Role of the conserved Sir3-BAH domain in nucleosome binding and silent chromatin assembly.

Silent chromatin domains in Saccharomyces cerevisiae represent examples of epigenetically heritable chromatin. The formation of these domains involves the recruitment of the SIR complex, composed of Sir2, Sir3, and Sir4, followed by iterative cycles of NAD-dependent histone deacetylation and spreading of SIR complexes over adjacent chromatin domains. We show here that the conserved bromo-adjacent homology (BAH) domain of Sir3 is a nucleosome- and histone-tail-binding domain and that its binding to nucleosomes is regulated by residues in the N terminus of histone H4 and the globular domain of histone H3 on the exposed surface of the nucleosome. Furthermore, using a partially purified system containing nucleosomes, the three Sir proteins, and NAD, we observe the formation of SIR-nucleosome filaments with a diameter of less than 20 nm. Together, these observations suggest that the SIR complex associates with an extended chromatin fiber through interactions with two different regions in the nucleosome.

[1]  D. Moazed,et al.  Common themes in mechanisms of gene silencing. , 2001, Molecular cell.

[2]  D. Tremethick,et al.  Higher-Order Structures of Chromatin: The Elusive 30 nm Fiber , 2007, Cell.

[3]  P. Philippsen,et al.  Additional modules for versatile and economical PCR‐based gene deletion and modification in Saccharomyces cerevisiae , 1998, Yeast.

[4]  M. Gartenberg,et al.  Yeast heterochromatin is a dynamic structure that requires silencers continuously. , 2000, Genes & development.

[5]  R. Sternglanz,et al.  Silent information regulator 2 family of NAD- dependent histone/protein deacetylases generates a unique product, 1-O-acetyl-ADP-ribose. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[6]  S. Gygi,et al.  Inhibition of homologous recombination by a cohesin-associated clamp complex recruited to the rDNA recombination enhancer. , 2006, Genes & development.

[7]  D. Moazed,et al.  Heterochromatin and Epigenetic Control of Gene Expression , 2003, Science.

[8]  K. Struhl,et al.  Lysine-79 of histone H3 is hypomethylated at silenced loci in yeast and mammalian cells: A potential mechanism for position-effect variegation , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[9]  K. Luo,et al.  SIR2 and SIR4 interactions differ in core and extended telomeric heterochromatin in yeast. , 1997, Genes & development.

[10]  Zhonggang Hou,et al.  Structure of the Sir3 protein bromo adjacent homology (BAH) domain from S. cerevisiae at 1.95 Å resolution , 2006, Protein science : a publication of the Protein Society.

[11]  T. Kouzarides.  Chromatin Modifications and Their Function , 2007, Cell.

[12]  C. Allis,et al.  Histone and chromatin cross-talk. , 2003, Current opinion in cell biology.

[13]  T. Richmond,et al.  X-ray structure of a tetranucleosome and its implications for the chromatin fibre , 2005, Nature.

[14]  Jasper Rine,et al.  Ordered nucleation and spreading of silenced chromatin in Saccharomyces cerevisiae. , 2002, Molecular biology of the cell.

[15]  Philip R. Gafken,et al.  Dot1p Modulates Silencing in Yeast by Methylation of the Nucleosome Core , 2002, Cell.

[16]  J. Broach,et al.  DNA in transcriptionally silent chromatin assumes a distinct topology that is sensitive to cell cycle progression , 1997, Molecular and cellular biology.

[17]  T. Jenuwein.  Re-SET-ting heterochromatin by histone methyltransferases. , 2001, Trends in cell biology.

[18]  A. Kimura,et al.  Chromosomal gradient of histone acetylation established by Sas2p and Sir2p functions as a shield against gene silencing , 2002, Nature Genetics.

[19]  Jasper Rine,et al.  Silent information regulator protein complexes in Saccharomyces cerevisiae: a SIR2/SIR4 complex and evidence for a regulatory domain in SIR4 that inhibits its interaction with SIR3. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[20]  M. Pazin,et al.  Histone H4-K16 Acetylation Controls Chromatin Structure and Protein Interactions , 2006, Science.

[21]  J. Boeke,et al.  Chemistry of gene silencing: the mechanism of NAD+-dependent deacetylation reactions. , 2001, Biochemistry.

[22]  J. Connelly,et al.  Structure and Function of the Saccharomyces cerevisiae Sir3 BAH Domain , 2006, Molecular and Cellular Biology.

[23]  L. Guarente,et al.  Transcriptional silencing and longevity protein Sir2 is an NAD-dependent histone deacetylase , 2000, Nature.

[24]  I. Herskowitz,et al.  Four genes responsible for a position effect on expression from HML and HMR in Saccharomyces cerevisiae. , 1987, Genetics.

[25]  Thomas Walz,et al.  Assembly of the SIR Complex and Its Regulation by O-Acetyl-ADP-Ribose, a Product of NAD-Dependent Histone Deacetylation , 2005, Cell.

[26]  J. Boeke,et al.  A core nucleosome surface crucial for transcriptional silencing , 2002, Nature Genetics.

[27]  B. Morgan,et al.  Genetic analysis of histone H4: essential role of lysines subject to reversible acetylation. , 1990, Science.

[28]  J. Davie,et al.  Histone H3 lysine 4 methylation is mediated by Set1 and required for cell growth and rDNA silencing in Saccharomyces cerevisiae. , 2001, Genes & development.

[29]  Tony Kouzarides,et al.  Methylation of H3 Lysine 4 at Euchromatin Promotes Sir3p Association with Heterochromatin* , 2004, Journal of Biological Chemistry.

[30]  Renato Paro,et al.  Epigenetic regulation of cellular memory by the Polycomb and Trithorax group proteins. , 2004, Annual review of genetics.

[31]  Jasper Rine,et al.  The establishment, inheritance, and function of silenced chromatin in Saccharomyces cerevisiae. , 2003, Annual review of biochemistry.

[32]  C. Allis,et al.  Translating the Histone Code , 2001, Science.

[33]  Kevin Struhl,et al.  Lysine methylation within the globular domain of histone H3 by Dot1 is important for telomeric silencing and Sir protein association. , 2002, Genes & development.

[34]  R. Sternglanz,et al.  The silencing protein SIR2 and its homologs are NAD-dependent protein deacetylases. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[35]  B. Turner,et al.  Essential and redundant functions of histone acetylation revealed by mutation of target lysines and loss of the Gcn5p acetyltransferase , 1998, The EMBO journal.

[36]  Nevan J. Krogan,et al.  COMPASS, a Histone H3 (Lysine 4) Methyltransferase Required for Telomeric Silencing of Gene Expression* , 2002, The Journal of Biological Chemistry.

[37]  M. Grunstein,et al.  Genetic evidence for an interaction between SIR3 and histone H4 in the repression of the silent mating loci in Saccharomyces cerevisiae. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[38]  S. Elgin,et al.  Epigenetic Codes for Heterochromatin Formation and Silencing Rounding up the Usual Suspects , 2002, Cell.

[39]  K. Struhl,et al.  Dynamics of global histone acetylation and deacetylation in vivo: rapid restoration of normal histone acetylation status upon removal of activators and repressors. , 2002, Genes & development.

[40]  L. Breeden,et al.  Characterization of a “silencer” in yeast: A DNA sequence with properties opposite to those of a transcriptional enhancer , 1985, Cell.

[41]  Steven P. Gygi,et al.  Budding Yeast Silencing Complexes and Regulation of Sir2 Activity by Protein-Protein Interactions , 2004, Molecular and Cellular Biology.

[42]  G. Karpen,et al.  The case for epigenetic effects on centromere identity and function. , 1997, Trends in genetics : TIG.

[43]  B. Cairns,et al.  Two functionally distinct forms of the RSC nucleosome-remodeling complex, containing essential AT hook, BAH, and bromodomains. , 1999, Molecular cell.

[44]  R. Kornberg,et al.  Twenty-Five Years of the Nucleosome, Fundamental Particle of the Eukaryote Chromosome , 1999, Cell.

[45]  W. Herr,et al.  Ethidium bromide provides a simple tool for identifying genuine DNA-independent protein associations. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[46]  P. Georgel,et al.  Sir3-dependent assembly of supramolecular chromatin structures in vitro , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[47]  Conrad C. Huang,et al.  UCSF Chimera—A visualization system for exploratory research and analysis , 2004, J. Comput. Chem..

[48]  Oscar M. Aparicio,et al.  Modifiers of position effect are shared between telomeric and silent mating-type loci in S. cerevisiae , 1991, Cell.

[49]  D. Moazed,et al.  Coupling of histone deacetylation to NAD breakdown by the yeast silencing protein Sir2: Evidence for acetyl transfer from substrate to an NAD breakdown product. , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[50]  Lisa Milne,et al.  Acetylation of the Yeast Histone H4 N Terminus Regulates Its Binding to Heterochromatin Protein SIR3* , 2002, The Journal of Biological Chemistry.

[51]  J. Broach,et al.  Transcriptional silencing in yeast is associated with reduced nucleosome acetylation. , 1993, Genes & development.

[52]  Hiten D. Madhani,et al.  Conserved Histone Variant H2A.Z Protects Euchromatin from the Ectopic Spread of Silent Heterochromatin , 2003, Cell.

[53]  Steven P. Gygi,et al.  Steps in Assembly of Silent Chromatin in Yeast: Sir3-Independent Binding of a Sir2/Sir4 Complex to Silencers and Role for Sir2-Dependent Deacetylation , 2002, Molecular and Cellular Biology.

[54]  K. Luo,et al.  Rap1-Sir4 binding independent of other Sir, yKu, or histone interactions initiates the assembly of telomeric heterochromatin in yeast. , 2002, Genes & development.

[55]  J. Connelly,et al.  Importance of the Sir3 N Terminus and Its Acetylation for Yeast Transcriptional Silencing , 2004, Genetics.

[56]  C. Nislow,et al.  SET1, a yeast member of the trithorax family, functions in transcriptional silencing and diverse cellular processes. , 1997, Molecular biology of the cell.

[57]  B. Séraphin,et al.  A generic protein purification method for protein complex characterization and proteome exploration , 1999, Nature Biotechnology.

[58]  Michael Grunstein,et al.  Sir2p and Sas2p opposingly regulate acetylation of yeast histone H4 lysine16 and spreading of heterochromatin , 2002, Nature Genetics.

[59]  J. Mornon,et al.  The BAH (bromo‐adjacent homology) domain: a link between DNA methylation, replication and transcriptional regulation , 1999, FEBS letters.

[60]  D. Moazed,et al.  A Nonhistone Protein-Protein Interaction Required for Assembly of the SIR Complex and Silent Chromatin , 2005, Molecular and Cellular Biology.

[61]  Stuart L. Schreiber,et al.  Methylation of histone H3 Lys 4 in coding regions of active genes , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[62]  T. Richmond,et al.  Crystal structure of the nucleosome core particle at 2.8 Å resolution , 1997, Nature.

[63]  S. Bell,et al.  The multidomain structure of Orc1 p reveals similarity to regulators of DNA replication and transcriptional silencing , 1995, Cell.

[64]  D. Shore,et al.  Evidence that a complex of SIR proteins interacts with the silencer and telomere-binding protein RAP1. , 1994, Genes & development.

[65]  R. Paro,et al.  The Polycomb protein shares a homologous domain with a heterochromatin-associated protein of Drosophila. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[66]  Kevin Struhl,et al.  Heterochromatin formation involves changes in histone modifications over multiple cell generations , 2005, The EMBO journal.

[67]  T. Richmond,et al.  Nucleosome Arrays Reveal the Two-Start Organization of the Chromatin Fiber , 2004, Science.

[68]  Andreas Hecht,et al.  Histone H3 and H4 N-termini interact with SIR3 and SIR4 proteins: A molecular model for the formation of heterochromatin in yeast , 1995, Cell.

[69]  B. Cairns,et al.  The Yaf9 Component of the SWR1 and NuA4 Complexes Is Required for Proper Gene Expression, Histone H4 Acetylation, and Htz1 Replacement near Telomeres , 2004, Molecular and Cellular Biology.

[70]  A. Klar,et al.  MAR1-a Regulator of the HMa and HMalpha Loci in SACCHAROMYCES CEREVISIAE. , 1979, Genetics.

[71]  M. Grunstein,et al.  Identification of a non‐basic domain in the histone H4 N‐terminus required for repression of the yeast silent mating loci. , 1992, The EMBO journal.

[72]  C. Allis,et al.  Histone acetyltransferases: preparation of substrates and assay procedures. , 1999, Methods in enzymology.

[73]  Ian M. Fingerman,et al.  Global Loss of Set1-mediated H3 Lys4 Trimethylation Is Associated with Silencing Defects in Saccharomyces cerevisiae* , 2005, Journal of Biological Chemistry.

[74]  G. Goodwin,et al.  Molecular cloning of polybromo, a nuclear protein containing multiple domains including five bromodomains, a truncated HMG-box, and two repeats of a novel domain. , 1996, Gene.