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PMID: 17101440 Published · ppublish English Comparative Study Evaluation Study Journal Article Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't

Accelerated nuclei preparation and methods for analysis of histone modifications in yeast.

Methods (San Diego, Calif.) ·Vol. 40 ·No. 4 ·2006-12-00 ·Pages 296-302

Kizer KO, Xiao T, Strahl BD

Abstract

The continuing identification of new histone post-translational modifications and ongoing discovery of their roles in nuclear processes has increased the demand for quick, efficient, and precise methods for their analysis. In the budding yeast Saccharomyces cerevisiae, a variety of methods exist for the characterization of histone modifications on a global scale. However, a wide gap in preparation time and histone purity exists between the most widely used extraction methods, which include a simple whole cell extraction (WCE) and an intensive histone extraction. In this work we evaluate various published WCE buffers for their relative effectiveness in the detection of histone modifications by Western blot analysis. We also present a precise, yet time-efficient method for the detection of subtle changes in histone modification levels. Lastly, we present a protocol for the rapid small-scale purification of nuclei that improves the performance of antibodies that do not work efficiently in WCE. These new methods are ideal for the analysis of histone modifications and could be applied to the analysis and improved detection of other nuclear proteins.

MeSH Terms
Buffers Cell Fractionation/methods Cell Nucleus/metabolism Gene Expression Regulation, Fungal Histone Code Histones/metabolism Karyometry/methods Nuclear Proteins/isolation & purification,metabolism Protein Processing, Post-Translational/physiology Saccharomyces cerevisiae/metabolism Saccharomyces cerevisiae Proteins/metabolism Titrimetry/methods
Chemicals
Buffers Histones Nuclear Proteins Saccharomyces cerevisiae Proteins
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Kizer Kelby O
Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, NC 27599, USA.
Xiao Tiaojiang
Strahl Brian D
References (38)
38 references, click to expand
  1. Repression domain of the yeast global repressor Tup1 interacts directly with histones H3 and H4.
    Genes Dev. 1996 May 15;10(10):1247-59 PMID: 8675011
  2. Transcriptional silencing in yeast is associated with reduced nucleosome acetylation.
    Genes Dev. 1993 Apr;7(4):592-604 PMID: 8458576
  3. A novel domain in Set2 mediates RNA polymerase II interaction and couples histone H3 K36 methylation with transcript elongation.
    Mol Cell Biol. 2005 Apr;25(8):3305-16 PMID: 15798214
  4. Histone H2B ubiquitylation controls processive methylation but not monomethylation by Dot1 and Set1.
    Mol Cell. 2005 Jul 22;19(2):271-7 PMID: 16039595
  5. Cross-talking histones: implications for the regulation of gene expression and DNA repair.
    Biochem Cell Biol. 2005 Aug;83(4):460-7 PMID: 16094449
  6. BUR kinase selectively regulates H3 K4 trimethylation and H2B ubiquitylation through recruitment of the PAF elongation complex.
    Curr Biol. 2005 Aug 23;15(16):1487-93 PMID: 16040246
  7. Molecular regulation of histone H3 trimethylation by COMPASS and the regulation of gene expression.
    Mol Cell. 2005 Sep 16;19(6):849-56 PMID: 16168379
  8. Global metabolite analysis of yeast: evaluation of sample preparation methods.
    Yeast. 2005 Oct 30;22(14):1155-69 PMID: 16240456
  9. The Bur1/Bur2 complex is required for histone H2B monoubiquitination by Rad6/Bre1 and histone methylation by COMPASS.
    Mol Cell. 2005 Nov 23;20(4):589-99 PMID: 16307922
  10. Histone demethylation by a family of JmjC domain-containing proteins.
    Nature. 2006 Feb 16;439(7078):811-6 PMID: 16362057
  11. Leaving a mark: the many footprints of the elongating RNA polymerase II.
    Curr Opin Genet Dev. 2006 Apr;16(2):184-90 PMID: 16503129
  12. The BUR1 cyclin-dependent protein kinase is required for the normal pattern of histone methylation by SET2.
    Mol Cell Biol. 2006 Apr;26(8):3029-38 PMID: 16581778
  13. The Spt4p subunit of yeast DSIF stimulates association of the Paf1 complex with elongating RNA polymerase II.
    Mol Cell Biol. 2006 Apr;26(8):3135-48 PMID: 16581788
  14. Yeast Hsl7 (histone synthetic lethal 7) catalyses the in vitro formation of omega-N(G)-monomethylarginine in calf thymus histone H2A.
    Biochem J. 2006 May 1;395(3):563-70 PMID: 16426232
  15. Chromatin modifications by methylation and ubiquitination: implications in the regulation of gene expression.
    Annu Rev Biochem. 2006;75:243-69 PMID: 16756492
  16. The transcriptional repressor JHDM3A demethylates trimethyl histone H3 lysine 9 and lysine 36.
    Nature. 2006 Jul 20;442(7100):312-6 PMID: 16732292
  17. Methylation of histone H3 at lysine 4 is highly conserved and correlates with transcriptionally active nuclei in Tetrahymena.
    Proc Natl Acad Sci U S A. 1999 Dec 21;96(26):14967-72 PMID: 10611321
  18. The language of covalent histone modifications.
    Nature. 2000 Jan 6;403(6765):41-5 PMID: 10638745
  19. Histone acetylation and an epigenetic code.
    Bioessays. 2000 Sep;22(9):836-45 PMID: 10944586
  20. Methylation of histone H4 at arginine 3 occurs in vivo and is mediated by the nuclear receptor coactivator PRMT1.
    Curr Biol. 2001 Jun 26;11(12):996-1000 PMID: 11448779
  21. Transcription regulation by histone methylation: interplay between different covalent modifications of the core histone tails.
    Genes Dev. 2001 Sep 15;15(18):2343-60 PMID: 11562345
  22. Histone H3 lysine 4 methylation is mediated by Set1 and required for cell growth and rDNA silencing in Saccharomyces cerevisiae.
    Genes Dev. 2001 Dec 15;15(24):3286-95 PMID: 11751634
  23. Kin28 is found within TFIIH and a Kin28-Ccl1-Tfb3 trimer complex with differential sensitivities to T-loop phosphorylation.
    Mol Cell Biol. 2002 Mar;22(5):1288-97 PMID: 11839796
  24. The many faces of histone lysine methylation.
    Curr Opin Cell Biol. 2002 Jun;14(3):286-98 PMID: 12067650
  25. Methylation of H3-lysine 79 is mediated by a new family of HMTases without a SET domain.
    Curr Biol. 2002 Jun 25;12(12):1052-8 PMID: 12123582
  26. Gene silencing: trans-histone regulatory pathway in chromatin.
    Nature. 2002 Aug 1;418(6897):498 PMID: 12152067
  27. Active genes are tri-methylated at K4 of histone H3.
    Nature. 2002 Sep 26;419(6905):407-11 PMID: 12353038
  28. Phosphorylation of RNA polymerase II CTD regulates H3 methylation in yeast.
    Genes Dev. 2003 Mar 1;17(5):654-63 PMID: 12629047
  29. In vivo assays to study histone ubiquitylation.
    Methods. 2003 Sep;31(1):59-66 PMID: 12893174
  30. Methylation of histone H3 K4 mediates association of the Isw1p ATPase with chromatin.
    Mol Cell. 2003 Nov;12(5):1325-32 PMID: 14636589
  31. Yeast chromatin. Preparation from isolated nuclei, histone composition and transcription capacity.
    Eur J Biochem. 1973 Feb 15;33(1):123-30 PMID: 4570761
  32. Structure of chromatin.
    Annu Rev Biochem. 1977;46:931-54 PMID: 332067
  33. Nucleosome structure.
    Annu Rev Biochem. 1980;49:1115-56 PMID: 6996562
  34. Saccharomyces cerevisiae plasmid, Scp or 2 mum: intracellular distribution, stability and nucleosomal-like packaging.
    Nucleic Acids Res. 1980 Aug 11;8(15):3371-91 PMID: 6255414
  35. Nuclease hypersensitive regions with adjacent positioned nucleosomes mark the gene boundaries of the PHO5/PHO3 locus in yeast.
    EMBO J. 1986 Oct;5(10):2681-7 PMID: 3023055
  36. Isolation and characterization of histones.
    Methods Enzymol. 1989;170:431-523 PMID: 2549339
  37. Higher-order structure of Saccharomyces cerevisiae chromatin.
    Proc Natl Acad Sci U S A. 1989 Nov;86(21):8266-70 PMID: 2682643
  38. Role of protein methylation in regulation of transcription.
    Endocr Rev. 2005 Apr;26(2):147-70 PMID: 15479858
Article Info
Journal
Methods (San Diego, Calif.)
Abbr.
Methods
ISSN
1046-2023
DOI
10.1016/j.ymeth.2006.06.022
Published
2006-12-00
Pages
296-302
Language
English
Region
United States
NLM ID
9426302
PMCID
PMC1698964
Subset
IM
Grants
NIGMS NIH HHS · R01 GM068088 · United States
NIGMS NIH HHS · GM68088 · United States
External Links
PubMed source DOI: 10.1016/j.ymeth.2006.06.022 PMC Full Text View journal details
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