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Jan 2017

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An Improved Method for Measuring Chromatin-binding Dynamics Using Time-dependent Formaldehyde Crosslinking
一种改进的使用时间依赖性甲醛交联测定染色质结合动力学的方法   

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Abstract

Formaldehyde crosslinking is widely used in combination with chromatin immunoprecipitation (ChIP) to measure the locations along DNA and relative levels of transcription factor (TF)-DNA interactions in vivo. However, the measurements that are typically made do not provide unambiguous information about the dynamic properties of these interactions. We have developed a method to estimate binding kinetic parameters from time-dependent formaldehyde crosslinking data, called crosslinking kinetics (CLK) analysis. Cultures of yeast cells are crosslinked with formaldehyde for various periods of time, yielding the relative ChIP signal at particular loci. We fit the data using the mass-action CLK model to extract kinetic parameters of the TF-chromatin interaction, including the on- and off-rates and crosslinking rate. From the on- and off-rate we obtain the occupancy and residence time. The following protocol is the second iteration of this method, CLKv2, updated with improved crosslinking and quenching conditions, more information about crosslinking rates, and systematic procedures for modeling the observed kinetic regimes. CLKv2 analysis has been applied to investigate the binding behavior of the TATA-binding protein (TBP), and a selected subset of other TFs. The protocol was developed using yeast cells, but may be applicable to cells from other organisms as well.

Keywords: Chromatin immunoprecipitation (ChIP) (染色质免疫沉淀(ChIP)), Protein dynamic (蛋白质动态), Protein cross-linking (蛋白质交联), Transcription factor (转录因子), Nucleic acid chemistry (核酸化学), Chromatin structure (染色质结构), Formaldehyde chemistry (甲醛化学)

Background

Transcription initiation is a complicated process that involves the cooperation and coordinated interaction of dozens of proteins on a chromatinized promoter (Kim et al., 2005; Encode Consortium, 2012; Rhee et al., 2012; Dowen et al., 2014). Many studies have investigated the assembly and regulation of the core transcriptional machinery in vitro (Zawel and Reinberg, 1992; Conaway and Conaway, 1993; Roeder, 1996; Hager et al., 2009; He et al., 2013; Cramer, 2014; Luse, 2014; Horn et al., 2016), but it has been more challenging to examine the stochastic nature of these processes in vivo. There are two general approaches used to measure chromatin-binding dynamics in vivo: microscopy and ChIP-based techniques (Coulon et al., 2013; Mueller et al., 2013). Microscopic techniques, such as fluorescence recovery after photobleaching (FRAP) or single molecule tracking (SMT), have high temporal resolution and have provided fundamental insight into chromatin binding dynamics, including results obtained by tracking individual molecules (Larson et al., 2009; Mueller et al., 2013; Morisaki et al., 2014). However, these approaches can be limited by photophysical effects such as photobleaching, and in addition, in the great majority of cases it is not possible to determine the identity of particular single copy loci where the measured interactions occur (Mueller et al., 2013). Alternatively, ChIP-based approaches provide precise chromatin location information. In Competition ChIP (CC), expression of a differentially tagged isoform of the TF of interest is induced and the relative levels of the constitutive and induced forms of the TF are monitored over time, yielding binding kinetic information through measurements of TF turnover at the sites of interest (van Werven et al., 2009; Lickwar et al., 2013). With advancements in modeling of CC data, residence times as short as 1.3 min have been measured (Zaidi et al., 2017). Relative dynamic measurements have also been made by conditional depletion of TFs from the nucleus using the Anchor Away technique (Haruki et al., 2008; Grimaldi et al., 2014), although specific mathematical models of the process have not yet been reported. The CLK method is complementary to these other ChIP-based approaches, exploiting the time dependence of formaldehyde crosslinking to derive binding kinetic parameters as well as fractional occupancy (Poorey et al., 2013). The first iteration of the CLK assay used ‘standard’ crosslinking and quenching conditions (1% formaldehyde and 250 mM glycine, respectively). Additional work has recently yielded experimental conditions that increase the crosslinking rate and improve quenching of the crosslinking reaction (Zaidi et al., 2017). These new conditions have resulted in a more robust method and the ability to model and analyze crosslinking kinetic data with more reliability and confidence.

Materials and Reagents

  1. Pipette tips
  2. Reusable cotton-plug top serological (glass) pipettes (can also use single-use plastic pipettes)
    10 ml pipettes (Fisher Scientific, catalog number: 13-675M )
    25 ml pipettes (Fisher Scientific, catalog number: 13-675N )
  3. Pipette sterilizing boxes (Fisher Scientific, catalog number: 03-465 )
  4. Nalgene PPCO centrifuge bottles with sealing closure (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 3141-0500 )
  5. 50 ml conical tubes 30 x 115 mm (Corning, Falcon®, catalog number: 352070 )
  6. 2.0 ml microcentrifuge conical screw cap tubes (FastPrep tubes, Fisher Scientific, catalog number: 02-681-344 )
  7. Microcentrifuge tube screw caps with O-Rings (for FastPrep tubes, Fisher Scientific, catalog number: 02-681-366 )
  8. Acid washed 425-600 μm glass beads (Sigma-Aldrich, catalog number: G8772 )
  9. 18 gauge needle (PrecisionGlide, BD, catalog number: 305195 )
  10. Disposable culture tubes, glass 13 x 100 mm (Fisher Scientific, catalog number: 14-961-27 )
  11. 1.5 ml microcentrifuge tubes graduated (Fisher Scientific, catalog number: 05-408-129 )
  12. Whatman filter paper (GE Healthcare, catalog number: 1003-917 )
  13. Autoradiography film (Genesee Scientific, catalog number: 30-101 )
  14. 1.5-1.7 ml polypropylene graduated tube with locking lid (Fisher Scientific, catalog number: 02-681-285 )
  15. Hard-Shell High-Profile 96-well semi-skirted PCR plates (Bio-Rad Laboratories, catalog number: HSS9641 )
  16. Tubular roll stock (to seal membrane in plastic before developing; Ampac, catalog number: TRS-95125-3 )
  17. Immobilon-P membrane (PVDF; Merck, catalog number: IPVH00010 )
  18. Microseal ‘B’ seal seals (Bio-Rad Laboratories, catalog number: MSB1001 )
  19. Stericup sterile vacuum filter units, 500 ml (Merck, catalog number: SCGPU05RE )
  20. Yeast strains, wild type (WT) and overexpression (OE) for each TF of interest (see Poorey et al., 2013 and Zaidi et al., 2017 for strain lists)
  21. Plasmids for TF overexpression and vector control for WT strain (see Poorey et al., 2013 and Zaidi et al., 2017 for plasmid lists)
  22. Locus specific primers (Invitrogen)
  23. In-Fusion HD cloning kit (Takara Bio, Clontech, catalog number: 639649 )
  24. Ice bucket
  25. 37% formaldehyde (Fisher Scientific, catalog number: F79 )
  26. Protein Assay Dye Reagent Concentrate (Bio-Rad Laboratories, catalog number: 500-0006 )
  27. nProtein A Sepharose 4 Fast Flow beads (GE Healthcare, catalog number: 17-5280-01 )
    Note: Ab-conjugated beads can be used instead, such as IgG Sepharose 6 Fast Flow (GE Healthcare, catalog number: 17-0969-01 ) and Sepharose 6 Fast Flow beads (GE Healthcare, catalog number: 17-0159-99 ).
  28. QIAQuick PCR purification kit (QIAGEN, catalog number: 28106 )
  29. iQ SYBR Green Supermix, 500 x 50 μl rxns (Bio-Rad Laboratories, catalog number: 1708882 )
  30. Instant non-fat dry milk (Carnation)
  31. Antibody (i.e., α-TBP, monoclonal, Abcam, catalog number: ab61411 )
  32. Secondary ECL-conjugated antibody (we use Amersham ECL mouse or rabbit IgG HRP-linked whole Ab, GE Healthcare, catalog numbers: NXA931 or NA934V )
  33. Amersham ECL Prime Western blotting detecting reagent (GE Healthcare, catalog number: RPN2232 )
  34. Yeast extract (BD, BactoTM, catalog number: 212750 )
  35. Bacto peptone (BD, BactoTM, catalog number: 211677 )
  36. Sugar source, i.e.,:
    D-(+)-Glucose (Sigma-Aldrich, catalog number: G7021 )
    D-(+)-Galactose (Sigma-Aldrich, catalog number: G5388 )
    D-(+)-Raffinose (MP Biomedicals, catalog number: 02102797 )
  37. Difco yeast nitrogen base without amino acids and ammonium sulfate (BD, DifcoTM, catalog number: 233520 )
    Note: Yeast nitrogen base without amino acids (with or without sugar source) can be used as an alternative and doesn’t require the addition of ammonium sulfate (i.e., Sigma-Aldrich, catalog number: Y0626 ).
  38. Amino acids:
    1. Adenine hemisulfate dihydrate (MP Biomedicals, catalog number: 02100195 )
    2. L-Histidine hydrochloride monohydrate (Acros Organics, catalog number: 411731000 )
    3. L-Lysine (Fisher Scientific, catalog number: BP386 )
    4. L-Tyrosine (Acros Organics, catalog number: 140641000 )
    5. L-Tryptophan (Fisher Scientific, catalog number: BP395 )
    6. Uracil (Affymetrix, catalog number: 23020 )
    7. L-Leucine (Acros Organics, catalog number: 125121000 )
    8. L-Methionine (Fisher Scientific, catalog number: BP388 )
    9. L-Arginine hydrochloride (Fisher Scientific, catalog number: BP372 )
    10. L-Serine (Fisher Scientific, catalog number: BP393 )
    11. Valine (Fisher Scientific, catalog number: BP397 )
    12. L-Threonine (MP Biomedicals, catalog number: 02103053 )
    13. L-Isoleucine (Fisher Scientific, catalog number: BP384 )
    14. L-Phenylalanine (Fisher Scientific, catalog number: BP391 )
    15. L-Cysteine hydrochloride monohydrate (Fisher Scientific, catalog number: BP376 )
    16. L-Aspartic Acid (Acros Organics, catalog number: 105041000 )
    17. L-Proline (Fisher Scientific, catalog number: BP392 )
    Note: Commercially available yeast synthetic drop-out medium supplements (Sigma-Aldrich) can be used instead of a home-made dropout mix.
  39. Bacto agar (BD, catalog number: 214010 )
  40. Glycine, 2 kg (Bio-Rad Laboratories, catalog number: 1610724 )
  41. Hydrochloric acid (Fisher Scientific, catalog number: A144SI-212 )
  42. Tris base (Sigma-Aldrich, catalog number: T1503 )
  43. Ammonium sulfate (Sigma-Aldrich, catalog number: A4418 )
  44. Magnesium chloride hexahydrate (Sigma-Aldrich, catalog number: M9272 )
  45. EDTA (Fisher Scientific, catalog number: BP120 )
  46. Glycerol (Fisher Scientific, catalog number: BP229 )
  47. β-Mercaptoethanol (Sigma-Aldrich, catalog number: M3148 )
  48. Protease inhibitors
    cOmplete protease inhibitor tablet EDTA-free (Roche Diagnostics, catalog number: 04693132001 )
    OR:
    Phenylmethylsulfonyl fluoride (Sigma-Aldrich, catalog number: P7626 )
    Benzamidine hydrochloride hydrate (Acros Organics, catalog number: 105241000 )
    Pepstatin A (Sigma-Aldrich, catalog number: P4265 )
    Leupeptin hemisulfate salt (Sigma-Aldrich, catalog number: L8511 )
    Chymostatin (Sigma-Aldrich, catalog number: C7268 )
  49. SDS (Sigma-Aldrich, catalog number: L3771 )
  50. DTT (Roche Diagnostics, catalog number: 03117006001 )
  51. Bromophenol blue (Bio-Rad Laboratories, catalog number: 1610404 )
  52. Coomassie blue
  53. Methanol (Fisher Scientific, catalog number: A452 )
  54. Acetic acid
  55. Sodium chloride (Fisher Scientific, catalog number: S640 )
  56. Tween 20 (Sigma-Aldrich, catalog number: P5927 )
    Note: This product has been discontinued.
  57. HEPES (Fisher Scientific, catalog number: BP310 )
  58. Triton X-100 (AMRESCO, catalog number: 0694 )
  59. Sodium deoxycholate (Sigma-Aldrich, catalog number: D6750 )
  60. Lithium chloride (Sigma-Aldrich, catalog number: L4408 )
  61. Nonidet P40 (Spectrum, catalog number: N1156 )
    Note: This product has been discontinued.
  62. Diethyl pyrocarbonate (DEPC), 97% pure (Acros Organics, catalog number: 170250250 )
  63. Acrylamide (Bio-Rad Laboratories, catalog number: 1610101 )
  64. Bis-acrylamide (Fisher Scientific, catalog number: BP171 )
  65. Ammonium persulfate (APS, Bio-Rad Laboratories, catalog number: 1610700 )
  66. TEMED (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 17919 )
  67. YPD media (see Recipes)
  68. 30% raffinose or galactose (see Recipes)
  69. SC media (Yeast synthetic media, see Recipes)
  70. Amino acid mix (see Recipes)
  71. 3 M glycine quench solution (see Recipes)
  72. Benoit’s buffer (see Recipes)
  73. Laemmli buffer (4x sample buffer, see Recipes)
  74. Coomassie stain (see Recipes)
  75. TBS (see Recipes)
  76. TBST (see Recipes)
  77. 140 mM ChIP lysis buffer (see Recipes)
  78. 500 mM ChIP lysis buffer (see Recipes)
  79. LiCl wash buffer (see Recipes)
  80. 1x TE (see Recipes)
  81. ChIP elution buffer (see Recipes)
  82. DEPC H2O (see Recipes)
  83. 30%/0.8% Bis-acrylamide solution (see Recipes)
  84. SDS-PAGE running buffer (see Recipes)
  85. Transfer buffer (see Recipes)

Equipment

  1. Glass culture tubes and caps, 18 x 150 mm (i.e., disposable borosilicate glass tubes with plain end, Fisher Scientific, catalog number: 14-961-32 and Diamond culture tube caps, 18 mm, Globe Scientific, catalog number: 118154 )
  2. Flasks (1 L, 250 ml; i.e., Pyrex narrow-neck heavy-duty glass Erlenmeyer flasks, Corning, PYREX®, catalog numbers: 4980-1L (1L) and 4980-250 (250 ml))
  3. Pipettes (P2, P2, P200, P1000)
  4. Pipet-Aid (Drummond Scientific, catalog number: 4-000-100 )
  5. Incubator, 30 °C (i.e., Isotemp CO2 incubator, Fisher Scientific, Fisherbrand, catalog number: 11-676-603 )
  6. Magnetic stir bars (Fisher Scientific, catalog number: 14-513-52 )
  7. Stirring hot plate (Fisher Scientific, catalog numbers: 11-510-49SH or SP88850200 )
  8. Timer
  9. Sorvall RC 5B centrifuge (GMI, model: Sorvall RC-5B )
  10. SLA-3000 rotor (Thermo Fisher Scientific, Thermo ScientificTM, model: SLA-3000 , catalog number: 07149)
  11. Eppendorf 5810 R benchtop centrifuge with plate buckets and 15 ml/50 ml adapters (Eppendorf, model: 5810R , catalog number: 5811000827)
  12. MP FastPrep-24 Bead beater (MP Biomedicals, model: FastPrep®-24 Classic, catalog number: 116004500 )
  13. Bunsen burner
  14. Branson Sonifier 250 with 1/8” microtip probe (Fisher Scientific, catalog numbers: 22-309782 and 22-309796 )
  15. Eppendorf 5415C or D benchtop centrifuge (Eppendorf, similar is catalog number: 022620304 )
  16. Shaker, 30 °C (i.e., Eppendorf, New Brunswick Scientific, model: Excella® E25 , catalog number: M1353-0002)
  17. 4 °C refrigerator
  18. -20 °C freezer
  19. -80 °C freezer
  20. Autoclave
  21. AutoMixer magnetic stir plate (Fisher Scientific, catalog number: 14-505-21 )
  22. Ultrospec 2100pro UV/Visible spectrophotometer (Biochrom, model: ULTROSPEC 2100® , catalog number: 80-2112-21)
  23. PowerPac Basic power supply (Bio-Rad Laboratories, catalog number: 1645050 )
  24. Mini-PROTEAN tetra cell with casting stand and frames, combs, short plates, and spacer plates (Bio-Rad Laboratories, catalog number: 1658006FC )
  25. Mini trans-blot electrophoretic transfer cell with holder cassettes, foam pads, and blue cooling unit (Bio-Rad Laboratories, catalog number: 1703930 )
  26. Shaker (Reliable Scientific, model: 55S )
  27. Autoradiography cassette (Fisher Biotech, catalog number: FBXC 810 )
  28. Labquake shaker rotisserie, 32 x 10 to 19 mm with clip bar (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 415110Q )
  29. Water baths (55 °C and 65 °C)
  30. MyiQ real-time instrument (Bio-Rad Laboratories, catalog number: 170-9770 )

Software

  1. ImageJ (NIH)
  2. Mathematica
  3. R or R-Studio

Procedure

This version of crosslinking kinetic (CLKv2) analysis is a modified ChIP procedure that yields kinetic measurements for transcription factor binding to specific loci by fitting ChIP data obtained from cells treated with formaldehyde for different periods of time. Before the samples can be collected for kinetic analysis, control experiments should be completed to optimize experimental conditions; the schematic in Figure 1 outlines the general workflow for this process. In this protocol, the required yeast strains are described first (Procedure A), followed by the detailed procedure for CLK data collection (Procedure B). CLK data collection conditions should be validated and may need to be optimized as described in Figure 1. The control experiments used for optimizing the procedure rely on the basic CLK methodology and so are described afterward (Procedure D and E) to minimize redundancy; Procedure C details quantification of the overexpression factor needed for modeling of the data.


Figure 1. CLKv2 workflow. Control experiments are performed to optimize the assay conditions before collection of CLK data and fitting to the CLK model.

  1. Yeast strain construction
    1. For the CLKv2 assay, two strains are used for the analysis of a TF of interest: a wild type (WT) strain and an overexpression (OE) strain. The overexpression strain is isogenic to the WT strain other than driving levels of the TF that are modestly higher (~3-5-fold) over the WT levels. Kinetic analysis of the WT and OE strains in parallel (described below) highly constrains fits of the data by revealing the mass action contribution to the increase in ChIP signal over time. The OE strain can be engineered by introducing into cells an additional copy of the TF gene on a plasmid or by integrating into the genome under control of the native promoter or an appropriate heterologous promoter. If the TF functions as a stable biochemical entity with more than one type of subunit, the OE strain ought to be engineered to drive balanced expression of each subunit, as for example, was done for the analysis of TFIIE (Zaidi et al., 2017). For a detailed description of OE strain and plasmid construction, see Poorey et al., 2013, Zaidi et al., 2017.
    2. To generate strains for kinetic analysis using an OE plasmid, transform WT S. cerevisiae cells in the strain background of interest with either the OE plasmid construct or an empty vector carrying the same selection marker. Select transformants on appropriate agar plates and restreak cells for single colonies. We typically use strains kept on plates for about one month at 4 °C; archive the WT and OE strains by storage at -80 °C using standard yeast glycerol stock methods (Amberg et al., 2005).

  2. CLK data collection
    As mentioned above, the experimental conditions required for analysis of a particular TF should be validated and may need to be optimized as described in Procedure D and E. This section describes the basic CLKv2 procedure as we optimized and recently described (Zaidi et al., 2017).
    Cell growth and cell sample collection
    1. Inoculate duplicate 5 ml YPD primary cultures with a colony of the desired WT or OE yeast strains; incubate cultures overnight at 30 °C with shaking.
      Note: If starting the primary cultures in selective media, cells may need to grow for ~24 h instead of ~16 h to obtain enough material to start the larger cultures in the next step.
    2. The next day, dilute each primary culture in 450 ml of the desired selective media (SC media with appropriate amino acid drop-out) and incubate with shaking at 30 °C until the OD600 is ~0.8 (~1.3 x 107 cells). Pellet cells by spinning for 7 min at 4,230 x g and 4 °C in Nalgene centrifuge bottles using an SLA-3000 rotor and Sorvall RC 5B centrifuge.
      Note: Growing conditions for copper-inducible strains are outlined in Poorey et al., 2013. If the TF of interest is under the control of a GAL-inducible promoter, the cells should be grown in selective media with 2% raffinose until OD600 is ~0.8 is reached; then pellet and resuspend the cells in YEP + 2% galactose and incubate for an hour at 30 °C with shaking.
    3. Resuspend each cell pellet in 450 ml YPD (or desired media); incubate at 30 °C with shaking for about one hour until the OD600 is ~1.0 (~1.9 x 107 cells).
    4. While the cells are growing, prepare the glycine quench solution. For each time point, 440 ml of 3 M glycine pH 5 is needed. Thus, 4 L of glycine will be required to quench the eight time point samples for one time course; see Recipes for instructions. Pour 440 ml of the 3 M glycine solution into each of eight 500 ml Nalgene centrifuge bottles.
    5. Pellet cells as in Step 2, then resuspend cells in 90 ml YPD (or desired media). This step yields a cell suspension that is concentrated five-fold over the initial culture.
      Note: The cells are concentrated in this way so that in subsequent steps quenching of formaldehyde crosslinking can be made more efficient by dilution of the cell suspension as well as by addition of glycine.
    6. Add a 10 ml glass or plastic pipette to a Pipette-Aid; this will be used for removal of an aliquot of the cell suspension at the first time point. Place flasks with 90 ml culture on a stir plate with a stir bar on medium speed and rapidly add 14 ml 37% formaldehyde to a final concentration of 5%; immediately start the timer. One way to rapidly add formaldehyde is to invert a pre-measured 14 ml aliquot contained in a 15 ml disposable conical tube into the stirring cell suspension. If formaldehyde is added with a Pipette-Aid, then a second Pipette-Aid should be fitted with a 10 ml pipette beforehand for removal of the first time point aliquot.
    7. At 5 sec, 20 sec, 60 sec (1 min), 120 sec (2 min), 300 sec (5 min), 600 sec (10 min), 900 sec (15 min), and 1,200 sec (20 min) remove a 10 ml aliquot of crosslinked culture with a Pipet-Aid and 10 ml glass pipette and quench by quickly adding the cell aliquot to a bottle containing the prepared glycine solution; cap each bottle immediately after adding the cell suspension and invert a few times to mix.
    8. Pellet cells by spinning at 4 °C for 7 min at 4,230 x g using an SLA-3000 rotor and Sorvall RC 5B centrifuge.
    9. Resuspend each cell pellet in 50 ml 4 °C TBS + 300 mM glycine pH 5 and transfer to a 50 ml conical tube; spin for 5 min at 3,220 x g and 4 °C in an Eppendorf 5810 R benchtop centrifuge. Discard supernatant.
    10. Wash each cell pellet with 50 ml 4 °C TBS; pellet cells as in the previous step.
    11. Resuspend each cell pellet in 1 ml TBS at 4 °C and transfer each sample to a FastPrep tube.
    12. Pellet cells by spinning for 2 min at 4 °C and 16,000 x g in an Eppendorf 5810 R benchtop centrifuge. Discard supernatant.
      Note: Samples can be stored at -80 °C at this step.

    Isolation of fragmented chromatin samples
    1. Resuspend each cell pellet in 600 μl 140 mM ChIP lysis buffer with protease inhibitors added. Add acid washed glass beads to each tube until just above the level of the liquid and screw cap on tightly.
    2. Bead-beat samples at 4.0 m/sec for 7 cycles, with 45 sec on and one minute off between cycles, using an MP FastPrep-24 Bead beater kept in the cold room.
    3. Poke a hole in the bottom of each FastPrep tube with a heated (using Bunsen burner flame) 18-gauge needle and then place each tube in a 13 x 100 mm glass tube. Spin for 3 min at 3,220 x g in an Eppendorf 5810 R benchtop centrifuge at 4 °C to transfer the liquid to the bottom of the glass tube.
    4. Briefly vortex the glass tubes containing the flow through material and transfer each cell suspension to an Eppendorf tube; place tubes on ice.
    5. Sonicate samples for 7 cycles with 5 pulses/cycle, 30% output, and 90% duty cycles using a Branson Sonifier 250 with microtip probe. Place tubes on dry ice between cycles.
      Note: By sonicating one sample after another for each of the 7 cycles, the samples will stay on dry ice long enough to keep them cool but not freeze. It may be necessary to alternate samples between wet and dry ice to keep the pellets from freezing if many samples are sonicated at once. The expected fragment size of the sonicated DNA is ~100-600 bp. The first time the experiment is performed, the size of the sonicated DNA should be checked by running samples on a 1% agarose gel with ethidium bromide followed by imaging with an appropriate system.
    6. Spin tubes for 5 min at 16,000 x g at 4 °C in an Eppendorf 5415C/D benchtop centrifuge.
    7. Remove the supernatant for each sample to a new 1.5 ml microcentrifuge tube; spin for 20 min at 16,000 x g at 4 °C in an Eppendorf 5415C/D benchtop centrifuge.
    8. Transfer each supernatant to a new 1.5 ml microcentrifuge tube and quantify protein with a Bradford assay using a 1 μg/μl bovine serum albumin standard and Bradford protein dye as recommended by the manufacturer of the Bradford reagent.
      Note: Samples can be stored at -80 °C at this step.

    Chromatin immunoprecipitation (ChIP)
    1. Set up ChIP reactions in microcentrifuge tubes with locking lids. For each time point sample, three tubes are needed: immunoprecipitation (IP), mock, and total (input). The IP is done in one step if antibody-conjugated beads are used (a) or two steps if the chromatin extract is incubated first with the antibody followed by incubation with Protein A beads (b).
      1. For antibody-conjugated beads: First prepare the beads used for the IP and mock samples. The IP beads have the desired antibody conjugated to them; the mock beads are either unconjugated beads of the same type used for the IP, or alternatively, if a tag is used for the TF of interest, chromatin from an otherwise identical untagged strain can be used with the same antibody conjugated beads used for the IP. Aliquot the total volume of beads required to process all the samples (45 μl/sample) by adding to a 1.5 ml microcentrifuge tube. Wash the beads three times with 1 ml 140 mM ChIP lysis buffer, with a quick spin in between washes to recover the beads (~6,000 x g for a few seconds). Resuspend the bead pellets in an equal volume 140 mM ChIP lysis buffer with protease inhibitors to make a 50% slurry. Aliquot 40 μl of the bead slurry into one tube for each IP and mock reaction. Add 1 mg of sample chromatin to each tube on ice and add 140 mM ChIP lysis buffer with protease inhibitors to bring the total volume to 500 μl. For the input samples, aliquot 0.1 mg of the sample chromatin to each tube. Store the input samples at -80 °C; place the IP and mock reaction tubes on a rotating shaker such as the Labquake rotisserie shaker overnight at 4 °C.
      2. For antibody incubation followed by bead incubation: For the IP samples, aliquot 1 mg of sample chromatin protein into each tube and add antibody for the TF of interest followed by 140 mM ChIP lysis buffer with protease inhibitors to bring the total volume to 500 μl. For the mock reactions, aliquot 1 mg of sample chromatin then 140 mM ChIP lysis buffer with protease inhibitors to bring the volume to 500 μl (no antibody added). For the input samples, aliquot 0.1 mg of the sample chromatin into each tube. Store the input samples overnight at -80 °C; place the IP and mock tubes on a Labquake rotisserie shaker overnight at 4 °C.
        Note: The amount of antibody added to the IP varies depending on its concentration and may need to be optimized before processing the CLK samples. Typically, ~5 μg antibody is used for ChIP with 1 mg sample chromatin protein.
    2. Skip to the next step if using antibody-conjugated beads. Prepare a 50% slurry of nSepharose Protein A beads in the same way the beads are prepared in Step 1a (Chromatin immunoprecipitation (ChIP)). Aliquot 40 μl of the bead slurry to a new tube for each IP or mock sample. Quick spin the chromatin samples in a benchtop microfuge and transfer each supernatant to a new tube with the nSepharose Protein A bead slurry. Incubate the chromatin-bead samples for 2 h at 4 °C with mixing as in Step 1.
    3. Quick spin tubes. Wash each bead pellet twice with 1 ml each of the following buffers; quick spin in a microfuge between washes:
      1. 140 mM ChIP lysis buffer
      2. 500 mM ChIP lysis buffer
      3. LiCl wash buffer
      4. 1x TE pH 8
    4. Remove excess liquid after the last wash and add 75 μl ChIP elution buffer to each tube. Mix beads and elution buffer by tapping the tube several times. Incubate samples at 65 °C for 10 min.
    5. Quick spin tubes in a microfuge and transfer the supernatant to a new locking lid tube.
    6. Add another 75 μl aliquot of ChIP elution buffer to each bead pellet. Mix by flicking the tube as above. Incubate samples again at 65 °C for 10 min.
    7. Quick spin the tubes in a microfuge and combine the supernatants in one tube. Incubate samples overnight at 65 °C.
    8. Thaw the input samples for a minute or two on the bench. Add 150 μl ChIP elution buffer, mix by briefly vortexing, and incubate the tubes overnight at 65 °C along with the IP and mock samples.
    9. The next day, clean up the DNA samples using the QiaQuick PCR purification kit by following the instructions provided by the manufacturer. Briefly, first, add 750 μl Buffer PB to each tube; mix and apply 450 μl of the sample to a column, then spin for 2 min at 9,300 x g using an Eppendorf 5415D benchtop centrifuge at room temperature.
    10. Apply the remainder of sample to each column and spin as in the previous step.
    11. Apply 750 μl Buffer PE to each column; spin as in the previous step.
    12. Discard the flow-through and then spin each column again to remove all residual ethanol.
    13. Place each column in a new microcentrifuge tube then apply 50 μl of 55 °C DEPC H2O to each column. Let the tubes sit on the bench for 1-2 min, then spin as in the previous step. Discard columns and either freeze samples at -80 °C or immediately perform real-time PCR analysis.

    Real-time PCR quantitation
    A standard curve is generated from serial dilutions of the input sample, which is then used to determine the ChIP levels for the IP and mock samples. The relative ChIP signal, described below, can be calculated for each time point and plotted before model fitting the data. It is critical to quantify the ChIP signal using a standard curve; using the threshold cycle number directly (for example) to quantify ChIP levels will not provide an accurate estimate of the quantity of the ChIP material unless it accounts for the log relationship with input.
    1. First, make a dilution series of input chromatin. Thaw the input samples on the benchtop for a few minutes, then quick spin in a microfuge and place on ice. We refer to the undiluted input as ‘125x’; five-fold dilutions of this material are made to generate 25x, 5x, and 1x input samples for the real-time PCR standard curve. To make the 25x standard, take 2 μl of the undiluted input and add to 8 μl DEPC H2O. Mix, quick spin, and make a dilution of the 25x sample in the same way to yield a 5x sample. With the 5x sample, make a 1x sample in the same way. Keep dilutions of input chromatin on ice.
    2. Prepare the real-time PCR reaction mix by adding 10 μl iQ SYBR Green Supermix (2x) for each reaction and forward and reverse primers to a final concentration of 0.2 μM; adjust the volume to 19 μl with DEPC H2O. As described below, we run technical triplicates for each sample, and the standards are run in duplicate. Make enough mix for a control reaction containing no sample as well.
    3. Real-time PCR reactions are run in wells of plates designed for the instrument. Pipette 19 μl of the reaction mix into each well. There will be 17 wells required for each time point: 3 for each IP, mock, and 5x input and 2 for each of the four standards. See Figure 2 below for an example plate set-up.


      Figure 2. Template for real-time PCR plate for four time point samples. Each experimental sample is run in triplicate (rows A, C, E, and G) and each of the four standards is run in duplicate (rows B, D, F, H). The same master mix is used for each reaction and one well (H12) has no sample added as a negative control.

    4. Thaw the IP and mock samples, then quick spin in a microfuge and place on ice. Add 1 μl of sample to each well as indicated except for the control well. We include the 5x input sample as an unknown as well as a standard; its estimated value as an unknown establishes how well the standard curve captures the ChIP signal quantitatively.
    5. Seal the plate with film and spin for 3 min at 1,810 x g/4 °C in an Eppendorf 5810 R benchtop centrifuge.
    6. Run the plate in a MyiQ or related instrument using a protocol optimized for the primer set, and with a melting curve to verify that a single product species is generated. The standard quantities are set to values of 125, 25, 5, and 1.
    7. To determine the relative ChIP signal at each time point, subtract the average mock signal from the average IP signal and divide by the average estimated 5x input signal. Plot the average of two data sets for each time point along with the standard deviation. The average values will be used for the next step of model fitting. A representative plot is shown in Figure 3.


      Figure 3. Example CLKv2 data for the TATA-binding protein (TBP) interaction with the URA1 locus. The average relative ChIP signal of two biological replicates is shown for the WT (blue circles) and OE (red circles) strains versus formaldehyde crosslinking time; the error bars represent the standard deviation. This is the raw data used to generate the model fit in Figure 8B in Zaidi et al., 2017.

  3. Transcription factor overexpression value quantification
    The overexpression factor is the level of the TF in the OE strain relative to the WT strain and is required for fitting the CLKv2 model to the formaldehyde incubation time-dependent ChIP data. This section describes how to estimate the overexpression factor by performing Western blots using extracts from the WT and OE strains.
    Cell growth and cell sample collection
    This protocol is similar to the detailed instructions in Procedure B, Cell growth and cell sample collection, with changes to the cell culture volume and wash buffers used as indicated below. In addition, the formaldehyde crosslinking and quenching steps are not necessary and therefore not performed for this section.
    1. Inoculate each of two 5 ml YPD primary cultures with a colony of the desired WT and OE yeast strains; incubate cultures overnight at 30 °C with shaking.
    2. The next day, dilute each primary culture in 50 ml of the desired selective media and incubate with shaking at 30 °C until the OD600 is ~0.8. Pellet cells in 2 x 50 ml conical tubes by spinning for 5 min at 3,220 x g and 4 °C in an Eppendorf 5810 R benchtop centrifuge.
    3. Resuspend each cell pellet in 50 ml YPD (or desired media); incubate at 30 °C with shaking for about one hour until the OD600 is ~1.0.
    4. Pellet cells as in Step 2.
    5. Combine cell pellets for each sample and resuspend in 50 ml 4 °C TBS and transfer to a 50 ml conical tube; spin for 5 min at 3,220 x g and 4 °C. Discard supernatant.
    6. Wash cell pellets with 10 ml 4 °C Benoit’s buffer with protease inhibitors and β-mercaptoethanol; pellet cells as in the previous step.
    7. Resuspend cell pellets in 1 ml Benoit’s buffer (with protease inhibitors and β-mercaptoethanol) and transfer each sample to a FastPrep tube.
    8. Pellet cells by spinning for 2 min at 4 °C in an Eppendorf 5810 R benchtop centrifuge. Discard supernatant.
      Note: Samples can be stored at -80 °C at this step.

    Preparation of whole cell extract
    This protocol is similar to the detailed instructions in Procedure B, Isolation of fragmented chromatin extract samples, with changes to the cell pellet resuspension volume, the buffer used, and the omission of sonication.
    1. Resuspend cell pellets in 300 μl Benoit’s buffer with protease inhibitors and β-mercaptoethanol. Add acid washed glass beads to each tube until just above the liquid. Tap tubes on the lab bench a few times to remove air bubbles.
    2. Bead-beat samples for 7 cycles, with 45 sec on and one minute off between cycles, using an MP FastPrep-24 Bead beater kept in the cold room.
    3. Poke a hole in the bottom of each FastPrep tube with a heated (using Bunsen burner flame) 18-gauge needle and then place each tube in a 13 x 100 mm glass tube. Spin for 3 min at 3,220 x g at 4 °C to transfer the liquid to the bottom of the glass tube.
    4. Briefly vortex the glass tubes containing the flow through material and transfer each cell suspension to an Eppendorf tube.
    5. Place samples on ice for 30 min.
    6. Spin tubes for 5 min at 16,000 x g at 4 °C.
    7. Remove the supernatant for each sample to a new 1.5 ml microcentrifuge tube; spin for 30 min at 16,000 x g at 4 °C.
    8. Transfer each supernatant to a new 1.5 ml microcentrifuge tube and quantify protein with a Bradford assay as described in Procedure B.
      Note: Samples can be stored at -80 °C at this step.

    Quantification of TF levels by Western blotting
    1. Pour an SDS-PAGE denaturing gel of appropriate concentration (8-14%) with 30%/0.8% Bis-acrylamide solution.
    2. Run the SDS-PAGE gel using, for example, a Bio-Rad mini-PROTEAN tetra cell filled to the corresponding marking with SDS-PAGE running buffer. Load a 2x serial dilution of sample for each strain on the same gel starting with 20 μg; the other amounts loaded will be 10 μg, 5 μg, and 2.5 μg. Each sample is mixed with Laemmli loading buffer and heated for 5 min at 95 °C to denature proteins before loading. Run the gel at 120 V for an appropriate amount of time to resolve polypeptide bands in the size range of interest.
      Note: More blots may need to be performed with different dilutions if the factor overexpression value is high.
    3. Transfer proteins from the gel to an Immobilon-P (PVDF) membrane overnight in transfer buffer using a Bio-Rad mini trans-blot electrophoretic system (or a related system) at 30 V and 4 °C.
      Note: Visually inspect the membrane the next day before blocking to make sure the ladder has transferred onto the membrane.
    4. The next day, block the membrane with an appropriate solution. We typically use 5% nonfat milk/TBST for 1 h on a shaker at room temperature.
    5. Incubate the membrane with primary antibody, typically an hour or more, and then wash the blot 4 times in TBST for 5 min per wash.
    6. Incubate the membrane with secondary antibody (incubation time is typically 1 h but may need to be optimized depending on the antibody), and then wash 4 times in TBST for 5 min per wash.
      Note: An ECL-conjugated HRP-linked secondary antibody can be used for Western development on film. However, fluorescently labeled antibodies, such as those conjugated with Cy2/3/5, are preferred as the fluorescence intensity can be quantified directly and fluorescence imaging has a wider dynamic range than film.
    7. Mix 500 μl of Solution A and 500 μl of Solution B from the ECL-Prime kit and spread evenly over blot; seal blot in plastic and expose to film.
      Note: A fluorescent secondary antibody can also be used instead of the chemiluminescent secondary, such as provided with the ECL-Plex kit. In this case, the blot can be scanned and quantified using a Typhoon scanner or other fluorescence imaging system and a digital copy of the image is saved.
    8. Scan the film using a standard color scanner; save images as TIFF files. As Western blots are notoriously difficult to quantify, we typically perform and quantify multiple Western blot images with multiple film exposure times. It is also a good idea to run gels with a dilution series of the extract to assess how the detection system responds to known changes in the relative level of the TF on the blot.
    9. Using ImageJ software, open the TIFF image and quantify the protein band(s) of interest in each lane and normalize to a loading control by drawing a box around each band with the rectangular selection tool and measuring the intensity inside each box. Determine the overexpression factor by dividing the quantified OE band by the corresponding WT band; do this for each dilution and average the values to get one OE factor. We estimate the OE value by averaging the effects observed in at least two biological replicate sets of samples.

  4. Measure the effect of formaldehyde crosslinking on the levels of the TF in the soluble protein pool
    An assumption in the CLK model is that the levels of the unbound TF do not change with increasing formaldehyde incubation time (Poorey et al., 2013). Before CLK analysis can be performed, it is therefore critical to measure the TF protein levels over the experimental time course to determine whether the TF is depleted from the nuclear pool of soluble proteins. Either whole cell extract or chromatin samples can be used; we’ve found for a number of TFs that either type of extract yields very similar results (Zaidi et al., 2017). The protocols for Procedure D and E are based on the CLKv2 data collection methods in Procedure B.
    Cell growth and cell sample collection
    This protocol is similar to the detailed instructions in Procedure B, Cell growth and cell sample collection, with changes to the cell culture volume and wash buffers used; a smaller volume is required for this optimization experiment and a different buffer is used for whole cell extract preparation.
    1. Inoculate each of two 5 ml YPD primary cultures with a colony of the desired WT yeast strain; incubate cultures overnight at 30 °C with shaking.
    2. The next day, dilute each primary culture in 300 ml of the desired selective media and incubate with shaking at 30 °C until the OD600 is ~0.8. Pellet cells by spinning for 7 min at 4,230 x g and 4 °C.
    3. Resuspend each cell pellet in 300 ml YPD (or desired media); incubate at 30 °C with shaking for about one hour until the OD600 is ~1.0.
    4. Prepare the glycine quench solution. 4 L of glycine will be required to quench the four time point samples in duplicate; see Recipes for instructions. Pour 440 ml of the 3 M glycine solution into each of 8 x 500 ml Nalgene centrifuge bottles.
    5. Pellet cells as in Step 2, then resuspend each pellet in 60 ml YPD (or desired media).
    6. Place each flask with 60 ml culture on a stir plate with a stir bar, then remove a 10 ml aliquot from each culture using a Pipet-Aid and glass pipette and quench in a bottle of 440 ml 3 M glycine for the zero minute control samples. Invert the bottles a few times to mix. Turn the stir bar speed to medium. To the remaining 50 ml culture, add 7.8 ml 37% formaldehyde, yielding a final concentration of 5%.
    7. At 5, 10, and 15 min (or other time points as needed), quickly remove a 10 ml aliquot of crosslinked culture and quench by adding to a 440 ml aliquot of glycine; cap and invert the bottle a few times to mix.
    8. Pellet cells by spinning at 4 °C for 7 min at 4,230 x g.
    9. Resuspend each cell pellet in 50 ml 4 °C TBS and transfer to a 50 ml conical tube; spin for 5 min at 3,220 x g and 4 °C. Discard supernatant.
    10. Wash cell pellets with 10 ml 4 °C Benoit’s buffer with protease inhibitors and β-mercaptoethanol; pellet cells as in the previous step.
    11. Resuspend cell pellets in 1 ml Benoit’s buffer (with protease inhibitors and β-mercaptoethanol) and transfer each sample to a FastPrep tube.
    12. Pellet cells by spinning for 2 min at 4 °C in an Eppendorf 5810 R benchtop centrifuge. Discard supernatant.
      Note: Samples can be stored at -80 °C at this step.

    Preparation of whole cell extract
    This section of the protocol is identical to the detailed instructions in Procedure C, Preparation of whole cell extract.

    Quantification of TF levels by Western blotting
    This protocol is similar to the detailed instructions under Procedure C, Quantification of TF levels by Western blotting, except for the amount of sample loaded and quantification output.
    1. Run an SDS-PAGE denaturing gel of appropriate concentration (8-14%). Load 15 μg protein for each sample after mixing with Laemmli loading buffer and heating for 5 min at 95 °C to denature proteins. Run the gel at 120 V for an appropriate amount of time.
    2. Transfer proteins from the gel to an Immobilon-P (PVDF) membrane overnight at 30 V and 4 °C.
    3. The next day, block the membrane with an appropriate solution optimized for the antibody of interest.
    4. Incubate the membrane with primary antibody and then wash the blot 4 times in TBST for 5 min per wash.
    5. Incubate the membrane with secondary antibody (incubation time is typically 1 h but may need to be optimized depending on the antibody), and then wash 4 times in TBST for 5 min per wash.
    6. Mix 500 μl of Solution A and 500 μl of Solution B from the ECL-Prime kit and spread evenly over blot; seal blot in plastic and expose to film.
    7. Scan the film.
    8. Using ImageJ software, open the TIFF image and quantify the protein band(s) of interest in each lane and normalize to the zero minute time point and/or a loading control. We estimate the effects of formaldehyde incubation time on soluble TF levels by averaging the effects observed in at least two biological replicate sets of samples.
      Note: The relative change in protein level with crosslinking time will guide the implementation of the assay in the next steps. See Zaidi et al., 2017 for a range of results with different factors. If the protein of interest is not depleted with increasing formaldehyde incubation time, then the CLKv2 assay can be conducted using this concentration of formaldehyde and over this range of formaldehyde incubation times. Alternatively, if the protein of interest is depleted from extracts with increasing formaldehyde incubation time, the options are to test a different (reduced) concentration of formaldehyde and/or to confine the crosslinking time course to the period in which the factor is not depleted. If the formaldehyde concentration is reduced, effort should be made to determine the maximum usable concentration in order to maximize the crosslinking rate, such as treating cells with a titration of formaldehyde concentrations at a later time point to find the optimal condition.

  5. Validate the quenching conditions
    Collection of order-of-addition samples
    It is important to verify that the quenching conditions quantitatively block formaldehyde crosslinking, and in this way ensure that the ChIP signals truly reflect the yield of crosslinked material following incubation with formaldehyde for a given time. We have found that the glycine quench protocol reported here effectively quenches 5% formaldehyde under the conditions described; the experiments in this section should be conducted to validate that this is true in your hands and under your specific conditions.
    This protocol is similar to the detailed instructions in Procedure B, Cell growth and cell sample collection, with changes to the cell culture volumes.
    1. Inoculate duplicate 5 ml YPD primary cultures with a colony of the desired WT yeast strain; incubate cultures overnight at 30 °C with shaking.
    2. The next day, dilute each primary cultures in 200 ml of the desired selective media and incubate with shaking at 30 °C until the OD600 is ~0.8. Pellet cells by spinning for 7 min at 4,230 x g and 4 °C as described above.
    3. Resuspend each cell pellet in 200 ml YPD (or desired media); incubate at 30 °C with shaking for about one hour until the culture OD600 is ~1.0.
    4. Prepare the glycine quench solution. 3 L of glycine will be required to quench the three time point samples in duplicate; see Recipes for instructions. Pour 440 ml of the 3 M glycine solution into each of 6 x 500 ml Nalgene centrifuge bottles.
    5. Pellet cells as in Step 2, then resuspend pellets in 60 ml YPD (or desired media). Transfer 10 ml culture into each of three 500 ml flasks.
    6. Place each flask on a stir plate with a stir bar on medium speed.
    7. Collect samples as follows:
      1. No formaldehyde: add the 440 ml glycine solution to one flask with 10 ml culture and mix for a few seconds; pour solution and cells back into the bottle, cap the bottle and invert a few times to mix thoroughly. This sample can remain on the bench at room temperature while the other samples are processed.
      2. Glycine before formaldehyde: to another flask containing a cell sample, add all 440 ml 3 M glycine pH 5 from the prepared bottle. Mix for a few seconds on a stir plate, then add 1.56 ml 37% formaldehyde (5% final concentration) and allow crosslinking to proceed for 8 min at room temperature on a stir plate. Pour the entire sample back into the bottle.
      3. Formaldehyde followed by glycine: to the last flask containing a cell sample, add 1.56 ml 37% formaldehyde (5% final concentration); mix by thoroughly swirling, and incubate on a stir plate for 8 min at room temperature. (The formaldehyde incubation time can be adjusted; 8 min is recommended here as it is in the regime in which the ChIP signal is typically still dependent on formaldehyde incubation time, i.e., not saturated, but incubation for several minutes is easy to handle with high temporal precision.) Add 440 ml 3 M glycine pH 5 to the sample and mix for a few minutes. Pour entire solution back into the bottle.
        Note: Two sets of samples are collected, but it is easiest to collect one set of samples first (a, b, c) and then the second set if both sets are collected on the same day.
    8. Pellet cells by centrifugation as in Step 2.
    9. Resuspend each cell pellet in 50 ml 4 °C TBS + 300 mM glycine pH 5 and transfer to a 50 ml conical tube; spin for 5 min at 3,220 x g and 4 °C. Discard supernatant.
    10. Wash each cell pellet with 50 ml 4 °C TBS; pellet cells as in the previous step.
    11. Resuspend each cell pellet in 1 ml TBS at 4 °C and transfer each sample to a FastPrep tube.
    12. Pellet cells by spinning for 2 min at 4 °C and 16,000 x g. Discard supernatant.
      Note: Samples can be stored at -80 °C at this step.

    Isolation of fragmented chromatin samples
    This section of the protocol is identical to the detailed instructions in Procedure B, Isolation of fragmented chromatin samples.

    Chromatin immunoprecipitation (ChIP)
    This section of the protocol is identical to the detailed instructions in Procedure B, Chromatin immunoprecipitation (ChIP).

    Real-time PCR
    This protocol is identical to the detailed instructions under Procedure B, Real-time PCR quantification.
    To determine the relative ChIP signal at each time point, subtract the average mock signal from the average IP signal and divide by the average estimated 5x input signal. We typically plot the results as a bar graph and perform t-tests to assess statistically significant differences in comparing different samples. With an efficient quench, the ‘glycine only’ and ‘glycine before formaldehyde’ ChIP signals should be quantitatively indistinguishable (Figure 4).


    Figure 4. Representative results for an order-of-addition experiment. G = glycine only, GF = glycine quench followed by formaldehyde addition, FG = formaldehyde crosslinking followed by glycine quench. In this example, the relative ChIP signal was calculated following immunoprecipitation of the Gal4 protein and real-time PCR readout for interaction at the GAL3 locus. Error bars represent the standard deviation from two biological replicates.

Data analysis

Model fitting of data

  1. Poorey et al. (2013, Science) developed the mass-action kinetic model that we used to analyze the data generated in that study and extract kinetic parameters. The procedure to fit the data to this model was systematically studied and re-formulated in Zaidi et al. (2017) based on the experimental regimes seen in the experimental data. In the following, we give a brief overview of the quantitative fitting procedure used to estimate kinetic parameters.
  2. In general, CLKv2 data can be represented by three types of fits: TF-limited, crosslink (XL)-limited, and linear XL-limited (see Figure 5). These categories are determined by the crosslinking rate (kxl*CFH), the overall on-rate (ka*CTF), and the off-rate (kd). In the TF-limited regime, ka*CTF<<kxl*CFH and kd<<kxl*CFH. On the other hand, in the XL-limited regime, ka*CTF>>kxl*CFH and kd>>kxl*CFH. The linear XL-limited regime is a sub-regime of the XL-limited model where the crosslinking rate is slow enough to be comparable to the latest crosslinking time points generated in the experiment (1,200 sec or more). We also observed data for which the on or the off-rate was comparable to the crosslinking rate, which we call the full-model regime.


    Figure 5. Schematic of potential crosslinking rates and TF-binding dynamics. CLKv2 data usually represent one of three possibilities, which are shown in panels A-C. The left side of the panel represents binding of a factor (gray shaded circles) to its binding site (denoted in green on DNA, symbolized as a thick horizontal line) in a cell (large gray circles). Four cells are shown for each time point. As time progresses from 0 to 1,200 sec, represented by black arrows, factors may become crosslinked to DNA with formaldehyde (yellow circles). The right side of the figure represents the resulting model fit for each data class. A. Data that are binding dynamics limited, where crosslinking occurs quickly (seconds) to capture bound TFs. The data are fit by a model with two distinguishable exponential phases, including an initial steep rise (often manifest as a non-zero y-intercept) followed by a shallow rise, shown in the plot on the right. B. Data that are crosslinking-limited, where crosslinking is slower than TF-binding rates (many minutes, ~600 sec in the panel). A single exponential fits the data, shown on the right with a y-intercept at zero. C. Data that are crosslinking-limited (linear), where the crosslinking rate is much slower than TF-binding rates (crosslinking time scale comparable to or greater than the longest experimental time point, 1,200 sec in the panel), is linear as a function of crosslinking time, shown on the right.

  3. Briefly, data that falls into the TF-limited category shows a quick exponential rise in the first few data points, often appearing as a non-zero y-intercept. The initial rise merges into a second exponential with a shallower slope, eventually flattening out to a saturation level. In this regime, the crosslinking rate (kxl), on-rate (ka), and the off-rate (kd) can be determined; residence time (t1/2), occupancy (θb) and dissociation constant (Kd) can then be calculated. Data that falls into the XL-limited category manifests a single exponential starting from zero that reaches saturation; in this case, crosslinking is slower than TF-binding dynamics. In this regime, ka and kd cannot be independently estimated and only Kd, kxl, and θb are obtained. For both the TF-limited and XL-limited data, we can estimate the saturation of the ChIP signal, which we use to normalize the data such that the late time data saturates around one, in line with the expectation that the crosslinked fraction of sites in a CLK experiment would go to one for long crosslinking times. The linear XL-limited plot is best approximated by a straight line from zero, consistent with the theoretical model for very slow crosslinking. The saturation level cannot be estimated, as the crosslinking rate is much slower than the TF-dynamics with no apparent saturation within the time scale of the experiment. In fact, the crosslinking time scale is greater than that of the last crosslinking time point in the linear XL-limited regime. In this regime, only Kd and θb can be estimated. For XL-limited fits, it is possible in some cases to put an approximate lower bound on ka and kd based on the crosslinking rate, kxl*CFH. Finally, we fit the full model to the data that did not unambiguously fall into any of the above three categories.
  4. Error in the extracted parameters was estimated by refitting the model to 1,000 iterations of simulated data for each locus. The simulated data for each crosslinking time point was sampled from a Gaussian distribution with the mean given by the numerical fit and the variance given by the mean of the squared residual between the experimental data and the fit.
  5. An F-test can be used for selecting between different fits when the data does not unambiguously fit into TF-limited or XL-limited regimes. In most cases considered in Zaidi et al., 2017, the simpler comparison of residuals (or adjusted R-squared) was sufficient to select the better fit. One feature that distinguishes the XL-limited regime from the TF-limited regime is the dependence of the former on the concentration of formaldehyde used for crosslinking. Seeing a dependence of the ChIP signal on formaldehyde concentration by repeating the experiment with a different concentration than used in the original experiment corroborates XL-limited dynamics. In addition to numerical error estimates, we assessed the significance of the fits by estimating p-values. For more details, see Zaidi et al., 2017.
  6.  Model fitting routines were programmed in Mathematica for the TF-limited, XL-limited, and full model fits, and in R for the XL-limited linear regime. Numerical parameter errors were also estimated in Mathematica. Custom fitting and error analysis scripts developed specifically for the CLKv2 analysis reported in Zaidi et al., 2017, are available upon request.

Notes

This protocol was developed with Saccharomyces cerevisiae cells using sterile conditions for cell growth. Sterile technique is not required once cell samples have been obtained for crosslinking.

Recipes

  1. YPD media (1 L)
    10 g Bacto yeast extract
    20 g Bacto peptone
    20 g dextrose (glucose)
    Add all components to ddH2O with final volume of 1 L
    Autoclave and store at room temperature
    Note: For galactose or raffinose media, substitute for dextrose at the same concentration (2%). However, these components should not be autoclaved and 66.7 ml is added from 30% liquid stock after autoclaving (see Recipe 2).
  2. 30% raffinose or galactose (100 ml)
    30 g sugar source
    Bring volume up to 100 ml with ddH2O in a glass bottle
    Microwave bottle for ~30 sec, then place on a stir plate with stir bar on medium speed until sugar is dissolved
    Filter sterilize and store at room temperature
  3. SC media (yeast synthetic media) (1 L)
    1.2 g yeast nitrogen base without amino acids and ammonium sulfate
    5 g ammonium sulfate
    1 g amino acid (dropout) mix
    Add all components to ddH2O with final volume of 950 ml
    Adjust pH to 7 with NaOH, then autoclave. After autoclaving, cool then add sugar to 2% and add back any amino acids that have been left out if using a dropout mix
    Store at room temperature
    Note: Yeast nitrogen base without amino acids and yeast synthetic drop-out medium supplements are both commercially available and can be used to simplify this recipe.
  4. Amino acid mix

    Place all components in a mortar and pestle; grind well then distribute into sterile 50 ml conical tubes
    Store at room temperature
    Note: To make a dropout mix (for use in selective media), leave out the desired components.
  5. 3 M glycine quench solution
    For each 1 L of solution:
    225.21 g glycine
    ~750 ml ddH2O
    Place water in a glass beaker on a heated stir plate. Turn stir bar to medium speed and add glycine. 3 M is close to the max solubility of glycine in water, so low heat with mixing is required for about an hour to completely dissolve the glycine. Once glycine is completely dissolved, let it cool and adjust pH to 5 using concentrated HCl. Bring the volume to 1 L with ddH2O. Keep glycine at room temperature once made; cooling it will cause the glycine to precipitate out of solution. If this happens, place glycine back on the heated stir plate with a low heat and stirring until dissolved again
  6. Benoit’s buffer
    200 mM Tris-HCl (pH 8.0)
    400 mM (NH4)2SO4
    10 mM MgCl2
    1 mM EDTA
    10% glycerol
    7 mM β-mercaptoethanol
    Protease inhibitors: Roche Complete Protease Inhibitor Cocktail Tablet OR 1.0 mM phenylmethylsulfonyl fluoride*
    2.0 mM benzamidine*
    2.0 μM pepstatin*
    0.6 μM leupeptin*
    2.0 μg of chymostatin*
    *Note: Per ml of buffer.
    Add Tris-glycerol components to ddH2O with desired final volume (usually make 500 ml at a time)
    Adjust pH to 8 and filter sterilize. Store at 4 °C
    Take out an aliquot of buffer for experiments. Add β-mercaptoethanol and protease inhibitors. Once β-mercaptoethanol is added, that aliquot can only be used for the day and must be remade fresh the next time it’s needed. Store the aliquot on ice/at 4 °C while using
    Note: If the protease inhibitor tablet is used, 25 ml buffer needs a ½ tablet and 50 ml takes a whole tablet.
  7. Laemmli buffer (4x sample buffer)
    0.2 M Tris-HCl pH 6.8
    40% glycerol
    277 mM SDS
    200 mM DTT
    3 mM bromophenol blue
    Add all components to ddH2O; aliquot into 1.5 ml microcentrifuge tubes and store at -20 °C
  8. Coomassie stain
    1.2 g Coomassie blue
    300 ml methanol
    60 ml acetic acid
    240 ml H2O
    Add all components; store at room temperature
  9. TBS
    50 mM Tris-HCl pH 7.5
    300 mM NaCl

    Add all components to ddH2O and bring to the desired volume (usually make 20 L at a time in a carboy)
    Store at room temperature
  10. TBST
    50 mM Tris-HCl pH 7.5
    300 mM NaCl
    0.05% Tween 20
    Add all components to ddH2O and bring to the desired volume (usually make 20 L at a time in a carboy)
    Store at room temperature
  11. 140 mM ChIP lysis buffer
    50 mM HEPES pH 7.5
    140 mM NaCl
    1% Triton X-100
    0.1% sodium deoxycholate
    Protease inhibitors: Roche Complete Protease Inhibitor Cocktail Tablet OR 1.0 mM phenylmethylsulfonyl fluoride*
    2.0 mM benzamidine*
    2.0 μM pepstatin*
    0.6 μM leupeptin*
    2.0 μg of chymostatin*
    *Note: Per ml of buffer.

    Add all components except protease inhibitors to ddH2O with desired final volume (usually 500 ml at a time)
    Filter sterilize the solution and store at room temperature
    Take out an aliquot of buffer for experiments. Add protease inhibitors. Aliquot can be used the same day that protease inhibitors are added but a new aliquot must be remade fresh the next time it’s needed. Store the aliquot on ice/at 4 °C while using
    Note: If the protease inhibitor tablet is used, 25 ml buffer needs a ½ tablet and 50 ml takes a whole tablet.
  12. 500 mM ChIP lysis buffer
    50 mM HEPES pH 7.5
    500 mM NaCl
    1% Triton X-100
    0.1% sodium deoxycholate
    Add all components to ddH2O with desired final volume (usually make 500 ml at a time)
    Filter sterilize. Store at room temperature
  13. LiCl wash buffer
    10 mM Tris pH 8.0
    250 mM LiCl
    0.5% NP-40
    0.5% sodium deoxycholate
    1 mM EDTA

    Add all components to ddH2O with desired final volume (usually make 500 ml at a time)
    Filter sterilize. Store at room temperature
  14. 1x TE
    10 mM Tris-HCl pH 8.0
    1 mM EDTA
    Add all components to ddH2O with desired final volume (usually make 500 ml at a time)
    Filter sterilize. Store at room temperature
  15. ChIP elution buffer
    50 mM Tris-HCl pH 8.0
    1% SDS
    10 mM EDTA
    Add all components to ddH2O with desired final volume (usually make 10 ml at a time)
    Store at room temperature. Solution should be remade each time a new experiment is done
  16. DEPC H2O
    1 ml diethyl pyrocarbonate
    1,000 ml H2O
    Mix DEPC with H2O in a screw cap bottle; incubate at room temperature in a hood for ~2 h with occasional stirring
    Aliquot into 100 ml bottles, then autoclave
    Store at room temperature for up to 12 months
  17. 30%/0.8% Bis-acrylamide solution
    For 500 ml:
    150 g acrylamide
    4 g Bis-acrylamide
    Add all components to ddH2O with desired final volume (usually make 500 ml at a time)
    Filter sterilize
    Store at 4 °C in a foil-wrapped bottle
  18. SDS-PAGE running buffer
    192 mM glycine
    25 mM Tris
    0.1% SDS
    Add all components to ddH2O and bring to the desired volume (usually make 20 L at a time in a carboy)
    Store at room temperature
  19. Transfer buffer
    25 mM Tris
    192 mM glycine
    15% methanol
    Add all components to ddH2O and bring to the desired volume (usually make 20 L at a time in a carboy)
    Store at room temperature

Acknowledgments

This work was supported by grants from the National Institutes of Health (R01 GM55763 to D.T.A, R21 GM110380 to S.B and D.T.A, and NCI Cancer Training Grant T32 CA009109-38 to E.A.H.) and a Wagner Fellowship to E.A.H. The protocols in this manuscript have been adapted from those reported in Poorey et al., 2013, Viswanathan et al., 2014 and Zaidi et al., 2017. The authors declare that they have no conflicts of interest or competing interests with the contents of this article.

References

  1. Amberg, D. C., Burke, D., and Strathern, J. N. (2005). Methods in Yeast Genetics: A Cold Spring Harbor laboratory course manual. Cold Spring Harbor Laboratory Press.
  2. Encode Consortium, E. P. (2012). An integrated encyclopedia of DNA elements in the human genome. Nature 489(7414): 57-74.
  3. Conaway, R. C. and Conaway, J. W. (1993). General initiation factors for RNA polymerase II. Annu Rev Biochem 62: 161-190.
  4. Coulon, A., Chow, C. C., Singer, R. H. and Larson, D. R. (2013). Eukaryotic transcriptional dynamics: from single molecules to cell populations. Nat Rev Genet 14(8): 572-584.
  5. Cramer, P. (2014). A tale of chromatin and transcription in 100 structures. Cell 159(5): 985-994.
  6. Dowen, J. M., Fan, Z. P., Hnisz, D., Ren, G., Abraham, B. J., Zhang, L. N., Weintraub, A. S., Schujiers, J., Lee, T. I., Zhao, K. and Young, R. A. (2014). Control of cell identity genes occurs in insulated neighborhoods in mammalian chromosomes. Cell 159(2): 374-387.
  7. Grimaldi, Y., Ferrari, P. and Strubin, M. (2014). Independent RNA polymerase II preinitiation complex dynamics and nucleosome turnover at promoter sites in vivo. Genome Res 24(1): 117-124.
  8. Hager, G. L., McNally, J. G. and Misteli, T. (2009). Transcription dynamics. Mol Cell 35(6): 741-753.
  9. Haruki, H., Nishikawa, J. and Laemmli, U. K. (2008). The anchor-away technique: Rapid, conditional establishment of yeast mutant phenotypes. Mol Cell 31(6): 925-932.
  10. He, Y., Fang, J., Taatjes, D. J. and Nogales, E. (2013). Structural visualization of key steps in human transcription initiation. Nature 495(7442): 481-486.
  11. Horn, A. E., Kugel, J. F. and Goodrich, J. A. (2016). Single molecule microscopy reveals mechanistic insight into RNA polymerase II preinitiation complex assembly and transcriptional activity. Nucleic Acids Res 44(15): 7132-7143.
  12. Kim, T. H., Barrera, L. O., Zheng, M., Qu, C., Singer, M. A., Richmond, T. A., Wu, Y., Green, R. D. and Ren, B. (2005). A high-resolution map of active promoters in the human genome. Nature 436(7052): 876-880.
  13. Larson, D. R., Singer, R. H. and Zenklusen, D. (2009). A single molecule view of gene expression. Trends Cell Biol 19(11): 630-637.
  14. Lickwar, C. R., Mueller, F. and Lieb, J. D. (2013). Genome-wide measurement of protein-DNA binding dynamics using competition ChIP. Nat Protoc 8(7): 1337-1353.
  15. Luse, D. S. (2014). The RNA polymerase II preinitiation complex. Through what pathway is the complex assembled? Transcription 5(1): e27050.
  16. Morisaki, T., Muller, W. G., Golob, N., Mazza, D. and McNally, J. G. (2014). Single-molecule analysis of transcription factor binding at transcription sites in live cells. Nat Commun 5: 4456.
  17. Mueller, F., Stasevich, T. J., Mazza, D. and McNally, J. G. (2013). Quantifying transcription factor kinetics: at work or at play? Crit Rev Biochem Mol Biol 48(5): 492-514.
  18. Poorey, K., Viswanathan, R., Carver, M. N., Karpova, T. S., Cirimotich, S. M., McNally, J. G., Bekiranov, S. and Auble, D. T. (2013). Measuring chromatin interaction dynamics on the second time scale at single-copy genes. Science 342(6156): 369-372.
  19. Rhee, H. S. and Pugh, B. F. (2012). Genome-wide structure and organization of eukaryotic pre-initiation complexes. Nature 483(7389): 295-301.
  20. Roeder, R. G. (1996). The role of general initiation factors in transcription by RNA polymerase II. Trends Biochem Sci 21(9): 327-335.
  21. van Werven, F. J., van Teeffelen, H. A., Holstege, F. C. and Timmers, H. T. (2009). Distinct promoter dynamics of the basal transcription factor TBP across the yeast genome. Nat Struct Mol Biol 16(10): 1043-1048.
  22. Viswanathan, R., Hoffman, E. A., Shetty, S. J., Bekiranov, S. and Auble, D. T. (2014). Analysis of chromatin binding dynamics using the crosslinking kinetics (CLK) method. Methods 70(2-3): 97-107.
  23. Zaidi, H., Hoffman, E. A., Shetty, S. J., Bekiranov, S. and Auble, D. T. (2017). Second-generation method for analysis of chromatin binding with formaldehyde-cross-linking kinetics. J Biol Chem 292(47): 19338-19355.
  24. Zawel, L. and Reinberg, D. (1992). Advances in RNA polymerase II transcription. Curr Opin Cell Biol 4(3): 488-495.

简介

甲醛交联广泛用于与染色质免疫沉淀(ChIP)相结合来测量沿着DNA的相对位置以及转录因子(TF)-DNA相互作用的体内相对水平。但是,通常所做的测量不能提供关于这些交互的动态属性的明确信息。我们已经开发了一种方法来评估来自时间依赖性甲醛交联数据的结合动力学参数,称为交联动力学(CLK)分析。酵母细胞的培养物与甲醛交联不同的时间段,在特定位点产生相对的ChIP信号。我们使用质量作用CLK模型来拟合数据,以提取TF-染色质相互作用的动力学参数,包括开关速率和交联速率。从停车费和停车费中我们可以获得停车和停车时间。以下方案是该方法的第二次迭代,CLKv2,更新了改进的交联和淬火条件,更多关于交联速率的信息以及对观察到的动力学模型建模的系统程序。已应用CLKv2分析来研究TATA结合蛋白(TBP)和其他TF的选定子集的结合行为。该协议使用酵母细胞开发,但也可适用于来自其他生物体的细胞。

【背景】转录起始是一个复杂的过程,涉及染色质化启动子上数十种蛋白的协作和协调相互作用(Kim等人,2005; Encode Consortium,2012; Rhee等人, ,2012; Dowen等人,2014年)。许多研究已经研究了体外核心转录机器的组装和调控(Zawel和Reinberg,1992; Conaway和Conaway,1993; Roeder,1996; Hager;等人) ,2009; He等人,2013; Cramer,2014; Luse,2014; Horn&lt; em&gt;等人,2016),但研究随机过程更具挑战性这些过程的性质在体内。用于测量体内染色质结合动力学的两种常用方法是:显微镜和基于ChIP的技术(Coulon等人,2013; Mueller等人, ,2013)。显微技术,如光漂白后的荧光恢复(FRAP)或单分子追踪(SMT),具有高时间分辨率,并提供了对染色质结合动力学的基本认识,包括通过追踪单个分子获得的结果(Larson等人< 2009年; Mueller等人,2013年; Morisaki等人,2014年)。然而,这些方法可能受到光漂白等光物理效应的限制,此外,在绝大多数情况下,不可能确定所测量的相互作用发生的特定单拷贝基因座的身份(Mueller et al。 ,2013)。另外,基于ChIP的方法提供精确的染色质位置信息。在竞争性ChIP(CC)中,诱导目的TF的差异标签同种型的表达,并且随时间监测TF的组成型和诱导型的相对水平,通过测量位点处的TF转换产生结合动力学信息(van Werven等人,2009; Lickwar等人,,2013)。随着CC数据建模的进步,停留时间缩短至1.3分钟(Zaidi et al。,2017)。还使用Anchor Away技术(Haruki等人,2008; Grimaldi等人,2014),通过从细胞核有条件地消耗TF来进行相对动态测量,尽管该过程的具体数学模型尚未报道。 CLK方法与这些其他基于ChIP的方法是互补的,利用甲醛交联的时间依赖性来推导结合动力学参数以及分数占用(Poorey等人,2013)。 CLK测定的第一次迭代使用“标准”交联和淬灭条件(分别为1%甲醛和250mM甘氨酸)。最近的其他工作已经产生了增加交联速率并改善交联反应猝灭的实验条件(Zaidi等人,2017)。这些新的条件导致了更稳健的方法以及更可靠和可靠地建模和分析交联动力学数据的能力。

关键字:染色质免疫沉淀(ChIP), 蛋白质动态, 蛋白质交联, 转录因子, 核酸化学, 染色质结构, 甲醛化学

材料和试剂

  1. 移液器吸头
  2. 可重复使用的棉塞顶部血清(玻璃)移液器(也可以使用一次性塑料移液器)
    10毫升移液器(Fisher Scientific,目录号:13-675M)
    25毫升移液器(Fisher Scientific,目录号:13-675N)
  3. 移液器消毒盒(Fisher Scientific,目录号:03-465)
  4. Nalgene PPCO带密封封闭的离心瓶(Thermo Fisher Scientific,Thermo Scientific TM,产品目录号:3141-0500)
  5. 50毫升30×115毫米的锥形管(Corning,Falcon ,产品目录号:352070)
  6. 2.0 ml微量离心机锥形螺旋盖管(FastPrep管,Fisher Scientific,目录号:02-681-344)
  7. 带O型圈的微量离心管螺旋盖(用于FastPrep管,Fisher Scientific,产品目录号:02-681-366)
  8. 酸洗425-600μm玻璃珠(Sigma-Aldrich,目录号:G8772)
  9. 18号针头(PrecisionGlide,BD,目录号:305195)

  10. 一次性培养管,玻璃13 x 100毫米(Fisher Scientific,目录号:14-961-27)

  11. 1.5 ml微量离心管(Fisher Scientific,目录号:05-408-129)
  12. Whatman滤纸(GE Healthcare,目录号:1003-917)
  13. 放射自显影胶片(Genesee Scientific,目录号:30-101)

  14. 1.5-1.7 ml带锁定盖的聚丙烯刻度管(Fisher Scientific,产品目录号:02-681-285)
  15. Hard-Shell High-Profile 96孔半裙边PCR板(Bio-Rad Laboratories,目录号:HSS9641)
  16. 管状卷材(在开发前用塑料密封隔膜; Ampac,目录号:TRS-95125-3)
  17. Immobilon-P膜(PVDF; Merck,目录号:IPVH00010)
  18. Microseal'B'密封件(Bio-Rad Laboratories,目录号:MSB1001)
  19. Stericup无菌真空过滤装置,500毫升(Merck,目录号:SCGPU05RE)
  20. 对于感兴趣的每种TF(酵母菌株,野生型(WT)和过表达(OE))(应用列表参见Poorey等人,2013和Zaidi等人,2017) )
  21. 用于TF过表达和WT菌株的载体对照的质粒(参见Poorey等人,2013和Zaidi等人,2017,质粒列表)
  22. 基因座特异性引物(Invitrogen)
  23. In-Fusion HD克隆试剂盒(Takara Bio,Clontech,目录号:639649)
  24. 冰桶
  25. 37%甲醛(Fisher Scientific,目录号:F79)
  26. 蛋白质分析染料试剂浓缩物(Bio-Rad Laboratories,目录号:500-0006)
  27. n蛋白A Sepharose 4 Fast Flow珠(GE Healthcare,目录号:17-5280-01)
    注意:可以使用Ab-缀合的珠代替,如IgG Sepharose 6 Fast Flow(GE Healthcare,目录号:17-0969-01)和Sepharose 6 Fast Flow珠(GE Healthcare,目录号:17-0159 -99)。
  28. QIAQuick PCR纯化试剂盒(QIAGEN,目录号:28106)
  29. iQ SYBR Green Supermix,500 x 50μlrxns(Bio-Rad Laboratories,目录号:1708882)
  30. 即时脱脂奶粉(康乃馨)
  31. 抗体(即,α-TBP,单克隆抗体,Abcam,目录号:ab61411)
  32. 次级ECL-缀合的抗体(我们使用Amersham ECL小鼠或兔IgG HRP-连接的整个Ab,GE Healthcare,目录号:NXA931或NA934V)
  33. Amersham ECL Prime Western印迹检测试剂(GE Healthcare,目录号:RPN2232)
  34. 酵母提取物(BD,Bacto TM,目录号:212750)
  35. 细菌蛋白胨(BD,Bacto TM,产品目录号:211677)
  36. 糖源,即 :
    D - (+) - 葡萄糖(Sigma-Aldrich,目录号:G7021)
    D - (+) - 半乳糖(Sigma-Aldrich,目录号:G5388)
    D - (+) - 棉子糖(MP Biomedicals,目录号:02102797)
  37. 不含氨基酸和硫酸铵的Difco酵母氮碱(BD,Difco TM,目录号:233520)
    注意:不含氨基酸的酵母氮碱(含或不含糖源)可用作替代品,并且不需要添加硫酸铵(即,Sigma-Aldrich,目录号:Y0626)。
  38. 氨基酸:
    1. 腺嘌呤半硫酸盐二水合物(MP Biomedicals,目录号:02100195)
    2. 组氨酸盐酸盐一水合物(Acros Organics,目录号:411731000)
    3. L-赖氨酸(Fisher Scientific,目录号:BP386)
    4. L-酪氨酸(Acros Organics,目录号:140641000)
    5. L-色氨酸(Fisher Scientific,目录号:BP395)
    6. 尿嘧啶(Affymetrix,目录号:23020)
    7. L-亮氨酸(Acros Organics,目录号:125121000)
    8. L-蛋氨酸(Fisher Scientific,目录号:BP388)
    9. L-精氨酸盐酸盐(Fisher Scientific,目录号:BP372)
    10. L-丝氨酸(Fisher Scientific,目录号:BP393)
    11. 缬氨酸(Fisher Scientific,目录号:BP397)
    12. L-苏氨酸(MP Biomedicals,目录号:02103053)
    13. L-异亮氨酸(Fisher Scientific,目录号:BP384)
    14. L-苯丙氨酸(Fisher Scientific,目录号:BP391)
    15. L-半胱氨酸盐酸盐一水合物(Fisher Scientific,目录号:BP376)
    16. L-天冬氨酸(Acros Organics,目录号:105041000)
    17. L-脯氨酸(Fisher Scientific,目录号:BP392)
    注意:可以使用市售的酵母合成辍学培养基补充剂(西格玛奥德里奇公司)代替自制的辍学混合物。
  39. 细菌琼脂(BD,目录号:214010)
  40. 甘氨酸,2公斤(Bio-Rad Laboratories,目录号:1610724)
  41. 盐酸(Fisher Scientific,目录号:A144SI-212)
  42. Tris碱(Sigma-Aldrich,目录号:T1503)
  43. 硫酸铵(Sigma-Aldrich,目录号:A4418)
  44. 氯化镁六水合物(Sigma-Aldrich,目录号:M9272)
  45. EDTA(Fisher Scientific,目录号:BP120)
  46. 甘油(Fisher Scientific,目录号:BP229)
  47. β-巯基乙醇(Sigma-Aldrich,目录号:M3148)
  48. 蛋白酶抑制剂
    完全蛋白酶抑制剂片剂不含EDTA(Roche Diagnostics,目录号:04693132001)
    OR:
    苯基甲基磺酰氟(Sigma-Aldrich,目录号:P7626)
    苯甲脒盐酸盐水合物(Acros Organics,目录号:105241000)
    胃蛋白酶抑制剂A(Sigma-Aldrich,目录号:P4265)
    亮肽素半硫酸盐(Sigma-Aldrich,目录号:L8511)
    Chymostatin(Sigma-Aldrich,目录号:C7268)
  49. SDS(Sigma-Aldrich,目录号:L3771)
  50. DTT(Roche Diagnostics,产品目录号:03117006001)
  51. 溴酚蓝(Bio-Rad Laboratories,目录号:1610404)
  52. 考马斯蓝
  53. 甲醇(Fisher Scientific,目录号:A452)
  54. 醋酸
  55. 氯化钠(Fisher Scientific,目录号:S640)
  56. 吐温20(Sigma-Aldrich,目录号:P5927)
    注意:此产品已停产。
  57. HEPES(Fisher Scientific,目录号:BP310)
  58. Triton X-100(AMRESCO,目录号:0694)
  59. 脱氧胆酸钠(Sigma-Aldrich,目录号:D6750)
  60. 氯化锂(Sigma-Aldrich,目录号:L4408)
  61. Nonidet P40(光谱,目录号:N1156)
    注意:此产品已停产。
  62. 焦碳酸二乙酯(DEPC),97%纯度(Acros Organics,目录号:170250250)
  63. 丙烯酰胺(Bio-Rad Laboratories,目录号:1610101)
  64. 双丙烯酰胺(Fisher Scientific,目录号:BP171)
  65. 过硫酸铵(APS,Bio-Rad Laboratories,目录号:1610700)
  66. TEMED(Thermo Fisher Scientific,Thermo Scientific TM,目录号:17919)
  67. YPD媒体(见食谱)
  68. 30%棉子糖或半乳糖(见食谱)
  69. SC培养基(酵母合成培养基,见食谱)
  70. 氨基酸混合(见食谱)
  71. 3M甘氨酸淬火溶液(见食谱)
  72. Benoit的缓冲区(见食谱)
  73. Laemmli缓冲液(4倍样品缓冲液,见食谱)
  74. 考马斯染色(见食谱)
  75. TBS(见食谱)
  76. TBST(见食谱)
  77. 140 mM ChIP裂解缓冲液(见食谱)
  78. 500 mM ChIP裂解缓冲液(见食谱)
  79. LiCl洗涤缓冲液(见食谱)
  80. 1x TE(见食谱)
  81. ChIP洗脱缓冲液(见食谱)
  82. DEPC H 2 O(见食谱)
  83. 30%/ 0.8%双丙烯酰胺溶液(见食谱)
  84. SDS-PAGE运行缓冲液(见食谱)
  85. 传输缓冲区(请参阅食谱)

设备

  1. 玻璃培养管和瓶盖,18x150mm( ie ,具有平端的一次性硼硅酸盐玻璃管,Fisher Scientific,目录号:14-961-32和Diamond培养管盖,18mm,Globe Scientific ,目录号:118154)
  2. 烧瓶(1L,250ml;即Emre,Pyrex窄颈重型玻璃锥形瓶,Corning,PYREX®,产品目录号:4980-1L(1L)和4980-250(250毫升))
  3. 移液器(P2,P2,P200,P1000)
  4. Pipet-Aid(Drummond Scientific,目录号:4-000-100)
  5. 培养箱,30℃(即,Isotemp CO 2培养箱,Fisher Scientific,Fisherbrand,目录号:11-676-603)。
  6. 磁力搅拌棒(Fisher Scientific,目录号:14-513-52)
  7. 搅拌热板(Fisher Scientific,目录号:11-510-49SH或SP88850200)
  8. 定时器
  9. Sorvall RC 5B离心机(GMI,型号:Sorvall RC-5B)
  10. SLA-3000转子(Thermo Fisher Scientific,Thermo Scientific TM,型号:SLA-3000,目录号:07149)
  11. Eppendorf 5810 R台式离心机带板桶和15 ml / 50 ml适配器(Eppendorf,型号:5810 R,目录号:5811000827)
  12. MP FastPrep-24打浆机(MP生物医学公司,型号:FastPrep® -24 Classic,目录号:116004500)
  13. 本生燃烧器
  14. 带有1/8“微尖端探头的Branson Sonifier 250(Fisher Scientific,目录号:22-309782和22-309796)
  15. Eppendorf 5415C或D台式离心机(Eppendorf,类似产品目录号:022620304)
  16. 振荡器,30℃(即Eppendorf,New Brunswick Scientific,型号:Excella E25,目录号:M1353-0002)。
  17. 4°C冰箱
  18. -20°C冷冻机
  19. -80°C冷冻机
  20. 高压灭菌器
  21. AutoMixer磁力搅拌板(Fisher Scientific,目录号:14-505-21)
  22. Ultrospec 2100pro UV /可见光分光光度计(Biochrom,型号:ULTROSPEC 2100,目录号:80-2112-21)
  23. PowerPac Basic电源(Bio-Rad Laboratories,产品目录号:1645050)
  24. 带铸造支架和框架的Mini-PROTEAN tetra cell,梳子,短板和隔板(Bio-Rad Laboratories,目录号:1658006FC)
  25. 带有保持盒,泡沫垫和蓝色冷却装置的迷你转印电泳转移细胞(Bio-Rad Laboratories,目录号:1703930)
  26. 振动筛(可靠的科学,型号:55S)
  27. 放射自显影盒(Fisher Biotech,目录号:FBXC 810)
  28. Labquake摇床旋转式烤箱,32 x 10至19 mm,带有剪条(Thermo Fisher Scientific,Thermo Scientific TM,产品目录号:415110Q)
  29. 水浴(55°C和65°C)
  30. MyiQ实时仪器(Bio-Rad Laboratories,目录号:170-9770)

软件

  1. ImageJ(NIH)
  2. Mathematica
  3. R或R-Studio

程序

该版本的交联动力学(CLKv2)分析是修改后的ChIP程序,其通过拟合从用甲醛处理的细胞获得的不同时间段的ChIP数据来产生与特定基因座结合的转录因子的动力学测量。在收集样品进行动力学分析之前,应完成对照实验以优化实验条件;图1中的示意图概述了此过程的一般工作流程。在该方案中,首先描述所需的酵母菌株(程序A),然后描述CLK数据收集的详细程序(程序B)。 CLK数据收集条件应经过验证,可能需要按照图1所述进行优化。用于优化程序的控制实验依赖于基本的CLK方法,因此后面(过程D和E)描述了最小化冗余;程序C详细说明了数据建模所需的过表达因子的量化。


图1. CLKv2工作流程在收集CLK数据并拟合CLK模型之前,进行对照实验以优化测定条件。

  1. 酵母菌株的构建
    1. 对于CLKv2测定,两种菌株用于分析感兴趣的TF:野生型(WT)菌株和过表达(OE)菌株。过度表达菌株与野生型菌株不同,而不是驱动TF水平,其在WT水平上略高(〜3-5倍)。平行的WT和OE菌株的动力学分析(下文所述)通过揭示质量作用对ChIP信号随时间增加的贡献来高度约束数据的拟合。 OE菌株可以通过向质粒中引入额外的TF基因拷贝或通过在天然启动子或合适的异源启动子控制下整合到基因组中来工程化。如果TF作为具有多于一种类型的亚基的稳定的生物化学实体发挥功能,则OE菌株应该被设计为驱动每个亚基的平衡表达,例如用于分析TFIIE(Zaidi等人。,2017)。 OE菌株和质粒构建的详细描述参见Poorey等人,2013,Zaidi等人,2017。
    2. 为了产生用于使用OE质粒的动力学分析的菌株,用感兴趣的菌株背景中的OE质粒构建体或携带相同选择标记的空载体转化WT酿酒酵母细胞。在适当的琼脂平板上选择转化体并对单菌落重新划线细胞。我们通常在4°C下使用保存在平板上的菌株约一个月;使用标准酵母甘油储备方法(Amberg等人,2005)通过在-80℃下储存将WT和OE菌株归档。

  2. CLK数据收集
    如上所述,分析特定TF所需的实验条件应该被验证,并且可能需要如程序D和E中所述进行优化。本节描述了我们优化和最近描述的基本CLKv2程序(Zaidi等人,2017)。
    细胞生长和细胞样本收集
    1. 用所需的WT或OE酵母菌株的菌落接种重复的5ml YPD原代培养物;在30°C摇动培养过夜。
      注意:如果在选择性培养基中启动原代培养物,细胞可能需要生长约24小时而不是约16小时,以获得足够的材料以在下一步中启动更大的培养物。
    2. 第二天,将每个原代培养物稀释于450ml所需的选择性培养基(具有合适的氨基酸缺失的SC培养基)中并在30℃下振荡孵育直至OD 600达到约0.8( 〜1.3×10 7个细胞)。使用SLA-3000转子和Sorvall RC 5B离心机,在Nalgene离心瓶中,在4,230×g和4℃下旋转7分钟沉淀细胞。
      注意:Poorey等,2013年概述了铜诱导菌株的生长条件。如果感兴趣的TF受GAL诱导型启动子的控制,细胞应该在含有2%棉子糖的选择性培养基中生长直至达到OD 600 为〜0.8;然后沉淀并重悬于YEP + 2%半乳糖中的细胞,并在30℃下摇动孵育1小时。
    3. 用450ml YPD(或所需培养基)重悬每个细胞沉淀;在30℃振荡孵育约1小时,直到OD 600为约1.0(〜1.9×10 7细胞)。
    4. 在细胞生长的同时,准备甘氨酸淬灭溶液。对于每个时间点,需要440ml的3M甘氨酸pH 5。因此,需要4L甘氨酸来猝灭一个时间过程的八个时间点样品;有关说明,请参阅食谱。将440毫升3M甘氨酸溶液倒入八个500毫升Nalgene离心瓶中。
    5. 如步骤2中的沉淀细胞,然后将细胞重悬于90ml YPD(或期望的培养基)中。这一步产生的细胞悬液浓缩五倍于初始培养。
      注意:以这种方式浓缩细胞,使得在随后的步骤中,通过稀释细胞悬浮液以及通过添加甘氨酸可以使甲醛交联的淬灭更加有效。
    6. 添加10毫升玻璃或塑料移液器到移液器 - 援助;这将用于在第一时间点除去细胞悬液的等分试样。将培养瓶中的90ml置于装有搅拌棒的搅拌盘上,中速加入14ml 37%甲醛至终浓度为5%;立即启动计时器。快速添加甲醛的一种方法是将预先测量的14毫升容纳在15毫升一次性锥形管中的等分试样倒入搅拌的细胞悬液中。如果使用移液器添加甲醛,则应事先安装第二支移液管,以预先取出第一个时间点的等分试样。
    7. 在5秒,20秒,60秒(1分钟),120秒(2分钟),300秒(5分钟),600秒(10分钟),900秒(15分钟)和1200秒(20分钟)用移液助剂和10ml玻璃吸液管将10ml等分的交联培养物快速加入到含有制备的甘氨酸溶液的瓶子中,在添加细胞悬液后立即盖上每个瓶子并倒转数次以混合。
    8. 使用SLA-3000转子和Sorvall RC 5B离心机,在4,230×g下,在4℃下旋转7分钟来沉淀细胞。
    9. 用50ml 4℃TBS + 300mM甘氨酸pH5重悬细胞沉淀并转移到50ml锥形管中;在Eppendorf 5810 R台式离心机中在3,220×g和4℃下旋转5分钟。弃上清。
    10. 用50ml 4℃TBS洗涤每个细胞沉淀;颗粒细胞如前一步一样。
    11. 在4°C下将每个细胞沉淀重悬于1 ml TBS中,并将每个样品转移到FastPrep管中。
    12. 通过在Eppendorf 5810 R台式离心机中在4℃下旋转2分钟而获得16,000×g的沉淀细胞。丢弃上清。
      注意:样品可以在-80°C储存在这一步。

    分离染色质样本
    1. 用加入蛋白酶抑制剂的600μl140mM ChIP裂解缓冲液重悬每个细胞沉淀。将酸洗过的玻璃珠加到每根管子上,直到液体和螺帽的高度紧紧地高于水平。
    2. 使用保存在冷室中的MP FastPrep-24 Bead Beater,以4.0米/秒的速度进行7次循环的Bead-beat样品,45秒钟和1分钟的循环间隔。
    3. 用加热的(使用本生灯火焰)18号针头在每个FastPrep管的底部打一个孔,然后将每个管放入13 x 100 mm的玻璃管中。
      在4°C的Eppendorf 5810 R台式离心机中以3,220×g的速度旋转3分钟以将液体转移到玻璃管的底部。
    4. 简单地涡旋含有流过物质的玻璃管并将每个细胞悬液转移到Eppendorf管中;将管放在冰上。
    5. 超声波样品进行7个循环,5个脉冲/周期,30%输出和90%占空比,使用带有微尖端探头的Branson Sonifier 250。循环之间将管置于干冰上。
      注意:通过对7个循环中的每一个循环超声处理一个样品,样品将留在干冰上足够长的时间以保持它们冷却但不冻结。如果许多样品一次超声处理,可能需要在湿冰和干冰之间交替样品以防止颗粒冻结。超声处理的DNA的预期片段大小为〜100-600bp。实验第一次进行时,超声处理的DNA的大小应通过在溴化乙锭的1%琼脂糖凝胶上运行样品,然后用适当系统进行成像来检查。
    6. 在Eppendorf 5415C / D台式离心机中,在4℃下在16,000×gg下离心5分钟。
    7. 将每个样品的上清液移至新的1.5ml微量离心管中;在Eppendorf 5415C / D台式离心机中于4℃在16,000×gg离心20分钟。
    8. 将每种上清液转移至新的1.5ml微量离心管中,并使用Bradford试剂制造商推荐的1μg/μl牛血清白蛋白标准和Bradford蛋白质染料通过Bradford测定法定量蛋白质。
      注意:样品可以在-80°C储存在这一步。

    染色质免疫沉淀(ChIP)
    1. 在带有锁定盖的微量离心管中设置ChIP反应。对于每个时间点样品,需要三个试管:免疫沉淀(IP),模拟和总量(输入)。如果使用抗体偶联珠粒(a),则一步完成IP,或如果染色质提取物首先与抗体孵育,然后与蛋白A珠粒孵育(b),则两步完成。
      1. 对于抗体缀合的珠子:首先准备用于IP和模拟样品的珠子。 IP珠具有与它们缀合的所需抗体;模拟珠子是用于IP的相同类型的未缀合珠子,或者,如果标签用于感兴趣的TF,则来自其他相同的未加标签的菌株的染色质可以与用于IP的相同抗体缀合的珠子一起使用。通过加入1.5 ml微量离心管分装处理所有样品所需的总量(45μl/样品)。用1ml 140mM ChIP裂解缓冲液洗珠3次,在洗涤之间快速旋转以回收珠粒(〜6,000×g数秒)。在含有蛋白酶抑制剂的等体积的140mM ChIP裂解缓冲液中重悬珠粒,制成50%浆液。将40μl的珠浆分装到一个管中,用于每个IP和模拟反应。在冰上的每个试管中加入1mg样品染色质,并添加140mM含蛋白酶抑制剂的ChIP裂解缓冲液以使总体积达到500μl。对于输入样品,将0.1mg样品染色质等分到每个管中。将输入样品保存在-80°C;将IP和模拟反应管置于旋转振荡器如Labquake旋转式振荡器上4°C过夜。
      2. 对于抗体孵育然后进行珠孵育:对于IP样品,将1mg样品染色质蛋白等分到每个管中,并添加抗体用于感兴趣的TF,然后加入140mM含蛋白酶抑制剂的ChIP裂解缓冲液以使总体积达到500μl。对于模拟反应,将1mg样品染色质等分至含有蛋白酶抑制剂的140mM ChIP裂解缓冲液以使体积达到500μl(不添加抗体)。对于输入样品,将0.1mg样品染色质等分到每个管中。将输入样品在-80℃保存过夜;
        将IP和模拟管放置在Labquake旋转式振动筛上4°C过夜。
        注:加入IP的抗体量取决于其浓度,可能需要在处理CLK样品前进行优化。通常情况下,约5μg抗体用于ChIP和1 mg样品染色质蛋白。
    2. 如果使用抗体结合珠,请跳到下一步。以与步骤1a中制备的珠相同的方式(染色质免疫沉淀(ChIP))制备50%的nSepharose蛋白A珠的浆液。将40μl珠浆液分装到每个IP或模拟样品的新管中。在台式微型离心机中快速旋转染色质样品,并将每种上清液与nSepharose蛋白A珠粒浆液一起转移到新管中。按照步骤1,在4°C孵育染色质珠样品2小时并混合。
    3. 快速旋转管。用1ml下列缓冲液洗涤每个珠子沉淀物两次;在洗涤之间的微型离心机中快速旋转:
      1. 140 mM ChIP裂解缓冲液
      2. 500 mM ChIP裂解缓冲液
      3. LiCl清洗缓冲液
      4. 1x TE pH 8
    4. 最后一次清洗后去除多余的液体,并向每个管中加入75μlChIP洗脱缓冲液。轻敲管子数次,混合珠子和洗脱缓冲液。
      在65°C孵育样品10分钟

    5. 在微型离心机中快速旋转离心管并将上清液转移到新的锁定盖管中。
    6. 将另外75μl等分的ChIP洗脱缓冲液添加至每个珠粒。如上所述通过轻弹管来混合。
      在65°C下再次孵育样品10分钟
    7. 在微离心管中快速旋转管并将上清液合并在一个管中。
      在65°C过夜孵育样本
    8. 在板凳上解冻输入样本一两分钟。加入150μlChIP洗脱缓冲液,通过短暂涡旋混合,并在65℃与IP和模拟样品一起温育过夜。
    9. 第二天,按照制造商提供的说明,使用QiaQuick PCR纯化试剂盒清理DNA样品。简而言之,首先,向每个管中加入750μl缓冲液PB;混合并将450μl样品加到柱上,然后在室温下使用Eppendorf 5415D台式离心机在9,300×gg下旋转2分钟。
    10. 将剩下的样本应用到每一列,然后像上一步那样旋转。
    11. 将750μl缓冲液PE加到每个柱子上;像上一步那样旋转。
    12. 丢弃流通,然后再旋转每个柱子,以去除所有残留的乙醇。
    13. 将每个色谱柱放入一个新的微量离心管中,然后在每个色谱柱上加入50μl55°C的DEPC H 2 O。让试管坐在试验台上1-2分钟,然后像上一步一样旋转。丢弃色谱柱并在-80°C下冷冻样品或立即进行实时PCR分析。

    实时PCR定量
    标准曲线由输入样品的连续稀释产生,然后用于确定IP和模拟样品的ChIP水平。可以为每个时间点计算下面描述的相对ChIP信号,并在模型拟合数据之前绘制。使用标准曲线量化ChIP信号至关重要;直接使用阈值循环数(例如)来量化ChIP水平将不会提供对ChIP材料数量的准确估计,除非它考虑到与输入的对数关系。
    1. 首先,制作输入染色质的稀释系列。在台式解冻输入样品几分钟,然后在微型离心机中快速旋转并置于冰上。我们将未稀释的输入称为“125x”;对该材料进行5倍稀释以产生用于实时PCR标准曲线的25x,5x和1x输入样品。为了制作25x标准品,取2μl未稀释的输入物并加入8μlDEPC H 2 O 2中。混合,快速旋转,并以相同的方式稀释25倍样品以产生5倍样品。对于5x样品,以相同的方式制作1x样品。保持输入染色质在冰上稀释。
    2. 通过为每个反应添加10μliQ SYBR Green Supermix(2x),并将正向和反向引物加至终浓度为0.2μM,制备实时PCR反应混合物;用DEPC H 2 O将体积调节至19μl。如下所述,我们为每个样品运行技术三重复,并且标准重复运行。为没有样品的对照反应制备足够的混合物。
    3. 实时PCR反应在为仪器设计的孔板中进行。吸取19μl反应混合物到每个孔中。每个时间点需要17个井:每个IP有3个,模拟和5个输入,每个标准有2个。
      请参见下面的图2

      图2.实时PCR板用于四个时间点样品的模板每个实验样品一式三份(行A,C,E和G)运行,并且四个标准中的每一个运行重复(行B,D,F,H)。每个反应使用相同的主混合物,一个孔(H12)没有添加样品作为阴性对照。

    4. 解冻IP和模拟样品,然后在微型离心机中快速旋转并置于冰上。按照指示向每个孔中加入1μl样品,除了对照孔。我们将5x输入样本作为未知数以及标准包括在内;其估计值作为未知值确定了标准曲线定量捕获ChIP信号的程度。
    5. 用薄膜密封平板并在Eppendorf 5810 R台式离心机中以1,810×g / 4℃旋转3分钟。
    6. 使用针对引物组优化的方案在MyiQ或相关仪器中运行平板,并用熔解曲线验证产生单一产物物种。标准数量设置为125,25,5和1的值。
    7. 为了确定每个时间点的相对ChIP信号,从平均IP信号中减去平均模拟信号并除以平均估计的5x输入信号。绘制每个时间点两个数据集的平均值以及标准偏差。平均值将用于模型拟合的下一步。一个代表性的情节如图3所示。


      图3. TATA结合蛋白(TBP)与URA1基因座相互作用的CLKv2数据示例。显示WT(蓝色圆圈)和OE(红色圆圈)菌株对甲醛交联时间的两个生物学重复的平均相对ChIP信号;误差线表示标准偏差。这是用于生成2017年Zaidi et。中的图8B所示模型的原始数据。

  3. 转录因子过表达值量化
    过表达因子是OE菌株中TF相对于WT菌株的水平,并且是将CLKv2模型拟合至甲醛温育时间依赖性ChIP数据所需的。本节介绍如何通过使用WT和OE菌株提取物进行蛋白质印迹来估计过表达因子。
    细胞生长和细胞样本收集
    该方案类似于程序B中的详细说明,<细胞生长和细胞样品收集 ,改变如下所示使用的细胞培养体积和洗涤缓冲液。另外,甲醛交联和淬火步骤

全细胞提取物的制备
该方案类似于程序B中的详细说明,分离碎片染色质提取物样品 ,改变细胞沉淀物重悬体积,使用的缓冲液以及省略超声。
  1. 将细胞沉淀重悬于含蛋白酶抑制剂和β-巯基乙醇的300μlBenoit缓冲液中。将酸洗过的玻璃珠加入到每个管中直到液体上方。几次在实验室工作台上轻敲管子以除去气泡。
  2. 使用保存在寒冷房间中的MP FastPrep-24 Bead打浆机,进行7个循环的珠状样品,45秒开启,每次循环间隔1分钟。
  3. 用加热的(使用本生灯火焰)18号针头在每个FastPrep管的底部打一个孔,然后将每个管放入13 x 100 mm的玻璃管中。在4℃下在3220×gg下旋转3分钟以将液体转移到玻璃管的底部。
  4. 简单地旋转包含流通物质的玻璃管并将每个细胞悬液转移到Eppendorf管中。
  5. 将样品放置在冰上30分钟。

  6. 在4℃以16,000×gg离心管5分钟
  7. 将每个样品的上清液移至新的1.5ml微量离心管中;
    在4℃以16,000×g克旋转30分钟。
  8. 将每个上清液转移至新的1.5ml微量离心管中,并如程序B所述用Bradford测定法定量蛋白质。
    注意:样品可以在-80°C储存在这一步。

通过Western印迹法定量TF水平

  1. 用30%/ 0.8%双丙烯酰胺溶液倒入适量浓度(8-14%)的SDS-PAGE变性凝胶。
  2. 使用例如Bio-Rad mini-PROTEAN tetra细胞,用SDS-PAGE运行缓冲液填充至相应的标记来运行SDS-PAGE凝胶。从20μg开始,在同一凝胶上加载每种菌株的2x连续稀释样品;其他加载量将为10μg,5μg和2.5μg。将每个样品与Laemmli上样缓冲液混合,并在95℃下加热5分钟以使蛋白质在加样前变性。在120V下运行凝胶适当的时间以解析目标大小范围内的多肽条带。
    注意:如果因子过表达值高,可能需要用不同的稀释度进行更多的印迹。

  3. 使用Bio-Rad mini trans-blot电泳系统(或相关系统)在30 V和4°C下将蛋白从凝胶转移至Immobilon-P(PVDF)膜过夜转移缓冲液中。
    注意:第二天在封闭之前目测检查膜,确保梯子已经转移到膜上。
  4. 第二天,用合适的溶液阻断膜。我们通常在室温下在摇床上使用5%脱脂牛奶/ TBST 1小时。
  5. 孵育与第一抗体膜,通常一个小时或更多,然后在TBST洗涤4次,每次洗5分钟。
  6. 用二抗孵育膜(孵育时间通常为1小时,但可能需要根据抗体进行优化),然后在TBST中洗涤4次,每次洗涤5分钟。
    注意:ECL缀合的HRP连接的二抗可用于西方膜上的发育。然而,优选荧光标记抗体,例如与Cy2 / 3/5偶联的抗体,因为荧光强度可以直接定量并且荧光成像具有比胶片更宽的动态范围。
  7. 将500μl溶液A和500μl来自ECL-Prime试剂盒的溶液B混合并均匀涂布在印迹上;用塑料密封印迹并暴露于胶片。
    注:荧光二抗也可用于替代化学发光二级抗体,如ECL-Plex试剂盒提供。在这种情况下,可以使用Typhoon扫描仪或其他荧光成像系统对印迹进行扫描和定量,并保存图像的数字副本。
  8. 使用标准彩色扫描仪扫描胶片;将图像保存为TIFF文件。由于Western印迹难以量化,我们通常使用多次胶片曝光时间来执行和定量多个蛋白质印迹图像。使用提取物的稀释系列来运行凝胶以评估检测系统如何响应印迹上TF相对水平的已知变化,这也是一个好主意。
  9. 使用ImageJ软件,打开TIFF图像并量化每条泳道中感兴趣的蛋白质条带,并使用矩形选择工具在每个条带周围画一个框,并测量每个框内的强度,从而归一化为加载控制。通过将量化的OE带除以相应的WT带来确定过表达因子;为每个稀释度做这个并且平均值得到一个OE因子。我们通过对在至少两个生物重复样本组中观察到的影响进行平均来估计OE值。

  • 测量甲醛交联对可溶性蛋白质库中TF水平的影响
    CLK模型中的一个假设是,未结合的TF的水平不随着甲醛培育时间的增加而变化(Poorey等,<2013>)。在进行CLK分析之前,因此在实验时间过程中测量TF蛋白质水平以确定TF是否从可溶性蛋白质核库中消耗是关键的。全细胞提取物或染色质样品均可使用;我们发现许多TFs的任何一种提取物都会产生非常相似的结果(Zaidi et al。,2017)。程序D和E的协议基于程序B中的CLKv2数据收集方法。
    细胞生长和细胞样本收集
    该方案类似于程序B中的详细说明,<细胞生长和细胞样品收集 ,其中细胞培养体积和洗涤缓冲液的使用发生变化;该优化实验需要更小的体积,并且使用不同的缓冲液来制备全细胞提取物。
    1. 用所需WT酵母菌株的菌落接种两个5ml YPD原代培养物中的每一个;在30°C摇动培养过夜。
    2. 第二天,将每种原代培养物稀释在300ml所需的选择性培养基中,并在30℃下振荡孵育直至OD 600达到〜0.8。
      在4,230×g和4℃下旋转7分钟沉淀细胞。
    3. 将每个细胞沉淀重悬于300ml YPD(或所需培养基)中;在30℃下振荡孵育约1小时,直到OD 600达到约1.0。
    4. 准备甘氨酸淬火溶液。需要4L甘氨酸来将四个时间点样品猝灭两次;有关说明,请参阅食谱。将440毫升3M甘氨酸溶液倒入每个8 x 500毫升Nalgene离心瓶中。
    5. 如步骤2中的沉淀细胞,然后将每个沉淀物重新悬浮在60ml YPD(或期望的培养基)中。
    6. 将每个烧瓶放置在带有搅拌棒的搅拌板上的60ml培养物中,然后使用移液助剂和玻璃吸管从每个培养物中取出10ml等分试样,并在440ml3M甘氨酸瓶中猝灭0分钟对照样品。倒转瓶子几次混合。将搅拌棒转速调至中等。向剩余的50ml培养物中加入7.8ml 37%甲醛,产生5%的最终浓度。
    7. 在5,10和15分钟(或根据需要的其他时间点),迅速取出10毫升等分的交联培养物,加入440毫升甘氨酸等分试样猝灭;盖上盖子倒转瓶子几次混合。

    8. 在4,230×g的条件下于4℃旋转7分钟沉淀细胞。
    9. 将每个细胞沉淀重悬于50ml 4℃TBS中,并转移至50ml锥形管中;在3220×g和4℃下旋转5分钟。弃上清。
    10. 用含有蛋白酶抑制剂和β-巯基乙醇的10ml 4℃Benoit缓冲液洗涤细胞团块;颗粒细胞如前一步一样。
    11. 用1 ml Benoit缓冲液(含蛋白酶抑制剂和β-巯基乙醇)重悬细胞沉淀,并将每个样品转移到FastPrep管中。
    12. 在Eppendorf 5810 R台式离心机中4°C旋转2分钟沉淀细胞。丢弃上清。
      注:样品可在-80°C储存在此步骤。

    全细胞提取物的制备
    该协议的这一部分与程序C 全细胞提取物的制备 中的详细说明相同。

    通过Western印迹定量TF水平
    该方案与程序C下的详细说明类似,除了样品加载量和定量输出量外,通过Western印迹定量TF水平 。
    1. 运行适当浓度(8-14%)的SDS-PAGE变性凝胶。与Laemmli上样缓冲液混合后,加入每种样品15μg蛋白质,并在95°C加热5 min以使蛋白质变性。在120 V下运行凝胶一段适当的时间。
    2. 将蛋白从凝胶转移至Immobilon-P(PVDF)膜,在30 V和4°C下过夜。
    3. 第二天,用针对感兴趣的抗体优化的合适溶液阻断膜。
    4. 孵育与第一抗体的膜,然后在TBST洗涤4次,每次洗5分钟。
    5. 用二抗孵育膜(孵育时间通常为1小时,但可能需要根据抗体进行优化),然后在TBST中洗涤4次,每次洗涤5分钟。
    6. 将500μl溶液A和500μl来自ECL-Prime试剂盒的溶液B混合并均匀涂布在印迹上;用塑料密封印迹并暴露于胶片。
    7. 扫描电影。
    8. 使用ImageJ软件,打开TIFF图像并量化每条泳道感兴趣的蛋白质条带,并归一化为零分钟时间点和/或加载对照。我们通过对至少两个生物重复样品组中观察到的影响进行平均,估算甲醛培养时间对可溶性TF水平的影响。
      注意:蛋白质水平与交联时间的相对变化将指导下一步实施测定。参见Zaidi等2017年的一系列不同因素的结果。如果感兴趣的蛋白质没有随着甲醛培育时间的增加而消耗,那么可以使用该浓度的甲醛并在此范围的甲醛培养时间下进行CLKv2测定。或者,如果感兴趣的蛋白质随着甲醛培养时间的增加而从提取物中消耗,则选择是测试不同(降低)的甲醛浓度和/或将交联时间过程限制在该因子未耗尽的时期。如果甲醛浓度降低,则应努力确定最大可用浓度以使交联速率最大化,例如在稍后的时间点通过滴定甲醛浓度来处理细胞以找到最佳条件。

  • 验证淬火条件
    收集附加订单样本
    验证淬灭条件定量阻断甲醛交联是重要的,并且以这种方式确保ChIP信号在与甲醛一起孵育给定时间后真实地反映交联材料的产量。我们发现这里报道的甘氨酸淬灭方案在所述条件下有效地猝灭5%的甲醛;应该进行本节中的实验,以验证您的手中和您的特定条件下是否属实。
    该方案类似于程序B中的详细说明<细胞生长和细胞样品收集 ,并改变细胞培养体积。
    1. 用所需WT酵母菌株的菌落接种重复的5ml YPD原代培养物;
      在30°C摇动培养过夜
    2. 第二天,将每种原代培养物稀释在200ml所需的选择性培养基中,并在30℃下振荡孵育直至OD 600达到〜0.8。如上所述,在4,230×g和4℃下旋转7分钟沉淀细胞。
    3. 用200ml YPD(或所需培养基)重悬每个细胞沉淀;在30℃下振荡孵育约1小时,直到培养物OD 600为约1.0。
    4. 准备甘氨酸淬火溶液。需要3L甘氨酸来将三个时间点样品猝灭两次;有关说明,请参阅食谱。将440毫升3M甘氨酸溶液倒入6×500毫升Nalgene离心瓶中。
    5. 如步骤2中的沉淀细胞,然后将沉淀重悬于60ml YPD(或期望的培养基)中。将10ml培养物转移到三个500ml烧瓶中的每一个中。

    6. 将每个烧瓶放在带有搅拌棒的搅拌盘上
    7. 收集样品如下:
      1. 无甲醛:将440毫升甘氨酸溶液加入一个含10毫升培养瓶的烧瓶中并混合几秒钟;将溶液和细胞倒入瓶中,盖上瓶子并倒转数次以彻底混合。
        该样品可以在室温下保留在工作台上,而其他样品则被处理。
      2. 甲醛前的甘氨酸:向含有细胞样品的另一个烧瓶中,从制备的瓶中加入全部440ml 3M甘氨酸pH5。在搅拌板上混合几秒钟,然后加入1.56ml 37%甲醛(终浓度5%),并在室温下在搅拌盘上交联8分钟。将整个样品倒入瓶中。
      3. 甲醛接着甘氨酸:到含有细胞样品的最后一个烧瓶中,加入1.56ml 37%甲醛(终浓度5%);充分涡旋混合,并在室温下在搅拌板上孵育8分钟。 (甲醛培养时间可以调整;这里推荐8分钟,因为它在ChIP信号通常仍然依赖于甲醛培养时间(即,,)不饱和的方案中,但是孵育几个分钟很容易处理,且时间精确度高)。向样品中加入440毫升3 M甘氨酸pH 5并混合几分钟。将整个溶液倒入瓶中。
        注意:收集两组样本,但最容易先收集一组样本(a,b,c),然后在同一天收集两组样本时最容易收集样本。 br />
    8. 按照步骤2通过离心沉淀细胞。
    9. 用50ml 4℃TBS + 300mM甘氨酸pH5重悬细胞沉淀并转移到50ml锥形管中;在3220×g和4℃下旋转5分钟。弃上清。
    10. 用50ml 4℃TBS洗涤每个细胞沉淀;颗粒细胞如前一步一样。
    11. 在4°C下将每个细胞沉淀重悬于1 ml TBS中,并将每个样品转移到FastPrep管中。
    12. 通过在4℃和16,000×gg下旋转2分钟来沉淀细胞。丢弃上清。
      注意:样品可以在-80°C储存在这一步。

    分离染色质碎片
    该协议的这一部分与程序B中的详细说明相同 分离染色质碎片 。

    染色质免疫沉淀(ChIP)
    该协议的这一部分与程序B中的详细说明<染色质免疫沉淀(ChIP) 相同。

    实时PCR
    该协议与程序B <实时PCR定量 下的详细说明相同。
    为了确定每个时间点的相对ChIP信号,从平均IP信号中减去平均模拟信号并除以平均估计的5x输入信号。我们通常将结果绘制成条形图并执行 t 测试来评估比较不同样品的统计显着性差异。通过高效淬灭,“仅甘氨酸”和“甲醛前的甘氨酸”ChIP信号应该在数量上难以区分(图4)。


    图4.添加次序实验的代表性结果G =仅甘氨酸,GF =甘氨酸淬灭,然后加入甲醛,FG =甲醛交联,接着甘氨酸淬灭。在该实施例中,在Gal4蛋白的免疫沉淀和实时PCR读出在GAL3基因座处的相互作用之后计算相对ChIP信号。误差线表示两种生物学重复的标准偏差。
  • 数据分析

    数据模型拟合

    1. Poorey等人(2013,Science)开发了用于分析该研究中生成的数据并提取动力学参数的质量作用动力学模型。根据实验数据中观察到的实验方案,在Zaidi et al。(2017)中系统地研究并重新拟定了将数据拟合至该模型的过程。下面我们简要介绍用于估算动力学参数的定量拟合程序。
    2. 一般来说,CLKv2数据可以用三种拟合来表示:TF限制,交联(XL)限制和线性XL限制(见图5)。这些类别由交联速率(k1×1×C×FH×),总体开启速率(k×1×C×H) TF )和解率(k d )。在TF受限制的情况下,k =下行链路TF <下行链路>下行链路和下行链路ķ<子> d &LT;&LT; k <子> XL * C <子> FH 。另一方面,在限制XL的状态下,k1×C1×TF1×k1×C1×FH和 d * C

      图5.潜在交联速率和TF-结合动力学的示意图。 CLKv2数据通常代表三种可能性中的一种,如面板A-C所示。小组的左侧表示一个因子(灰色阴影圆圈)与细胞(大灰色圆圈)中其结合位点(在DNA上用绿色表示,以粗横线表示)的结合。每个时间点显示四个单元格。随着时间的推移从0到1200秒,用黑色箭头表示,因素可能会与甲醛交联成DNA(黄色圆圈)。该图的右侧表示适用于每个数据类的最终模型。 A.结合动力学受限的数据,其中交联快速发生(秒)以捕获结合的TF。数据由具有两个可区分的指数阶段的模型拟合,包括初始陡峭上升(通常表现为非零y截距),接着是浅升高,如右图所示。 B.交联受限的数据,其中交联比TF-结合速率慢(数分钟,图中约600秒)。单一指数拟合数据,右侧显示y截距为零。 C.交联受限(线性)的数据,其中交联速率比TF结合速率慢得多(交联时间尺度相当于或大于最长的实验时间点,图中为1200秒),与交联时间呈线性关系交联时间的功能,如右图所示。

    3. 简而言之,属于TF限制类别的数据显示前几个数据点的指数快速上升,通常表现为非零y截距。最初的上涨合并成一个斜率较浅的第二指数,最终趋于饱和。在这种情况下,交联速率(k1×1),结合速率(k1)和解离速率(k1•d2)可以确定;然后可以计算出停留时间(t 1/2),占有率(θb b)和解离常数(K d⊥)。属于XL限制类别的数据显示从零开始的单指数,达到饱和;在这种情况下,交联比TF结合动力学慢。在这种情况下,k a和k d不能独立地估计,只有K d,k x1和θb> b 。对于TF限制和XL限制的数据,我们可以估计ChIP信号的饱和度,我们使用该信号来对数据进行归一化,以使后期数据饱和一个,符合期望交联部分的位点在CLK实验中会进入长时间的交联时间。线性XL限制曲线最好用从0开始的直线进行近似,与非常缓慢交联的理论模型一致。饱和水平不能被估算,因为在实验的时间范围内交联速度比TF-动态慢得多,没有明显的饱和。事实上,交联时间尺度大于线性XL限制区域中的最后交联时间点的交联时间尺度。在这种情况下,只能估计出K d和θb。对于XL限制的拟合,在某些情况下,可以基于交联速率k 1,k 1,k 1,k 1,k 1, /子> * C <子> FH 。最后,我们将完整模型拟合到没有明确地落入上述三类中的任何一个的数据中。
    4. 通过将模型重新设置为每个基因座的1,000次模拟数据迭代来估计提取的参数中的误差。每个交联时间点的模拟数据是从高斯分布采样的,其中均值由数值拟合给出,方差由实验数据和拟合之间的平方残差的平均值给出。
    5. 当数据不明确地适用于TF限制或XL限制机制时,可以使用F检验在不同拟合之间进行选择。在2017年Zaidi 等人中考虑的大多数情况下,残差(或调整的R平方)的简单比较足以选择更好的拟合。将XL限制体系与TF限制体系区分开来的一个特征是前者对用于交联的甲醛浓度的依赖性。通过用与原始实验中使用的浓度不同的浓度重复实验来观察ChIP信号对甲醛浓度的依赖性,证实了XL限制的动力学。除了数值误差估计之外,我们还通过估计p值来评估拟合的显着性。有关更多详情,请参阅Zaidi 等,2017。
    6. &nbsp;模型拟合程序在Mathematica中编程为TF限制,XL限制和完整模型拟合,R为XL限制线性机制。 Mathematica还估计了数值参数误差。根据Zaidi et al。 2017年的报告专门为CLKv2分析开发的定制拟合和误差分析脚本可根据要求提供。

    笔记

    该方案使用无菌条件进行细胞生长,用酿酒酵母细胞开发。一旦细胞样品已经获得交联,无菌技术就不需要了。

    食谱

    1. YPD媒体(1升)
      10克细菌酵母提取物
      20克细菌蛋白胨
      20克葡萄糖(葡萄糖)
      将所有组件添加到ddH 2 O,最终音量为1 L

      高压灭菌并在室温下储存 注:对于半乳糖或棉子糖培养基,用相同浓度(2%)代替葡萄糖。然而,这些成分不应该高压灭菌,高压灭菌后从30%液体中加入66.7 ml(见配方2)。
    2. 30%棉子糖或半乳糖(100毫升)
      30克糖源
      用ddH 2 O在玻璃瓶中将体积加至100ml
      微波瓶约30秒,然后以搅拌棒中速置于搅拌盘上直至糖溶解
      过滤消毒并在室温下保存
    3. SC培养基(酵母合成培养基)(1L)
      1.2克无氨基酸和硫酸铵的酵母氮碱
      5克硫酸铵
      1克氨基酸(辍学)混合
      将所有组分添加到ddH 2 O中,终体积为950 ml
      用NaOH调节pH至7,然后高压灭菌。高压灭菌后,冷却,然后添加糖至2%,并添加任何氨基酸,如果使用辍学混合物,将被遗漏。
      在室温下储存
      注意:不含氨基酸的酵母氮源和酵母合成的退出培养基补充剂均可在市场上买到,并可用于简化此配方。
    4. 氨基酸混合

      将所有组分置于研钵和研杵中;研磨好然后分散到无菌的50毫升锥形管中
      在室温下储存
      注意:要制作一个压缩混音(用于选择性媒体),不要使用所需的组件。
    5. 3M甘氨酸淬火溶液
      对于每个1升的解决方案:
      225.21克甘氨酸
      〜750ml ddH 2 O
      将水放在加热的搅拌盘上的玻璃烧杯中。将搅拌棒转到中速并加入甘氨酸。 3 M接近甘氨酸在水中的最大溶解度,因此需要低热量混合约1小时以完全溶解甘氨酸。一旦甘氨酸完全溶解后,让其冷却并使用浓HCl将pH调节至5。用ddH <2> O将体积加至1L。甘氨酸一旦制成室温;冷却它会导致甘氨酸从溶液中沉淀出来。如果发生这种情况,请将甘氨酸置于加热的搅拌盘上,用低热量搅拌直至再次溶解。
    6. Benoit的缓冲区
      200 mM Tris-HCl(pH 8.0)
      400毫摩尔(NH 4)2 SO 4 4 10mM MgCl 2 2/2 1 mM EDTA
      10%甘油
      7 mMβ-巯基乙醇
      蛋白酶抑制剂:罗氏完全蛋白酶抑制剂鸡尾酒片剂1.0mM苯甲基磺酰氟*
      2.0 mM苯甲脒*
      2.0μM胃酶抑素*
      0.6μM亮抑酶肽*
      2.0μg胰凝乳蛋白酶抑制剂*
      注:每毫升缓冲液
      将Tris-甘油组分加入到具有所需最终体积的ddH 2 O中(通常一次制成500ml)。
      调整pH值至8并过滤灭菌。在4°C储存
      取出等分试样的缓冲液。加入β-巯基乙醇和蛋白酶抑制剂。一旦加入β-巯基乙醇,该等分试样只能在当天使用,并且必须在下次需要时重新制作。使用
      将等分试样保存在冰上/ 4°C 注意:如果使用蛋白酶抑制剂片剂,25ml缓冲液需要1/2片剂,50ml需要整片。
    7. Laemmli缓冲液(4x样品缓冲液)
      0.2M Tris-HCl pH 6.8
      40%甘油
      277 mM SDS
      200 mM DTT
      3 mM溴酚蓝
      将所有组件添加到ddH 2 O;分装到1.5毫升微量离心管中并储存在-20°C。
    8. 考马斯染色
      1.2克考马斯蓝
      300毫升甲醇
      60毫升醋酸
      240ml H 2 O
      添加所有组件;在室温下储存
    9. TBS
      50mM Tris-HCl pH 7.5
      300 mM NaCl
      将所有组分添加到ddH 2 O中并使其达到所需的体积(通常在一个大瓶中一次制成20 L)
      在室温下储存
    10. TBST
      50mM Tris-HCl pH 7.5
      300 mM NaCl
      0.05%吐温20
      将所有组分添加到ddH 2 O中并使其达到所需的体积(通常在一个大瓶中一次制成20 L)
      在室温下储存
    11. 140 mM ChIP裂解缓冲液
      50 mM HEPES pH 7.5
      140 mM NaCl
      1%Triton X-100
      0.1%脱氧胆酸钠
      蛋白酶抑制剂:罗氏完全蛋白酶抑制剂鸡尾酒片剂1.0mM苯甲基磺酰氟*
      2.0 mM苯甲脒*
      2.0μM胃酶抑素*
      0.6μM亮抑酶肽*
      2.0μg胰凝乳蛋白酶抑制剂*
      注:每毫升缓冲液。
      将除蛋白酶抑制剂以外的所有组分加入到具有所需最终体积(通常每次500ml)的ddH 2 O中。
      过滤消毒溶液并在室温下保存
      取出等分试样的缓冲液。加入蛋白酶抑制剂。可以在加入蛋白酶抑制剂的同一天使用等分试样,但在下次需要时新的等分试样必须重新制成新的等分试样。使用
      将等分试样保存在冰上/ 4°C 注意:如果使用蛋白酶抑制剂片剂,25ml缓冲液需要1/2片剂,50ml需要整片。
    12. 500 mM ChIP裂解缓冲液
      50 mM HEPES pH 7.5
      500 mM NaCl
      1%Triton X-100
      0.1%脱氧胆酸钠
      将所有组分加入到具有所需最终体积的ddH 2 O中(通常一次制成500ml)
      过滤消毒。在室温下储存
    13. LiCl清洗缓冲液
      10 mM Tris pH 8.0
      250 mM LiCl
      0.5%NP-40
      0.5%脱氧胆酸钠
      1 mM EDTA
      将所有组分加入到具有所需最终体积的ddH 2 O中(通常一次制成500ml)
      过滤消毒。在室温下储存
    14. 1x TE
      10 mM Tris-HCl pH 8.0
      1 mM EDTA
      将所有组分加入到具有所需最终体积的ddH 2 O中(通常一次制成500ml)
      过滤消毒。在室温下储存
    15. ChIP洗脱缓冲液
      50 mM Tris-HCl pH 8.0
      1%SDS
      10 mM EDTA
      将所有组分加入ddH 2 O中并加入所需的最终体积(通常每次10ml)
      在室温下储存。
      每次新实验完成后,都应该重新制作解决方案
    16. DEPC H 2 O
      1 ml焦碳酸二乙酯
      1000毫升H 2 O
      将DEPC与H 2 O在螺帽瓶中混合;偶尔搅拌,在室温下在室温下孵育〜2小时。
      分装成100毫升瓶,然后高压灭菌器

      在室温下储存长达12个月
    17. 30%/ 0.8%双丙烯酰胺溶液
      对于500毫升:
      150克丙烯酰胺
      4克双丙烯酰胺
      将所有组分加入到具有所需最终体积的ddH 2 O中(通常一次制成500ml)
      过滤消毒
      4°C储存在铝箔包装的瓶子里
    18. SDS-PAGE运行缓冲液
      192 mM甘氨酸
      25 mM Tris
      0.1%SDS
      将所有组分添加到ddH 2 O中并使其达到所需的体积(通常在一个大瓶中一次制成20 L)
      在室温下储存
    19. 传输缓冲区
      25 mM Tris
      192 mM甘氨酸
      15%甲醇
      将所有组分添加到ddH 2 O中并使其达到所需的体积(通常在一个大瓶中一次制成20 L)
      在室温下储存

    致谢

    这项工作得到了美国国立卫生研究院(R01 GM55763至D.T.A,R21 GM110380至S.B和D.T.A,以及NCI癌症培训基金T32 CA009109-38至E.A.H.)和Wagner奖学金至E.A.H的资助。本手册中的协议已根据Poorey等人2013年发布的Viswanathan等人2014年和Zaidi等人的报道进行了改编。 >,2017年。作者声明他们与本文的内容没有利益冲突或利益冲突。

    参考

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    2. Encode Consortium,E.P。(2012)。 人类基因组中DNA元素的综合百科全书 489(7414):57-74。
    3. Conaway,R.C。和Conaway,J.W。(1993)。 RNA聚合酶II的一般起始因子 62:161-190。
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    5. Cramer,P.(2014)。 100个结构中的染色质和转录故事 Cell 159(5):985-994。
    6. Dowen,JM,Fan,ZP,Hnisz,D.,Ren,G.,Abraham,BJ,Zhang,LN,Weintraub,AS,Schujiers,J.,Lee,TI,Zhao,K.and Young,RA(2014) 。 细胞识别基因的控制发生在哺乳动物染色体绝缘区域 细胞 159(2):374-387。
    7. Grimaldi,Y.,Ferrari,P.和Strubin,M.(2014)。 独立RNA聚合酶II预先启动复合物动态和启动子位点在体内的核小体转换。 Genome Res 24(1):117-124。
    8. Hager,G.L.,McNally,J.G。和Misteli,T。(2009)。 转录动态。 Mol Ce ll 35(6):741-753。
    9. Haruki,H.,Nishikawa,J。和Laemmli,U.K。(2008)。 锚定技术:快速,有条件地建立酵母突变表型。 > Mol Cell 31(6):925-932。
    10. 他,Y.,Fang,J.,Taatjes,D.J。和Nogales,E。(2013)。 人类转录启动关键步骤的结构可视化 Nature 495(7442):481-486。
    11. Horn,A.E.,Kugel,J.F。和Goodrich,J.A。(2016)。 单分子显微镜揭示对RNA聚合酶II预起始复合物组装和转录活性的机械洞察力。 Nucleic Acids Res 44(15):7132-7143。
    12. Kim,T.H.,Barrera,L.O.,Zheng,M.,Qu,C.,Singer,M.A.,Richmond,T.A.,Wu,Y.,Green,R.D.and Ren,B。(2005)。 人类基因组中活跃启动子的高分辨图谱 Nature 436(7052):876-880。
    13. Larson,D.R。,Singer,R.H。和Zenklusen,D。(2009)。 单分子基因表达谱。 Trends Cell Biol 19(11):630-637。
    14. Lickwar,C. R.,Mueller,F.和Lieb,J. D.(2013年)。 利用竞争性ChIP全基因组测量蛋白质-DNA结合动力学。 Nat Protoc 8(7):1337-1353。
    15. Luse,D.S。(2014)。 RNA聚合酶II预引发复合物。通过什么途径来组装? 转录 5(1):e27050。
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    引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
    1. Hoffman, E. A., Zaidi, H., Shetty, S. J., Bekiranov, S. and Auble, D. T. (2018). An Improved Method for Measuring Chromatin-binding Dynamics Using Time-dependent Formaldehyde Crosslinking. Bio-protocol 8(4): e2905. DOI: 10.21769/BioProtoc.2905.
    2. Zaidi, H., Hoffman, E. A., Shetty, S. J., Bekiranov, S. and Auble, D. T. (2017). Second-generation method for analysis of chromatin binding with formaldehyde-cross-linking kinetics. J Biol Chem 292(47): 19338-19355.
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