参见作者原研究论文

本实验方案简略版
Apr 2020

本文章节


 

Chromatin Immunoprecipitation (ChIP) to Assess Histone Marks in Auxin-treated Arabidopsis thaliana Inflorescence Tissue
染色质免疫沉淀法(ChIP)检测生长素处理拟南芥花序组织中的组蛋白标记   

引用 收藏 提问与回复 分享您的反馈 Cited by

Abstract

Chromatin immunoprecipitation coupled with quantitative PCR (ChIP-qPCR) or high-throughput sequencing (ChIP-seq) has become the gold standard for the identification of binding sites of DNA binding proteins and the localization of histone modification on a locus-specific or genome-wide scale, respectively. ChIP experiments can be divided into seven critical steps: (A) sample collection, (B) crosslinking of proteins to DNA, (C) nuclear extraction, (D) chromatin isolation and fragmentation by sonication, (E) immunoprecipitation of histone marks by appropriate antibodies, (F) DNA recovery, and (G) identification of precipitated protein-associated DNA by qPCR or high-throughput sequencing. Here, we describe a time-efficient protocol that can be used for ChIP-qPCR experiments to study the localization of histone modifications in young inflorescences of the model plants Arabidopsis thaliana.

Keywords: ChIP (染色质免疫沉淀), Arabidopsis thaliana (拟南芥), Epigenetic mark (表观遗传标记), Histone modification (组蛋白修饰), Inflorescence (花序)

Background

Eukaryotic genomes are organised in chromosomes in which DNA associates with histones to form chromatin. Tight interactions between histones and DNA impedes the accessibility of DNA to other factors. Consequently, the location of histones relative to important regulatory DNA sequences and the strength of histone-DNA contacts can either hide or expose genes providing yet another layer of gene regulation. In chromatin, both histones and DNA can be chemically modified (Zhou et al., 2010; Schübeler, 2015). Depending on the physical properties of the modification, the chromatin state can either prevent or enhance the transcription of the underlying genes (Kouzarides, 2007; Yang et al., 2014; Wu et al., 2015). In plants, the epigenetic state of the chromatin has been shown to be a crucial determinant of gene expression in response to developmental or environmental stimuli (Yang et al., 2014; Wu et al., 2015; Chung et al., 2019; Kuhn et al., 2020).

The plant hormone auxin regulates almost all aspect of a plant’s life cycle. A mechanism for auxin perception has been established involving a nuclear signalling pathway. This pathway is based on the de-repression of Auxin Response Factors (ARFs) via auxin-induced degradation of Aux/IAA transcriptional repressors. Aux/IAAs inhibit ARF activity towards target genes by recruiting the co-repressor TOPLESS (TPL) to repress auxin-dependent gene expression. TPL/TPRs mediate their repressive effect by attracting histone deacetylases (HDACs) (Krogan et al., 2012; Weijers and Wagner, 2016). We have identified an alternative auxin signalling mechanism which is essential for gynoecium development in Arabidopsis thaliana. In this noncanonical pathway, auxin directly affects the activity of complexes involving an atypical ARF, called ETTIN (ETT/ARF3) towards downstream targets (Simonini et al., 2016 and 2017, Kuhn et al., 2020). Recently, we found that direct auxin binding by ETT leads to dissociation from co-repressor proteins of the TPL/TPR family followed by induction of gene expression. Moreover, we showed that this correlates with ETT-dependent auxin-responsive histone acetylation of the target genes (Kuhn et al., 2020).

Chromatin immunoprecipitation coupled with quantitative PCR (ChIP-qPCR) has become the gold standard for the identification and characterisation of histone modifications at given gene loci. Since ETT is primarily involved in floral reproductive development, we optimized existing ChIP protocols (Yang et al., 2014, Qüesta et al., 2016) to study the effect of auxin on histone modifications in inflorescence tissue. The protocol that we present here has been successfully applied to evaluate several epigenetic marks including H3K27Ac (Kuhn et al., 2020) in Arabidopsis inflorescence tissue.

Materials and Reagents

  1. Spray bottle TurnNSpray 500 ml (R&L Slaughter, catalog number: 215-2962)

  2. Corning 50 ml centrifuge tubes (Sigma-Aldrich, catalog number: CLS430829-500E)

  3. Elastic rubber bands

  4. Paper towel

  5. Aluminium Foil

  6. Funnel (6 cm diameter)

  7. 2 ml DNA low binding (LoBind) tubes (Eppendorf, catalog number: 0030108078)

  8. 1.5 ml DNA low binding (LoBind) tubes (Eppendorf, catalog number: 022431021)

  9. Arabidopsis thaliana

  10. Indole-3-acetic acid (Sigma-Aldrich, catalog number: I5148)

  11. Silwet L-77 (De Sangosse Ltd., catalog number: 0640)

  12. Miracloth (MERK, catalog number: 475855)

  13. Liquid nitrogen

  14. 10x Phosphate-buffered saline (PBS) (Sigma-Aldrich, catalog number: 11666789001)

  15. Formaldehyde (Sigma-Aldrich, catalog number: F8775)

  16. Glycine (Thermo Fisher Scientific, catalog number: G/0800/60)

  17. Sterile water

  18. Succrose (Thermo Fisher Scientific, catalog number: S/8600/60)

  19. Ficoll 400 (Sigma-Aldrich, catalog number: F8636)

  20. Dextran from Leuconostoc spp. (Sigma-Aldrich, catalog number: 31389)

  21. HEPES (Sigma-Aldrich, catalog number: H4034)

  22. Magnesium chloride (MgCl2) (Sigma-Aldrich, catalog number: M8266)

  23. Triton X-100 (Sigma-Aldrich, catalog number: T9284)

  24. DL-Dithiothreitol (DTT; Sigma-Aldrich, catalog number: D0632)

  25. cOmplete EDTA-free protease inhibitor cocktail (Roche Molecular Systems, catalog number: 4693132001)

  26. Tris(hydroxymethyl)aminomethane (TRIS; Sigmal-Aldrich, catalog number: B2005)

  27. Ethylenediaminetetraacetic acid (EDTA; Sigma-Aldrich, catalog number: E6758)

  28. 20% Sodium Dodecyl Sulfate (SDS) solution (Severn Biotech; catalog number: 20-4002-05)

  29. Sodium Chloride (NaCl) (Sigma-Aldrich, catalog number: S7653)

  30. Dynabeads Protein A (Invitrogen, catalog number: 10001D)

  31. Anti-H3 Antibody (Rabbit polyclonal Antibody; Abcam, catalog number: ab1791)

  32. Anti-H3K27ac Antibody (Rabbit polyclonal Antibody; Abcam, catalog number: ab4729)

  33. Sodium bicarbonate (NaHCO3) (Sigma-Aldrich, catalog number: S5761)

  34. Proteinase K (Roche Molecular Systems, catalog number: 40693800)

  35. Phenol:Chloroform:Isoamyl alcohol (25:24:1) (Sigma-Aldrich, catalog number: 77617)

  36. Sodium acetate (NaAc) (Sigma-Aldrich, catalog number: S7670)

  37. Ethanol absolute (VWR Chemicals, catalog number: 20821.330)

  38. LightCycler 480 Multiwell Plate 96, white (Roche Molecular Systems, catalog number: 04729692001)

  39. LightCycler® 480 Sealing Foil (Roche Molecular Systems, catalog number: 04729757001)

  40. Oligo-nucleotide primers (Integrated DNA Technologies)

  41. SYBR Green JumpStart Taq Ready Mix (Sigma-Aldrich, catalog number: S4438)

  42. RNase A (Thermo Fisher Scientific, catalog number: EN0531)

  43. Agarose (Melford Biolaboratories Ltd., catalog number: A20080-1000.0)

  44. 100 bp DNA ladder (New England Biolabs Ltd., catalog number: N3221S)

  45. Stock Solutions (see Recipes)

    2 M Glycine

    0.5 M HEPES KOH pH 7.4

    1 M MgCl2

    1 M Tris-HCl pH 8

    0.5 M EDTA

    5 M NaCl

    3 M NaAc

    1 M DTT

    20% (v/v) Triton X-100

    10% (v/v) SDS

  46. ChIP Buffers (see Recipes)

    Honda Buffer

    Nuclei Lysis Buffer

    ChIP Dilution Buffer

    Low Salt Wash Buffer

    High Salt Wash Buffer

    TE Buffer

    Elution Buffer

Equipment

  1. Tweezers (DUMONT BIOLOGICAL TYPE 5 SS-1, TAAB Laboratories Equipment, catalog number: T083)

  2. Glass beaker 100 ml

  3. Mortar and pistil

  4. Bel-Art Space Saver Vacuum Desiccator (Thermo Fisher Scientific, catalog number: 11852732)

  5. Vacuum rump (Edwards, model: IEC34-1)

  6. Tube rotor (Stuart Scientific, model: SB1)

  7. Centrifuge (Eppendorf, model: 5810R)

  8. Swing-bucket rotor with 50 ml tube adaptors (Eppendorf, model: A-4-81)

  9. Fixed-angle rotor for 48 x 1.5/2.0 ml tubes (Eppendorf, model: FA-45-48-11)

  10. Sonicator (Diagenode, Model Bioruptor Plus)

  11. Magnetic separator rack (GE Healthcare, MagRack6, catalog number 28-9489-64)

  12. Thermomixer (Eppendorf, Model Eppendorf ThermoMixer C)

  13. CFX96 touch real-time PCR detection system (Bio-Rad)

  14. Vacuum concentrator (Eppendorf, model: Concentator Plus)

  15. Gel electrophoresis system (Thermo Electric Corporation, model: CSSU1214)

  16. Power pack (Kikusui Electronics Corporation, model: PAB 350-0.2)

Procedure

  1. Sample treatment and collection

    This section describes the plant cultivation and treatment procedures for Arabidopsis thaliana and Capsella rubella inflorescence tissue subjected to auxin treatments. Cultivation and treatment procedures may differ for other species, tissues and treatments.

    1. Grow Arabidopsis plants on soil at 22 °C in long day conditions (16 h day/8 h dark) until inflorescences emerge (3-4 weeks).

    2. Treat inflorescences by spraying the full tray of plants with a solution containing 100 µM Indole-3-acetic acid (IAA, auxin) and 0.015% Silwet L-77 or MOCK (only 0.015% Silwet L-77) in water (Note 1).

    3. Return treated plants to the growth room for two hours.

    4. Using tweezers collect 3 g of young inflorescence tissue in a 50 ml Falcon Tube (Note 2).

    5. Collect a total of three biological replicates (3 times 3 g) for one experiment (Note 3).


  2. Crosslinking

    1. Transfer the collected samples onto Miracloth squares (~5 x 5 cm), fold into small bags and close them securely with an elastic band (Note 4).

    2. Place samples in a glass beaker (100 ml) and add 50 ml 1% Formaldehyde solution (Note 5).

      Note: Formaldehyde is toxic, work in fume cabinet.

    3. Place the glass beakers in the vacuum desiccator and turn it on.

    4. Crosslink samples in 1% formaldehyde solution for 3 times 5 min, releasing the vacuum after 5 min and reapply when fully released (Note 6).

    5. Quench the reaction by adding 2 M glycine solution to a final concentration of 125 mM and apply vacuum for 5 min.

    6. Release vacuum and discard quenched 1% Formaldehyde solution.

    7. Wash samples twice with 1x PBS and twice with water.

    8. Blot them dry on paper towel and then fold the tissue into aluminium foil.

    9. Flash freeze packets in liquid nitrogen.

    *Stopping point (store at -80 °C).


Note: Unless stated otherwise all subsequent steps should be carried out on ice using pre-cooled buffers.

  1. Nuclear extraction

    1. Using pre-cooled mortar and pistil, grind the plant material in liquid nitrogen into fine powder.

    2. Suspend ground up plant material in 25 ml Honda Buffer and homogenise for 10 min at 4 °C on a rotor.

    3. Filter through one layer of Miracloth (~7 x 7 cm) using a funnel into new 50 ml Falcon tube. Squeeze the Miracloth to maximise the yield.

    4. Centrifuge at 850 x g for 20 min (4 °C).

    5. Gently resuspend the pellet in 1 ml Honda Buffer (Note 7).

    6. Centrifuge at 850 x g for 5 min (4 °C).

    7. Repeat step 5 and 6 twice to clear the extract.

    *Stopping point (pellet can be stored overnight at -20 °C).


  1. Chromatin fragmentation by sonication

    1. Centrifuge 4,000 x g for 1 min (4 °C).

    2. Remove residual Honda Buffer and resuspend pellet in 300 μl of Nuclei Lysis Buffer.

    3. Split the sample in two 2 ml Lobind tubes (~250 μl per tube).

    4. Take a 25 μl of this new suspension for your non-sonicated control and store at -20 °C.

    5. Sonicate 3 times for 5 min on medium strength using a Bioruptor water bath sonicator (Diagenode) using a 30 s ON/OFF cycle, to fragment the chromatin. Mix samples between each 5 min period (Note 8).

    6. Centrifuge the samples for 10 min, 4,000 x g, 4 °C.

    7. Transfer the supernatant to fresh 2 ml Lobind tubes.

    8. Take a 25 μl to check sonication efficiency and store at -20 °C (Note 9).

    *Stopping point (samples can be stored overnight at -20 °C).


  1. Immunoprecipitation (IP)

    1. Dilute the samples ten-fold with ChIP Dilution Buffer (~2 ml).

      Note: Take 20 μl sonicated homogenate as Input control.

    2. Prepare the antibody coated magnetic beads.

      1. Take 15 μl of Protein A magnetic beads for each IP and wash three times with 1 ml ChIP dilution buffer.

      2. Resuspend magnetic beads in 50 μl ChIP Dilution Buffer per IP.

      3. Bind 1 μg of antibody per IP to the beads.

      4. To IP H3 we use the anti-H3 antibody and to IP H3K27Ac we use the anti-H3K27ac Antibody.

      5. Incubate on a rotor for an hour at 4 °C.

    3. Add 50 μl anti-H3 antibody-coated beads to one half of the samples and 50 μl antibody-coated beads to the other half of the samples.

    4. Incubate on a rotor at 4 °C for at least 4 h.

      *Stopping point (this step can also be done overnight).

    5. Wash the beads sequentially (each time 5 min at 4 °C incubation on the rotor) with 1 ml with:

      1. Twice Low Salt Washing Buffer.

      2. Twice High Salt Washing Buffer.

      3. Twice TE Buffer.

      Note: For each of these washes, leave on the magnetic stand for one minute at a time.

    6. After the last TE wash transfer the sample to a fresh 1.5 ml Lobind tube then separate on the magnetic stand and remove TE.


  1. DNA recovery

    1. Elute from beads twice with 200 μl pre-heated Elution Buffer. Incubate for 15 min at 65 °C with 800 rpm agitation. Collect the two elutions (400 μl total volume) in one fresh 1.5 ml Lobind tube and discard the magnetic antibody coated beads.

    2. Bring the Input samples to 400 μl using Elution Buffer.

    3. To reverse crosslink, add 16 μl 5M NaCl to the eluate and incubate at 65 °C and 600 rpm agitation overnight.

    4. After reverse crosslinking, add 3 μl Proteinase K (20 mg/ml) and incubate for 1 h at 45 °C.

    5. To extract DNA, add 400 μl Phenol:Chloroform:Isoamyl alcohol (25:24:1), vortex for 30 s and centrifuge at 4,000 x g for 10 min at room temperature.

    6. Transfer the aqueous layer (~400 μl) in a fresh 1.5 ml Lobind tube.

    7. To precipitate DNA, add 0.1 volume (40 μl) 3 M NaAc and 2 volumes (800 μl) 100% ethanol (mix well) and incubate at -20 °C for 1.5 h.

    8. Centrifuge at 4,000 x g for 30 min (4 °C).

    9. Wash pellet with 500 μl ice cold 70% ethanol, centrifuge for 10 min at 4,000 x g (4 °C).

    10. Remove all traces of ethanol and dry the pellet in vacuum concentrator for 10 min (65 °C).

    11. Resuspend the pellet in the required volume of nuclease free water (Note 10).


  1. Identification of precipitated protein-associated DNA by qPCR

    1. Setup qPCR reactions in triplicate per antibody, treatment and primer pair as follows (Note 11):

      Per reaction (10 μl final volume): 3 μl DNA, 5 μl SYBR Green JumpStart Taq ReadyMix (Sigma), 0.125 μl Forward Primer (10 μM), 0.125 μl Reverse Primer (10 μM), 1.75 μl water.

    2. Run the reaction on qPCR machine using the following conditions: 95 °C for 2 min followed by 60 cycles at 95 °C for 10 s, 60 °C for 10 s and 72 °C for 30 s.

    3. Analyse the data as described under “Data analysis”.

Data analysis

Note: We have analysed the data published in Kuhn et al. (2020) using described protocol.

  1. Once the Ct (Cycle threshold) values have been obtained calculate the mean between the three technical replicates.

  2. Calculate the ratio between each IP with the corresponding Input for all target regions:

    MEAN = 2-(MEAN(IP)-MEAN(Input)) to transform to linear scale.

  3. Calculate the enrichment of the Histone H3 modification of interest (e.g., H3K27Ac) as the ratio between the H3K27Ac and H3 for each biological replicate at each target region.

  4. Calculate mean and standard deviation between all biological replicates at each target region.

  5. Analyse the statistical differences between hormone treated and MOCK samples at each target region using two-way ANOVA with post hoc Bonferroni multiple comparison test.

  6. Plot the mean and standard deviation of the ratio (H3K27Ac/H3) along the length of the examined target region.

Notes

  1. Indole-3-acetic acid (IAA) is insoluble in water and needs to be freshly prepared in ethanol to a stock concentration of 100 mM. Mock treatment has to contain the same amount of ethanol as the auxin treatment. When treating the plants, make sure that you spray equally with treatment and mock solution. Treatment of ~100 Arabidopsis plants requires ~100 ml solution.

  2. We only harvested inflorescence tissue that contained closed flower buds. Flowers that were opened or young siliques were removed.

  3. To obtain 3 g sample inflorescences of ~100 plants need to be harvested. We recommend to collect one pair of biological replicates (one replicate per genotype, mock and IAA-treatment) at the same time and to repeat collection three times to obtain three replicates for an experiment. We recommend having two people collecting to reduce the time.

  4. If including more than one genotype or treatment, it is advised to use one glass beaker per treatment and genotype to prevent mixing of samples.

  5. Crosslinking time may need optimisation depending on protein and tissue. The optimal crosslink time crosslinks your protein of interest to the DNA but does not block the epitope. This can be tested by Western blotting.

  6. Do not start timing the 5 min before the first bubbles emerge.

  7. This can be done with a 100 μl pipette. We recommend cutting the pipette tip to prevent damaging the nuclei.

  8. Sonication conditions can differ between sonicators and depend on samples. Generally four parameters are essential for the efficiency of the DNA fragmentation: 1) the temperature of the sample (we recommend to maintain a temperature of 4 °C throughout the sonication), 2) the concentration of formaldehyde used for crosslinking (the higher the concentration leads increases the sonication time), 3) the volume in which sonication is performed (we recommend not to sonicate more than 250 μl of lysate in a 1.5 ml tube), 4) the concentration of SDS in the lysis buffer (more SDS leads to easier lysis during sonication).

  9. Sonication test:

    Once your sonication conditions are well established, you can skip this step. Bring sonication and non-sonication aliquots to 100 μl volume with elution buffer.

    1. Add 1 μl of 10 mg/ml RNase A, mix well and incubate for 1 h at 37 °C.

    2. Add 100 μl of Phenol:Chloroform:Isoamyl alcohol (25:24:1), vortex for 30 s and centrifuge 4,000 x g for 10 min at room temperature.

    3. Transfer the aqueous phase to a fresh 1.5 ml Lobind tube and add 0.1 volumes (10 μl) of 3 M NaAc (pH 5.5) and 2 volumes (200 μl) of 100% EtOH. Vortex and incubate for 1.5h at -20 °C.

    4. Centrifuge for 4,000 x g for 20 min at 4 °C.

    5. Remove the supernatant and dry the pellet for 10 min in vacuum concentrator (65 °C).

    6. Resuspend the pellet in 10 μl water.

    7. Run samples on a 1.5% Agarose gel.

    8. Visualise the gel. The DNA should be visible as a smear between 100 bp and 500 bp (Figure 1). The smaller the DNA is fragmented the higher the resolution of the experiment.



    Figure 1. Sonication test. Marker (NEB 100 bp ladder); Sample showing the bulk of DNA fragments range from 100-500 bp.


  10. The volume of water depends on the number of qPCR reactions that will be run (3 μl DNA per reaction).

  11. Primers should be designed to amplify fragments (the ideal fragment length is ~100 bp) with regular spacing across the whole gene locus of interest. The resolution of the experiments increases with increasing amplicon density. Before use, all qPCR primers should be tested for their efficiency and specificity. The amplification efficiency of primers can be tested using a genomic DNA dilution series (1/4 to 1/4096) (Figure 2A). The specificity of primers was confirmed by analysing the melting curves (65 °C to 95 °C) (Figure 2B). qPCR reactions should be prepared and performed as described in the experimental procedures (G1-3).



    Figure 2. Example a suitable qPCR primer. A. Amplification efficiency of a suitable primer should show a correlation between amplification and dilution (R2) of 0.95-1 and a primer efficiency (10-1/slope) of 2 ± 0.1. B. Melt peak indicating high specificity of the primer pair.

Recipes

  1. Stock Solutions

    1. 2 M Glycine
      Autoclave

    2. 0.5 M HEPES KOH pH 7.4

      Autoclave

    3. 1 M MgCl2

      Autoclave

    4. 1 M Tris-HCl pH 8
      Autoclave

    5. 0.5 M EDTA
      Autoclave

    6. 5 M NaCl
      Autoclave

    7. 3 M NaAc
      Autoclave

    8. 1 M DTT

      Weigh 1.54 g of DTT and dissolve in 10 ml water

      Filter sterilize after all DTT is dissolved and make 1 ml aliquots

      Store at -20 °C

    9. 20% (v/v) Triton X-100

      Dilute Triton X-100 five times with water to obtain 20% Triton X-100

    10. 10% (v/v) SDS

      Dilute 20% (v/v) SDS with 1 volume water to obtain 10% (v/v) SDS solution


  2. ChIP Buffers

    1. Honda Buffer

      0.44 M sucrose

      1.25% (w/v) Ficoll

      2.5% (w/v) Dextran T40

      20 mM HEPES KOH pH 7.4

      0.5% (v/v) Triton X-100

      10 mM MgCl2

      0.5 mM DTT

      1 tablet per 50 ml cOmplete EDTA-free protease inhibitor cocktail (Roche)

    2. Nuclei Lysis Buffer

      50 mM Tris-HCl pH 8.0

      10 mM EDTA

      1% (v/v) SDS

      1 tablet per 50 ml cOmplete EDTA-free protease inhibitor cocktail (Roche)

    3. ChIP Dilution Buffer

      16.7 mM Tris-HCl pH 8.0

      1.2 mM EDTA

      1.1% (v/v) Triton X-100

      167 mM NaCl

      1 tablet per 50 ml cOmplete EDTA-free protease inhibitor cocktail (Roche)

    4. Low Salt Wash Buffer

      20 mM Tris-HCl pH 8.0

      2 mM EDTA

      150 mM NaCl

      0.1% (v/v) SDS

      1% (v/v) Triton X-100

      1 tablet per 50 ml cOmplete EDTA-free protease inhibitor cocktail (Roche)

    5. High Salt Wash Buffer

      20 mM Tris-HCl pH 8.0

      2 mM EDTA

      500 mM NaCl

      0.1% (v/v) SDS

      1% (v/v) Triton X-100

      1 tablet per 50 ml cOmplete EDTA-free protease inhibitor cocktail (Roche)

    6. TE Buffer

      10 mM Tris-HCl pH 8.0

      1 mM EDTA

      1 tablet per 50 ml cOmplete EDTA-free protease inhibitor cocktail (Roche)

    7. Elution Buffer

      0.1 M NaHCO3

      1% (v/v) SDS

Acknowledgments

We are grateful to Yang Dong and Emilie Knight for critical comments on the protocol. We also express our gratitude to Caroline Dean, Hongchun Yang and Julia Qüesta for their helpful advice throughout the development of this protocol. This protocol was adapted from Kuhn et al., 2020 . This work was supported by grant BB/S002901/1 to L.Ø., the Norwich Research Park Biosciences Doctoral Training Partnership [grant number BB/M011216/1 to A.K.] and by the Institute Strategic Programme grant (BB/P013511/1) to the John Innes Centre all from the UKRI Biotechnological and Biological Sciences Research Council.

Competing interests

The authors declare no competing interests.

References

  1. Chung, Y., Zhu, Y., Wu, M. F., Simonini, S., Kuhn, A., Armenta-Medina, A., Jin, R., Ostergaard, L., Gillmor, C. S. and Wagner, D. (2019). Auxin Response Factors promote organogenesis by chromatin-mediated repression of the pluripotency gene SHOOTMERISTEMLESS. Nat Commun 10(1): 886.
  2. Kouzarides, T. (2007). Chromatin modifications and their function. Cell 128(4): 693-705.
  3. Krogan, N. T., Hogan, K. and Long, J. A. (2012). APETALA2 negatively regulates multiple floral organ identity genes in Arabidopsis by recruiting the co-repressor TOPLESS and the histone deacetylase HDA19. Development 139(22): 4180-4190.
  4. Kuhn, A., Ramans Harborough, S., McLaughlin, H. M., Natarajan, B., Verstraeten, I., Friml, J., Kepinski, S. and Ostergaard, L. (2020). Direct ETTIN-auxin interaction controls chromatin states in gynoecium development. Elife 9: e51787.
  5. Qüesta, J. I., Song, J., Geraldo, N., An, H. and Dean, C. (2016). Arabidopsis transcriptional repressor VAL1 triggers Polycomb silencing at FLC during vernalization. Science 353(6298): 485-488.
  6. Schübeler, D. (2015). Function and information content of DNA methylation. Nature 517(7534): 321-326.
  7. Simonini, S., Deb, J., Moubayidin, L., Stephenson, P., Valluru, M., Freire-Rios, A., Sorefan, K., Weijers, D., Friml, J. and Ostergaard, L. (2016). A noncanonical auxin-sensing mechanism is required for organ morphogenesis in Arabidopsis. Genes Dev 30(20): 2286-2296.
  8. Simonini, S., Bencivenga, S., Trick, M. and Ostergaard, L. (2017). Auxin-Induced Modulation of ETTIN Activity Orchestrates Gene Expression in Arabidopsis. Plant Cell 29(8): 1864-1882.
  9. Weijers, D. and Wagner, D. (2016). Transcriptional Responses to the Auxin Hormone. Annu Rev Plant Biol  67: 539-574.
  10. Wu, M. F., Yamaguchi, N., Xiao, J., Bargmann, B., Estelle, M., Sang, Y. and Wagner, D. (2015). Auxin-regulated chromatin switch directs acquisition of flower primordium founder fate. Elife 4: e09269.
  11. Yang, H., Howard, M. and Dean, C. (2014). Antagonistic roles for H3K36me3 and H3K27me3 in the cold-induced epigenetic switch at Arabidopsis FLC. Curr Biol 24(15): 1793-1797.
  12. Zhou, V. W., Goren, A. and Bernstein, B. E. (2011). Charting histone modifications and the functional organization of mammalian genomes. Nat Rev Genet 12(1): 7-18.

简介

[摘要]染色质免疫沉淀与定量PCR(ChIP -qPCR)或高通量测序(ChIP-seq )结合已成为鉴定DNA结合蛋白结合位点和在特定基因座上定位组蛋白修饰的金标准。或全基因组规模。ChIP实验可分为七个关键步骤:(A)样品收集,(B)蛋白质与DNA交联,(C)核提取,(D)染色质分离和f 超声处理的碎片化;(E)通过适当的抗体对组蛋白标记的免疫沉淀;(F)DNA的回收;(G)通过qPCR或高通量测序鉴定沉淀的蛋白质相关DNA。在这里,我们描述了一种可用于ChIP -qPCR实验的省时协议,以研究模型植物拟南芥幼花序中组蛋白修饰的定位。


[背景]真核基因组中的染色体中,其与组蛋白DNA结合形成染色质组织的。组蛋白与DNA之间的紧密相互作用阻碍了DNA与其他因素的可及性。因此,组蛋白相对于重要调控DNA序列的位置和组蛋白-DNA接触的强度可以隐藏或暴露提供另一层基因调控的基因。在染色质中,组蛋白和DNA均可被化学修饰(Zhou等,2010 ;Schübeler ,2015)。根据修饰的物理性质,染色质状态可以阻止或增强基础基因的转录(Kouzarides ,2007; Yang等,2014; Wu等,2015)。在植物中,染色质的表观遗传状态已被证明是响应发育或环境刺激的基因表达的关键决定因素(Yang等人,2014 ; Wu等人,2015 ; Chung等人,2019 ; Kuhn等人,2020)。

植物激素生长素调节着植物生命周期的几乎所有方面。已经建立了涉及核信号传导途径的生长素感知机制。该途径基于生长素诱导的Aux / IAA转录阻遏物降解引起的生长素反应因子(ARF)的抑制。Aux / IAA通过募集协同阻遏物TOPLESS(TPL)来抑制生长素依赖性基因表达来抑制ARF对靶基因的活性。TPL / TPR通过吸引组蛋白脱乙酰基酶(HDAC)介导其抑制作用(Krogan等,2012;Weijers和Wagner ,2016)。我们已经确定了另一种生长素信号传导机制,这对拟南芥的妇科发育是必不可少的。在这种非经典途径中,生长素直接影响涉及一种非典型ARF,称为双头怪(ETT / ARF3)朝向下游靶复合物的活性(Simonini等人,2016和2017年,库恩等人,2020)。最近,我们发现ETT与生长素的直接结合导致TPL / TPR家族的共阻遏蛋白解离,随后诱导基因表达。此外,我们表明这与靶基因的ETT依赖的生长素反应性组蛋白乙酰化有关(Kuhn等,2020)。

染色质免疫沉淀与定量PCR(ChIP -qPCR)结合已成为鉴定和表征给定基因位点组蛋白修饰的金标准。由于ETT主要参与花卉生殖发育,因此我们优化了现有的ChIP方案(Yang等,2014;Qüesta等,2016),以研究生长素对花序组织中组蛋白修饰的影响。我们在此提出的方案已成功应用于评估拟南芥花序组织中的几种表观遗传标记,包括H3K27Ac(Kuhn等人,2020年)。

关键字:染色质免疫沉淀, 拟南芥, 表观遗传标记, 组蛋白修饰, 花序

材料和试剂
1.喷雾瓶TurnNSpray 500毫升(R&L屠宰,目录号:215-2962)     
2.康宁50毫升离心管(Sigma - Aldrich,目录号:CLS430829-500E )     
3.弹性橡皮筋     
4.纸巾     
5.铝箔     
6.漏斗(直径6厘米)     
7. 2 ml DNA低结合(LoBind )管(Eppendorf,目录号:0030108078)     
8. 1.5 ml DNA低结合(LoBind )管(Eppendorf,目录号:022431021)     
9.拟南芥     
10.吲哚-3-乙酸(Sigma - Aldrich,目录号:I5148) 
11. Silwet L-77(De Sangosse Ltd.,目录号:0640) 
12.的Miracloth (MERK,目录号:475855) 
13.液氮 
14. 10倍磷酸盐缓冲盐水(PBS)(Sigma - Aldrich,目录号:11666789001) 
15.甲醛(Sigma - Aldrich,目录号:F8775) 
16.甘氨酸(Thermo Fisher Scientific,目录号:G / 0800/60) 
17.无菌水 
18.蔗糖(Thermo Fisher Scientific,目录号:S / 8600/60 ) 
19. Ficoll 400(Sigma - Aldrich,目录号:F8636) 
20.来自Leuconostoc spp的葡聚糖。(Sigma - Aldrich,目录号:31389) 
21. HEPES(Sigma - Aldrich,目录号:H4034) 
22.氯化镁(MgCl 2 ; Sigma - Aldrich,目录号:M8266) 
23. Triton X-100(Sigma - Aldrich,目录号:T9284) 
24. DL-二硫苏糖醇(DTT; Sigma - Aldrich,目录号:D0632) 
25.不含EDTA的蛋白酶抑制剂混合物(Roche Molecular Systems,目录号:4693132001) 
26.三(羟甲基)氨基甲烷(TRIS;Sigmal - Aldrich,目录号:B2005) 
27.乙二胺四乙酸(EDTA; Sigma - Aldrich,目录号:E6758) 
28. 20%十二烷基硫酸钠(SDS)溶液(Severn Biotech;目录号:20-4002-05) 
29.氯化钠(NaCl; Sigma - Aldrich,目录号:S7653) 
30. Dynabeads蛋白A(Invitrogen,目录号:10001D) 
31.抗H3抗体(兔多克隆抗体; Abcam,目录号:ab1791) 
32.抗H3K27ac抗体(兔多克隆抗体; Abcam,目录号:ab4729) 
33.碳酸氢钠(NaHCO 3 ,Sigma - Aldrich,目录号:S5761) 
34.蛋白酶K(Roche Molecular Systems,目录号:40693800) 
35.苯酚:氯仿:异戊醇(25:24:1)(Sigma - Aldrich,目录号:77617) 
36.乙酸钠(NaAc ,Sigma - Aldrich,目录号:S7670) 
37.无水乙醇(VWR Chemicals,目录号:20821.330) 
38. LightCycler 480多孔板96,白色(Roche Molecular Systems,目录号:04729692001) 
39.的LightCycler ® 480密封膜(罗氏分子系统,目录号:04729757001) 
40.寡核苷酸引物(整合DNA技术) 
41. S YBR绿色JumpStart Taq预混料(Sigma - Aldrich,目录号:S4438) 
42. RNase A(Thermo Fisher Scientific,目录号:EN0531) 
43.琼脂糖(Melford Biolaboratories Ltd.,目录号:A20080-1000.0) 
44. 100 bp DNA Ladder(New England Biolabs Ltd.,目录号:N3221S) 
45.库存解决方案(请参阅食谱) 
2 M甘氨酸
0.5 M HEPES KOH pH 7.4
1 M氯化镁2
1 M Tris-HCl pH 8
50万EDTA
5 M氯化钠
3 M NaAc
1 M DTT
20%(v / v)海卫X-100
10%(v / v)安全数据表
46. ChIP缓冲区(请参阅食谱) 
本田缓冲器
核裂解缓冲液
芯片稀释缓冲液
低盐洗涤缓冲液
高盐洗涤缓冲液
TE缓冲液
洗脱缓冲液

设备

1.镊子(DUMONT生物型5 SS-1,TAAB实验室设备,目录号:T083)     
2.玻璃烧杯100毫升     
3.砂浆和雌蕊     
4. Bel-Art节省空间的真空干燥器(Thermo Fisher Scientific,目录号:11852732)     
5.真空泵(爱德华兹,型号:IEC34-1)     
6.管式转子(Stuart Scientific,型号:SB1)     
7.离心机(埃彭多夫,型号:5810R)     
8.带有50 ml管接头的摆桶式转子(Eppendorf,型号:A-4-81)     
9.用于48 x 1.5 / 2.0 ml管的固定角度转子(Eppendorf,型号:FA-45-48-11)     
10.超声波仪(Diagenode ,型号Bioruptor加) 
11.电磁分离器机架(GE Healthcare,MagRack6,目录号28-9489-64) 
12. Thermomixer(Eppendorf,型号Eppendorf ThermoMixer C) 
13. CFX96触摸实时PCR检测系统(Bio - Rad) 
14. V acuum浓缩仪(Eppendorf,米Odel等:Concentator加) 
15.凝胶电泳系统(Thermo Electric Corporation,型号:CSSU1214) 
16.电源组(菊水电子公司,型号:PAB 350-0.2) 

程序

样品处理和收集
本节描述的植物栽培和治疗程序拟南芥和荠菜风疹经受生长素处理花序组织。其他物种,组织和处理的栽培和处理程序可能有所不同。
1.在长日条件下(16小时/ 8小时黑暗)在2 2 °C的土壤上种植拟南芥植物,直到花序出现(3-4周)。     
2.款待花序通过用含100μM吲哚-3-乙酸(IAA,茁长素)和0.015%的溶液喷雾的植物的满料盘的Silwet L-77(德Sangosse Ltd。制造)或MOCK(仅0.015%的Silwet L- 77)在水中(注1 )。     
3.将处理过的植物放回生长室两个小时。     
4.用镊子在50 ml的Falcon管中收集3 g的年轻花序组织(注2 )。     
5.一次实验总共收集3个生物重复样品(3 g 3 g)(注3 )。     

交联
1.将收集到的样本转移到Miracloth正方形(约5 x 5厘米)上,折叠成小袋子,并用松紧带将其牢固关闭(注4 )。     
2.将样品放入玻璃烧杯(100毫升)中,然后加入50毫升1%甲醛溶液(注5 )。     
注意:甲醛是有毒的,请在通风柜中工作。
3.将玻璃烧杯放入真空干燥器中,然后将其打开。     
4.将样品在1%甲醛溶液中交联3次,每次5分钟,在5分钟后释放真空,并在完全释放后重新应用(注释6 )。     
5.通过添加2 M甘氨酸溶液至终浓度125 mM淬灭反应,并施加真空5分钟。     
6.释放真空并弃去1%甲醛溶液。     
7.用1x PBS洗涤样品两次,用水洗涤两次。     
8.用纸巾将它们吸干,然后将薄纸折成铝箔。     
9.用液氮快速冷冻小包。     
*停止点(存储在-80 °C)。

注意:除非另有说明,否则所有后续步骤均应使用预冷却的缓冲液在冰上进行。

核提取
1.使用预冷的研钵和雌蕊,将植物材料在液氮中研磨成细粉。     
2.将磨碎的植物材料悬浮在25 ml本田缓冲液中,并在4 °C的转子上匀浆10分钟。     
3.使用漏斗将一层Miracloth (〜7 x 7 cm )过滤到新的50 ml Falcon管中。挤压Miracloth,以最大程度地提高产量。     
4.以850 x g离心20分钟(4 °C )。     
5.轻轻地将沉淀重悬于1 ml本田缓冲液中(注7 )。     
6.以850 x g离心5分钟(4 °C )。     
7.重复步骤5和6两次以清除提取物。     
*停止点(小丸可在-20 °C下保存过夜)。

超声破碎染色质
1.离心4 ,000 ×g离心1分钟(4 ℃下)。     
2.在300清除残留本田缓冲液,重悬沉淀微升核裂解缓冲液中。     
3.拆分在TW样品ø2毫升的Lobind管(〜250微升每管)。     
4.取25μl这种新悬浮液进行非超声处理,并保存在-20 °C下。     
5.使用Bioruptor水浴超声仪(Diagenode )以30秒的ON / OFF周期在中等强度下超声3次,持续5分钟,以使染色质破碎。在每5分钟之间混合样品(注8 )。     
6.将样品在4000 xg ,4 °C下离心10分钟。     
7.将上清液转移到新鲜的2 ml Lobind管中。     
8.取25μl检查超声处理效率,并保存在-20 °C (注9 )。     
*临界点(样品可以在-20 °C下保存过夜)。

免疫沉淀(IP)
1.用ChIP稀释缓冲液(〜2 ml)将样品稀释十倍。     
注:取20微升超声匀浆作为输入控制。
2.准备抗体包被的磁珠。     
  采取15微升的每个IP蛋白A磁珠并用1ml洗涤三次的ChIP稀释缓冲液。
  在重悬磁珠50微升的ChIP每个IP稀释缓冲液。
  绑定1微克每IP抗体与珠的。
  对于IP H3,我们使用抗H3抗体,对于IP H3K27Ac,我们使用抗H3K27ac抗体。
  在转子上于4 °C孵育一个小时。
3.加入50微升抗H3抗体-包被的珠粒的样品的一半和50微升抗体-包被的珠粒的样品的另一半。     
4.在转子上于4 °C孵育至少4 h。     
*停止点(此步骤也可以在一夜之间完成)。
5.依次用1 ml洗涤磁珠(每次在4 °C的转子上孵育5分钟,每次):     
两次低盐洗涤缓冲液。
两次高盐洗涤缓冲液。
两次TE缓冲区。
注意:对于每种清洗,一次在磁力架上停留一分钟。
6.最后一次TE清洗后,将样品转移到新的1.5 ml Lobind管中,然后在磁力架上分离并取出TE。     

DNA回收
1.用200μl预热洗脱缓冲液从珠上洗脱两次。在65 °C下以800 rpm的搅拌温育15分钟。在一个新鲜的1.5 ml Lobind管中收集两种洗脱液(总体积为400μl ),并丢弃涂有磁性抗体的磁珠。     
2.使用洗脱缓冲液使输入样品达到400μl 。     
3.要反转的交联中,添加16微升5M的NaCl在65至洗脱液和孵化℃,600rpm下搅拌过夜。     
4.反向交联后,添加3μl蛋白酶K(20 mg / ml)并在45°C孵育1小时。     
5.要提取DNA,请添加400μl苯酚:氯仿:异戊醇(25:24:1),涡旋振荡30秒,然后在室温下以4000 xg离心10分钟。     
6.转移水层(〜400微升在新的1.5ml)的Lobind管。     
7.为了沉淀DNA,加0.1体积(40微升)的3M醋酸钠和2倍体积(800微升在-20℃)的100%乙醇(拌匀)并孵育1.5小时。     
8.以4000 xg离心30分钟(4°C)。     
9.洗涤沉淀用500微升冰冷的70%乙醇,离心在4000rpm 10分钟XG (4℃)。     
10.除去所有痕量乙醇,并在真空浓缩器中将沉淀干燥10分钟(65°C)。 
11.将沉淀重悬在所需体积的无核酸酶水中(注10 )。 
通过qPCR鉴定沉淀的蛋白质相关DNA
1.每个抗体,处理和引物对一式三份设置qPCR反应,如下所示(注11 ):     
每反应(10微升最终体积):3微升DNA,5微升小号YBR绿色的JumpStart的Taq预拌(Sigma公司),0.125微升正向引物(10 μM ),0.125微升反向引物(10 μM ),1.75微升的水。
2.使用以下条件在qPCR仪上运行反应:95°C 2分钟,然后在95°C 10 s,60°C 10 s和72°C 30 s进行60个循环。     
3.按照“数据分析”中所述分析数据。     

数据分析

注意:我们已经分析了Kuhn等人发表的数据。(2020)使用描述的协议。
1.一旦获得Ct(循环阈值)值,就计算三个技术重复之间的平均值。     
2.计算所有目标区域的每个IP与相应输入之间的比率:     
MEAN = 2- (MEAN(IP)-MEAN(Input))转换为线性比例。
3.计算所关注的组蛋白H3修饰(富集例如,H3K27Ac)作为H3之间的比率K27Ac在每个目标区域中的每个生物复制和H3。     
4.计算每个目标区域所有生物重复之间的均值和标准差。     
5.使用事后Bonfe rroni多重比较测试,采用双向方差分析分析每个目标区域的激素处理样品和MOCK样品之间的统计差异。     
6.沿着检查的目标区域的长度绘制比率(H3 K27Ac / H3)的平均值和标准偏差。     

笔记

1.吲哚-3-乙酸(IAA)不溶于水,需要在乙醇中新鲜制备到储备浓度为100 mM。模拟处理必须包含与生长素处理相同量的乙醇。处理植物时,请确保均匀地喷洒处理剂和模拟溶液。处理约100种拟南芥植物需要约100 ml溶液。     
2. W¯¯包含封闭的花蕾Ë只收获花序组织。打开的花朵或年轻的角果被移走了。     
3.要获得3 g样品,需要收获约100株植物的花序。我们建议同时收集一对生物学复制品(每个基因型,模拟和IAA处理重复一个),并重复收集三次以得到三个重复品进行实验。我们建议由两个人来收集以减少时间。     
4.如果包括多于一个的基因型或治疗,它建议使用一个玻璃烧杯中,每个处理和基因型,以防止样品的混合。     
5.交联ING时间视蛋白和组织需要优化。最佳的交联时间可使您感兴趣的蛋白质与DNA交联,但不会阻止表位。可以通过蛋白质印迹法进行测试。     
6.不要在第一个气泡出现前5分钟开始计时。     
7.这可以用100μl移液器完成。我们建议切割移液器吸头以防止损坏细胞核。     
8.超声条件在超声仪之间可能会有所不同,并取决于样品。通常,四个参数对于DNA片段化的效率至关重要:1)样品的温度(我们建议在整个超声处理过程中保持4°C的温度),2)用于交联的甲醛浓度(浓度导致增加超声处理时间),3)在其中进行超声处理的体积(我们建议不要声处理超过250微升裂解物在1.5ml试管),4)SDS中的裂解缓冲液的浓度(更多SDS引线以便在超声处理时更容易溶解)。     
9.超声测试:     
一旦确定好超声处理条件,就可以跳过此步骤。用洗脱缓冲液将超声和非超声等分试样移至100μl体积。
一种。加入1μl10 mg / ml RNase A,混合,在37°C孵育1 h。                     
b。加入100微升的苯酚:氯仿:异戊醇(25:24:1),涡旋30秒,离心4 ,000 ×g下在室温下10分钟。                   
C。将水相转移到新鲜的1.5 ml Lobind管中,并加入0.1体积(10μl )的3 M NaAc (pH 5.5)和2体积(200μl )的100%乙醇。涡旋并在-20°C下孵育1.5h。                     
d。离心4 ,000 ×g下在4℃下20分钟。                   
e。除去上清液,并在真空浓缩器(65°C)中干燥沉淀10分钟。                     
F。将沉淀重悬于10μl水中。                     
G。在1.5%琼脂糖凝胶上运行样品。                   
H。可视化凝胶。DNA应该显示为100 bp至500 bp之间的涂片(图1)。DNA片段越小,实验的分辨率越高。                   

图1 。超声TES牛逼。标记(NEB 100 bp梯形图); 显示大部分DNA片段的样品范围为100-500 bp。

10.水的量取决于将要运行的qPCR反应的数量(3微升每个反应DNA)。 
11.应该设计引物来扩增片段s(理想的片段长度为〜100 bp),并在整个目标基因位点之间有规则的间隔。实验的分辨率随着扩增子密度的增加而增加。使用前,应对所有qPCR引物进行效率和特异性测试。可以使用基因组DNA稀释系列(1/4至1/4096)(Figu re 2 A )测试引物的扩增效率。通过分析解链曲线(65°C至95°C)确认了引物的特异性(图2 B )。q PCR反应应按照实验程序(G1-3)中所述进行准备和进行。 

图2.合适的qPCR引物实例。一。合适的底漆的扩增效率应该显示扩增和稀释(R之间的相关性2的0.95)- 1和引物效率(10 -1 /斜率的2±)0.1。乙。熔解峰表明引物对的高特异性。

菜谱

库存解决方案
1. 2 M甘氨酸高压釜                                                                                     
2. 0.5 M HEPES KOH pH 7.4                                                                                   
高压釜
3. 1 M氯化镁2                                                                                   
高压釜
4. 1 M Tris-HCl pH 8高压釜                                                                                     
5. 0.5 M EDTA高压釜                                                                                     
6. 5 M氯化钠                                                                                   
7. Autocla已经                                                                                   
8. 3 M NaAc高压釜                                                                                     
9. 1百万吨DTT                                                                                   
称取1.54 g DTT,溶于10 ml水中
全部DTT溶解后,过滤除菌并制成1毫升等分试样
储存在-20 °C
10. 20%(v / v)的Triton X-100                                                                                 
用水将Triton X-100稀释五倍,以获得20%Triton X-100
11. 10%(v / v)安全数据表                                                                                 
用1体积的水稀释20%(v / v)SDS,以获得10%(v / v)SDS溶液

芯片缓冲液
1.本田缓冲器     
0.44 M蔗糖
1.25%(w / v)菲科尔
2.5%(w / v)葡聚糖T40
20毫米HEPES KOH pH 7.4
0.5%(v / v)海卫X-100
10毫米MgCl 2
0.5毫米DTT
每50毫升无EDTA的蛋白酶抑制剂鸡尾酒(Roche)1片
2.细胞核裂解缓冲液     
50 mM的Tris-HCl pH 8.0
10毫米EDTA
1%(v / v)安全数据表
每50毫升无EDTA的蛋白酶抑制剂鸡尾酒(Roche)1片
3. ChIP稀释缓冲液     
16.7 mM的Tris-HCl pH 8.0
1.2毫米EDTA
1.1%(v / v)海卫X-100
167毫米氯化钠
每50毫升无EDTA的蛋白酶抑制剂鸡尾酒(Roche)1片
4.低盐洗涤缓冲液     
20毫米Tris-HCl pH 8.0
2毫米EDTA
150毫米氯化钠
0.1%(v / v)安全数据表
1%(v / v)海卫X-100
每50毫升无EDTA的蛋白酶抑制剂鸡尾酒(Roche)1片
5.高盐洗涤缓冲液     
20毫米Tris-HCl pH 8.0
2毫米EDTA
500毫米氯化钠
0.1%(v / v)安全数据表
1%(v / v)海卫X-100
每50毫升无EDTA的蛋白酶抑制剂鸡尾酒(Roche)1片
6. TE缓冲液     
10毫米Tris-HCl pH 8.0
1毫米EDTA
每50毫升无EDTA的蛋白酶抑制剂鸡尾酒(Roche)1片
7.洗脱缓冲液     
0.1 M碳酸氢钠3
1%(v / v)安全数据表

致谢

我们感谢Yang Dong和Emilie Knight对协议提出批评。我们也表示感谢卡罗琳·迪恩,洪春洋和Julia QUESTA为他们提供有用的建议在本协议的发展。该协议改编自Kuhn等。,2020年。这项工作得到了L.Ø.的赠款BB / S002901 / 1,诺里奇研究园生物科学的博士培训合作伙伴[AK的赠款编号BB / M011216 / 1]和研究所战略计划赠款(BB / P013511 / 1)都归UKRI生物技术和生物科学研究委员会的John Innes中心所有。

利益争夺

作者宣称没有利益冲突。

参考文献

Chung,Y.,Zhu,Y.,Wu,MF,Simonini ,S.,Kuhn,A.,Armenta-Medina,A.,Jin ,R.,Ostergaard ,L.,Gillmor,CS and Wagner,D.( 2019)。生长素反应因子通过染色质介导的多能性基因SHOOTMERISTEMLESLES的阻遏促进器官发生。Nat Commun 10(1):886。
Kouzarides ,T。(2007)。染色质修饰及其功能。细胞128(4):693-705。
新罕布什尔州克罗根(Krogan ),霍根(Hogan)和长安(JA)(2012)。APETALA2通过募集协同阻遏物TOPLESS和组蛋白脱乙酰基酶HDA19负调控拟南芥中的多个花器官同一性基因。发展139(22):4180-4190。
库恩,A.,Ramans伯勒,S.,麦克劳克林,HM,纳塔拉詹,B.,Verstraeten ,I.,Friml,J.,Kepinski ,S。和奥斯特加德,L。(2020)。ETTIN-生长素的直接相互作用控制了妇生殖发育中的染色质状态。Elife 9:e51787。
Qüesta ,JI,Song,J.,Geraldo,N.,An,H. and Dean,C.(2016年)。拟南芥转录阻遏物VAL1在春化过程中触发FLC的Polycomb沉默。科学353(6298):485-488。
Schübeler ,D.(2015年)。DNA甲基化的功能和信息含量。自然517(7534):321-326。
Simonini ,S.,德布,J.,Moubayidin ,L.,Stephenson的,P.,Valluru ,M.,弗莱雷-Rios的,A.,Sorefan ,K.,Weijers ,D.,Friml,J。和奥斯特加德,L (2016)。拟南芥器官形态发生需要一种非规范的生长素传感机制。基因开发30(20):2286-2296。
Simonini ,S.,Bencivenga ,S.,招,M。和奥斯特加德,L。(2017)。生长素诱导的ETTIN活性调节调节拟南芥中的基因表达。植物细胞29(8):1864-1882。
Weijers ,D.和Wagner,D.(2016)。对生长素激素的转录反应。Annu启植物生物学67:539-574。
Wu,MF,Yamaguchi,N.,Xiao,J.,Bargmann ,B.,Estelle,M.,Sang,Y. and Wagner,D.(2015)。生长素调节的染色质开关指导花原基创建者命运的获取。Elife 4:e09269。
Yang,H.,Howard,M. and Dean,C.(2014年)。H3K36me3和H3K27me3在拟南芥FLC冷诱导的表观遗传开关中的拮抗作用。Curr Biol 24(15):1793-1797。
Zhou,大众,Goren,A。和Bernstein,BE(2011)。绘制组蛋白修饰和哺乳动物基因组的功能组织图。Nat Rev Genet 12(1):7-18。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright Kuhn and Østergaard. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Kuhn, A. and Østergaard, L. (2020). Chromatin Immunoprecipitation (ChIP) to Assess Histone Marks in Auxin-treated Arabidopsis thaliana Inflorescence Tissue. Bio-protocol 10(23): e3832. DOI: 10.21769/BioProtoc.3832.
  2. Kuhn, A., Ramans Harborough, S., McLaughlin, H. M., Natarajan, B., Verstraeten, I., Friml, J., Kepinski, S. and Ostergaard, L. (2020). Direct ETTIN-auxin interaction controls chromatin states in gynoecium development. Elife 9: e51787.
提问与回复
提交问题/评论即表示您同意遵守我们的服务条款。如果您发现恶意或不符合我们的条款的言论,请联系我们:eb@bio-protocol.org。

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。