HULC, a Histone H2B Ubiquitinating Complex, Modulates Heterochromatin Independent of Histone Methylation in Fission Yeast*

Heterochromatin in fission yeast is targeted dynamically by opposing chromatin-modifying activities capable of alleviating or promoting transcriptional gene silencing. In this study, we report the biochemical and genetic characterization of a ubiquitin-conjugating enzyme Rhp6 (a homolog of budding yeast Rad6), which has been shown to negatively affect stability of heterochromatic structures. We show that Rhp6 is a component of the multisubunit protein complex (termed HULC) that also contains two RING finger proteins Rfp1 and Rfp2, sharing homology with budding yeast Bre1 protein and a unique serine-rich protein Shf1. HULC is required for ubiquitination of histone H2B at lysine 119 (H2B-K119), and it localizes to heterochromatic sequences. Moreover, our analyses suggest that Rhp6-induced changes in heterochromatic silencing are mediated predominantly through H2B ubiquitination (ubH2B), and they correlate with increased RNA polymerase II levels at repeat elements embedded within heterochromatin domains. Interestingly, heterochromatic derepression caused by Rhp6 occurs independently of the involvement of HULC subunits and ubH2B in methylation of histone H3 at lysine 4 (H3K4me). These analyses implicate ubH2B in modulation of heterochromatin, which has important implications for dynamics and many functions associated with heterochromatic structures.

[1]  D. Reinberg,et al.  Histone H2B Monoubiquitination Functions Cooperatively with FACT to Regulate Elongation by RNA Polymerase II , 2006, Cell.

[2]  T. Jenuwein,et al.  The many faces of histone lysine methylation. , 2002, Current opinion in cell biology.

[3]  C. Ponting,et al.  Regulation of chromatin structure by site-specific histone H3 methyltransferases , 2000, Nature.

[4]  Andrew J. Bannister,et al.  Selective recognition of methylated lysine 9 on histone H3 by the HP1 chromo domain , 2001, Nature.

[5]  S. Grewal,et al.  A Chromodomain Protein, Swi6, Performs Imprinting Functions in Fission Yeast during Mitosis and Meiosis , 2000, Cell.

[6]  Ira M. Hall,et al.  Establishment and Maintenance of a Heterochromatin Domain , 2002, Science.

[7]  S. Fang,et al.  RING fingers mediate ubiquitin-conjugating enzyme (E2)-dependent ubiquitination. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[8]  Eun Shik Choi,et al.  Two Ubiquitin-Conjugating Enzymes, Rhp6 and UbcX, Regulate Heterochromatin Silencing in Schizosaccharomyces pombe , 2002, Molecular and Cellular Biology.

[9]  Cyrus Martin,et al.  The diverse functions of histone lysine methylation , 2005, Nature Reviews Molecular Cell Biology.

[10]  Yi Zhang,et al.  Tudor, MBT and chromo domains gauge the degree of lysine methylation , 2006, EMBO reports.

[11]  M. Yanagida,et al.  Histone H2B mutations in inner region affect ubiquitination, centromere function, silencing and chromosome segregation , 2006, The EMBO journal.

[12]  Ming-Ming Zhou,et al.  Bromodomain: an acetyl‐lysine binding domain , 2002, FEBS letters.

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

[14]  John R. Yates,et al.  Chd1 chromodomain links histone H3 methylation with SAGA- and SLIK-dependent acetylation , 2005, Nature.

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

[16]  Ira M. Hall,et al.  Regulation of Heterochromatic Silencing and Histone H3 Lysine-9 Methylation by RNAi , 2002, Science.

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

[18]  A. Shevchenko,et al.  High Conservation of the Set1/Rad6 Axis of Histone 3 Lysine 4 Methylation in Budding and Fission Yeasts* , 2003, The Journal of Biological Chemistry.

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

[20]  M. Osley,et al.  Histone ubiquitylation and the regulation of transcription. , 2006, Results and problems in cell differentiation.

[21]  Yi Zhang.  Transcriptional regulation by histone ubiquitination and deubiquitination. , 2003, Genes & development.

[22]  T. Sugiyama,et al.  Comprehensive analysis of heterochromatin- and RNAi-mediated epigenetic control of the fission yeast genome , 2005, Nature Genetics.

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

[24]  A. Klar,et al.  A Novel Function of the DNA Repair Gene rhp6 in Mating-Type Silencing by Chromatin Remodeling in Fission Yeast , 1998, Molecular and Cellular Biology.

[25]  A. Caudy,et al.  Functional Divergence between Histone Deacetylases in Fission Yeast by Distinct Cellular Localization and In Vivo Specificity , 2002, Molecular and Cellular Biology.

[26]  Brian D. Strahl,et al.  Role of Histone H3 Lysine 9 Methylation in Epigenetic Control of Heterochromatin Assembly , 2001, Science.

[27]  D. Reinberg,et al.  Facts about FACT and transcript elongation through chromatin. , 2004, Current opinion in genetics & development.

[28]  E. Nimmo,et al.  Mutations derepressing silent centromeric domains in fission yeast disrupt chromosome segregation. , 1995, Genes & development.

[29]  J. Mellor.  It Takes a PHD to Read the Histone Code , 2006, Cell.

[30]  N. Krogan,et al.  Maintenance of low histone ubiquitylation by Ubp10 correlates with telomere-proximal Sir2 association and gene silencing. , 2005, Molecular cell.

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

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

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

[34]  S. Grewal,et al.  A Novel jmjC Domain Protein Modulates Heterochromatization in Fission Yeast , 2003, Molecular and Cellular Biology.

[35]  P. Sung,et al.  Specific complex formation between yeast RAD6 and RAD18 proteins: a potential mechanism for targeting RAD6 ubiquitin-conjugating activity to DNA damage sites. , 1994, Genes & development.

[36]  C. Pickart,et al.  Mechanisms underlying ubiquitination. , 2001, Annual review of biochemistry.

[37]  G. Thon,et al.  The Fission Yeast Ubiquitin-Conjugating Enzymes UbcP3, Ubc15, and Rhp6 Affect Transcriptional Silencing of the Mating-Type Region , 2002, Eukaryotic Cell.

[38]  SHREC, an Effector Complex for Heterochromatic Transcriptional Silencing , 2007, Cell.

[39]  S. Grewal,et al.  Ubiquitin ligase component Cul4 associates with Clr4 histone methyltransferase to assemble heterochromatin , 2005, Nature Cell Biology.

[40]  G. Schotta,et al.  The indexing potential of histone lysine methylation. , 2004, Novartis Foundation symposium.

[41]  T. Sugiyama,et al.  The nucleation and maintenance of heterochromatin by a histone deacetylase in fission yeast. , 2005, Molecular cell.

[42]  S. Forsburg,et al.  Conserved Locus-Specific Silencing Functions of Schizosaccharomyces pombe sir2+ , 2005, Genetics.

[43]  L. Prakash,et al.  The rhp6+ gene of Schizosaccharomyces pombe: a structural and functional homolog of the RAD6 gene from the distantly related yeast Saccharomyces cerevisiae. , 1990, The EMBO journal.

[44]  Karl Mechtler,et al.  Methylation of histone H3 lysine 9 creates a binding site for HP1 proteins , 2001, Nature.

[45]  M. Zofall,et al.  Swi6/HP1 recruits a JmjC domain protein to facilitate transcription of heterochromatic repeats. , 2006, Molecular cell.

[46]  T. Sugiyama,et al.  RITS acts in cis to promote RNA interference–mediated transcriptional and post-transcriptional silencing , 2004, Nature Genetics.

[47]  R. Martienssen,et al.  RNA Polymerase II Is Required for RNAi-Dependent Heterochromatin Assembly , 2005, Science.

[48]  Jagmohan Singh,et al.  Identification of Uhp1, a Ubiquitinated Histone-like Protein, as a Target/Mediator of Rhp6 in Mating-type Silencing in Fission Yeast* , 2003, The Journal of Biological Chemistry.

[49]  Thomas A. Milne,et al.  A PHD finger of NURF couples histone H3 lysine 4 trimethylation with chromatin remodelling , 2006, Nature.

[50]  Songtao Jia,et al.  RNAi-Mediated Targeting of Heterochromatin by the RITS Complex , 2004, Science.

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

[52]  S. Berger,et al.  Histone H2B ubiquitylation and deubiquitylation in genomic regulation. , 2004, Cold Spring Harbor symposia on quantitative biology.

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

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

[55]  A. Varshavsky,et al.  The N-end rule is mediated by the UBC2(RAD6) ubiquitin-conjugating enzyme. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[56]  L. Prakash.  The structure and function of RAD6 and RAD18 DNA repair genes of Saccharomyces cerevisiae. , 1989, Genome.

[57]  A. Wolffe,et al.  Structure and function of the core histone N-termini: more than meets the eye. , 1998, Biochemistry.

[58]  S. Berger,et al.  Histone modifications in transcriptional regulation. , 2002, Current opinion in genetics & development.

[59]  Yi Zhang,et al.  Recognition of Histone H3 Lysine-4 Methylation by the Double Tudor Domain of JMJD2A , 2006, Science.

[60]  S. Grewal,et al.  Histone deacetylase homologs regulate epigenetic inheritance of transcriptional silencing and chromosome segregation in fission yeast. , 1998, Genetics.

[61]  S. Grewal,et al.  Histone H3 lysine 4 methylation is mediated by Set1 and promotes maintenance of active chromatin states in fission yeast , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[62]  M. Hochstrasser.  Ubiquitin-dependent protein degradation. , 1996, Annual review of genetics.

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

[64]  Tony Kouzarides,et al.  cis-Acting DNA from Fission Yeast Centromeres Mediates Histone H3 Methylation and Recruitment of Silencing Factors and Cohesin to an Ectopic Site , 2002, Current Biology.

[65]  S. Elgin,et al.  The contradictory definitions of heterochromatin: transcription and silencing , 2006, Chromosoma.

[66]  B M Turner,et al.  Histone acetylation and an epigenetic code. , 2000, BioEssays : news and reviews in molecular, cellular and developmental biology.

[67]  C. Bonilla,et al.  RNA Pol II subunit Rpb7 promotes centromeric transcription and RNAi-directed chromatin silencing. , 2005, Genes & development.

[68]  T. Urano,et al.  A chromodomain protein, Chp1, is required for the establishment of heterochromatin in fission yeast , 2004, The EMBO journal.

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

[70]  D. Moazed,et al.  Sir2 Regulates Histone H3 Lysine 9 Methylation and Heterochromatin Assembly in Fission Yeast , 2003, Current Biology.

[71]  P. Grant,et al.  Effector Proteins for Methylated Histones: An Expanding Family , 2005, Cell cycle.

[72]  K. Ekwall.  Genome-wide analysis of HDAC function. , 2005, Trends in genetics : TIG.