Four histone variants mark the boundaries of polycistronic transcription units in Trypanosoma brucei.

Unusually for a eukaryote, genes transcribed by RNA polymerase II (pol II) in Trypanosoma brucei are arranged in polycistronic transcription units. With one exception, no pol II promoter motifs have been identified, and how transcription is initiated remains an enigma. T. brucei has four histone variants: H2AZ, H2BV, H3V, and H4V. Using chromatin immunoprecipitation (ChIP) and sequencing (ChIP-seq) to examine the genome-wide distribution of chromatin components, we show that histones H4K10ac, H2AZ, H2BV, and the bromodomain factor BDF3 are enriched up to 300-fold at probable pol II transcription start sites (TSSs). We also show that nucleosomes containing H2AZ and H2BV are less stable than canonical nucleosomes. Our analysis also identifies >60 unexpected TSS candidates and reveals the presence of long guanine runs at probable TSSs. Apparently unique to trypanosomes, additional histone variants H3V and H4V are enriched at probable pol II transcription termination sites. Our findings suggest that histone modifications and histone variants play crucial roles in transcription initiation and termination in trypanosomes and that destabilization of nucleosomes by histone variants is an evolutionarily ancient and general mechanism of transcription initiation, demonstrated in an organism in which general pol II transcription factors have been elusive.

[1]  C. Jin,et al.  Nucleosome stability mediated by histone variants H3.3 and H2A.Z. , 2007, Genes & development.

[2]  G. Cross,et al.  Histone H2AZ dimerizes with a novel variant H2B and is enriched at repetitive DNA in Trypanosoma brucei , 2005, Journal of Cell Science.

[3]  C. Clayton,et al.  Post-transcriptional control of hsp70 mRNA in Trypanosoma brucei. , 1996, Molecular and biochemical parasitology.

[4]  M. Carrington,et al.  Polymerase chain reaction-based gene disruption in Trypanosoma brucei. , 1997, Molecular and biochemical parasitology.

[5]  E. Ullu,et al.  Structure of the Trypanosoma brucei U6 snRNA gene promoter. , 1997, Molecular and biochemical parasitology.

[6]  L. Pillus.  MYSTs mark chromatin for chromosomal functions. , 2008, Current opinion in cell biology.

[7]  Steven Henikoff,et al.  Nucleosome destabilization in the epigenetic regulation of gene expression , 2008, Nature Reviews Genetics.

[8]  M. Lee.  An RNA polymerase II promoter in the hsp70 locus of Trypanosoma brucei , 1996, Molecular and cellular biology.

[9]  G. Cross,et al.  Trypanosomal TBP Functions with the Multisubunit Transcription Factor tSNAP To Direct Spliced-Leader RNA Gene Expression , 2005, Molecular and Cellular Biology.

[10]  Philipp Korber,et al.  SWRred Not Shaken Mixing the Histones , 2004, Cell.

[11]  Richard A Young,et al.  Chromatin immunoprecipitation and microarray-based analysis of protein location , 2006, Nature Protocols.

[12]  G. Cross,et al.  Unusual histone modifications in Trypanosoma brucei , 2006, FEBS letters.

[13]  G. Cross,et al.  Trypanosome H2Bv replaces H2B in nucleosomes enriched for H3 K4 and K76 trimethylation. , 2008, Biochemical and biophysical research communications.

[14]  P. D’haeseleer.  How does DNA sequence motif discovery work? , 2006, Nature Biotechnology.

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

[16]  S. Henikoff,et al.  Genome-scale profiling of histone H3.3 replacement patterns , 2005, Nature Genetics.

[17]  Lei Zeng,et al.  Structure and ligand of a histone acetyltransferase bromodomain , 1999, Nature.

[18]  C. Clayton,et al.  Life without transcriptional control? From fly to man and back again , 2002, The EMBO journal.

[19]  G. Cross,et al.  Analysis of the Trypanosoma brucei cell cycle by quantitative DAPI imaging. , 2008, Molecular and biochemical parasitology.

[20]  G. Cross,et al.  Acetylation of histone H4K4 is cell cycle regulated and mediated by HAT3 in Trypanosoma brucei , 2007, Molecular microbiology.

[21]  P. Hanawalt,et al.  G4-forming Sequences in the Non-transcribed DNA Strand Pose Blocks to T7 RNA Polymerase and Mammalian RNA Polymerase II* , 2008, Journal of Biological Chemistry.

[22]  S. Kunz,et al.  A vector series for rapid PCR-mediated C-terminal in situ tagging of Trypanosoma brucei genes. , 2006, Molecular and biochemical parasitology.

[23]  S. Schreiber,et al.  Histone Variant H2A.Z Marks the 5′ Ends of Both Active and Inactive Genes in Euchromatin , 2006, Cell.

[24]  Gene W. Yeo,et al.  Divergent Transcription from Active Promoters , 2008, Science.

[25]  Daniel Nilsson,et al.  Comparative Genomics of Trypanosomatid Parasitic Protozoa , 2005, Science.

[26]  Dustin E. Schones,et al.  High-Resolution Profiling of Histone Methylations in the Human Genome , 2007, Cell.

[27]  C. Clayton,et al.  Testing promoter activity in the trypanosome genome: isolation of a metacyclic-type VSG promoter, and unexpected insights into RNA polymerase II transcription. , 1998, Experimental parasitology.

[28]  H Hirumi,et al.  Continuous cultivation of Trypanosoma brucei blood stream forms in a medium containing a low concentration of serum protein without feeder cell layers. , 1989, The Journal of parasitology.

[29]  Christine Clayton,et al.  A doubly inducible system for RNA interference and rapid RNAi plasmid construction in Trypanosoma brucei. , 2005, Molecular and biochemical parasitology.

[30]  S. Grewal,et al.  A Role for TFIIIC Transcription Factor Complex in Genome Organization , 2006, Cell.

[31]  Samer Khateb,et al.  Homodimeric MyoD Preferentially Binds Tetraplex Structures of Regulatory Sequences of Muscle-specific Genes* , 2005, Journal of Biological Chemistry.

[32]  Á. Rada-Iglesias,et al.  Histone Acetylation and Methylation at Sites Initiating Divergent Polycistronic Transcription in Trypanosoma cruzi* , 2008, Journal of Biological Chemistry.

[33]  M. Johnston,et al.  tRNA genes as transcriptional repressor elements , 1994, Molecular and cellular biology.

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

[35]  M. Groudine,et al.  Chromosomal subunits in active genes have an altered conformation. , 1976, Science.

[36]  Tu N. Nguyen,et al.  Characterization of a Multisubunit Transcription Factor Complex Essential for Spliced-Leader RNA Gene Transcription in Trypanosoma brucei , 2005, Molecular and Cellular Biology.

[37]  Kenneth Stuart,et al.  Transcription of Leishmania major Friedlin chromosome 1 initiates in both directions within a single region. , 2003, Molecular cell.

[38]  K. Gull,et al.  A novel epitope tag system to study protein targeting and organelle biogenesis in Trypanosoma brucei. , 1996, Molecular and biochemical parasitology.

[39]  W. J. Kent,et al.  BLAT--the BLAST-like alignment tool. , 2002, Genome research.

[40]  E. Ullu,et al.  Upstream tRNA genes are essential for expression of small nuclear and cytoplasmic RNA genes in trypanosomes , 1994, Molecular and cellular biology.

[41]  G. Cross,et al.  Two essential MYST-family proteins display distinct roles in histone H4K10 acetylation and telomeric silencing in trypanosomes , 2008, Molecular Microbiology.

[42]  A. Sentenac,et al.  Participation of the TATA factor in transcription of the yeast U6 gene by RNA polymerase C. , 1991, Science.

[43]  E. Ullu,et al.  Physical and transcriptional analysis of the Trypanosoma brucei genome reveals a typical eukaryotic arrangement with close interspersionof RNA polymerase II- and III-transcribed genes. , 1998, Nucleic acids research.

[44]  R. Brun,et al.  Cultivation and in vitro cloning or procyclic culture forms of Trypanosoma brucei in a semi-defined medium. Short communication. , 1979, Acta tropica.

[45]  G. Cross,et al.  Histone modifications in Trypanosoma brucei. , 2007, Molecular and biochemical parasitology.

[46]  Shulamit Michaeli,et al.  MINIREVIEWS trans and cis Splicing in Trypanosomatids : Mechanism , Factors , and Regulation , 2003 .

[47]  G. Cross,et al.  CRE recombinase-based positive-negative selection systems for genetic manipulation in Trypanosoma brucei. , 2008, Molecular and biochemical parasitology.

[48]  E. Pays,et al.  The actin gene promoter of Trypanosoma brucei. , 1991, Nucleic acids research.

[49]  Mathieu Blanchette,et al.  Variant Histone H2A.Z Is Globally Localized to the Promoters of Inactive Yeast Genes and Regulates Nucleosome Positioning , 2005, PLoS biology.

[50]  D. Engelke,et al.  A CBF5 mutation that disrupts nucleolar localization of early tRNA biosynthesis in yeast also suppresses tRNA gene-mediated transcriptional silencing. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[51]  Shankar Balasubramanian,et al.  G-quadruplexes in promoters throughout the human genome , 2006, Nucleic acids research.

[52]  J. Workman,et al.  Function and Selectivity of Bromodomains in Anchoring Chromatin-Modifying Complexes to Promoter Nucleosomes , 2002, Cell.

[53]  B. Cairns,et al.  Genome-Wide Dynamics of Htz1, a Histone H2A Variant that Poises Repressed/Basal Promoters for Activation through Histone Loss , 2005, Cell.

[54]  J. Workman,et al.  SAS-mediated acetylation of histone H4 Lys 16 is required for H2A.Z incorporation at subtelomeric regions in Saccharomyces cerevisiae. , 2006, Genes & development.

[55]  E. Birney,et al.  Trawler: de novo regulatory motif discovery pipeline for chromatin immunoprecipitation , 2007, Nature Methods.

[56]  William Stafford Noble,et al.  Assessing computational tools for the discovery of transcription factor binding sites , 2005, Nature Biotechnology.

[57]  A. Shilatifard.  Molecular implementation and physiological roles for histone H3 lysine 4 (H3K4) methylation. , 2008, Current opinion in cell biology.

[58]  Fred Winston,et al.  The bromodomain: a chromatin-targeting module? , 1999, Nature Structural Biology.

[59]  Heather J Munden,et al.  The Genome of the Kinetoplastid Parasite, Leishmania major , 2005, Science.