SUMOylation by the E3 Ligase TbSIZ1/PIAS1 Positively Regulates VSG Expression in Trypanosoma brucei

Bloodstream form trypanosomes avoid the host immune response by switching the expression of their surface proteins between Variant Surface Glycoproteins (VSG), only one of which is expressed at any given time. Monoallelic transcription of the telomeric VSG Expression Site (ES) by RNA polymerase I (RNA pol I) localizes to a unique nuclear body named the ESB. Most work has focused on silencing mechanisms of inactive VSG-ESs, but the mechanisms involved in transcriptional activation of a single VSG-ES remain largely unknown. Here, we identify a highly SUMOylated focus (HSF) in the nucleus of the bloodstream form that partially colocalizes with the ESB and the active VSG-ES locus. SUMOylation of chromatin-associated proteins was enriched along the active VSG-ES transcriptional unit, in contrast to silent VSG-ES or rDNA, suggesting that it is a distinct feature of VSG-ES monoallelic expression. In addition, sequences upstream of the active VSG-ES promoter were highly enriched in SUMOylated proteins. We identified TbSIZ1/PIAS1 as the SUMO E3 ligase responsible for SUMOylation in the active VSG-ES chromatin. Reduction of SUMO-conjugated proteins by TbSIZ1 knockdown decreased the recruitment of RNA pol I to the VSG-ES and the VSG-ES-derived transcripts. Furthermore, cells depleted of SUMO conjugated proteins by TbUBC9 and TbSUMO knockdown confirmed the positive function of SUMO for VSG-ES expression. In addition, the largest subunit of RNA pol I TbRPA1 was SUMOylated in a TbSIZ-dependent manner. Our results show a positive mechanism associated with active VSG-ES expression via post-translational modification, and indicate that chromatin SUMOylation plays an important role in the regulation of VSG-ES. Thus, protein SUMOylation is linked to active gene expression in this protozoan parasite that diverged early in evolution.

[1]  L. Vanhamme,et al.  Transcription is initiated on silent variant surface glycoprotein expression sites despite monoallelic expression in Trypanosoma brucei , 2014, Proceedings of the National Academy of Sciences.

[2]  Mark C. Field,et al.  Antigenic variation in African trypanosomes: the importance of chromosomal and nuclear context in VSG expression control , 2013, Cellular microbiology.

[3]  C. Clayton,et al.  SUMOylation in Trypanosoma brucei , 2013, PeerJ.

[4]  Huilin Zhou,et al.  Distinct SUMO Ligases Cooperate with Esc2 and Slx5 to Suppress Duplication-Mediated Genome Rearrangements , 2013, PLoS genetics.

[5]  F. Melchior,et al.  Sumoylation: a regulatory protein modification in health and disease. , 2013, Annual review of biochemistry.

[6]  Samir Karaca,et al.  Detecting endogenous SUMO targets in mammalian cells and tissues , 2013, Nature Structural &Molecular Biology.

[7]  Julien Guizetti,et al.  Silence, activate, poise and switch! Mechanisms of antigenic variation in Plasmodium falciparum , 2013, Cellular microbiology.

[8]  G. Rudenko,et al.  TDP1 is an HMG chromatin protein facilitating RNA polymerase I transcription in African trypanosomes , 2013, Nucleic acids research.

[9]  M. Matunis,et al.  SUMO: a multifaceted modifier of chromatin structure and function. , 2013, Developmental cell.

[10]  S. Jentsch,et al.  Protein Group Modification and Synergy in the SUMO Pathway as Exemplified in DNA Repair , 2012, Cell.

[11]  M. Bermudez-lopez,et al.  A SUMO-Dependent Step during Establishment of Sister Chromatid Cohesion , 2012, Current Biology.

[12]  Kun Huang,et al.  Chromatin modification by SUMO-1 stimulates the promoters of translation machinery genes , 2012, Nucleic acids research.

[13]  David Landeira,et al.  Role of RPB7 in RNA pol I transcription in Trypanosoma brucei. , 2011, Molecular and biochemical parasitology.

[14]  Sung Hee Park,et al.  Transcription by the multifunctional RNA polymerase I in Trypanosoma brucei functions independently of RPB7. , 2011, Molecular and biochemical parasitology.

[15]  Hyungwon Choi,et al.  SUMOylation Pathway in Trypanosoma cruzi: Functional Characterization and Proteomic Analysis of Target Proteins* , 2011, Molecular & Cellular Proteomics.

[16]  Bin Zhang,et al.  Biogenesis and function of nuclear bodies. , 2011, Trends in genetics : TIG.

[17]  G. Rudenko,et al.  TbISWI Regulates Multiple Polymerase I (Pol I)-Transcribed Loci and Is Present at Pol II Transcription Boundaries in Trypanosoma brucei , 2011, Eukaryotic Cell.

[18]  P. I. Lorenzo,et al.  PIAS1 interacts with FLASH and enhances its co-activation of c-Myb , 2011, Molecular Cancer.

[19]  Jaclyn R. Gareau,et al.  The SUMO pathway: emerging mechanisms that shape specificity, conjugation and recognition , 2010, Nature Reviews Molecular Cell Biology.

[20]  G. Rudenko,et al.  The FACT subunit TbSpt16 is involved in cell cycle specific control of VSG expression sites in Trypanosoma brucei , 2010, Molecular microbiology.

[21]  M. Taylor,et al.  Centromere-associated topoisomerase activity in bloodstream form Trypanosoma brucei , 2010, Nucleic acids research.

[22]  Ruth Nussinov,et al.  A Mechanistic View of the Role of E3 in Sumoylation , 2010, PLoS Comput. Biol..

[23]  J. Manley,et al.  SUMO functions in constitutive transcription and during activation of inducible genes in yeast. , 2010, Genes & development.

[24]  S. Liao,et al.  The small ubiquitin-like modifier (SUMO) is essential in cell cycle regulation in Trypanosoma brucei. , 2010, Experimental cell research.

[25]  G. Rudenko,et al.  Active VSG Expression Sites in Trypanosoma brucei Are Depleted of Nucleosomes , 2009, Eukaryotic Cell.

[26]  G. Cross,et al.  Nucleosomes Are Depleted at the VSG Expression Site Transcribed by RNA Polymerase I in African Trypanosomes , 2009, Eukaryotic Cell.

[27]  David Landeira,et al.  Cohesin regulates VSG monoallelic expression in trypanosomes , 2009, The Journal of cell biology.

[28]  G. Cross,et al.  Epigenetic regulation in African trypanosomes: a new kid on the block , 2009, Nature Reviews Microbiology.

[29]  J. Reyes,et al.  SUMO association with repressor complexes, emerging routes for transcriptional control. , 2009, Biochimica et biophysica acta.

[30]  L. Figueiredo,et al.  RAP1 Is Essential for Silencing Telomeric Variant Surface Glycoprotein Genes in Trypanosoma brucei , 2009, Cell.

[31]  David Landeira,et al.  RNA pol II subunit RPB7 is required for RNA pol I‐mediated transcription in Trypanosoma brucei , 2009, EMBO reports.

[32]  Christiane Hertz-Fowler,et al.  Telomeric Expression Sites Are Highly Conserved in Trypanosoma brucei , 2008, PloS one.

[33]  A. Strunnikov,et al.  Cooperation of Sumoylated Chromosomal Proteins in rDNA Maintenance , 2008, PLoS genetics.

[34]  G. Cross,et al.  A Histone Methyltransferase Modulates Antigenic Variation in African Trypanosomes , 2008, PLoS biology.

[35]  M. J. Lyst,et al.  A role for SUMO modification in transcriptional repression and activation. , 2007, Biochemical Society transactions.

[36]  Patrick Heun,et al.  SUMOrganization of the nucleus. , 2007, Current opinion in cell biology.

[37]  David Landeira,et al.  Nuclear architecture underlying gene expression in Trypanosoma brucei. , 2007, Trends in microbiology.

[38]  David Landeira,et al.  Nuclear repositioning of the VSG promoter during developmental silencing in Trypanosoma brucei , 2007, The Journal of cell biology.

[39]  E. J. Lee,et al.  SUMO-specific protease SUSP4 positively regulates p53 by promoting Mdm2 self-ubiquitination , 2006, Nature Cell Biology.

[40]  D. Sterner,et al.  Histone sumoylation is a negative regulator in Saccharomyces cerevisiae and shows dynamic interplay with positive-acting histone modifications. , 2006, Genes & development.

[41]  A. Sharrocks.  PIAS proteins and transcriptional regulation--more than just SUMO E3 ligases? , 2006, Genes & development.

[42]  Xiaolan Zhao,et al.  A SUMO ligase is part of a nuclear multiprotein complex that affects DNA repair and chromosomal organization. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[43]  G. Cross,et al.  A variant histone H3 is enriched at telomeres in Trypanosoma brucei , 2004, Journal of Cell Science.

[44]  John R Yates,et al.  Global Analysis of Protein Sumoylation in Saccharomyces cerevisiae* , 2004, Journal of Biological Chemistry.

[45]  A. Dejean,et al.  Role of the fission yeast SUMO E3 ligase Pli1p in centromere and telomere maintenance , 2004, The EMBO journal.

[46]  L. Vanhamme,et al.  Antigenic variation in Trypanosoma brucei: facts, challenges and mysteries. , 2004, Current opinion in microbiology.

[47]  Erica S. Johnson,et al.  Protein modification by SUMO. , 2004, Annual review of biochemistry.

[48]  R. McCulloch.  Antigenic variation in African trypanosomes: monitoring progress. , 2004, Trends in parasitology.

[49]  S. Müller,et al.  PIAS/SUMO: new partners in transcriptional regulation , 2003, Cellular and Molecular Life Sciences CMLS.

[50]  F. Melchior,et al.  SUMO: ligases, isopeptidases and nuclear pores. , 2003, Trends in biochemical sciences.

[51]  R. Eisenman,et al.  Histone sumoylation is associated with transcriptional repression , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[52]  M. Kagey,et al.  The Polycomb Protein Pc2 Is a SUMO E3 , 2003, Cell.

[53]  E. Miska,et al.  The SUMO E3 ligase RanBP2 promotes modification of the HDAC4 deacetylase , 2002, The EMBO journal.

[54]  P. Borst.  Antigenic Variation and Allelic Exclusion , 2002, Cell.

[55]  Keith Gull,et al.  A pol I transcriptional body associated with VSG mono-allelic expression in Trypanosoma brucei , 2001, Nature.

[56]  Erica S. Johnson,et al.  An E3-like Factor that Promotes SUMO Conjugation to the Yeast Septins , 2001, Cell.

[57]  Zefeng Wang,et al.  Inhibition of Trypanosoma brucei Gene Expression by RNA Interference Using an Integratable Vector with Opposing T7 Promoters* , 2000, The Journal of Biological Chemistry.

[58]  G. Cross,et al.  Trypanosoma brucei , 1998 .

[59]  F. Melchior,et al.  A Small Ubiquitin-Related Polypeptide Involved in Targeting RanGAP1 to Nuclear Pore Complex Protein RanBP2 , 1997, Cell.

[60]  G. Blobel,et al.  A novel ubiquitin-like modification modulates the partitioning of the Ran-GTPase-activating protein RanGAP1 between the cytosol and the nuclear pore complex , 1996, The Journal of cell biology.

[61]  T. Nash,et al.  Antigenic variation in Giardia lamblia. , 1988, Journal of immunology.