Genome‐Wide Analysis of Nucleosome Positions, Occupancy, and Accessibility in Yeast: Nucleosome Mapping, High‐Resolution Histone ChIP, and NCAM

Jairo Rodriguez1, Jeffrey N. McKnight1, Toshio Tsukiyama2

1 These authors contributed equally to this work, 2 Basic Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, Washington
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 21.28
DOI:  10.1002/0471142727.mb2128s108
Online Posting Date:  October, 2014
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Abstract

Because histones bind DNA very tightly, the location on DNA and the level of occupancy of a given DNA sequence by nucleosomes can profoundly affect accessibility of non‐histone proteins to chromatin, affecting virtually all DNA‐dependent processes, such as transcription, DNA repair, DNA replication and recombination. Therefore, it is often necessary to determine positions and occupancy of nucleosomes to understand how DNA‐dependent processes are regulated. Recent technological advances made such analyses feasible on a genome‐wide scale at high resolution. In addition, we have recently developed a method to measure nuclease accessibility of nucleosomes on a global scale. This unit describes methods to map nucleosome positions, to determine nucleosome density, and to determine nuclease accessibility of nucleosomes using deep sequencing. Curr. Protoc. Mol. Biol. 108:21.28.1‐21.28.16. © 2014 by John Wiley & Sons, Inc.

Keywords: nucleosome positioning; nucleosome occupancy; chromatin accessibility; deep sequencing; genome‐wide analysis

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Determining Nucleosome Positions Using Micrococcal Nuclease Digestion and High‐Throughput Sequencing (MNase‐Seq)
  • Basic Protocol 2: Chromatin Immunoprecipitation of Histone H3 from Yeast Cells
  • Basic Protocol 3: Normalized Chromatin Accessibility to MNase (NCAM) to Determine Nucleosome Occupancy
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Determining Nucleosome Positions Using Micrococcal Nuclease Digestion and High‐Throughput Sequencing (MNase‐Seq)

  Materials
  • Overnight, saturated culture of S. cerevisiae
  • 37% formaldehyde
  • 2.5 M glycine
  • Spheroblast solution (see recipe)
  • 100T zymolyase (AMSBIO, cat. no. 120493‐1)
  • MNase digestion buffer (see recipe)
  • Exonuclease III (NEB, cat. no. M0206)
  • 20 U/µl micrococcal nuclease (Worthington, cat. no. LS004798), prepared in 10 mM Tris, pH 7.4, store at −80°C
  • MNase stop buffer (see recipe)
  • 20 mg/ml Proteinase K
  • 25:24:1 Phenol:Chloroform:Isoamyl Alcohol (Sigma‐Aldrich)
  • 3 M sodium acetate, pH 5.2
  • 20 mg/ml glycogen
  • Ice‐cold ethanol (100% and 70%)
  • NEBuffers 2 and 3
  • 10 mg/ml RNase A (Sigma‐Aldrich, cat. no. 9001‐99‐4)
  • QIAquick PCR purification kit (Qiagen), includes buffer EB
  • Alkaline phosphatase (NEB, cat. no. M0290S)
  • QIAquick gel extraction kit (Qiagen, cat. no. 28704)
  • TruSeq DNA LT Sample Prep Kit (Illumina, cat. no. RS‐122‐2001), includes resuspension buffer, end repair mix, A‐tail mix, ligation mix, adapters, stop ligation buffer, PCR primer cocktail, and PCR master mix.
  • MinElute PCR Purification Kit (Qiagen, cat. no. 28004)
  • Low‐melt agarose (GeneMate, cat. no. E3109‐125)
  • Thermal cycler
  • Additional reagents and equipment for growing and manipulating yeast (units 13.1 and 13.2)

Basic Protocol 2: Chromatin Immunoprecipitation of Histone H3 from Yeast Cells

  Materials
  • Yeast cells
  • YPD (see recipe)
  • Fix solution, 10× (see recipe)
  • 2.5 M glycine
  • Ice‐cold TBS (see recipe)
  • ChIP breaking buffer (see recipe)
  • 0.5‐mm, acid‐washed glass beads (Sigma, cat. no. G8772)
  • FA buffer (see recipe), ice cold and room temperature
  • Protease inhibitor cocktail, 100× (see recipe)
  • Protein G‐coupled Dynabeads (Life Technologies, cat. no. 2015‐08)
  • 0.1 M sodium phosphate buffer, pH 7.0 (see recipe)
  • Anti‐C‐terminal histone H3 antibody (Active Motif, cat. no. 39163)
  • PBST buffer (see recipe)
  • FA‐HS buffer (see recipe)
  • RIPA buffer (see recipe)
  • Stop buffer, 2× (see recipe)
  • 10 mg/ml Proteinase K
  • 10 mg/ml RNAse A
  • TBE, 10× (see recipe)
  • MinElute PCR purification kit (Qiagen, cat. no. 28004)
  • 15‐ and 50‐ml polypropylene conical tubes
  • Refrigerated swing‐bucket centrifuge
  • 2‐ml screw‐cap flat bottom microcentrifuge tubes (Sarstedt, cat. no. 72.693.005)
  • Mini beadbeater 96 (Biospec)
  • 20‐G needles
  • Lid‐lock clips (Sorenson, cat. no. 11870)
  • 15‐ml polystyrene conical tubes
  • Bioruptor sonication bath with cool bath and pump (Diagenode, model UCD‐200)
  • 1.5‐ml microcentrifuge tubes
  • 1.5‐ml siliconized microcentrifuge tubes (RPI, cat. no. 145450)
  • Refrigerated benchtop centrifuge
  • Magnetic tube rack (Millipore, cat. no. LSKMAGS08)
  • Thermomixer (Eppendorf, cat. no. 05‐400‐205)
  • Rotator for microcentrifuge tubes (Cole Parmer, model 7637‐01)
  • 65°C incubator
  • Additional reagents and equipment for growing and manipulating yeast (units 13.1 and 13.2)
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Figures

Videos

Literature Cited

Literature Cited
  Cheadle, C., Vawter, M.P., Freed, W.J., and Becker, K.G. 2003. Analysis of microarray data using Z score transformation. J. Mol. Diagn. 5:73–81.
  Lee, W., Tillo, D., Bray, N., Morse, R.H., Davis, R.W., Hughes, T.R., and Nislow, C. 2007. A high‐resolution atlas of nucleosome occupancy in yeast. Nat. Genet. 39:1235–1244.
  Nagalakshmi, U., Wang, Z., Waern, K., Shou, C., Raha, D., Gerstein, M., and Snyder, M. 2008. The transcriptional landscape of the yeast genome defined by RNA sequencing. Science 320:1344–1349.
  Nikitina, T., Wang, D., Gomberg, M., Grigoryev, S.A., and Zhurkin, V.B. 2013. Combined micrococcal nuclease and exonuclease III digestion reveals precise positions of the nucleosome core/linker junctions: Implications for high‐resolution nucleosome mapping. J. Mol. Biol. 425:1946–1960.
  Rizzo, J.M., Bard, J.E., and Buck, M.J. 2012. Standardized collection of MNase‐seq experiments enables unbiased dataset comparisons. BMC Mol. Biol. 13:15.
  Rodriguez, J. and Tsukiyama, T. 2013. ATR‐like kinase Mec1 facilitates both chromatin accessibility at DNA replication forks and replication fork progression during replication stress. Genes Dev. 27:74–86.
  Shivaswamy, S., Bhinge, A., Zhao, Y., Jones, S., Hirst, M., and Iyer, V.R. 2008. Dynamic remodeling of individual nucleosomes across a eukaryotic genome in response to transcriptional perturbation. PLOS Biol. 6:e65.
  Weiner, A., Hughes, A., Yassour M., Rando, O.J., and Friedman, N. 2010. High‐resolution nucleosome mapping reveals transcription‐dependent promoter packaging. Genome Res. 20:90–100.
  Yuan, G.C., Liu, Y.J., Dion, M.F., Slack, M.D., Wu, L.F., Altschuler, S.J., and Rando, O.J. 2005. Genome‐scale identification of nucleosome positions in S. cerevisiae. Science 309:626–630.
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