Control of transcriptional elongation and cotranscriptional histone modification by the yeast BUR kinase substrate Spt5

Elongation by RNA polymerase II (RNAPII) is a finely regulated process in which many elongation factors contribute to gene regulation. Among these factors are the polymerase-associated factor (PAF) complex, which associates with RNAPII, and several cyclin-dependent kinases, including positive transcription elongation factor b (P-TEFb) in humans and BUR kinase (Bur1–Bur2) and C-terminal domain (CTD) kinase 1 (CTDK1) in Saccharomyces cerevisiae. An important target of P-TEFb and CTDK1, but not BUR kinase, is the CTD of the Rpb1 subunit of RNAPII. Although the essential BUR kinase phosphorylates Rad6, which is required for histone H2B ubiquitination on K123, Rad6 is not essential, leaving a critical substrate(s) of BUR kinase unidentified. Here we show that BUR kinase is important for the phosphorylation in vivo of Spt5, a subunit of the essential yeast RNAPII elongation factor Spt4/Spt5, whose human orthologue is DRB sensitivity-inducing factor. BUR kinase can also phosphorylate the C-terminal region (CTR) of Spt5 in vitro. Like BUR kinase, the Spt5 CTR is important for promoting elongation by RNAPII and recruiting the PAF complex to transcribed regions. Also like BUR kinase and the PAF complex, the Spt5 CTR is important for histone H2B K123 monoubiquitination and histone H3 K4 and K36 trimethylation during transcription elongation. Our results suggest that the Spt5 CTR, which contains 15 repeats of a hexapeptide whose consensus sequence is S[T/A]WGG[A/Q], is a substrate of BUR kinase and a platform for the association of proteins that promote both transcription elongation and histone modification in transcribed regions.

[1]  John R Yates,et al.  Deubiquitination of Histone H2B by a Yeast Acetyltransferase Complex Regulates Transcription* , 2004, Journal of Biological Chemistry.

[2]  D. Bentley,et al.  Dynamic association of capping enzymes with transcribing RNA polymerase II. , 2000, Genes & development.

[3]  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.

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

[5]  J. Lis,et al.  Coordination of transcription, RNA processing, and surveillance by P-TEFb kinase on heat shock genes. , 2004, Molecular cell.

[6]  N. Krogan,et al.  Histone H2B Ubiquitylation Is Associated with Elongating RNA Polymerase II , 2005, Molecular and Cellular Biology.

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

[8]  D. Price,et al.  Control of RNA Polymerase II Elongation Potential by a Novel Carboxyl-terminal Domain Kinase* , 1996, The Journal of Biological Chemistry.

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

[10]  Nevan J. Krogan,et al.  Cotranscriptional Set2 Methylation of Histone H3 Lysine 36 Recruits a Repressive Rpd3 Complex , 2005, Cell.

[11]  D. Reines.  Use of RNA yeast polymerase II mutants in studying transcription elongation. , 2003, Methods in enzymology.

[12]  N. Krogan,et al.  Transitions in RNA polymerase II elongation complexes at the 3′ ends of genes , 2004, The EMBO journal.

[13]  Mark Johnston,et al.  The Bur1/Bur2 complex is required for histone H2B monoubiquitination by Rad6/Bre1 and histone methylation by COMPASS. , 2005, Molecular cell.

[14]  D. Price.  P-TEFb, a Cyclin-Dependent Kinase Controlling Elongation by RNA Polymerase II , 2000, Molecular and Cellular Biology.

[15]  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.

[16]  G. Hartzog,et al.  Phosphorylation of the RNA Polymerase II Carboxy-Terminal Domain by the Bur1 Cyclin-Dependent Kinase , 2001, Molecular and Cellular Biology.

[17]  J. Corden,et al.  Yeast Carboxyl-terminal Domain Kinase I Positively and Negatively Regulates RNA Polymerase II Carboxyl-terminal Domain Phosphorylation* , 1999, Journal of Biological Chemistry.

[18]  Mark Gerber,et al.  Transcriptional Elongation by RNA Polymerase II and Histone Methylation* , 2003, Journal of Biological Chemistry.

[19]  G. Cagney,et al.  RNA Polymerase II Elongation Factors of Saccharomyces cerevisiae: a Targeted Proteomics Approach , 2002, Molecular and Cellular Biology.

[20]  M. Gallardo,et al.  Molecular evidence indicating that the yeast PAF complex is required for transcription elongation , 2004, EMBO reports.

[21]  D. Price.  Poised polymerases: on your mark...get set...go! , 2008, Molecular cell.

[22]  Craig D. Kaplan,et al.  Transcription Elongation Factors Repress Transcription Initiation from Cryptic Sites , 2003, Science.

[23]  Karl Henry,et al.  Rad6 plays a role in transcriptional activation through ubiquitylation of histone H2B. , 2004, Genes & development.

[24]  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.

[25]  Ali Shilatifard,et al.  Transcriptional activation via sequential histone H2B ubiquitylation and deubiquitylation, mediated by SAGA-associated Ubp8. , 2003, Genes & development.

[26]  John T. Lis,et al.  Breaking barriers to transcription elongation , 2006, Nature Reviews Molecular Cell Biology.

[27]  Hiroshi Handa,et al.  P-TEFb-mediated phosphorylation of hSpt5 C-terminal repeats is critical for processive transcription elongation. , 2006, Molecular cell.

[28]  K. Arndt,et al.  Regulation of histone modification and cryptic transcription by the Bur1 and Paf1 complexes , 2007, The EMBO journal.

[29]  B. Peterlin,et al.  Controlling the elongation phase of transcription with P-TEFb. , 2006, Molecular cell.

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

[31]  Richard A Young,et al.  Exchange of RNA polymerase II initiation and elongation factors during gene expression in vivo. , 2002, Molecular cell.

[32]  E. Cho,et al.  Different phosphorylated forms of RNA polymerase II and associated mRNA processing factors during transcription. , 2000, Genes & development.

[33]  Michael Hampsey,et al.  Tails of Intrigue Phosphorylation of RNA Polymerase II Mediates Histone Methylation , 2003, Cell.

[34]  S. Buratowski,et al.  Phosphorylation of serine 2 within the RNA polymerase II C-terminal domain couples transcription and 3' end processing. , 2004, Molecular cell.

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

[36]  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.

[37]  P. Grant,et al.  Set2 Is a Nucleosomal Histone H3-Selective Methyltransferase That Mediates Transcriptional Repression , 2002, Molecular and Cellular Biology.

[38]  T. Hughes,et al.  BUR Kinase Selectively Regulates H3 K4 Trimethylation and H2B Ubiquitylation through Recruitment of the PAF Elongation Complex , 2005, Current Biology.

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

[40]  K. Struhl,et al.  The transition from transcriptional initiation to elongation. , 2008, Current opinion in genetics & development.

[41]  K. Mulder,et al.  Regulation of histone H3K4 tri-methylation and PAF complex recruitment by the Ccr4-Not complex , 2007, Nucleic acids research.

[42]  A. Hinnebusch,et al.  The Spt4p Subunit of Yeast DSIF Stimulates Association of the Paf1 Complex with Elongating RNA Polymerase II , 2006, Molecular and Cellular Biology.

[43]  M. Keogh,et al.  Bur1 Kinase Is Required for Efficient Transcription Elongation by RNA Polymerase II , 2003, Molecular and Cellular Biology.

[44]  F. Holstege,et al.  An unusual eukaryotic protein phosphatase required for transcription by RNA polymerase II and CTD dephosphorylation in S. cerevisiae. , 1999, Molecular cell.

[45]  N. Krogan,et al.  The Saccharomyces cerevisiae histone H2A variant Htz1 is acetylated by NuA4. , 2006, Genes & development.

[46]  H. Phatnani,et al.  Phosphorylation and functions of the RNA polymerase II CTD. , 2006, Genes & development.

[47]  A. Shilatifard,et al.  Bur1/Bur2 and the Ctk Complex in Yeast: The Split Personality of Mammalian P-TEFb , 2006, Cell cycle.

[48]  R. Sternglanz,et al.  The Bur1 Cyclin-Dependent Protein Kinase Is Required for the Normal Pattern of Histone Methylation bySet2 , 2006, Molecular and Cellular Biology.

[49]  Michael Hampsey,et al.  Ssu72 Is an RNA polymerase II CTD phosphatase. , 2004, Molecular cell.

[50]  Bing Li,et al.  Histone H3 Methylation by Set2 Directs Deacetylation of Coding Regions by Rpd3S to Suppress Spurious Intragenic Transcription , 2005, Cell.

[51]  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.

[52]  S. Squazzo,et al.  The Paf1 complex physically and functionally associates with transcription elongation factors in vivo , 2002, The EMBO journal.

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

[54]  Kevin Struhl,et al.  Eaf3 chromodomain interaction with methylated H3-K36 links histone deacetylation to Pol II elongation. , 2005, Molecular cell.

[55]  T. Hughes,et al.  Organization and Function of APT, a Subcomplex of the Yeast Cleavage and Polyadenylation Factor Involved in the Formation of mRNA and Small Nucleolar RNA 3′-Ends* , 2003, Journal of Biological Chemistry.