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Nucleosome Positioning Assay
核小体定位分析实验   

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Abstract

The basic unit of chromatin is the nucleosome, a histone octamer with 147 base pairs of DNA wrapped around it. Positions of nucleosomes relative to each other and to DNA elements have a strong impact on chromatin structure and gene activity and are tightly regulated at multiple levels, i.e., DNA sequence, transcription factor binding, histone modifications and variants, and chromatin remodeling enzymes (Bell et al., 2011; Hughes and Rando, 2014). Nucleosome positions in cells or isolated nuclei can be detected by partial nuclease digestion of native or cross-linked chromatin followed by ligation-mediated polymerase chain reaction (LM-PCR) (McPherson et al., 1993; Soutoglou and Talianidis, 2002). This protocol describes a nucleosome positioning assay using Micrococcal Nuclease (MNase) digestion of formaldehyde-fixed chromatin followed by LM-PCR. We exemplify the nucleosome positioning assay for the promoter of genes encoding ribosomal RNA (rRNA genes or rDNA) in mice, which has two mutually exclusive configurations. The rDNA promoter harbors either an upstream nucleosome (NucU) covering nucleotides -157 to -2 relative to the transcription start site, or a downstream nucleosome (NucD) at position -132 to +22 (Li et al., 2006; Xie et al., 2012). Radioactive labeling of LM-PCR products followed by denaturing urea-polyacrylamide gel electrophoresis allows resolution and relative quantification of both configurations. As depicted in the diagram in Figure 1, the nucleosome positioning assay is a versatile low to medium throughput method to map discrete nucleosome positions with high precision in a semi-quantitative manner.


Figure 1. Flow chart depicting the nucleosome positioning assay. The diagram shows how the assay is used to detect the ratio between upstream (NucU) and downstream (NucD) nucleosome positions at the mouse rDNA promoter. After all steps have been performed, the LM-PCR yields two radiolabeled products that differ in size and correspond to NucU and NucD. Signal intensities of the bands reflect the relative abundance of each nucleosome position in the original sample.

Keywords: Chromatin(染色质), Nucleosome positioning(核小体定位), Micrococcal nuclease(微球菌核酸酶), LM-PCR(LM-PCR)

Background

Chromatin accessibility is regulated by nucleosome packaging, which therefore directs DNA-templated reactions such as transcription, DNA repair, recombination and replication. Dynamic positioning of nucleosomes depends on DNA sequence, transcription factor binding, histone modifications, histone variants and chromatin remodeling enzymes, and is used by cells to regulate genome activity (Bell et al., 2011; Hughes and Rando, 2014). The inaccessibility of nucleosomal DNA facilitates probing of nucleosome positions in cells by digesting nucleosome-free chromatin regions with nucleases like DNase I and MNase. These, and similar approaches are nowadays frequently combined with deep sequencing methods and provide thereby a genome-wide picture of nucleosome positioning (Tsompana and Buck, 2014). However, DNase-seq and MNase-seq assays are relative labor- and cost-intensive and might be immoderate for analysis of the nucleosomal architecture of a specific genomic region. In such a case, MNase digestion of chromatin followed by LM-PCR provides a simple and straightforward alternative, which allows interrogation of nucleosome positions at a given genomic site. Here we describe this gene-centric nucleosome positioning assay and demonstrate its application for analysis of the two nucleosome configurations at the mouse rRNA gene promoter (Li et al., 2006; Xie et al., 2012; Zhao et al., 2016a and 2016b).

Materials and Reagents

  1. Pipette tips (TipOne filter tips, STARLAB INTERNATIONAL)
  2. 10 cm-dish
  3. Tubes (Eppendorf Safe-lock microcentrifuge tubes) (Eppendorf, catalog number: 0030120086 )
  4. Cell scraper (Sigma-Aldrich, catalog number: SIAL0010 )
  5. Syringe
  6. Whatman paper (Whatman, catalog number: 10547922 )
  7. Plastic wrap
  8. Clean razor blades
  9. Immortalized mouse embryonic fibroblast cell line NIH/3T3 (ATCC, catalog number: CRL-1658 )
  10. Formaldehyde solution (Sigma-Aldrich, catalog number: F8775 )
  11. Glycine (Sigma-Aldrich, catalog number: G7126 )
  12. Phosphate buffered saline (PBS)
  13. 0.2 M ethylene glycol-bis(2-aminoethylether)-N,N,N’,N’-tetraacetic acid (EGTA), adjust the pH to 8.0 with NaOH (Sigma-Aldrich, catalog number: E3889 )
  14. 0.5 M ethylenediaminetetraacetic acid (EDTA), adjust the pH to 8.0 with NaOH (Sigma-Aldrich, catalog number: E5134 )
  15. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653 )
  16. Phenol:chloroform:isoamyl alcohol (25:24:1, v/v) (Carl Roth, catalog number: A156.1 )
  17. Sodium acetate (pH 5.2)
  18. 70% ethanol
  19. QIAquick Gel Extraction Kit (QIAGEN, catalog number: 28706 )
  20. Quick Blunting Kit (New England Biolabs, catalog number: E1210L )
  21. QIAquick PCR Purification Kit (QIAGEN, catalog number: 28106 )
  22. T4 DNA ligase (New England Biolabs, catalog number: M0202S )
  23. T4 polynucleotide kinase (New England Biolabs, catalog number: M0201S )
  24. PCR-primer specific for the nucleosomal region of interest. For the mouse rDNA promoter we used mrDNA (-63/-36): GATCACAAGCATAAAAGAGACAGGGAGG
  25. QIAquick Nucleotide Removal Kit (QIAGEN, catalog number: 28306 )
  26. [γ-32P]-ATP (3,000 Ci/mmol, 10 mCi/ml) (PerkinElmer, catalog number: BLU002001MC )
  27. GoTaq G2 Hot-Start Green PCR Master Mix (Promega, catalog number: M742A )
  28. Linker primers: linker S: 5’-gaattcagatc-3’, linker L: 5’-gcggtgacccgggagatctgaattc-3’
  29. DMSO (Sigma-Aldrich, catalog number: D8418 )
  30. Sucrose (Sigma-Aldrich, catalog number: 84097 )
  31. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9541 )
  32. 1 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) adjust the pH to 7.9 with NaOH (Applichem, catalog number: A1069 )
  33. Potassium phosphate dibasic (K2HPO4·3H2O) (Carl Roth, catalog number: 6878.1 )
  34. Magnesium chloride (MgCl2·6H2O) (Applichem, catalog number: 131396.1211 )
  35. Calcium chloride (CaCl2) (Sigma-Aldrich, catalog number: 499609 )
  36. L-α-lysophosphatidylcholine (Sigma-Aldrich, catalog number: L4129 )
  37. Micrococcal Nuclease (MNase) (New England Biolabs, catalog number: M0247S )
  38. Tris base
  39. Boric acid
  40. Formamide
  41. Xylene cyanol
  42. Bromophenol blue
  43. SDS (Sigma-Aldrich, catalog number: 74255 )
  44. Rotiphorese sequencing gel concentrate (Carl Roth, catalog number: 3043.1 )
  45. Rotiphorese sequencing gel diluents (Carl Roth, catalog number: 3047.1 )
  46. Ammonium persulfate (APS) (Carl Roth, catalog number: 9592.2 )
  47. TEMED (Carl Roth, catalog number: 2367.3 )
  48. Permeabilization buffer (see Recipes)
  49. MNase digestion buffer (see Recipes)
  50. TE buffer (see Recipes)
  51. 10x TBE buffer (see Recipes)
  52. Formamide loading buffer (see Recipes)
  53. Denaturing polyacrylamide gel (6%) (see Recipes)

Equipment

  1. Pipette
  2. Standard microcentrifuge
  3. NanoDrop 2000 UV-Vis spectrophotometer (Thermo Fisher ScientificTM, model: NanoDrop 2000 )
  4. PCR machine
  5. Electrophoresis equipment with power supply
  6. Vacuum pump
  7. Gel dryer (Bio-Rad Laboratories, model: 583 )
    Note: This product has been discontinued.
  8. Phosphor imaging instrument (FujiFilm, model: FLA-3000 )
  9. Imaging plate (GE Healthcare, model: BAS-IP MS 2025 E )

Software

  1. Quantification software (Raytest, AIDA Image Analyzer)

Procedure

  1. Purification of mononucleosomal DNA from cells
    1. Fix NIH/3T3 cells on a 10 cm-dish (80% confluency) by adding 270 μl of 37% formaldehyde solution to 10 ml culture medium (1% formaldehyde final) for 10 min at room temperature. Quench excessive formaldehyde by adding 1/20 volume of 2.5 M glycine solution (125 mM glycine final) for 5 min.
    2. After two washes with PBS, permeabilize cells at 37 °C for 1 min in permeabilization buffer on the culture dish.
    3. Remove the permeabilization buffer and directly incubate cells in MNase digestion buffer for 20 min at room temperature. Inactivate MNase by adding EDTA and EGTA to a final concentration of 10 mM each. Discard the solution and harvest cells in PBS by scraping and transfer them to a microcentrifuge tube.
    4. To revert formaldehyde-mediated crosslinking by heating, incubate cells in PBS containing 300 mM NaCl at 65 °C for 6 h. Mix DNA solution with the same volume of phenol:chloroform:isoamyl solution and spin in a standard microcentrifuge (13,523 x g, room temperature) for 5 min. Transfer the upper aqueous phase into a fresh tube and repeat extraction. Add 1/10 volume of 3 M sodium acetate (pH 5.2) and 2.5 volumes of ethanol to the DNA solution and precipitate DNA at -20 °C for 30 min. Spin down the precipitate in a standard microcentrifuge (13,523 x g, 4 °C) for 15 min, carefully remove supernatant, and wash once with 70% ethanol. Air dry the pellet and dissolve in 50 μl TE buffer.
    5. To isolate mononucleosome-sized fragments, separate DNA on 2% agarose gels. Excise the ~150 bp band from the gel and purify DNA with a QIAquick Gel Extraction Kit according to manufacturer’s instruction. Elute in 30-50 μl elution buffer.
    6. Measure DNA concentration on a NanoDrop 2000 spectrophotometer and store at -20 °C. The yield of mononucleosomal DNA from a 10 cm-dish of NIH/3T3 cells is between 4-8 μg.

  2. Ligation of linkers to mononucleosomal DNA
    1. To generate blunted and phosphorylated DNA ends, set up the reaction as described below using the Quick Blunting Kit. Incubate at room temperature for 30 min followed by enzyme inactivation at 70 °C for 10 min.
      10x blunting buffer
      2.5 μl
      1 mM dNTP
      2.5 μl
      Enzyme mix
      0.5 μl
      DNA
      2.0 μg
      H2O
      to 25 μl
    2. Dilute linkers S and L in TE buffer at a final concentration of 20 µM each (mass concentration of ~0.3 µg/µl). Anneal S and L by the following program on a PCR machine:
      95 °C, 15 min
      65 °C, 15 min (slope 0.01 °C/sec)
      55 °C, 15 min (slope 0.01 °C/sec)
      45 °C, 15 min (slope 0.01 °C/sec)
      37 °C, 15 min (slope 0.01 °C/sec)
      25 °C, 15 min (slope 0.01 °C/sec)
      4 °C, 5 min
    3. To ligate double-stranded linker L+S (from step B2) to mononucleosomal DNA (from step B1) set up the reaction as described below, and incubate the reaction at 16 °C overnight. Inactivate the enzyme at 65 °C for 20 min, purify the DNA with the QIAquick PCR Purification Kit and elute in 20 µl TE buffer.
      10x ligation buffer
      2 μl
      100 mM ATP
      1 μl
      T4 DNA ligase
      1 μl
      DNA (from step B1)
      1 μg
      Linker L+S (from step B2)
      1 μg
      H2O to
      20 μl

  3. LM-PCR
    1. Label the PCR primer specific for the nucleosomal region of interest radioactively by setting up the reaction below and incubating for 1 h at 37 °C. After inactivating PNK at 65 °C for 20 min purify the primer with the QIAquick Nucleotide Removal Kit and elute in 20 µl TE buffer.
      10x PNK buffer
      2 μl
      PNK
      3 μl
      [γ-32P]-ATP (3.3 µM)
      9 μl
      Primer (20 μM)
      1 μl
      H2O
      8 μl
    2. Set up LM-PCR reaction containing linker-ligated mononucleosomal DNA (from step B3), linker primer L and the 32P-labeled specific primer (from step C1) as follows:
      GoTaq PCR Master Mix
      7.5 μl
      DNA (from step B3)
      2 μl
      Linker L (10 μM)
      0.5 μl
      32P-primer (from step C1)
      2 μl
      DMSO
      0.5 μl
      H2O
      2.5 μl
    3. Run the following ‘Touchdown’-PCR program:
      Denaturation 94 °C 5 min
      94 °C 30 sec, 65 °C 30 sec, 72 °C 30 sec; x 3 cycles
      94 °C 30 sec, 64 °C 30 sec, 72 °C 30 sec; x 3 cycles
      94 °C 30 sec, 63 °C 30 sec, 72 °C 30 sec; x 3 cycles
      94 °C 30 sec, 62 °C 30 sec, 72 °C 30 sec; x 3 cycles
      94 °C 30 sec, 61 °C 30 sec, 72 °C 30 sec; x 3 cycles
      94 °C 30 sec, 60 °C 30 sec, 72 °C 30 sec; x 15 cycles
      Complete extension 72 °C 10 min

  4. Gel electrophoresis
    1. Prepare 6% denaturing urea-polyacrylamide gel (see Recipes).
    2. Assemble electrophoresis and pre-run the gel in 1x TBE for 30 min at 250 V. The pre-run allows equilibration of the gel/buffer system and heats up the gel to the operating temperature of 45-55 °C.
    3. After the pre-run, rinse gel pockets by flushing with fresh buffer using a syringe.
    4. Add an equal volume of formamide loading buffer to the LM-PCR reaction (from step C3) and incubate for 5 min at 95 °C, then quickly chill on ice. This step should be carried out during the pre-run of the gel so that the LM-PCR/loading buffer mix is on ice while rinsing the gel pockets. Thereby the time between pre-run and sample loading is minimized to avoid cooling Load 5 µl of the LM-PCR/loading buffer mix. Restart the electrophoresis and run for 1.5-2 h.
    5. Disassemble the gel set-up, put the gel on a Whatman paper and cover the opposite gel side with plastic wrap. It does not matter which side of the gel faces down. However, it is helpful to clip off one upper corner of the gel to locate its position after drying.
    6. Dry the gel at 80 °C for 1-2 h under vacuum.
    7. Expose gel to phosphor imaging plate overnight.
    8. Scan screen with phosphor imaging instrument and quantify bands representing different nucleosomal positions with appropriate software (e.g., AIDA Image Analyzer).

Data analysis

Detection of the radioactive signals from the LM-PCR products representing different nucleosome positions enables a relative, semi-quantitative analysis. Quantify the signal intensities with appropriate software and relate them within one sample/lane to each other. For instance, for the two nucleosome configurations at the rDNA promoter, NucU and NucD, we used the NucU/NucD ratio to compare changes between different physiological conditions (Zhao et al., 2016a and 2016b). Using the internal ratios of nucleosome positions for comparison rather than comparing the same positions between samples diminishes the effect of handling variations between samples and thus provides more robust results. Verify the reproducibility of the data by repeating the LM-PCR at least twice (technical replicates) and by performing the whole procedure at least three times with individually prepared nucleosomal DNA samples (biological replicates). Moreover, using a different gene-specific primer in the LM-PCR is also a good way to validate the results. If comparing internal ratios of nucleosome positions between two differently treated samples of cells, use a paired two-tailed Student’s t-test to calculate the statistical significance.

Recipes

  1. Permeabilization buffer
    150 mM sucrose
    80 mM KCl
    35 mM HEPES (pH 7.4)
    5 mM K2HPO4
    5 mM MgCl2
    0.5 mM CaCl2
    0.05% L-α-lysophosphatidylcholine
  2. MNase digestion buffer
    150 mM sucrose
    50 mM NaCl
    50 mM Tris-HCl (pH 7.4)
    2 mM CaCl2
    50 U/ml MNase
  3. TE buffer
    20 mM Tris-HCl (pH 8.0)
    2 mM EDTA
  4. 10x TBE buffer (1 L)
    108 g of Tris base
    55 g of boric acid
    40 ml of 0.5 M of EDTA (pH 8.0)
  5. Formamide loading buffer
    95% formamide
    0.025% xylene cyanol
    0.025% bromophenol blue
    18 mM EDTA
    0.025% SDS
  6. Denaturing polyacrylamide gel (6%)

Acknowledgments

We thank Ingrid Grummt for comments on the manuscript. This work was funded by the Thuringian country program ProExzellenz (RegenerAging–FSU-I-03/14) of the Thuringian Ministry for Research (TMWWDG), Deutsche Forschungsgemeinschaft (SFB 1036), ‘CellNetworks’ (EcTop Survey 2014), and the Baden-Württemberg Stiftung.
The protocol was adapted from previous work (Li et al., 2006; Zhao et al., 2016a and 2016b).

References

  1. Bell O, Tiwari VK, Thoma NH, Schubeler D (2011). Determinants and dynamics of genome accessibility. Nat Rev Genet 12(8): 554-564
  2. Hughes, A. L. and Rando, O. J. (2014). Mechanisms underlying nucleosome positioning in vivo. Annu Rev Biophys 43: 41-63.
  3. Li, J., Langst, G. and Grummt, I. (2006). NoRC-dependent nucleosome positioning silences rRNA genes. EMBO J 25(24): 5735-5741.
  4. McPherson, C. E., Shim, E. Y., Friedman, D. S. and Zaret, K. S. (1993). An active tissue-specific enhancer and bound transcription factors existing in a precisely positioned nucleosomal array. Cell 75(2): 387-398.
  5. Soutoglou, E. and Talianidis, I. (2002). Coordination of PIC assembly and chromatin remodeling during differentiation-induced gene activation. Science 295(5561): 1901-1904.
  6. Tsompana, M. and Buck, M. J. (2014). Chromatin accessibility: a window into the genome. Epigenetics Chromatin 7(1): 33
  7. Xie, W., Ling, T., Zhou, Y., Feng, W., Zhu, Q., Stunnenberg, H. G., Grummt, I. and Tao, W. (2012). The chromatin remodeling complex NuRD establishes the poised state of rRNA genes characterized by bivalent histone modifications and altered nucleosome positions. Proc Natl Acad Sci U S A 109(21): 8161-8166.
  8. Zhao, Z., Dammert, M. A., Grummt, I. and Bierhoff, H. (2016a). lncRNA-induced nucleosome repositioning reinforces transcriptional repression of rRNA genes upon hypotonic stress. Cell Rep 14(8): 1876-1882.
  9. Zhao, Z., Dammert, M. A., Hoppe, S., Bierhoff, H. and Grummt, I. (2016b). Heat shock represses rRNA synthesis by inactivation of TIF-IA and lncRNA-dependent changes in nucleosome positioning. Nucleic Acids Res 44(17): 8144-8152.

简介

染色质的基本单位是核小体,一个组织蛋白八聚体,其中包含147个碱基对的DNA。核小体相对于彼此和DNA元件的位置对染色质结构和基因活性具有强烈的影响,并且在多个水平(例如,DNA序列,转录因子结合,组蛋白修饰和变体)和染色质重塑酶(Bell et al。,2011; Hughes和Rando,2014)。可以通过天然或交联染色质的部分核酸酶消化,然后连接介导的聚合酶链反应(LM-PCR)(McPherson等人,1993; Soutoglou)检测细胞或分离的核中的核小体位置和Talianidis,2002)。该方案描述了使用微球菌核酸酶(MNase)消化甲醛固定染色质,然后进行LM-PCR的核小体定位测定。我们举例说明了在小鼠中编码核糖体RNA(rRNA基因或rDNA)的基因启动子的核小体定位测定,其具有两个相互排斥的配置。 rDNA启动子含有相对于转录起始位点的核苷酸-157至-2或位于-132位的下游核小体(NucD )的上游核小体(NucU )至+22(Li等人,2006; Xie等人,2012)。 LM-PCR产物的放射性标记,然后变性尿素 - 聚丙烯酰胺凝胶电泳,允许两种配置的分辨和相对定量。如图1所示,核小体定位测定是通用的低至中等通量的方法,以半定量方式以高精度映射离散的核小体位置。


图1.描绘核小体定位测定的流程图。该图显示了该检测法如何用于检测上游(NucU )和下游之间的比例(NucD )小鼠rDNA启动子的核小体位置。在所有步骤完成后,LM-PCR产生两个尺寸不同并且对应于NucU和NucD的放射性标记产物。条带的信号强度反映了原始样品中每个核小体位置的相对丰度。

背景 染色质可及性受核小体包装的调节,因此DNA指导DNA模板反应,如转录,DNA修复,重组和复制。核小体的动态定位取决于DNA序列,转录因子结合,组蛋白修饰,组蛋白变体和染色质重塑酶,并被细胞用于调节基因组活性(Bell等人,2011; Hughes和Rando ,2014)。核小体DNA的无法通过用核酸酶如DNA酶I和MNase消化无核小体的染色质区域来促进细胞中核小体位置的探测。这些和类似的方法现在常常与深度测序方法相结合,从而提供核基因组定位的全基因组图像(Tsompana and Buck,2014)。然而,DNase-seq和MNase-seq测定是相对劳动和成本密集的,并且可能对于分析特定基因组区域的核小体结构是无意义的。在这种情况下,染色质的MNase消化后接LM-PCR提供了简单直接的替代方案,其允许询问给定基因组位点处的核小体位置。在这里,我们描述这种以基因为核心的核小体定位测定,并证明其在小鼠rRNA基因启动子上分析两种核小体构型的应用(Li等人,2006; Xie等人,2012; Zhao等人,2016a和2016b)。

关键字:染色质, 核小体定位, 微球菌核酸酶, LM-PCR

材料和试剂

  1. 移液器提示(TipOne过滤器技巧,STARLAB INTERNATIONAL)
  2. 10厘米盘
  3. 管(Eppendorf安全锁微量离心管)(Eppendorf,目录号:0030120086)
  4. 细胞刮刀(Sigma-Aldrich,目录号:SIAL0010)
  5. 注射器
  6. Whatman论文(Whatman,目录号:10547922)
  7. 塑料包装
  8. 清洁剃刀刀片
  9. 不间断小鼠超声成纤维细胞系NIH/3T3(ATCC,目录号:CRL-1658)
  10. 甲醛溶液(Sigma-Aldrich,目录号:F8775)
  11. 甘氨酸(Sigma-Aldrich,目录号:G7126)
  12. 磷酸盐缓冲盐水(PBS)
  13. 0.2M乙二醇 - 双(2-氨基乙醚)-N,N,N',N'-四乙酸(EGTA),用NaOH调节pH至8.0(Sigma-Aldrich,目录号:E3889)
  14. 0.5M乙二胺四乙酸(EDTA),用NaOH调节pH至8.0(Sigma-Aldrich,目录号:E5134)
  15. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S7653)
  16. 苯酚:氯仿:异戊醇(25:24:1,v/v)(Carl Roth,目录号:A156.1)
  17. 乙酸钠(pH 5.2)
  18. 70%乙醇
  19. QIAquick凝胶提取试剂盒(QIAGEN,目录号:28706)
  20. Quick Blunting Kit(New England Biolabs,目录号:E1210L)
  21. QIAquick PCR纯化试剂盒(QIAGEN,目录号:28106)
  22. T4 DNA连接酶(New England Biolabs,目录号:M0202S)
  23. T4多核苷酸激酶(New England Biolabs,目录号:M0201S)
  24. 对于感兴趣的核小体区域特异的PCR引物。对于小鼠rDNA启动子,我们使用mrDNA(-63/-36):GATCACAAGCATAAAAGAGACAGGGAGG
  25. QIAquick核苷酸去除试剂盒(QIAGEN,目录号:28306)
  26. [300℃/mmol,10mCi/ml](PerkinElmer,目录号:BLU002001MC)
  27. Go Taq G2 Hot-Start Green PCR Master Mix(Promega,目录号:M742A)
  28. 接头引物:接头S:5'-gaattcagatc-3',接头L:5'-gcggtgacccgggagatctgaattc-3'
  29. DMSO(Sigma-Aldrich,目录号:D8418)
  30. 蔗糖(Sigma-Aldrich,目录号:84097)
  31. 氯化钾(KCl)(Sigma-Aldrich,目录号:P9541)
  32. 1 M 4-(2-羟乙基)-1-哌嗪乙磺酸(HEPES)用NaOH调节pH至7.9(应用目录号:A1069)
  33. 磷酸氢二钾(K 2 HPO 4·3H 2 O)(Carl Roth,目录号:6878.1)
  34. 氯化镁(MgCl 2·6H 2 O)(应用目录号:131396.1211)
  35. 氯化钙(CaCl 2)(Sigma-Aldrich,目录号:499609)
  36. L-α-溶血磷脂酰胆碱(Sigma-Aldrich,目录号:L4129)
  37. 微球菌核酸酶(MNase)(New England Biolabs,目录号:M0247S)
  38. 三碱基
  39. 硼酸
  40. 甲酰胺
  41. 二甲苯cyanol
  42. 溴酚蓝
  43. SDS(Sigma-Aldrich,目录号:74255)
  44. Rotiphorese测序凝胶浓缩物(Carl Roth,目录号:3043.1)
  45. Rotiphorese测序凝胶稀释剂(Carl Roth,目录号:3047.1)
  46. 过硫酸铵(APS)(Carl Roth,目录号:9592.2)
  47. TEMED(Carl Roth,目录号:2367.3)
  48. 渗透缓冲液(见配方)
  49. MNase消化缓冲液(参见食谱)
  50. TE缓冲(见配方)
  51. 10x TBE缓冲区(见配方)
  52. 甲酰胺加载缓冲液(参见食谱)
  53. 变性聚丙烯酰胺凝胶(6%)(见配方)

设备

  1. 移液器
  2. 标准微量离心机
  3. NanoDrop 2000 UV-Vis分光光度计(Thermo Fisher Scientific TM,型号:NanoDrop 2000)
  4. PCR机器
  5. 带电源的电泳设备
  6. 真空泵
  7. 凝胶干燥器(Bio-Rad Laboratories,型号:583)
    注意:本产品已停产。
  8. 荧光成像仪(FujiFilm,型号:FLA-3000)
  9. 成像板(GE Healthcare,型号:BAS-IP MS 2025 E)

软件

  1. 量化软件(Raytest,AIDA Image Analyzer)

程序

  1. 从细胞中纯化单核细胞DNA
    1. 通过在室温下向10ml培养基(1%甲醛终浓度)中加入270μl37%甲醛溶液10分钟,将NIH/3T3细胞固定在10cm皿(80%汇合点)上10分钟。通过加入1/20体积的2.5M甘氨酸溶液(125mM甘氨酸最终)5分钟来淬灭过量的甲醛。
    2. 用PBS洗两次后,在培养皿的透化缓冲液中,37℃下将细胞透化1分钟。
    3. 去除透化缓冲液,并在室温下直接将细胞在MNase消化缓冲液中孵育20分钟。通过加入EDTA和EGTA终止浓度为10mM的MNase。弃去溶液,并通过刮擦收集PBS中的细胞并将其转移到微量离心管中
    4. 通过加热还原甲醛介导的交联,在含有300mM NaCl的PBS中在65℃孵育细胞6小时。将DNA溶液与相同体积的苯酚:氯仿:异戊基溶液混合,并在标准微量离心机(13,523×g,室温)下旋转5分钟。将上层水相转移到新鲜管中并重复萃取。向DNA溶液中加入1/10体积的3M乙酸钠(pH 5.2)和2.5体积乙醇,并在-20℃下沉淀DNA 30分钟。在标准微量离心机(13,523×g,4℃)中将沉淀物旋转15分钟,小心地除去上清液,并用70%乙醇洗涤一次。空气干燥沉淀并溶于50μlTE缓冲液中
    5. 为了分离单核细胞大小的片段,在2%琼脂糖凝胶上分离DNA。从凝胶上清除〜150bp的条带,并根据制造商的说明书用QIAquick Gel Extraction Kit纯化DNA。在30-50μl洗脱缓冲液中洗脱。
    6. 测量NanoDrop 2000分光光度计上的DNA浓度,并保存在-20°C。来自NIH/3T3细胞10cm的单核细胞体DNA的产量在4-8μg之间。

  2. 连接到单核细胞DNA的接头
    1. 为了产生钝化和磷酸化的DNA末端,使用Quick Blunting Kit如下所述设置反应。在室温下孵育30分钟,然后在70℃酶灭10分钟
      10x钝化缓冲区
      2.5μl
      1 mM dNTP
      2.5μl
      酶混合物
      0.5μl
      DNA
      2.0μg
      H 2 O
      至25μl
    2. 在TE缓冲液中稀释接头S和L,最终浓度为20μM(质量浓度为〜0.3μg/μl)。退火S和L通过以下程序在PCR机上:
      95℃,15分钟
      65℃,15分钟(斜率0.01℃/秒)
      55℃,15分钟(斜率0.01℃/秒)
      45℃,15分钟(斜率0.01℃/秒)
      37℃,15分钟(斜率0.01℃/秒)
      25℃,15分钟(斜率0.01℃/秒)
      4℃,5分钟
    3. 为了将双链接头L + S(来自步骤B2)连接到单核细胞体DNA(来自步骤B1),如下所述设置反应,并将反应在16℃温育过夜。在65℃灭菌20分钟酶,用QIAquick PCR纯化试剂盒纯化DNA,并在20μlTE缓冲液中洗脱。
      10x连接缓冲液
      2μl
      100 mM ATP
      1μl
      T4 DNA连接酶
      1μl
      DNA(来自步骤B1)
      1μg
      链接器L + S(来自步骤B2)
      1μg
      H 2 O到
      20μl

  3. LM-PCR
    1. 通过设置下面的反应并在37℃下孵育1小时,对放射性核素体区域特异性PCR引物进行标记。在65℃下灭活PNK 20分钟后,用QIAquick Nucleotide Removal Kit纯化引物,并在20μlTE缓冲液中洗脱。
      10x PNK缓冲区
      2μl
      PNK
      3μl
      [γ- 32 P] -ATP(3.3μM)
      9μl
      引物(20μM)
      1μl
      H 2 O
      8μl
    2. 设置含有接头连接的单核细胞DNA(来自步骤B3),接头引物L和 32标记的特异性引物(来自步骤C1)的LM-PCR反应如下:
      Go Taq PCR Master Mix
      7.5μl
      DNA(来自步骤B3)
      2μl
      链接器L(10μM)
      0.5μl
      32 P引物(来自步骤C1)
      2μl
      DMSO
      0.5μl
      H 2 O
      2.5μl
    3. 运行以下"Touchdown"PCR程序:
      变性94℃5分钟
      94℃30秒,65℃30秒,72℃30秒; x 3个周期
      94℃30秒,64℃30秒,72℃30秒; x 3个周期
      94℃30秒,63℃30秒,72℃30秒; x 3个周期
      94℃30秒,62℃30秒,72℃30秒; x 3个周期
      94℃30秒,61℃30秒,72℃30秒; x 3个周期
      94℃30秒,60℃30秒,72℃30秒; x 15个周期
      完成扩展72°C 10分钟

  4. 凝胶电泳
    1. 准备6%变性尿素 - 聚丙烯酰胺凝胶(参见食谱)
    2. 组装电泳并在250℃下预先在1×TBE中预处理凝胶30分钟。预运行允许凝胶/缓冲系统平衡,并将凝胶加热至45-55℃的工作温度。
    3. 预运行后,使用注射器用新鲜缓冲液冲洗冲洗凝胶袋。
    4. 将等体积的甲酰胺加载缓冲液加入到LM-PCR反应(来自步骤C3)中,并在95℃下孵育5分钟,然后在冰上快速冷却。该步骤应在凝胶预运行期间进行,以使LM-PCR /加载缓冲液混合物在冰上同时冲洗凝胶袋。因此,预运行和样品加载之间的时间被最小化以避免冷却加载5μl的LM-PCR /加载缓冲液混合物。重新开始电泳并运行1.5-2小时。
    5. 拆卸凝胶装置,将凝胶放在Whatman纸上,用塑料包装盖住相对的凝胶面。凝胶的哪一边面朝下都没关系。然而,夹住凝胶的一个上角以确定干燥后的位置是有帮助的。
    6. 在80℃下真空干燥凝胶1-2小时
    7. 在磷光体成像板上暴露凝胶过夜
    8. 使用荧光成像仪扫描屏幕,并使用适当的软件(例如,AIDA Image Analyzer)量化表示不同核小体位置的条带。

数据分析

检测来自代表不同核小体位置的LM-PCR产物的放射性信号可进行相对的半定量分析。用适当的软件量化信号强度,并将它们在一个样品/泳道内相互关联。例如,对于rDNA启动子,NucU和NucD 两个核小体构型,我们使用NucU/NucD 比来比较不同的生理条件(Zhao等,,2016a和2016b)。使用核小体位置的内部比例进行比较而不是比较样本之间的相同位置减少了处理样品之间变化的影响,从而提供了更强大的结果。通过重复LM-PCR至少两次(技术重复)和通过使用单独制备的核小体DNA样品(生物重复)至少三次进行整个程序来验证数据的重复性。此外,在LM-PCR中使用不同的基因特异性引物也是验证结果的好方法。如果比较两个不同处理的细胞样本之间的核小体位置的内部比例,则使用配对的双尾Student's t检验来计算统计学显着性。

食谱

  1. 渗透缓冲液
    150 mM蔗糖
    80 mM KCl
    35 mM HEPES(pH 7.4)
    5mM K 2 HPO 4
    5mM MgCl 2
    0.5mM CaCl 2
    0.05%L-α-溶血磷脂酰胆碱
  2. MNase消化缓冲液
    150 mM蔗糖
    50 mM NaCl
    50mM Tris-HCl(pH7.4)
    2mM CaCl 2
    50 U/ml MNase
  3. TE缓冲区
    20mM Tris-HCl(pH8.0)
    2 mM EDTA
  4. 10x TBE缓冲区(1 L)
    108克Tris碱
    55克硼酸
    40ml的0.5M EDTA(pH8.0)
  5. 甲酰胺加载缓冲液
    95%甲酰胺
    0.025%二甲苯氰胺
    0.025%溴酚蓝
    18 mM EDTA
    0.025%SDS
  6. 变性聚丙烯酰胺凝胶(6%)

致谢

我们感谢Ingrid Grummt对手稿的意见。这项工作由图林根国家计划(图尔根研究部(TMWWDG),德意志集团1036),"细胞网络(2014年EcTop调查报告)"和巴登的图林根国家计划ProExzellenz(RegenerAging-FSU-I-03/14) - 符腾堡州基金会。
该协议从以前的工作(Li等人,2006; Zhao等人,2016a和2016b)进行了改编。

参考

  1. Bell O,Tiwari VK,Thoma NH,Schubeler D(2011)。  基因组可及性的决定因素和动态。 Nat Rev Genet 12(8):554-564
  2. Hughes,AL和Rando,OJ(2014)。机制底层核小体定位。 Annu Rev Biophys 43:41-63。
  3. Li,J.,Langst,G。和Grummt,I.(2006)。 NoRC依赖的核小体定位沉默rRNA基因。 EMBO J 25(24):5735-5741。
  4. McPherson,CE,Shim,EY,Friedman,DS和Zaret,KS(1993)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/8402920 "target ="_ blank">存在于精确定位的核小体阵列中的活性组织特异性增强子和结合转录因子。细胞<75>(2):387-398。
  5. Soutoglou,E.和Talianidis,I。(2002)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/11884757"target ="_ blank" >在分化诱导的基因激活期间PIC组装和染色质重塑的协调。 科学 295(5561):1901-1904。
  6. Tsompana,M. and Buck,MJ(2014)。  染色质可及性:进入基因组的窗口。表观遗传学染色质 7(1):33
  7. Xie,W.,Ling,T.,Zhou,Y.,Feng,W.,Zhu,Q.,Stunnenberg,HG,Grummt,I. and Tao,W。(2012)。< a class = -insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/22570494"target ="_ blank">染色质重塑复合物NuRD建立以二价组蛋白修饰和改变的核小体为特征的rRNA基因的稳态位置。 Proc Natl Acad Sci USA 109(21):8161-8166。
  8. Zhao,Z.,Dammert,MA,Grummt,I。和Bierhoff,H。(2016a)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pnP/26904956"target ="_ blank"> lncRNA诱导的核小体重新定位强化了在低应力时rRNA基因的转录抑制。细胞Rep 14(8):1876-1882。 >
  9. Zhao,Z.,Dammert,MA,Hoppe,S.,Bierhoff,H。和Grummt,I.(2016b)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm热激冲击通过TIF-IA的失活和核小体定位中的nncRNA依赖性变化来抑制rRNA合成。核酸研究 44 (17):8144-8152。
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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: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Zhao, Z. and Bierhoff, H. (2017). Nucleosome Positioning Assay. Bio-protocol 7(10): e2285. DOI: 10.21769/BioProtoc.2285.
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