the machine for a reading that reveals the concentration of the original 1 μl sample. Once the concentration of DNA samples is determined, proceed to library preparation.

  2. ChIP Library Preparation: NEXTflexTM ChIP-Seq Kit (Illumina Compatible) (Bioo Scientific Corp.)
    For quantifying H4R3me2s enrichment on the viral KSHV genome, we used 10 ng of ChIP/Input DNA and chose to follow ‘Option 3’ of manufacturer’s instructions exactly for library preparation. A brief overview of the ChIP-Kit protocol is as follows:
    1. End Repair of DNA samples, during this step, ends of DNA are blunted and 5’ phosphorylated.
    2. Gel-free size selection clean up, using magnetic beads DNA fragments below 300 bp and above 400 bp are discarded while fragments between 300-400 bp long are purified for the next step of library preparation.
    3. 3’ Adenylation, during this step 3’ ends are A-tailed.
    4. Adapter Ligation, during this step specific barcode DNA sequences are ligated to sequencing samples to enrich library concentration downstream and enable multiplexed sequencing runs.
    5. Clean up, during this step DNA sequences with barcodes ligated on are purified in preparation for the next step. This is important to remove excess adapter sequences or adapter dimers, which can unfavorably disrupt the next PCR step in the protocol.
    6. PCR amplification, during this step PCR is performed to enrich adapter-ligated products. To reduce PCR bias, the least number of cycles possible is recommended.
      Note: For the library preparation PCR amplification, kit step F3, 16 repeat cycles were used for preparing the libraries used in our previous study, ‘KSHV encoded ORF59 modulates histone arginine methylation of the viral genome to promote viral reactivation’. As the kit recommends, the number of PCR cycles may be optimized depending on starting material concentration and quantity.
    7. Clean up, this is the last clean-up and purification step of the library preparation protocol. We recommend using 23 μl of Resuspension Buffer for the final elution and collecting 20 μl of the final sample as opposed to the kit’s instructions (33 μl Resuspension Buffer, collecting 30 μl final sample) as this yields a more concentrated end product.

  3. ChIP library validation and quantification
    1. To evaluate library quality, we suggest using the Agilent Bioanalyzer and running the High Sensitivity DNA chip. Properly prepared libraries yield one band of approximately 300-350 bp in size.
    2. To accurately quantify library sample concentration, we suggest using the KAPA Library Quantification Kit and following manufacturer’s instructions exactly. Accurate quantification of library concentration is essential for optimal clustering and maximizing the number of reads for each sample during the sequencing run.

  4. Sequencing
    1. High coverage sequencing means that each base is covered by a greater degree of aligned sequence reads, and thus the base calls assume a higher degree of confidence. According to Illumina’s instructions, coverage of approximately 100x is recommended for ChIP-Seq applications. Next-generation sequencing is a highly-standardized, validated process and we recommend following the prescribed procedures.
    2. For further information on Next Generation sequencing depth and coverage, manufacturer’s recommendations may be followed. Illumina’s recommendations can be accessed at https://www.illumina.com/science/education/sequencing-coverage.html for a detailed description of these important considerations.

Data analysis

  1. FastQ data generated from the NextSeq (Illumina, San Diego, CA) was annotated and the sequence reads were analyzed using CLC workbench 10.0.1 (QIAGEN, Germantown, MD). The sequence reads were downloaded from the Illumina Base Space repository that the Nevada Genomics Center uploaded. These files can be accessed through internet imported to the sequences analysis software, CLC Workbench in our case using the ‘Import Illumina Reads’ option.
  2. The reads obtained from input, control IgG antibody and specific antibody samples were mapped to the KSHV genome using ‘map reads to the reference’ operation of the CLC workbench.
  3. Mapped reads were subjected for identifying the enriched peaks in ChIP samples after comparing with the respective input samples peaks using the ‘ChIP-seq’ tool of the CLC Workbench. Relative enrichment is presented as a peak score calculated using minimum peak-calling P-value of 0.05. (Please refer to ‘KSHV encoded ORF59 modulates histone arginine methylation of the viral genome to promote viral reactivation’ by Strahan et al., 2017, publicly available at PLoS Pathogens).
  4. One challenge unique to assessing the relative abundance of this chromatin mark on the KSHV viral genome was that despite of high sequencing depth for each sample, only about 1% of the total ChIP-Seq reads mapped to the viral genome. This illustrates the importance of deep sequencing coverage for evaluating viral chromatin conformation.

Recipes

  1. Cell lysis buffer, 50 ml
    500 μl 0.5 M PIPES, pH 8.0
    2.5 ml 1.7 M KCl
    2.5 ml 10% NP-40
    Sterile ddH2O to 50 ml, store at room temperature
    Add protease inhibitors fresh just before use
  2. ChIP dilution buffer, 50 ml
    125 μl 0.5 M EDTA pH 8.0
    835 μl 1 M Tris-HCl pH 8.1
    1.67 ml 5 M NaCl
    Sterile ddH2O to 50 ml, store at room temperature
    Add protease inhibitors fresh just before use
  3. ChIP Magnetic A + G beads
    1. Combine 300 μl Protein A magnetic beads with 300 μl Protein G magnetic beads and wash with 1x TE 3 times
      Note: Place tubes against a magnetic stand, remove supernatant, remove from magnetic stand, add 1 ml TE, repeat.
    2. Resuspend bead slurry in 1x TE with 1 mg/ml sonicated salmon sperm DNA, and 1 mg/ml BSA and rotate overnight 4 °C
      Note: Sonicated salmon sperm DNA is used as a blocking agent to reduce non-specific background DNA binding.
    3. Next day, wash beads 1 time with TE, then resuspend in a final volume of 500 μl TE, beads are now ready for ChIP assay
  4. ChIP Low Salt Wash, 50 ml
    5 ml 10% Triton X-100
    500 μl 10% SDS
    200 μl 0.5 M EDTA pH 8.0
    1 ml 1 M Tris-HCl pH 8.1
    1.5 ml 5 M NaCl
    Sterile ddH2O to 50 ml, store at room temperature
    Add protease inhibitors fresh just before use and store on ice
    Discard after washing steps
  5. ChIP elution buffer
    1 ml 10% SDS
    1 ml 1 M NaHCO3 (freshly prepared)
    Sterile ddH2O to 10 ml, store at room temperature and discard excess after use

Acknowledgments

This work was supported by the National Institute of Health (CA174459 and AI105000). This protocol was adapted to determine H4R3me2s enrichment on the KSHV genome from a combination of previous ChIP-Seq protocols and the LowCell number ChIP-protocol from Diagenode Inc. The authors would like to acknowledge the cooperation and help from the Nevada Genomics Center (University of Nevada, Reno, USA) in performing the Illumina sequencing runs.
Conflict of Interest: Authors declare no conflict of interests and competing interests, which may impact design and implementation of the protocol described above.

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  16. Strahan, R. C., McDowell-Sargent, M., Uppal, T., Purushothaman, P. and Verma, S. C. (2017). KSHV encoded ORF59 modulates histone arginine methylation of the viral genome to promote viral reactivation. PLoS Pathog 13(7): e1006482.
  17. Toth, Z., Brulois, K. and Jung, J. U. (2013). The chromatin landscape of Kaposi's sarcoma-associated herpesvirus. Viruses 5(5): 1346-1373.
  18. Toth, Z., Brulois, K., Lee, H. R., Izumiya, Y., Tepper, C., Kung, H. J. and Jung, J. U. (2013). Biphasic euchromatin-to-heterochromatin transition on the KSHV genome following de novo infection. PLoS Pathog 9(12): e1003813.
  19. Toth, Z., Maglinte, D. T., Lee, S. H., Lee, H. R., Wong, L. Y., Brulois, K. F., Lee, S., Buckley, J. D., Laird, P. W., Marquez, V. E. and Jung, J. U. (2010). Epigenetic analysis of KSHV latent and lytic genomes. PLoS Pathog 6(7): e1001013.
  20. Uppal, T., Banerjee, S., Sun, Z., Verma, S. C. and Robertson, E. S. (2014). KSHV LANA--the master regulator of KSHV latency. Viruses 6(12): 4961-4998.
  21. Uppal, T., Jha, H. C., Verma, S. C. and Robertson, E. S. (2015). Chromatinization of the KSHV genome during the KSHV life cycle. Cancers (Basel) 7(1): 112-142.
  22. Zhang, T., Cooper, S. and Brockdorff, N. (2015). The interplay of histone modifications - writers that read. EMBO Rep 16(11): 1467-1481.

简介

组蛋白尾部的翻译后修饰有助于染色质的三维结构,并在确定附近基因的相对表达中起重要作用。一种这样的修饰是精氨酸残基的对称二甲基化,其可能对基因表达展现出不同的作用,包括阻断转录激活剂的结合或招募抑制效应分子。最近的ChIP-Seq研究已经证明不同组蛋白修饰之间在基因调节中的相互作用的重要性。因此,为了全面了解这些表观遗传标记的共同努力,必须利用ChIP-Seq来鉴定染色质上的特定富集。利用表观遗传机制进行基因调控,并且通过ChIP-Seq以全面,不偏倚的方式评估多种组蛋白修饰的相对丰度,我们可以获得关于病毒复制和发病机理的复杂机制的更好的见解。

【背景】卡波西肉瘤相关疱疹病毒(KSHV)是一种致癌性人类病毒,在其生命周期中有两个不同阶段。在最初感染后,KSHV在宿主中建立持续的终生感染,这对免疫受损的个体来说特别有问题。 KSHV可引起HIV / AIDS患者中的各种肿瘤,包括卡波西肉瘤和多发性B细胞淋巴瘤(Chang等,1994; Cesarman等,1995; Russo ,1996; Soulier ,1995)。 KSHV拥有大约165,000bp的大型基因组,编码近90种不同的开放阅读框,具有逃避宿主免疫监视系统,改变宿主细胞生长途径和产生感染性后代病毒粒子的充足工具。

在潜伏阶段期间,仅有一部分病毒基因被表达,其在本质上是致癌的并且还有助于病毒附加体复制和传递到分裂的肿瘤细胞中(Uppal等人,2014; Purushothaman 等人,2016)。然而,包括病毒合并感染(HIV)和其他刺激如低氧,氧化应激或免疫抑制药物在内的许多因素可能会引发病毒转移到生命周期的活动裂解阶段,导致感染后代的产生病毒粒子从宿主细胞表面流出以感染周围组织(Purushothaman等人,2015)。这些复杂的过程以协调的方式进行,在转换到病毒生命周期的裂解(产生病毒)阶段期间,以顺序方式表达特定基因(Purushothaman等人,2015; Aneja和Yuan,2017)。

KSHV采用表观遗传机制来仔细调节将病毒维持在生命周期特定阶段所需的差异基因表达。在感染并进入人细胞后,病毒基因组获得与宿主基因组相似的细胞组蛋白,并且持续存在常染色质(转录 - 允许)或异染色质(转录 - 抑制)状态(Toth等人 ,2013; Uppal 等人,2015)。通过这些手段,KSHV能够将潜伏期中的基因表达限制在仅仅最小的基因子集中,并且准备快速转变为裂解复制期。 KSHV诱导的恶性肿瘤的发展涉及生命周期的两个阶段,因此研究人员必须清楚了解这些表观遗传变化如何调控病毒基因表达的机制。

基因表达的表观遗传调节依赖于染色质的构象变化,这改变了某些蛋白转录的可用性(Bernstein等人,2007)。研究得最多的生物体之一诱导染色质构象变化是通过组蛋白尾部修饰。组蛋白残基可以通过将不同分子添加到组蛋白上的特定残基上进行“修饰”(Bannister and Kouzarides,2011)。虽然已经确定了几种修饰,但是最常被研究的两种类型是赖氨酸残基乙酰化或赖氨酸残基甲基化,并且它们可以决定那些被修饰的组蛋白占据的基因的转录状态(Zhang等人 ,2015)。例如,赖氨酸乙酰化通常被认为是上调基因表达的“活化”标记。另一个激活标记是组蛋白3,赖氨酸4三甲基化(H3K4me3),对组蛋白H3在赖氨酸27上的另一种修饰(H3K27me3)是'抑制'标记,导致转录沉默(Bannister and Kouzarides,2011)。

已经在KSHV基因组上评估了组蛋白赖氨酸乙酰化(H3ac),H3K4me3和H3K27me3水平,但尚未确定裂解再激活期间其他组蛋白修饰的景观(Gunther和Grundhoff,2010; Toth等人, 2010年和2013年)。因此,当我们实验室进行的蛋白质组学相互作用研究表明裂解性病毒蛋白ORF59可能参与染色质重塑,尤其是精氨酸甲基化时,我们开始研究组蛋白精氨酸甲基化。我们最近的研究'KSHV编码的ORF59调节病毒基因组的组蛋白精氨酸甲基化以促进病毒再激活'检查了整个病毒基因组中特定组蛋白修饰H4R3me2s(组蛋白4精氨酸3对称二甲基化)的富集(Strahan等人,2017)。 H4R3me首先涉及精氨酸的单甲基化,接着以对称或不对称方式(分别为H4R3me2s或H4R3me2a)添加另一个甲基(Di Lorenzo and Bedford,2011)。精氨酸甲基化对于包括RNA加工,DNA修复,转录,信号转导和染色质重塑在内的多种细胞过程是重要的(Pahlich等人,2006)。尽管在精氨酸残基上添加甲基增加了阻断氢键的疏水性,但总体电荷没有改变,因此核酸或其他蛋白质之间的结合保持不受干扰(Pahlich等人,2006)。各种蛋白质精氨酸甲基转移酶(PRMT)在多个亚细胞位置表达以修饰核和细胞质蛋白质的蛋白质精氨酸残基(Bedford和Clarke,2009)。

有趣的是,不对称修饰的H4R3me2和对称的H4R3me2之间的构象差异非常明显地影响转录激活/抑制。对称修饰的H4R3me2有利于抑制基因表达和转录沉默,而相反,不对称修饰的H4R3me2有利于基因转录的上调(Di Lorenzo和Bedford,2011)。

为了特异性捕获对称修饰的H4R3me2s染色质,首先必须验证用于ChIP-Seq目的的抗体不会与H4R3me2a发生交叉反应。获得抗H4R3me2s抗体,并且在用于从KSHV感染的细胞中免疫沉淀染色质之前测试对对称修饰的H4R3me2的特异性。确认其特异性后,我们继续从潜在的和裂解性地重新激活KSHV阳性TRExBCBL1-RTA细胞中进行ChIP-Seq。使用TRExBCBL1-RTA细胞(KSHV感染的细胞系)的优点是通过四环素/多西环素诱导复制和转录激活物(RTA)的表达,这对于引发溶解性再激活是必需的和充分的(Nakamura等人, ,2003)。 ChIP测定与先前完成的ChIP-Seq测定类似地进行,其包括以下基本步骤:收获细胞并与甲醛交联,分离并剪切染色质,免疫沉淀感兴趣的DNA-蛋白质复合物,纯化DNA并制备测序来自免疫沉淀和各自输入DNA的文库(图1)。为了更全面地了解病毒蛋白ORF59的染色质重塑作用,我们需要测试KSHV基因组中几种不同因子的相对富集,包括H4R3me2s,ORF59,PRMT5,COPR5。每个这些ChIP都重复三次,并将样品合并用于文库制备。 ChIP-Seq实验传统上每个样品使用约2000万个细胞;然而,为了量化KSHV基因组上的H4R3me2s(以及其他因子的富集),我们选择使用每个样品500万个细胞的改良的低细胞ChIP方案(Park,2009)。为此,使用Diagenode Bioruptor Pico Pico来优化ChIP测定的染色质剪切步骤以提高DNA免疫沉淀的效率。

“”src
图1. H4R3me2s ChIP的流程图描绘来自潜伏和裂解性KSHV阳性细胞的细胞交联以保持DNA-蛋白质相互作用,并将DNA剪切成小片段。然后将DNA片段免疫沉淀,纯化,并进行下一代测序。

在评估病毒基因组的染色质结构时面临的另一个独特挑战是H4R3me2s ChIP测定分离与H4R3me2结合的病毒和细胞宿主DNA;此外,裂解性重新激活后,病毒基因组倍增,导致两个样本之间病毒DNA水平大幅度不同(具有大致相同数量的细胞)(图2)。出于这个原因,我们在裂解重新激活期间(12小时)在非常早的时间点评估H4R3me2s水平,然后病毒基因组拷贝有机会积累并可能偏向下游ChIP-Seq。作为这些仔细调整的结果,我们能够成功量化在生命周期的两个不同阶段期间病毒基因组上抑制性染色质标记的相对富集,并展示对于早期病毒蛋白ORF59的新的染色质重塑作用。

“”src
图2. H4R3me2s在KSHV裂解性再激活中的作用示意图描述H4R3me2s是转录沉默异染色质的一个丰富标志,必须去除以利于活性基因转录。

关键字:KSHV, 病毒染色质, ChIP-Seq, 精氨酸甲基化

材料和试剂

  1. 物料
    1. 移液器吸头
    2. 1.5 ml Bioruptor Pico Microtubes with Caps(Diagenode,目录号:C30010016)
    3. Illumina NextSeq 500中等输出KT v2(150个周期)(Illumina,目录号:FC-404-2001)
      注意:研究人员应该为他们的实验规格选择最合适的流通池。为了对KSHV基因组上的精氨酸甲基化进行测序,这个特定的流动细胞已经足够了。

  2. 化学品/储备溶液
    1. 蛋白酶抑制剂(亮肽素,抑肽酶,氟化钠,胃蛋白酶抑制剂和苯甲基磺酰氟)(Sigma-Aldrich,目录号:S8830)
    2. Pierce 16%甲醛(w / v),无甲醇(Thermo Fisher Scientific,Thermo Scientific TM,目录号:28906)
    3. 1 M甘氨酸
    4. Bioruptor Pico超声珠(Diagenode,目录号:C01020031)
    5. RNA酶A,20mg / ml
    6. 1.5%琼脂糖凝胶
    7. 特定ChIP级抗体
      注:为了定量KSHV基因组上的H4R3me2s,使用了以下ChIP级抗体:兔抗H4R3me2(活性基序,目录号:61187),兔抗组蛋白H4(活性基序,目录号:61299),兔抗 - 对照IgG(ChIP级 - 细胞信号传导技术,目录号:2729)。
    8. 1x TE
    9. 5 M NaCl
    10. 7.5 M醋酸铵
    11. 100%乙醇
    12. GlycoBlue共沉淀剂(Thermo Fisher Scientific,Invitrogen TM,目录号:AM9516)
    13. 蛋白酶K
    14. QIAGEN MinElute PCR纯化试剂盒(QIAGEN,目录号:28004)
    15. Qubit dsDNA HS分析试剂盒(Thermo Fisher Scientific,Invitrogen TM,目录号:Q32851)
    16. NEXTflex TM ChIP Seq试剂盒(Illumina兼容)(Bioo Scientific,目录号:NOVA-5143-02)
    17. NEXTflex TM ChIP Seq条形码-12(Illumina兼容)(Bioo Scientific,目录号:NOVA-514120)
    18. KAPA Library Quantification Complete Kit(Universal),试剂盒编码KK4824(Kapa Biosystems,产品目录号:07960140001)
    19. 安捷伦生物分析仪高灵敏度DNA芯片试剂盒(安捷伦科技公司,产品目录号:5067-4626)
    20. 0.5 M PIPES,pH 8.0
    21. 1.7 M KCl
    22. 10%Nonidet-P40(NP-40)
    23. 0.5M EDTA,pH8.0
    24. 1 M Tris-HCl pH 8.1
    25. 磁蛋白A和G珠子
      1. 蛋白A Mag Sepharose(GE Healthcare,目录号:28944006)
      2. 蛋白G Mag Sepharose(GE Healthcare,目录号:28944008)
    26. 剪下的10mg / ml鲑鱼精子DNA(Thermo Fisher Scientific,Invitrogen TM,目录号:AM9680)
    27. 10%Triton X-100
    28. 10%SDS
    29. 1M NaHCO 3

  3. 缓冲区
    1. 1xPBS(137mM NaCl,10mM磷酸盐,2.7mM KCl,pH7.4)
    2. 缓冲液D染色质剪切缓冲液(Diagenode,目录号:C01020030)
    3. 细胞裂解缓冲液(见食谱)
    4. ChIP稀释缓冲液(见食谱)
    5. ChIP磁性A + G珠(见食谱)
    6. ChIP低盐洗(见食谱)
    7. ChIP洗脱缓冲液(见食谱)

设备

  1. 移液器
  2. 台式Eppendorf离心机,冷藏和非冷藏
  3. Bioruptor®Pico声处理设备(Diagenode,目录号:B01060001)
  4. 磁座
  5. 水浴
  6. 管旋转器
  7. 热循环仪
  8. Qubit荧光计(赛默飞世尔科技)
  9. Agilent 2100生物分析仪(Agilent Technologies,型号:Agilent 2100,目录号:G2939BA)
  10. 定量PCR仪
  11. 漩涡
  12. Illumina NextSeq 500(Illumina,型号:NextSeq 500)
    注意:用于这些研究的Illumina NextSeq机器是内华达州基因组学中心的财产,该中心为本研究执行所有测序运行。

软件

  1. CLC工作台10.0.1(许可来自德国马里兰州Germantown的QIAGEN)

程序

  1. 染色质免疫沉淀分析
    1. 收获大约2000万个细胞并离心成颗粒。吸出培养基,然后重悬于含有蛋白酶抑制剂(1μg/ ml亮抑蛋白酶肽,1μg/ ml抑肽酶,1μg/ ml氟化钠,1μg/ ml胃酶抑素和1mM苯甲基磺酰氟)的5ml冰冷PBS中,并重悬于离心机再次洗涤细胞。
    2. 将细胞沉淀重新悬浮于PBS中的10ml 1%甲醛中,室温孵育10分钟,轻轻摇动。
      注意:如果使用16%无甲醛的甲醛溶液,将1 ml + 15 ml PBS混合以获得1%甲醛溶液以交联细胞。
    3. 立即将甘氨酸加入终浓度为125 mM的样品中以终止交联反应,并在室温下摇动样品5分钟。
      注意:对于10ml交联细胞悬液,加入1.25ml 1M甘氨酸以获得125mM的终浓度。
    4. 将细胞以70μgxg离心5分钟,再用5ml含蛋白酶抑制剂的冰冷PBS洗涤一次。
    5. 从细胞沉淀中吸出PBS并将细胞重悬于1ml细胞裂解缓冲液[5mM哌嗪-N,N'-双(2-乙磺酸)(PIPES)-KOH(pH8.0)-85mM KCl-0.5%NP-40;配方1]补充蛋白酶抑制剂并在冰上孵育10分钟。
    6. 在4℃下800×g离心5分钟以收集细胞核,从细胞团中吸出上清液。
    7. 同时,通过适当添加约100mg Bioruptor超声处理珠和标记管来制备1.5ml超声处理管。
    8. 用缓冲液D染色质剪切缓冲液(Diagenode Inc.)重悬沉淀,用Bioruptor Pico仪器(Diagenode Inc.)准备染色质剪切。对于2000万个细胞,将细胞核重悬于1.8ml缓冲液D中,并将300μl分配到6个制备的超声处理管中。
    9. 使用Bioruptor Pico超声处理装置进行超声处理,每个循环30秒/ 30秒使用开/关时间进行60个循环。
      注意:超声参数可以进一步优化条件以获得理想的染色质剪切,其中获得约300bp长的片段。有关优化超声处理效率的进一步建议,请参阅Diagenode手册。
    10. 用4,000×g 离心超声处理管在台式离心机中1分钟以除去细胞碎片并将上清液转移到清洁的管中。超声处理后上清液应清除干净。如果需要,可以从该步骤中取少量15-20μl等分试样并用1μlRNase(20mg / ml原液)处理30分钟,然后通过在65℃孵育至少4小时来逆转交联,或者过夜并在1.5%琼脂糖凝胶上解析以检查剪切效率(参见图3的剪切结果实例)。


      图3. KSHV阳性细胞系的染色质剪切泳道1,100bp标记。泳道2-5是来自TRExBCBL1-RTA细胞的剪切对照,泳道2和4是潜在未诱导样品,泳道3和5是裂解活化的细胞。泳道2和3在ChIP测定中表现出有效的剪切作用,而泳道4和5显示出一些对于ChIP-Seq测定而言太大的残余DNA片段(涂抹)。泳道2和3使用Bioruptor Pico Pico剪切30秒开/关循环60分钟;
      使用相同的参数剪切4-5线,但时间较短(30分钟)。

    11. 加入1-3倍体积的ChIP稀释缓冲液(1.2mM EDTA,16.7mM Tris,pH8.1,167mM NaCl和蛋白酶抑制剂;方案2)以剪切染色质样品并将体积分成两份,一份用于IgG对照ChIP,其他用于特异性抗体。
      注:对于来自KSHV细胞的H4R3me2s ChIP,我们使用3倍稀释的ChIP稀释缓冲液和2μg特异性抗体。取决于抗体效率,剪切染色质的较低稀释度(1倍或2倍体积的ChIP稀释缓冲液)可用于ChIP测定。
    12. 预先清除样品并加入15μl/ ml ChIP磁性A + G珠(配方3),并在4℃下旋转30分钟。
      注意:如果抗体特异性非常好,则可以省略预清除步骤,但是为了减少进行ChIP-Seq时IgG样本中的背景信号,我们建议保留此步骤。
    13. 保留10%的ChIP裂解物用于输入部分,将样品储存在4°C。
    14. 将磁珠收集到磁力架上并将ChIP裂解物转移至新鲜试管中。加入IgG或特异性抗体(2μg或根据供应商的说明)并在4°C下旋转芯片样品。
    15. 第二天,通过加入25μl/ ml ChIP磁性A + G珠并收集DNA-蛋白质复合物并在4℃下旋转2小时。
    16. 用1ml ChIP低盐洗涤缓冲液(0.1%SDS-1.0%Triton X-100-2mM EDTA-20mM Tris [pH8.1] -150mM NaCl;配方4)洗涤ChIP样品各3次,并用1x TE。
      注意:请参阅图4以了解磁珠清洗程序的可视表示。


      图4.磁珠清洗程序。 :一种。用洗涤缓冲液轻轻颠倒磁珠浆液,以均匀混合并清洗珠子。 B.将样品放置在磁性架上,静置直到浆液显得清澈,因为所有磁珠都沉积在靠近磁铁的管道边缘。小心地从珠子中除去上清液并进行进一步的洗涤或DNA洗脱步骤。该方法去除了在测序期间造成背景噪声的松散结合的非特异性DNA片段。

    17. 通过加入150μl新制备的洗脱缓冲液(1%SDS-0.1M NaHCO 3 3;配方5)从磁珠洗脱染色质复合物并非常温和地涡旋15分钟。
      使用磁力架收集洗脱液并将上清液转移到清洁管中。
      注意:为了洗脱,将旋涡器设置为最低速度并保证样品连续涡旋15分钟。或者,样品可以旋转15分钟以达到相同的轻微搅动效果。洗脱步骤必须在室温下进行。
    18. 重复步骤A17共2个洗脱步骤,并且总共300μl洗脱的染色质。另外,将ChIP输入样品加入到300μl的总体积中,如果需要的话加入洗脱缓冲液。
    19. 通过加入18μl5M NaCl(0.3M NaCl终浓度)和1μlRNase A(10mg / ml)反向交联,并将样品在65℃孵育至少4小时或过夜。
      注意:如果反向交联时间较短(4 h),我们建议在65°C水浴中孵育样品以确保有效加热样品。如果在一夜之间反转交联,设置为65°C的杂交炉就足够了。
    20. 反向交联完成后,通过加入1/15体积7.5M乙酸铵和2体积100%乙醇并在-80℃温育至少30分钟来沉淀样品。
      注意:我们建议在此阶段向样品中加入一种共沉淀剂,如1微升GlycoBlue,以便更容易地观察颗粒。
    21. 在4℃台式离心机中以最大速度离心15分钟以沉淀DNA。
      。将管子打开半干燥约5分钟
    22. 用100μl1x TE重悬沉淀,加2μl蛋白酶K(10 mg / ml),45°C孵育2 h。
    23. 根据制造商的说明使用QIAGEN MinElute PCR纯化试剂盒纯化DNA。
      注意:或者,可以在该步骤中进行苯酚 - 氯仿提取,并且可以如步骤A20那样沉淀所产生的DNA。然后向每个样品中加入1ml 70%乙醇,以最大速度离心,4℃15分钟,弃去乙醇,重复总计3次洗涤。让DNA颗粒短暂风干,然后重新悬浮于20-30μl无核酸酶水中进行文库制备和测序。
    24. 按照制造商的说明使用Qubit高灵敏度DNA荧光计定量DNA浓度。简而言之,将Qubit dsDNA HS试剂与适当体积的Qubit dsDNA HS缓冲液混合以制备Qubit dsDNA HS工作溶液。将1μl样品与199μlQubit dsDNA HS工作溶液混合,并将含有样品的管放入机器中读取显示原始1μl样品浓度的读数。一旦确定了DNA样本的浓度,请继续进行文库准备。

  2. ChIP文库制备:NEXTflex TM ChIP-Seq试剂盒(Illumina兼容)(Bioo Scientific Corp.)
    为了量化病毒KSHV基因组上H4R3me2s的富集,我们使用了10ng ChIP /输入DNA,并选择遵循制造商说明的'选项3'准确地用于文库制备。 ChIP-Kit协议简要概述如下:
    1. 末端修复DNA样本,在这一步骤中,DNA末端被钝化并被5'磷酸化。
    2. 使用磁珠将300bp以上和400bp以下的DNA片段丢弃,而将300-400bp长的片段纯化以用于下一步文库制备。
    3. 3'腺苷酸化,在这个步骤3'末端是A尾。
    4. 适配器连接,在此步骤中,将特定的条形码DNA序列连接到测序样品以丰富下游的文库浓度并实现多路测序运行。
    5. 清理,在这一步骤中,将连接有条形码的DNA序列纯化以备下一步骤之用。这对去除多余的衔接子序列或衔接子二聚体非常重要,这会不利于破坏协议中下一个PCR步骤。
    6. PCR扩增,在此步骤中进行PCR以富集接头连接产物。为了减少PCR偏差,建议尽可能少的循环次数。
      注:对于文库制备PCR扩增,试剂盒步骤F3,16重复循环用于制备我们先前研究中使用的文库,'KSHV编码的ORF59调节病毒基因组的组蛋白甲基化以促进病毒再活化'。根据试剂盒的建议,PCR循环次数可根据原料浓度和数量进行优化。
    7. 清理,这是库准备协议的最后清理和净化步骤。我们建议使用23μl重悬缓冲液进行最终洗脱,并收集20μl最终样品,而不是试剂盒说明书(33μl重悬缓冲液,收集30μl最终样品),因为这会产生更浓缩的终产物。

  3. ChIP库验证和量化
    1. 为了评估文库质量,我们建议使用安捷伦生物分析仪并运行高灵敏度DNA芯片。正确准备的文库产生一个大小约为300-350 bp的条带。
    2. 为了准确量化文库样本浓度,我们建议使用KAPA文库量化试剂盒并严格按照制造商的说明操作。精确定量文库浓度对于优化聚类和在测序运行过程中使每个样本的读数数量最大化至关重要。

  4. 测序
    1. 高覆盖率测序意味着每个碱基都被更大程度的对齐序列读取所覆盖,因此碱基识别电位具有更高的置信度。根据Illumina的说明,ChIP-Seq应用推荐使用约100倍的覆盖率。新一代测序是一种高度标准化的验证过程,我们建议遵循规定的程序。
    2. 有关下一代测序深度和覆盖范围的更多信息,可以遵循制造商的建议。 Illumina的建议可以通过 https://www.illumina.com/science/education /sequencing-coverage.html 了解这些重要注意事项的详细说明。

数据分析

  1. 由NextSeq(Illumina,San Diego,CA)产生的FastQ数据被注释并且使用CLC workbench 10.0.1(QIAGEN,Germantown,MD)分析序列读数。序列读数是从内华达基因组学中心上传的Illumina Base Space库下载的。这些文件可以通过互联网导入序列分析软件,在我们的案例中使用“导入Illumina读取”选项的CLC Workbench。
  2. 从输入的对照IgG抗体和特异性抗体样品获得的读数使用CLC工作台的'参照'参考'操作映射到KSHV基因组。
  3. 在使用CLC Workbench的'ChIP-seq'工具与各自的输入样品峰进行比较后,对映射的读数进行识别ChIP样品中的富集峰。相对富集呈现为使用0.05的最小呼叫峰值计算得到的峰值分数。 (请参阅' KSHV编码的ORF59调节病毒的组蛋白精氨酸甲基化基因组以促进病毒再活化',由Strahan等人于2017年在PLoS Pathogens公开提供)。
  4. 评估KSHV病毒基因组上染色质标记相对丰度的唯一挑战是,尽管每个样品的测序深度都很高,但只有约1%的ChIP-Seq读数与病毒基因组相对应。这说明了深度测序覆盖评估病毒染色质构象的重要性。

食谱

  1. 细胞裂解缓冲液,50毫升
    500μl0.5 M PIPES,pH 8.0
    2.5毫升1.7 M KCl
    2.5毫升10%NP-40
    无菌ddH 2 O至50毫升,室温储存

    在使用前加入新鲜的蛋白酶抑制剂
  2. ChIP稀释缓冲液,50毫升
    125μl0.5 M EDTA pH 8.0
    835μl1 M Tris-HCl pH 8.1
    1.67毫升5M NaCl
    无菌ddH 2 O至50毫升,室温储存

    在使用前加入新鲜的蛋白酶抑制剂
  3. ChIP磁性A + G珠子
    1. 将300μlProtein A磁珠与300μlProtein G磁珠合并,并用1×TE 3次洗涤 注意:将试管置于磁力架上,取出上清液,从磁力架上取下,加1 ml TE,重复。
    2. 用1 mg / ml超声处理过的鲑鱼精子DNA和1 mg / ml BSA在1x TE中重悬珠浆并在4°C下旋转过夜。
      注意:超声波处理的鲑鱼精子DNA被用作阻断剂以减少非特异性背景DNA结合。
    3. 第二天,用TE洗珠1次,然后以500μlTE的终体积重新悬浮,珠子现在可用于ChIP测定。
  4. ChIP低盐洗,50毫升
    5毫升10%Triton X-100
    500μl10%SDS
    200μl0.5 M EDTA pH 8.0
    1毫升1M Tris-HCl pH 8.1
    1.5 ml 5 M NaCl
    无菌ddH 2 O至50毫升,室温储存
    在使用前添加新鲜的蛋白酶抑制剂并储存在冰上
    洗涤步骤后丢弃
  5. ChIP洗脱缓冲液
    1毫升10%SDS
    1ml 1M NaHCO 3(新鲜制备)
    无菌ddH 2 O至10毫升,在室温下储存并在使用后丢弃过量

致谢

这项工作得到了国家卫生研究院(CA174459和AI105000)的支持。该协议适用于从先前的ChIP-Seq方案和Diagenode Inc.的LowCell号ChIP方案的组合中确定H4R3me2s在KSHV基因组上的富集。作者想感谢来自内华达州基因组学中心(University of内华达州,里诺,美国)进行Illumina测序运行。
利益冲突:作者声明不存在利益冲突和利益冲突,这可能会影响上述协议的设计和实施。

参考

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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用:Strahan, R. C., Hiura, K. S. and Verma, S. C. (2018). Quantifying Symmetrically Methylated H4R3 on the Kaposi’s Sarcoma-associated Herpesvirus (KSHV) Genome by ChIP-Seq. Bio-protocol 8(6): e2781. DOI: 10.21769/BioProtoc.2781.
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