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A central challenge of the post-genomic era is to understand how the 30,000 to 40,000 unique genes in the human genome are selectively expressed or silenced to coordinate cellular growth and differentiation. The packaging of eukaryotic genomes in a complex of DNA, histones, and nonhistone proteins called chromatin provides a surprisingly sophisticated system that plays a critical role in controlling the flow of genetic information. This packaging system has evolved to index our genomes such that certain genes become readily accessible to the transcription machinery, while other genes are reversibly silenced. Moreover, chromatin-based mechanisms of gene regulation, often involving domains of covalent modifications of DNA and histones, can be inherited from one generation to the next. The heritability of chromatin states in the absence of DNA mutation has contributed greatly to the current excitement in the field of epigenetics.

The past 5 years have witnessed an explosion of new research on chromatin biology and biochemistry. Chromatin structure and function are now widely recognized as being critical to regulating gene expression, maintaining genomic stability, and ensuring faithful chromosome transmission. Moreover, links between chromatin metabolism and disease are beginning to emerge. The identification of altered DNA methylation and histone acetylase activity in human cancers, the use of histone deacetylase inhibitors in the treatment of leukemia, and the tumor suppressor activities of ATP-dependent chromatin remodeling enzymes are examples that likely represent just the tip of the iceberg.This 3-volume set of Methods in Enzymology provides nearly one hundred procedures covering the full range of tools—bioinformatics, structural biology, biophysics, biochemistry, genetics, and cell biology—employed in chromatin research. Volume 375 includes a histone database, methods for preparation of histones, histone variants, modified histones and defined chromatin segments, protocols for nucleosome reconstitution and analysis, and cytological methods for imaging chromatin functions in vivo. Volume 376 includes electron microscopy and biophysical protocols for visualizing chromatin and detecting chromatin interactions, enzymological assays for histone modifying enzymes, and immunochemical protocols for the in situ detection of histone modifications and chromatin proteins. Volume 377 includes genetic assays of histones and chromatin regulators, methods for the preparation and analysis of histone modifying and ATP-dependent chromatin remodeling enzymes, and assays for transcription and DNA repair on chromatin templates. We are exceedingly grateful to the very large number of colleagues representing the field’s leading laboratories, who have taken the time and effort to make their technical expertise available in this series.

As such, the field is attracting new investigators who enter with little first hand experience with the standard assays used to dissect chromatin structure and function. In addition, even seasoned veterans are overwhelmed by the rapid introduction of new chromatin technologies. Accordingly, we sought to bring together a useful ‘‘go-to’’ set of chromatin-based methods that would update and complement two previous publications in this series, Volume 170 (Nucleosomes) and Volume 304 (Chromatin). While many of the classic protocols in those volumes remain as timely now as when they were written, it is our hope the present series will fill in the gaps for the next several years.

Ed. Carl Wu, C. David Allis

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Table of Contents

1. Functional analyses of chromatin modifications in yeast. (3-55)
   Jacobson SJ, Laurenson PM, Pillus L.
2. Genetic analysis of chromatin remodeling using budding yeast as a model. (55-60)
   Steger DJ, O’Shea EK.
3. Introduction to Trx-G and Pc-G genes. (61-70)
   Kennison JA.
4. Genetic and cytological analysis of Drosophila chromatin-remodeling factors. (70-85)
   Corona DF, Armstrong JA, Tamkun JW.
5. Genetic analysis of H1 linker histone subtypes and their functions in mice. (85-107)
   Fan Y, Skoultchi AI.
6. The use of mass spectrometry for the analysis of histone modifications. (111-30)
   Bonaldi T, Regula JT, Imhof A.
7. Histone modification patterns during gene activation. (130-53)
   Lo WS, Henry KW, Schwartz MF, Berger SL.
8. Identification and analysis of native HAT complexes. (154-67)
   McMahon SJ, Doyon Y, Cote J, Grant PA.
9. Histone deacetylases: purification of the enzymes, substrates, and assay conditions. (167-79)
   Rezai-Zadeh N, Tsai SC, Wen YD, Yao YL, Yang WM, Seto E.
10. Measurement of mammalian histone deacetylase activity. (180-96)
    Verdin E, Dequiedt F, Fischle W, Frye R, Marshall B, North B.
11. Analysis of histone phosphorylation: coupling intracellular signaling to chromatin remodeling. (197-212)
    Loury R, Sassone-Corsi P.
12. Purification of histone methyltransferases from HeLa cells. (213-26)
    Fang J, Wang H, Zhang Y.
13. Global proteomic analysis of S. cerevisiae (GPS) to identify proteins required for histone modifications. (227-34)
    Schneider J, Dover J, Johnston M, Shilatifard A.
14. Purification of Sir2 proteins from yeast. (234-54)
    Gangadharan S, Ghidelli S, Kamakaka RT.
15. Purification and biochemical properties of the Drosophila TAC1 complex. (255-66)
    Petruk S, Sedkov Y, Smith ST, Krajewski W, Nakamura T, Canaani E, Croce CM, Mazo A.
16. Isolation and characterization of CHRASCH, a polycomb-containing silencing complex. (267-82)
    Huang DH, Chang YL.
17. Biochemical analysis of mammalian polycomb group protein complexes and the identification of genetic elements that block polycomb-mediated gene repression. (282-96)
    Sewalt RG, Kwaks TH, Hamer K, Otte AP.
18. Purification and functional analysis of the mammalian SWI/SNF-family of chromatin-remodeling complexes. (299-316)
    Chi T, Yan Z, Xue Y, Wang W.
19. Isolation and assay of the RSC chromatin-remodeling complex from Saccharomyces cerevisiae. (316-22)
    Lorch Y, Kornberg RD.
20. DNA translocation and nucleosome remodeling assays by the RSC chromatin remodeling complex. (322-43)
    Wittmeyer J, Saha A, Cairns B.
21. A nucleosome sliding assay for chromatin remodeling factors. (344-53)
    Eberharter A, Langst G, Becker PB.
22. Methods for analysis of nucleosome sliding by Drosophila NURF. (353-63)
    Hamiche A, Xiao H.
23. Methods for preparation and assays for Xenopus ISWI complexes. (364-75)
    Palmer MB, Elgar S, Wade PA.
24. Functional analysis of ISWI complexes in mammalian cells. (376-89)
    Bozhenok L, Poot R, Collins N, Varga-Weisz P.
25. Preparation and assays for mammalian ISWI complexes. (389-401)
    Barak OG, Shiekhattar R.
26. Preparation and analysis of the INO80 complex. (401-12)
    Shen X.
27. Assay of Z-DNA induction by chromatin remodeling factors. (412-20)
    Liu H, Zhao K.
28. Immuno-depletion and purification strategies to study chromatin-remodeling factors in vitro. (421-42)
    Chalkley GE, Verrijzer CP.
29. Transcription through the nucleosome by mRNA-producing RNA polymerases. (445-60)
    Walter W, Kashlev M, Studitsky VM.
30. Reconstitution and transcriptional analysis of chromatin in vitro. (460-74)
    An W, Roeder RG.
31. Techniques used to study transcription on chromatin templates. (474-99)
    Loyola A, He S, Oh S, McCafferty DG, Reinberg D.
32. Analysis of DNA repair on nucleosome templates. (499-507)
    Beard BC, Smerdon MJ.

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