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Measuring Nucleosome Assembly Activity in vitro with the Nucleosome Assembly and Quantification (NAQ) Assay
核小体组装和定量测定法(NAQ)测定体外核小体组装活性   

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参见作者原研究论文

本实验方案简略版
eLIFE
Mar 2017

Abstract

Nucleosomes organize the eukaryotic genome into chromatin. In cells, nucleosome assembly relies on the activity of histone chaperones, proteins with high binding affinity to histones. At least a subset of histone chaperones promotes histone deposition in vivo. However, it has been challenging to characterize this activity, due to the lack of quantitative assays.

Here we developed a quantitative nucleosome assembly (NAQ) assay to measure the amount of nucleosome formation in vitro. This assay relies on a Micrococcal nuclease (MNase) digestion step that yields DNA fragments protected by the deposited histone proteins. A subsequent run on the Bioanalyzer machine allows the accurate quantification of the fragments (length and amount), relative to a loading control. This allows us to measure nucleosome formation by following the signature DNA length of ~150 bp. This assay finally enables the characterization of the nucleosome assembly activity of different histone chaperones, a step forward in the understanding of the functional roles of these proteins in vivo.

Keywords: Nucleosome assembly (核小体组装), Histone chaperones (组蛋白伴侣), Chromatin (染色质), Micrococcal nuclease (微球菌核酸酶), Quantification (定量)

Background

The eukaryotic genome is organized into nucleosomes. Nucleosomes are modular and dynamic structures composed of an octameric core of histone proteins, wrapped by 147 bp of DNA (Luger et al., 1997). Nucleosome assembly begins with the deposition of one (H3-H4)2 tetramer onto DNA to form a tetrasome. Subsequent incorporation of H2A-H2B dimers forms a hexasome, and finally the nucleosome. Histones are highly positively charged small proteins that primarily exist as histone dimers at physiological salt concentrations. Because of their charges, histones require chaperones which shuttle them from the cytoplasm to the nucleus, and then aid their deposition onto, or removal from DNA (Gurard-Levin et al., 2014).

Histone chaperones are grouped in families of structurally unrelated proteins, all characterized by high binding affinity for histones (Laskey et al., 1978). In this way, they shield the histone charges and prevent their non-specific interaction with DNA and other cellular factors (Elsässer and D’Arcy, 2013). How histone chaperones participate in these different roles, and the degree of division of labor among histone chaperones remain largely unknown.

This is due to the lack of mechanistic knowledge of histone chaperone function, in particular as histone deposition factors onto DNA. It is therefore critical to develop assays that can measure histone deposition activity, i.e., nucleosome assembly, to be able to fully understand the functions of this class of proteins and the dynamics of histones in cells.

Because histone chaperones are not enzymes per se, it has been challenging to develop reliable assays to measure their histone deposition activity. Most existing assays have used native gel analysis to assay nucleosome assembly (Muthurajan et al., 2016). The readout in these assays is prone to misinterpretation, as histones and DNA can form a variety of complexes and native gel analysis is not sufficient to accurately distinguish between the different histone-DNA complexes.

We have developed a nucleosome assembly and quantitation (NAQ) assay that measures the amount of nucleosome particles formation in vitro. This assay relies on the activity of Micrococcal Nuclease (MNase), an enzyme that digests DNA that is not bound by histone proteins. The subsequent purification of the DNA fragments provides a footprint of the histone-DNA complexes in solution. The characteristic protection of ~150 bp DNA is a signature of intact nucleosome species and can be measured using a Bioanalyzer apparatus (Muthurajan et al., 2016). Data normalization to a loading control DNA allows us to compare and accurately quantify the amount of nucleosomes formed in different samples. The NAQ assay has been successfully used to measure the activity of the chromatin assembly factor 1 (CAF-1) in vitro (Mattiroli et al., 2017a and 2017b), and has the potential to reveal the differential contribution of histone chaperones to nucleosome assembly in cells. This will pave the way for the complete understanding of their functional roles in nucleosome dynamics.

Materials and Reagents

  1. Low retention pipette tips (USA Scientific)
  2. PCR tubes (USA Scientific, catalog number: 1402-4308 )
  3. 1.5 ml tubes (Fisher Scientific, catalog number: 05-408-129 )
  4. 207 bp DNA (procedure explained in Dyer et al., 2004)
  5. Loading control DNA [of length between 400 and 1,000 bp, we use a 621 bp DNA (procedure explained in Muthurajan et al., 2016)]
  6. Refolded H2A-H2B (procedure explained in Dyer et al., 2004) or H2A-H2B labeled with ATTO 647N (procedure explained in Muthurajan et al., 2016).
  7. Refolded (H3-H4)2 (procedure explained in Dyer et al., 2004)
  8. Recombinant histone chaperone
  9. Salt-assembled nucleosome (procedures explained in Dyer et al., 2004)
  10. 50% glycerol (autoclaved and stored at room temperature)
  11. 50 bp DNA ladder (Gold Bio, catalog number: D100-500 )
  12. 10x MNase buffer (New England Biolabs, provided with M0247S )
  13. 100x BSA (New England Biolabs, catalog number: B9001 )
    Note: This product has been discontinued.
  14. Micrococcal nuclease (MNase) (New England Biolabs, catalog number: M0247S )
  15. MinElute kit (QIAGEN, catalog number: 28006 )
  16. Proteinase K, 20 mg/ml solution (BioExpress, catalog number: E195-5ML )
  17. Tris (2-Carboxyethyl) phosphine Hydrochloride (TCEP) (Gold Bio, catalog number: TCEP100 )
  18. Sodium hydroxide (NaOH) (Fisher Scientific, catalog number: S318-3 )
  19. Tris base (Fisher Scientific, catalog number: BP152-5 )
  20. Sodium chloride (NaCl) (Fisher Scientific, catalog number: S271-10 )
  21. 500 mM EDTA solution at pH ~8 (stored at room temperature)
  22. Tween-20 (Fisher Scientific, catalog number: BP337 )
  23. SYBR Gold nucleic acid gel stain (Thermo Fisher Scientific, InvitrogenTM, catalog number: S11494 )
  24. Boric acid (Acros Organics, catalog number: 180570025 )
  25. Ammonium persulfate (AMRESCO, catalog number: 0486 )
  26. 30% acrylamide 37.5:1 (Life science Products, catalog number: EC-890 )
  27. Tetramethylethylenediamine (TEMED) (Fisher Scientific, catalog number: BP150-20 )
  28. Bromophenol blue (Fisher Scientific, catalog number: B392-5 )
  29. Xylene cyanol FF (Sigma Aldrich, catalog number: X4126 )
  30. Sodium acetate (Fisher Scientific, catalog number: S210-500 )
  31. Acetic acid (Avantor Performance Materials, catalog number: V193-46 )
  32. 1 M TCEP (Tris (2-Carboxyethyl) phosphine Hydrochloride; see Recipes)
  33. NA buffer (see Recipes)
  34. SYBR Gold stain solution (see Recipes)
  35. 10x TBE (Tris/Borate/EDTA; see Recipes)
  36. 25% APS (Ammonium Persulfate; see Recipes)
  37. 6% PAGE gels (see Recipes)
  38. 10% PAGE gels (see Recipes)
  39. DNA sample buffer (see Recipes)
  40. 3 M Na acetate pH 5.0 solution (see Recipes)

Equipment

  1. Pipettes (Gilson)
  2. PAGE running apparatus (Hoefer, model: SE250 )
  3. Thermoblock (Fisher Scientific, catalog number: 11-718 )
  4. Centrifuge (Eppendorf, model: 5417 C )
  5. Typhoon FLA 9500 (GE Healthcare, model: Typhoon FLA 9500, catalog number: 28996943 )
  6. Bioanalyzer (Agilent)
  7. DNA 1000 chip for Bioanalyzer (Agilent Technologies, catalog number: 5067-1504 )

Software

  1. Agilent Expert 2100 Software
  2. Excel Software

Procedure

The workflow of the NAQ assay is shown in Figure 1:


Figure 1. Workflow of the NAQ assay procedure. In gray are the optional steps.

  1. Nucleosome assembly reaction
    1. Prepare a 40 µl reaction in NA buffer (see Recipes) containing:
      200 nM histone chaperone
      200 nM (H3-H4)2 (tetramer concentration)
      400 nM H2A-H2B (dimer concentration)
      Notes:
      1. It is important to pipette the histone chaperone first, as the histones are destabilized at low salt concentration.
      2. Histones are added subsequently in any order. Octamer preparations (Dyer et al., 2004) (200 nM) can also be used instead of separate (H3-H4)2 and H2A-H2B addition.
      3. Keep the stock solutions at 4 °C, but the reaction at room temperature (20 °C).
      4. We normally set up reactions for multiple histone chaperone concentrations, usually between 100 and 800 nM (or 0.5-4 times the concentration of histones and DNA components).
      5. Always include a ‘no chaperone’ reaction (where only histones are present), a chaperone only reaction (where no histones are present) and a salt-reconstituted nucleosome reaction (pre-assembled on 207 bp DNA [Dyer et al., 2004]). These are set up in parallel and treated exactly like the assembly reactions.
    2. Incubate for 15 min at room temperature.
    3. Add 200 nM of 207 bp DNA, and mix by pipetting.
    4. Incubate for 30 min at room temperature.
    5. Proceed with the following steps and do not store the assembly reactions.

  2. Native PAGE analysis of assembled nucleosomes
    10 µl of the assembly reaction can be used for native PAGE analysis for validation purposes:
    1. Add 2 µl of 50% glycerol stock.
    2. Load 4 µl of the final mix onto a 6% native PAGE (see Recipes) and run in 0.2x TBE buffer (pre-run gel at 150 V at 4 °C for 1 h, see Recipes).
    3. Include a lane with a DNA ladder (50 bp DNA ladder).
    4. Run samples at 150 V for 70 min at 4 °C.
    5. Stain with SYBR GOLD stain solution (see Recipes) for 20 min at room temperature.
    6. Image with Typhoon: Cy2/488 and A647 (if using ATTO 647N labeled H2A-H2B), with PMT (photomultiplier tube) at 500 V and resolution at 100 µm.
    An example of native PAGE analysis of assembled nucleosomes is shown in Figure 2.


    Figure 2. Example of 6% PAGE analysis of the purified DNA. Nuc stands for salt-assembled nucleosomes. tCAF-1 is an active construct of the histone chaperone CAF-1 (Mattiroli et al., 2017b).

  3. Micrococcal Nuclease digestion and DNA purification
    1. Take 25 µl of the nucleosome assembly reaction and mix with 10 µl of 10x MNase buffer, 1 µl 100x BSA, 1 µl of MNase (stock at 25 U/µl) and 63 µl of water.
    2. Incubate for 10 min in a thermoblock at 37 °C
    3. Quench the reaction by adding 10 µl of 500 mM EDTA (final EDTA concentration ~50 mM).
    4. Optional: Treat the sample with 25 µg of Proteinase K (1.25 µl of a 20 mg/ml solution) for 20 min at 50 °C.
    5. Add 550 µl of PB buffer from MinElute kit (QIAGEN) and 10 µl of 3 M Na acetate pH 5.0 solution.
    6. Incubate for 10 min at room temperature.
    7. Add 50 ng of loading control DNA (stock at 25 ng/µl).
    8. Apply sample to the spin column.
    9. Centrifuge for 1 min at 16,000 x g at room temperature.
    10. Discard flow-through.
    11. Wash membrane with 100 µl of PB buffer.
    12. Centrifuge for 1 min at 16,000 x g at room temperature.
    13. Discard flow-through.
    14. Wash membrane with 700 µl of PE buffer.
    15. Centrifuge for 1 min at 16,000 x g at room temperature.
    16. Discard flow-through.
    17. Centrifuge for 1 min at 16,000 x g at room temperature.
    18. Discard flow-through.
    19. Apply 10 µl of ddH2O to the spin column (make sure the tip of the pipette is in the center of the membrane).
    20. Incubate for 10 min at room temperature.
    21. Place spin column into a clean Eppendorf tube.
    22. Centrifuge for 1 min at 16,000 x g at room temperature.

  4. Purified DNA analysis on native PAGE
    2.5 µl of the purified DNA can be used for native PAGE analysis for validation purposes:
    1. Add 2.5 µl of 2x DNA sample buffer.
    2. Load 5 µl of the final mix onto a 10% native PAGE (see Recipes) and run in 0.5x TBE buffer.
    3. Include a lane with 50 bp DNA ladder.
    4. Run samples at 200 V for 50 min at room temperature.
    5. Stain with SYBR GOLD stain solution for 10 min at room temperature.
    6. Image with Typhoon: Cy2/488 with PMT (photomultiplier tube) at 500 V and resolution at 100 µm.
    An example of native PAGE analysis of the purified DNA is shown in Figure 3.


    Figure 3. Example of 10% PAGE analysis of the purified DNA. Nuc stands for salt-assembled nucleosomes. tCAF-1 is an active construct of the histone chaperone CAF-1 (Mattiroli et al., 2017b).

  5. Bioanalyzer run
    Inject 1 µl of the purified DNA (at least 15-20 ng of DNA) into a DNA1000 chip in a Bioanalyzer machine.

Data analysis

The output of the Bioanalyzer run (Figure 4) is checked in the Expert 2100 Software to keep the signal threshold to 20 RFU (relative fluorescence units).


Figure 4. Example of Bioanalyzer output data. Nuc stands for salt-assembled nucleosomes. tCAF-1 is an active construct of the histone chaperone CAF-1 (Mattiroli et al., 2017b).

The data is then analyzed in Excel. We use an example to explain the analysis procedure (Figures 5 and 6).


Figure 5. Example of output Bioanalyzer data of two samples. Sample 1 (columns A-C). Sample 2 (columns E-G). Column I explains the interpreted nature of the bands identified.

  1. Sum the molarity of the fragments with size between 126-165 bp (orange) to obtain 126-165 bp fragments [nM] (column L in Figure 6)
    Sample 1: 16.6 + 13.8 + 29.1 + 19.5 + 28.6 = 107.5 nM
    Sample 2: 13.6 + 11.6 + 22.3 + 13.2 + 21.3 = 82 nM
  2. Perform loading control DNA correction to obtain loading control [nM] (column M in Figure 6)
    Molarity of the loading control (C18 or G18) x Size [bp] of the loading control (A18 or E18)/621 bp (exact length of the loading control DNA)
    Sample 1: 5.9 x 743/621= 7.06 nM
    Sample 2: 6.8 x 752/621= 8.23 nM
  3. Normalize the amount of 126-165 bp fragments [nM] (column L in Figure 6) to the loading control [nM] (column M in Figure 6) to obtain the Normalized values used for sample comparison (column N in Figure 6).
    Sample 1: 107.5/7.06 = 15.24
    Sample 2: 82/8.23 = 9.96


    Figure 6. Analysis table example

These values are then plotted in Excel or other software (GraphPad Prism) to show the amount of nucleosome formation in each sample and to allow comparisons.
These values can be expressed as percentage of nucleosomal DNA fragments, calculated by running a known amount of untreated pure 207 bp DNA into the Bioanalyzer to deduce the conversion factor for the theoretical maximum DNA amount.
For example:
If the analysis of untreated 207 bp DNA yields a Normalized nM value of 35.
Sample 1 above will contain the following percentage (%) of nucleosome protected DNA:
% nucleosomal DNA fragmentsSample 1 = 100 x normalized valueSample 1/normalized valueuntreated DNA
% nucleosomal DNA fragmentsSample 1 = 100 x 15.24/35 = 42.8%

Notes

  1. Fluorescently-labeled H2A-H2B histones can be used in this assay to facilitate the identification of the nucleosome band in the assembly gel analysis.
  2. H2A-H2B associate with a tetrasome in absence of histone chaperones (Mattiroli et al., 2017b).
  3. The Bioanalyzer machine estimates the fragments size based on the elution time of the peaks and using the upper and lower marker bands as references. This leads to errors in the absolute measure of the fragment bp length. Use a salt-assembled nucleosome control to check for this variation in each experiment.

Recipes

  1. 1 M TCEP
    0.2866 g of TCEP (Tris (2-Carboxyethyl) phosphine Hydrochloride)
    360 µl of 10 M NaOH
    ddH2O to 1.5 ml
    Use fresh or store at -20 °C for use within one week (limit freeze/thaw cycles)
  2. NA buffer
    25 mM Tris pH 7.5 (pH measured at room temperature)
    150 mM NaCl
    1 mM EDTA
    0.02% Tween 20
    Store at 4 °C and add a final concentration of 0.5 mM TCEP just before use
  3. SYBR Gold stain solution
    Dissolve 3 µl of SYBR Gold gel stain into 50 ml of ddH2O or 0.2x TBE buffer
  4. 10x TBE buffer (Tris/Borate/EDTA)
    890 mM Tris
    890 mM boric acid
    20 mM EDTA pH 8.0
  5. 25% APS
    Dissolve 0.25 g of ammonium persulfate in 1 ml of ddH2O
    Store at 4 °C for not longer than 1 week
  6. 6% PAGE (final concentrations)
    6% acrylamide
    0.2x TBE
    0.05% APS
    0.08% TEMED
  7. 10% PAGE (final concentrations)
    10% acrylamide
    1x TBE
    0.1% APS
    0.1% TEMED
  8. DNA sample buffer
    30% (v/v) glycerol
    0.25% (w/v) bromophenol blue
    0.25% (w/v) xylene cyanol FF
    Store at 4 °C
  9. 3 M Na acetate pH 5.0 solution
    3 M sodium acetate
    Adjust pH with acetic acid to pH 5.0.
    Store at room temperature

Acknowledgments

We thank Serge Bergeron for optimization of the assembly part of the protocol. F.M. is funded by EMBO (ALTF 1267-2013) and the Dutch Cancer Society (KWF 2014-6649). Research in the Luger lab is funded by the Howard Hughes Medical Institute and NIH (GM067777). The authors declare no conflicts of interest or competing interests.

References

  1. Dyer, P. N., Edayathumangalam, R. S., White, C. L., Bao, Y., Chakravarthy, S., Muthurajan, U. M. and Luger, K. (2004). Reconstitution of nucleosome core particles from recombinant histones and DNA. Methods Enzymol 375: 23-44.
  2. Elsässer, S. J. and D’Arcy, S. (2013). Towards a mechanism for histone chaperones. Biochim Biophys Acta 1819(3-4): 211-221.
  3. Gurard-Levin, Z. A., Quivy, J. P. and Almouzni, G. (2014). Histone chaperones: assisting histone traffic and nucleosome dynamics. Annu Rev Biochem 83: 487-517.
  4. Laskey, R. A., Honda, B. M., Mills, A. D. and Finch, J. T. (1978). Nucleosomes are assembled by an acidic protein which binds histones and transfers them to DNA. Nature 275(5679): 416-420.
  5. Luger, K., Mader, A. W., Richmond, R. K., Sargent, D. F. and Richmond, T. J. (1997). Crystal structure of the nucleosome core particle at 2.8 A resolution. Nature 389(6648): 251-260.
  6. Mattiroli, F., Gu, Y., Balsbaugh, J. L., Ahn, N. G. and Luger, K. (2017a). The Cac2 subunit is essential for productive histone binding and nucleosome assembly in CAF-1. Sci Rep 7: 46274.
  7. Mattiroli, F., Gu, Y., Yadav, T., Balsbaugh, J. L., Harris, M. R., Findlay, E. S., Liu, Y., Radebaugh, C. A., Stargell, L. A., Ahn, N. G., Whitehouse, I. and Luger, K. (2017b). DNA-mediated association of two histone-bound complexes of yeast Chromatin Assembly Factor-1 (CAF-1) drives tetrasome assembly in the wake of DNA replication. Elife 6.
  8. Muthurajan, U., Mattiroli, F., Bergeron, S., Zhou, K., Gu, Y., Chakravarthy, S., Dyer, P., Irving, T. and Luger, K. (2016). In vitro chromatin assembly: Strategies and quality control. Methods Enzymol 573: 3-41.

简介

核小体将真核生物基因组组装成染色质。在细胞中,核小体装配依赖于组蛋白分子伴侣的活性,对组蛋白具有高结合亲和力的蛋白。至少有一部分组蛋白伴侣促进组蛋白在体内的沉积。然而,由于缺乏定量分析,鉴定这种活性一直是一个挑战。

在这里,我们开发了一种定量核小体装配(NAQ)测定来测量体外核小体形成的量。该测定依赖于微球菌核酸酶(MNase)。随后在生物分析仪上运行,可以准确量化相对于加样对照的片段(长度和数量)。这使我们能够测量约150bp的DNA长度。该测定最终实现了不同组蛋白分子伴侣的核小体装配活性的表征,这是理解这些蛋白体内功能作用的一个步骤。

【背景】真核生物基因组被组织成核小体。核小体是由组蛋白八聚体核心组成的模块化和动态结构,由147bp的DNA包裹(Luger等人,1997)。核小体组装始于一个(H3-H4)2四聚体沉积到DNA上以形成四体体。随后的H2A-H2B二聚体结合形成六聚体,最后形成核小体。组蛋白高度带电,因为它们以生理盐浓度存在于组蛋白二聚体中。由于它们的作用,组蛋白需要分子伴侣将它们从细胞质穿梭到细胞核,然后辅助它们沉积到DNA上或从DNA上去除(Gurard-Levin等人,2014)。

组蛋白分子伴侣分组在结构上不相关的蛋白质家族中,所有这些蛋白质的特征在于对组蛋白的高度结合亲和力(Laskey等,1978)。通过这种方式,他们屏蔽了组蛋白的电荷,阻止了与DNA和其他细胞因子的非特异性相互作用(Elsässerand D'Arcy,2013)。组蛋白分子伴侣如何参与这些不同的角色,组蛋白伴侣分工的程度在很大程度上还不清楚。

这是由于缺乏关于组蛋白伴侣功能的机械知识,特别是作为DNA上的组蛋白沉积因子。组蛋白沉积活性,即核小体装配,被充分理解为这类蛋白质的功能和细胞中组蛋白的动力学。

由于组蛋白伴侣本身不是酶,所以它们一直在发展其组蛋白沉积活性。大多数现有的分析已被用于天然凝胶分析以组装核小体装配(Muthurajan等,2016)。在这些测定中的读数易于误解,因为组蛋白和DNA可以在多种组织DNA复合物中发现。

我们已经开发出一种名词 ucleosome 一 ssembly和 q uantitation(NAQ)测定没有措施核小体的量颗粒形成的体外的。该测定依赖于微球菌核酸酶(Micrococcal Nuclease,MNase)的活性,所述酶是消化未被组蛋白蛋白结合的DNA的酶。 DNA片段的随后纯化提供了溶液中组蛋白DNA复合物的足迹。 〜150bp DNA的特征性保护是完整核小体种类的特征,并且可以使用生物分析仪(Bioanalyzer)设备(Muthurajan等人,2016)进行测量。数据归一化到加载对照DNA量化不同样品中形成的核小体的量。所述NAQ测定已被成功地用于测量染色质装配因子的活性1(CAF-1)的体外(Mattiroli 等人,2017A和2017b),和有可能揭示组蛋白分子伴侣对细胞内核小体装配的不同贡献。这将为全面了解其在核小体动力学中的功能角色铺平道路。

关键字:核小体组装, 组蛋白伴侣, 染色质, 微球菌核酸酶, 定量

材料和试剂

  1. 低保留枪头(美国科学)
  2. PCR管(USA Scientific,目录号:1402-4308)。
  3. 1.5毫升管(Fisher Scientific,目录号:05-408-129)。
  4. 207bp DNA(在Dyer等人,2004中解释的程序)
  5. 载入控制DNA [400和1000碱基对之间的长度的,我们使用一个621 bp的DNA(程序Muthurajan 等人解释”。,2016)]
  6. 重折叠的H2A-H2B(过程中的说明 '中代尔等人,2004年)或H2A-H2B用ATTO 647N标记(过程中的说明' 中Muthurajan 等人,2016)。< br />
  7. 重折叠(H3-H4)<子> 2 (步骤中代尔等人解释”。,2004年)
  8. 重组组蛋白伴侣
  9. 盐组装的核小体(在Dyer等人,2004中解释的程序)
  10. 50%甘油(高压灭菌并在室温下储存)

  11. 50 bp DNA梯(Gold Bio,产品目录号:D100-500)
  12. 10x MNase缓冲液(新英格兰生物实验室,提供M0247S)。
  13. 100x BSA(新英格兰生物实验室,目录号:B9001)
    注:此产品已停产。
  14. 微球菌核酸酶(MNase)(New England Biolabs,目录号:M0247S)。
  15. MinElute试剂盒(QIAGEN,产品目录号:28006)
  16. 蛋白酶K,20mg / ml溶液(BioExpress,目录号:E195-5ML)。
  17. 三(2-羧乙基)膦盐酸盐(TCEP)(Gold Bio,目录号:TCEP100)。
  18. 氢氧化钠(NaOH)(Fisher Scientific,目录号:S318-3)。
  19. Tris碱(Fisher Scientific,目录号:BP152-5)。
  20. 氯化钠(NaCl)(Fisher Scientific,目录号:S271-10)。
  21. 在pH〜8的500mM EDTA溶液(在室温下储存)。
  22. Tween-20(Fisher Scientific,目录号:BP337)。
  23. SYBR金核酸凝胶染色(赛默飞世尔科技,Invitrogen公司 TM ,目录号:S11494)
  24. 硼酸(Acros Organics,目录号:180570025)。
  25. 过硫酸铵(AMRESCO,目录号:0486)
  26. 30%丙烯酰胺37.5:1(生命科学产品,目录号:EC-890)
  27. 四甲基乙二胺(TEMED)(Fisher Scientific,目录号:BP150-20)。
  28. 溴酚蓝(Fisher Scientific,目录号:B392-5)。
  29. 二甲苯蓝FF(Sigma Aldrich,目录号:X4126)
  30. 醋酸钠(Fisher Scientific,目录号:S210-500)。
  31. 醋酸(Avantor性能材料,目录号:V193-46)。
  32. 1M TCEP(三(2-羧乙基)膦盐酸盐;见食谱)。
  33. NA缓冲液(见食谱)
  34. SYBR Gold染色液(见食谱)
  35. 10倍TBE(Tris /硼酸盐/ EDTA;见食谱)
  36. 25%APS(过硫酸铵;见食谱)
  37. 6%PAGE凝胶(见食谱)
  38. 10%PAGE凝胶(见食谱)
  39. DNA样品缓冲液(见食谱)
  40. 3M醋酸钠pH 5.0溶液(见食谱)。

设备

  1. 移液器(吉尔森)
  2. PAGE运行设备(Hoefer,型号:SE250)
  3. Thermoblock(Fisher Scientific,目录号:11-718)
  4. 离心机(Eppendorf,型号:5417 C)
  5. 台风FLA 9500(GE Healthcare,型号:Typhoon FLA 9500,目录号:28996943)
  6. 生物分析仪(安捷伦)
  7. 用于生物分析仪的DNA 1000芯片(Agilent Technologies,目录号:5067-1504)

软件

  1. Agilent Expert 2100软件
  2. Excel软件

程序

图1显示了NAQ分析的工作流程:


图1. NAQ分析程序的工作流程灰色是可选的步骤。

  1. 核小体装配反应
    1. 准备在NA缓冲液(见食谱)含有的40μL反应:
      200 nM组蛋白伴侣
      200nM(H3-H4)2(四聚体浓度)。
      400 nM H2A-H2B(二聚体浓度)
      说明:
      1. 首先吸取组蛋白分子伴侣是重要的,因为组蛋白在低盐浓度下不稳定。
      2. 随后以任何顺序添加组蛋白。因此可以使用八聚体制剂(Dyer等人,2004)(200nM)代替分开的(H3-H4)H2A和H2A -H2B另外。
      3. 将原液保持在4°C,但在室温(20°C)下反应。
      4. 我们通常为多种组蛋白伴侣分子浓度建立反应,通常在100到800 nM之间(或组蛋白和DNA组分浓度的0.5到4倍)。
      5. 始终包括“无分子伴侣”反应(只有组蛋白存在),仅分子伴侣反应和盐重建核小体反应(预组装在207 bp DNA上[Dyer et al。 ,2004])。这些都是平行设置的,并且像组装反应一样处理。

    2. 在室温下孵育15分钟
    3. 加入200nM的207bp DNA,并通过移液混合。
    4. 在室温下孵育30分钟。
    5. 继续执行以下步骤,不要存储装配反应。

  2. 组装的核小体的原生PAGE分析
    为了验证目的,可以使用10μl装配反应进行天然PAGE分析:
    1. 加2μl50%的甘油。
    2. 负载4微升最终混合物的到6%非变性PAGE(见配方)和在0.2X TBE缓冲液中进行(预运行凝胶在150V在4℃下1个小时,见配方)。
    3. 包括一个DNA梯(50 bp的DNA梯)。

    4. 在150 V下运行样品70分钟
    5. SYBR GOLD染色溶液(见配方)在室温下染色20分钟。
    6. 图像的台风:的Cy2 / 488和A647(如果使用ATTO 647N标记H2A-H2B)中,用PMT(光电倍增管)在500 V和分辨率在100微米。
    图2显示了装配的核小体的天然PAGE分析的一个例子。

    “”
    图2.纯化的DNA的6%PAGE分析的实例。 Nuc代表盐组装的核小体。 TCAF-1是组蛋白的活性构建体伴侣CAF-1(Mattiroli 等人,2017b)

  3. 微球菌核酸酶消化和DNA纯化
    1. 采取25微升的核小体装配反应的,并用10微升10X MNase缓冲液,1微升100×BSA,MNase(股票在25U /微升)和63微升水的1微升混合。

    2. 在37℃的恒温箱中孵育10分钟
    3. 加入10μl的500mM EDTA(最终的EDTA浓度〜50mM)。
    4. 可选:在50℃下
    5. 加入来自MinElute试剂盒(QIAGEN)的550μlPB缓冲液和10μl3M醋酸钠pH 5.0溶液。
    6. 在室温下孵育10分钟。
    7. 加入50 ng的上样对照DNA(25 ng /μl)。
    8. 将样品应用于旋转柱。

    9. 在室温下以16,000×gg离心1分钟
    10. 放弃流通。
    11. 用100μlPB缓冲液洗膜。
    12. 在室温下以16,000×gg离心1分钟。
    13. 放弃流通。
    14. 用700μlPE缓冲液洗膜。
    15. 在室温下以16,000×gg离心1分钟。
    16. 放弃流通。
    17. 在室温下以16,000×gg离心1分钟。
    18. 放弃流通。
    19. 向旋转柱上滴加10μlddH 2 O(确保移液管的尖端位于膜的中心)。

    20. 在室温下孵育10分钟
    21. 将旋转放入干净的Eppendorf管中。

    22. 在室温下以16,000×gg离心1分钟
  4. 纯天然PAGE的纯化DNA分析 为了验证的目的,2.5μl纯化的DNA可以用于天然PAGE分析:
    1. 加入2.5μl2×DNA样品缓冲液。
    2. 将5μl最终混合物加载到10%非变性PAGE上(见食谱),并在0.5x TBE缓冲液中运行。
    3. 包括一个带有50 bp DNA梯的泳道。

    4. 在室温下以200V运行50分钟
    5. SYBR GOLD染色溶液在室温下染色10分钟。
    6. 图像与台风:Cy2 / 488与PMT(光电倍增管)在500 V和分辨率在100微米。
    图3显示了纯化DNA的天然PAGE分析实例。


    的纯化的DNA的10%PAGE分析的图3.实施例。国统代表盐组装核小体。 TCAF-1是组蛋白的活性构建体伴侣CAF-1(Mattiroli 等人,2017b)

  5. 生物分析仪运行
    注入1微升纯化的DNA(至少15-20纳克DNA的)的入生物分析仪机DNA1000芯片。

数据分析

生物分析仪运行的输出(图4)在Expert 2100软件中检查,以保持信号阈值为20RFU(相对荧光单位)。


图4.生物分析仪输出数据的例子Nuc状态用于盐组装的核小体。 tCAF-1是组蛋白分子伴侣CAF-1的活性构建体(Mattiroli等人,2017b)。

数据然后在Excel中分析。我们用一个例子来解释分析过程(图5和图6)。


图5.两个样品的输出生物分析仪数据示例样品1(A-C列)。样品2(E-G列)。第一栏解释了确定的乐队的解释性质。

  1. 126-165bp(橙色)以获得126-165bp的片段[nM](图6中的L列) 样品1:16.6 + 13.8 + 29.1 + 19.5 + 28.6 = 107.5nM
    样品2:13.6±11.6±22.3±13.2±21.3 = 82nM
  2. 执行加载控制DNA校正以获得加载控制[nM](图6中的列M) 上样对照(C18或G18)的摩尔浓度x上样对照(A18或E18)/ 621bp(上样对照DNA的确切长度)的大小[bp]。
    样品1:5.9×743/627 = 7.06nM
    样品2:6.8 x 752/621 = 8.23 nM
  3. 将126-165bp片段[nM]的量(图6中的L列)标准化为上样对照[nM](图6中的列M),以获得用于样品比较的<归一化值。图6中的N列)。
    样本1:107.5 / 7.06 = 15.24
    样本2:82 / 8.23 = 9.96


    图6.分析表格示例

然后将这些值绘制在Excel或其他软件(GraphPad Prism)中,以显示每个样品中核小体形成的量并进行比较。
这些值可以表示为核小体DNA片段的百分比,通过将已知量的未经处理的纯207bp的DNA运行到生物分析仪中来推导出理论最大DNA量的转换因子。
例如:
如果分析未经处理的207bp DNA产生35的标准化nM值。
上面的样本1想要包含核小体保护DNA的以下百分比(%):
%核小体DNA片段样品1 = 100×归一化值样品1 /归一化值未处理DNA
%核小体DNA片段样品1 = 100×15.24 / 35 = 42.8%

笔记

  1. 在该测定中可使用荧光标记的H2A-H2B组蛋白来促进鉴定装配凝胶分析中的核小体条带。
  2. 在不存在组蛋白伴侣的情况下,H2A-H2B与四体相关联(Mattiroli等人,2017b)。
  3. 生物分析仪根据峰的时间估算片段大小,并使用上下标记带作为参考。这些导致了片段bp长度的绝对测量误差。
    。使用盐组装核小体来控制每个实验中的这种变化

食谱

  1. 1 M TCEP
    0.2866克TCEP(三(2-羧乙基)膦盐酸盐)。
    360μl的10N NaOH
    ddH 2 O至1.5ml
    使用新鲜或在-20°C储存一周(限制冻结/解冻周期)
  2. NA缓冲区
    25mM Tris pH 7.5(在室温下测量的pH)。
    150 mM NaCl。
    1mM EDTA。
    0.02%吐温20

    在4°C储存并在使用前加入终浓度为0.5mM的TCEP
  3. SYBR Gold染色液
    将3μlSYBR Gold凝胶染色溶解于50 ml ddH 0.2 O或0.2x TBE缓冲液中
  4. 10倍TBE缓冲液(Tris / Borates / EDTA)
    890 mM Tris。
    890毫摩尔硼酸
    20 mM EDTA pH 8.0
  5. 25%APS
    将0.25g过硫酸铵溶于1ml ddH 2 O中 在4°C储存不超过1周
  6. 6%PAGE(终浓度)
    6%丙烯酰胺
    0.2x TBE
    0.05%APS
    0.08%TEMED
  7. 10%PAGE(终浓度)
    10%丙烯酰胺
    1x TBE
    0.1%APS
    0.1%TEMED
  8. DNA样品缓冲液
    30%(v / v)甘油
    0.25%(w / v)溴酚蓝
    0.25%(w / v)二甲苯蓝FF FF 在4°C储存
  9. 3M醋酸钠pH 5.0溶液。
    3M醋酸钠
    用乙酸调节pH至pH 5.0。
    在室温下储存。

确认

我们感谢Serge Bergeron对协议部分的优化。 F.M.由EMBO(ALTF 1267年至2013年)和荷兰癌症协会(KWF 2014-6649)资助。 Luger实验室的研究由Howard Hughes医学研究所和NIH(GM067777)资助。作者声明不存在利益冲突或利益冲突。

参考

  1. 代尔,P. N.,Edayathumangalam,R.S。,白色,C. L.,宝,Y.,Chakravarthy,S.,Muthurajan,U. M.和卢格,K。(2004)。从重组组蛋白和DNA。 酶学方法<核小体核心颗粒的重建/ 375:23-44。
  2. Elsässer,S.J.和D'Arcy,S。(2013)。 走向组蛋白伴侣蛋白的机制。 生物化学生物物理学学报 1819(3-4):211-221。
  3. Gurard莱,Z. A.,Quivy,J. P.和Almouzni,G。(2014)。 组蛋白伴侣。辅助组蛋白的流量和核小体动力学代码 Annu启生物化学 83:487-517。
  4. Laskey,R.A.,本田,B. M.,米尔斯,A.D。和Finch,J.T。(1978)。 核小体可以通过在结合组蛋白,并将它们传送到DNA酸性蛋白组装。 <自然 275(5679):416-420。
  5. 卢格,K.马德尔,A. W.,里士满,R. K.,萨金特,D.F。和里士满,T.J。(1997)。核小体核心颗粒的晶体结构在2.8的分辨率。 自然 389(6648):251-260。
  6. Mattiroli,F.,谷,Y.,Balsbaugh,J. L.,安,N. G.和卢格,K。(2017A)。 CAC的2亚基是用于生产性组蛋白结合和核小体装配在CAF-1是必不可少的。 Sci Rep 7:46274。
  7. Mattiroli,F.,顾,Y.,亚达夫T.,Balsbaugh,JL,哈里斯,MR,芬德利,ES,刘,Y.,Radebaugh,CA,Stargell,LA,安贞焕,NG,白宫和一鲁格,K.(2017b)。的酵母染色质装配因子1(CAF-1两组蛋白结合的复合物的 DNA介导的缔合)在DNA复制后驱动tetrasome组装。 6.
  8. Muthurajan,U.,Mattiroli,F.伯杰龙,S.,周,K.,谷,Y.,Chakravarthy,S.,代尔,P.,欧文,T。和卢格,K。(2016)。 体外染色质装配。策略和质量控制 Methods Enzymol 573:3-41。
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免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright Mattiroli et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Mattiroli, F., Gu, Y. and Luger, K. (2018). Measuring Nucleosome Assembly Activity in vitro with the Nucleosome Assembly and Quantification (NAQ) Assay. Bio-protocol 8(3): e2714. DOI: 10.21769/BioProtoc.2714.
  2. Mattiroli, F., Gu, Y., Yadav, T., Balsbaugh, J. L., Harris, M. R., Findlay, E. S., Liu, Y., Radebaugh, C. A., Stargell, L. A., Ahn, N. G., Whitehouse, I. and Luger, K. (2017). DNA-mediated association of two histone-bound complexes of yeast Chromatin Assembly Factor-1 (CAF-1) drives tetrasome assembly in the wake of DNA replication. Elife 6.
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