Transcriptional activation via sequential histone H2B ubiquitylation and deubiquitylation, mediated by SAGA-associated Ubp8.

Gene activation and repression regulated by acetylation and deacetylation represent a paradigm for the function of histone modifications. We provide evidence that, in contrast, histone H2B monoubiquitylation and its deubiquitylation are both involved in gene activation. Substitution of the H2B ubiquitylation site at Lys 123 (K123) lowered transcription of certain genes regulated by the acetylation complex SAGA. Gene-associated H2B ubiquitylation was transient, increasing early during activation, and then decreasing coincident with significant RNA accumulation. We show that Ubp8, a component of the SAGA acetylation complex, is required for SAGA-mediated deubiquitylation of histone H2B in vitro. Loss of Ubp8 in vivo increased both gene-associated and overall cellular levels of ubiquitylated H2B. Deletion of Ubp8 lowered transcription of SAGA-regulated genes, and the severity of this defect was exacerbated by codeletion of the Gcn5 acetyltransferase within SAGA. In addition, disruption of either ubiquitylation or Ubp8-mediated deubiquitylation of H2B resulted in altered levels of gene-associated H3 Lys 4 methylation and Lys 36 methylation, which have both been linked to transcription. These results suggest that the histone H2B ubiquitylation state is dynamic during transcription, and that the sequence of histone modifications helps to control transcription.

[1]  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.

[2]  D. Moras,et al.  The Human TFIID Components TAFII135 and TAFII20 and the Yeast SAGA Components ADA1 and TAFII68 Heterodimerize to Form Histone-Like Pairs , 2000, Molecular and Cellular Biology.

[3]  A. Dudley,et al.  The Spt components of SAGA facilitate TBP binding to a promoter at a post-activator-binding step in vivo. , 1999, Genes & development.

[4]  J Seth Strattan,et al.  Nucleosomes unfold completely at a transcriptionally active promoter. , 2003, Molecular cell.

[5]  L. Murphy,et al.  Level of ubiquitinated histone H2B in chromatin is coupled to ongoing transcription. , 1990, Biochemistry.

[6]  John R. Yates,et al.  The Novel SLIK Histone Acetyltransferase Complex Functions in the Yeast Retrograde Response Pathway , 2002, Molecular and Cellular Biology.

[7]  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.

[8]  Kevin Struhl,et al.  The Rtf1 Component of the Paf1 Transcriptional Elongation Complex Is Required for Ubiquitination of Histone H2B* , 2003, Journal of Biological Chemistry.

[9]  M. Green,et al.  SAGA is an essential in vivo target of the yeast acidic activator Gal4p. , 2001, Genes & development.

[10]  Yi Zhang,et al.  Bre1, an E3 ubiquitin ligase required for recruitment and substrate selection of Rad6 at a promoter. , 2003, Molecular cell.

[11]  P. Grant,et al.  The something about silencing protein, Sas3, is the catalytic subunit of NuA3, a yTAF(II)30-containing HAT complex that interacts with the Spt16 subunit of the yeast CP (Cdc68/Pob3)-FACT complex. , 2000, Genes & development.

[12]  C. Brandl,et al.  TOM1p, a yeast hect-domain protein which mediates transcriptional regulation through the ADA/SAGA coactivator complexes. , 1998, Journal of molecular biology.

[13]  R. Conaway,et al.  Emerging Roles of Ubiquitin in Transcription Regulation , 2002, Science.

[14]  Steven P. Gygi,et al.  Association of the Histone Methyltransferase Set2 with RNA Polymerase II Plays a Role in Transcription Elongation* , 2002, The Journal of Biological Chemistry.

[15]  M. Johnston,et al.  The Paf1 complex is required for histone H3 methylation by COMPASS and Dot1p: linking transcriptional elongation to histone methylation. , 2003, Molecular cell.

[16]  B. Séraphin,et al.  The tandem affinity purification (TAP) method: a general procedure of protein complex purification. , 2001, Methods.

[17]  D. Sterner,et al.  SALSA, a variant of yeast SAGA, contains truncated Spt7, which correlates with activated transcription , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[18]  Andrew J. Link,et al.  Proteomics of the Eukaryotic Transcription Machinery: Identification of Proteins Associated with Components of Yeast TFIID by Multidimensional Mass Spectrometry , 2002, Molecular and Cellular Biology.

[19]  John R Yates,et al.  A Subset of TAFIIs Are Integral Components of the SAGA Complex Required for Nucleosome Acetylation and Transcriptional Stimulation , 1998, Cell.

[20]  A. Caudy,et al.  Regulation of Transcriptional Activation Domain Function by Ubiquitin , 2001, Science.

[21]  Kevin Struhl,et al.  Targeted recruitment of Set1 histone methylase by elongating Pol II provides a localized mark and memory of recent transcriptional activity. , 2003, Molecular cell.

[22]  B. Raboy,et al.  RAD6 gene product of Saccharomyces cerevisiae requires a putative ubiquitin protein ligase (E3) for the ubiquitination of certain proteins. , 1991, The Journal of biological chemistry.

[23]  R Ohba,et al.  Yeast Gcn5 functions in two multisubunit complexes to acetylate nucleosomal histones: characterization of an Ada complex and the SAGA (Spt/Ada) complex. , 1997, Genes & development.

[24]  Michael Grunstein,et al.  Requirement of Hos2 Histone Deacetylase for Gene Activity in Yeast , 2002, Science.

[25]  Gary D Bader,et al.  Systematic identification of protein complexes in Saccharomyces cerevisiae by mass spectrometry , 2002, Nature.

[26]  J. Yates,et al.  The Set2 Histone Methyltransferase Functions through the Phosphorylated Carboxyl-terminal Domain of RNA Polymerase II* , 2003, The Journal of Biological Chemistry.

[27]  Muyang Li,et al.  Crystal Structure of a UBP-Family Deubiquitinating Enzyme in Isolation and in Complex with Ubiquitin Aldehyde , 2002, Cell.

[28]  John R. Yates,et al.  The ADA Complex Is a Distinct Histone Acetyltransferase Complex in Saccharomyces cerevisiae , 1999, Molecular and Cellular Biology.

[29]  M. Osley,et al.  Rad6-dependent ubiquitination of histone H2B in yeast. , 2000, Science.

[30]  Charles Boone,et al.  A conserved RING finger protein required for histone H2B monoubiquitination and cell size control. , 2003, Molecular cell.

[31]  P. Komarnitsky,et al.  ADR1 Activation Domains Contact the Histone Acetyltransferase GCN5 and the Core Transcriptional Factor TFIIB* , 1996, The Journal of Biological Chemistry.

[32]  E. Bradbury,et al.  Reversible histone modification and the chromosome cell cycle , 1992 .

[33]  A. Amerik,et al.  Analysis of the Deubiquitinating Enzymes of the Yeast Saccharomyces cerevisiae , 2000, Biological chemistry.

[34]  Fred Winston,et al.  Functional Organization of the Yeast SAGA Complex: Distinct Components Involved in Structural Integrity, Nucleosome Acetylation, and TATA-Binding Protein Interaction , 1999, Molecular and Cellular Biology.

[35]  Mark Johnston,et al.  The Paf1 Complex Is Essential for Histone Monoubiquitination by the Rad6-Bre1 Complex, Which Signals for Histone Methylation by COMPASS and Dot1p* , 2003, Journal of Biological Chemistry.

[36]  G. Cagney,et al.  Methylation of Histone H3 by Set2 in Saccharomyces cerevisiae Is Linked to Transcriptional Elongation by RNA Polymerase II , 2003, Molecular and Cellular Biology.

[37]  Stuart L. Schreiber,et al.  Active genes are tri-methylated at K4 of histone H3 , 2002, Nature.

[38]  Christoph H Borchers,et al.  Phosphorylation of RNA polymerase II CTD regulates H3 methylation in yeast. , 2003, Genes & development.

[39]  F. Winston,et al.  Evidence that Set1, a Factor Required for Methylation of Histone H3, Regulates rDNA Silencing in S. cerevisiae by a Sir2-Independent Mechanism , 2002, Current Biology.

[40]  Ioannis Xenarios,et al.  Microarray Deacetylation Maps Determine Genome-Wide Functions for Yeast Histone Deacetylases , 2002, Cell.

[41]  J R Yates,et al.  The ATM-related cofactor Tra1 is a component of the purified SAGA complex. , 1998, Molecular cell.

[42]  C. Allis,et al.  Ubiquitinated histone H2B is preferentially located in transcriptionally active chromatin. , 1989, Biochemistry.

[43]  F. Winston,et al.  The S. cerevisiae SAGA complex functions in vivo as a coactivator for transcriptional activation by Gal4. , 2001, Genes & development.

[44]  Brian D. Strahl,et al.  Gene silencing: Trans-histone regulatory pathway in chromatin , 2002, Nature.

[45]  C. Peterson.  HDAC's at work: everyone doing their part. , 2002, Molecular cell.

[46]  S. Berger,et al.  Phosphorylation of serine 10 in histone H3 is functionally linked in vitro and in vivo to Gcn5-mediated acetylation at lysine 14. , 2000, Molecular cell.

[47]  C. Peterson,et al.  Role for ADA/GCN5 products in antagonizing chromatin-mediated transcriptional repression , 1997, Molecular and cellular biology.

[48]  P. Sung,et al.  The RAD6 protein of Saccharomyces cerevisiae polyubiquitinates histones, and its acidic domain mediates this activity. , 1988, Genes & development.

[49]  Mark Johnston,et al.  Methylation of Histone H3 by COMPASS Requires Ubiquitination of Histone H2B by Rad6* , 2002, The Journal of Biological Chemistry.

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

[51]  Michael R. Green,et al.  Dissecting the Regulatory Circuitry of a Eukaryotic Genome , 1998, Cell.

[52]  C. Allis,et al.  The language of covalent histone modifications , 2000, Nature.

[53]  Ken-ichi Noma,et al.  Transitions in Distinct Histone H3 Methylation Patterns at the Heterochromatin Domain Boundaries , 2001, Science.

[54]  C. Brandl,et al.  Transcriptional Activation by Yeast PDR1p Is Inhibited by Its Association with NGG1p/ADA3p* , 1996, The Journal of Biological Chemistry.

[55]  Zu-Wen Sun,et al.  Ubiquitination of histone H2B regulates H3 methylation and gene silencing in yeast , 2002, Nature.

[56]  P. Grant,et al.  Histone H3 specific acetyltransferases are essential for cell cycle progression. , 2001, Genes & development.

[57]  Hans-Joachim Schüller,et al.  Transcriptional control of nonfermentative metabolism in the yeast Saccharomyces cerevisiae , 2003, Current Genetics.

[58]  C. Allis,et al.  Correlation Between Histone Lysine Methylation and Developmental Changes at the Chicken β-Globin Locus , 2001, Science.

[59]  H. Petropoulos,et al.  The E2 Ubiquitin Conjugase Rad6 Is Required for the ArgR/Mcm1 Repression of ARG1 Transcription , 2002, Molecular and Cellular Biology.

[60]  P. Bork,et al.  Functional organization of the yeast proteome by systematic analysis of protein complexes , 2002, Nature.

[61]  P. Wade,et al.  Paf1p, an RNA polymerase II-associated factor in Saccharomyces cerevisiae, may have both positive and negative roles in transcription , 1996, Molecular and cellular biology.

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

[63]  C. Allis,et al.  Evidence that Set 1 , a Factor Required for Methylation of Histone H 3 , Regulates rDNA Silencing , 2002 .

[64]  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.

[65]  L. Tora,et al.  Histone Folds Mediate Selective Heterodimerization of Yeast TAFII25 with TFIID Components yTAFII47 and yTAFII65 and with SAGA Component ySPT7 , 2001, Molecular and Cellular Biology.

[66]  Mihail Sarov,et al.  The histone 3 lysine 36 methyltransferase, SET2, is involved in transcriptional elongation. , 2003, Nucleic acids research.

[67]  D. Sterner,et al.  Inhibition of TATA-Binding Protein Function by SAGA Subunits Spt3 and Spt8 at Gcn4-Activated Promoters , 2000, Molecular and Cellular Biology.

[68]  H. Reinke,et al.  Histones are first hyperacetylated and then lose contact with the activated PHO5 promoter. , 2003, Molecular cell.

[69]  F. Winston,et al.  Analysis of Spt7 Function in the Saccharomyces cerevisiae SAGA Coactivator Complex , 2002, Molecular and Cellular Biology.