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Peer-reviewed

Nucleosome Positioning Assay

核小体定位分析实验

ZZ Zhongliang Zhao
HB Holger Bierhoff

发布: 2017年05月20日第7卷第10期 DOI: 10.21769/BioProtoc.2285 浏览次数: 11366

评审: Antoine de MorreeXiaoyi ZhengToshitsugu Fujita

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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) search Nucleosome positioning (核小体定位) search Micrococcal nuclease (微球菌核酸酶) search LM-PCR (LM-PCR) search

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

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文章信息

版权信息

© 2017 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Zhao, Z. and Bierhoff, H. (2017). Nucleosome Positioning Assay. Bio-protocol 7(10): e2285. DOI: 10.21769/BioProtoc.2285.

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分类

癌症生物学 > 通用技术 > 生物化学试验

分子生物学 > DNA > DNA 结构

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