Trypanosoma brucei is a protozoan parasite that lacks many transcription factors found in other eukaryotes, such as those whose binding demarcates enhancers. T. brucei retains histone variants and modifications, however, and it is hypothesized that it relies on epigenetic marks to define transcription-related boundaries. The histone H3 variant (H3.V) and an alternate nucleotide, base J (？-D-glucosyl-hydroxymethyluracil), are two chromatin marks found at both transcription termination sites (TTSs) and telomeres. Here, we report that the absence of both base J and H3.V result in transcription readthrough and the appearance of antisense transcripts near TTSs. Additionally, we find that maintaining the transcriptional silencing of pol I-transcribed telomeric Variant Surface Glycoprotein (VSG) genes appears to be dependent on deposition of H3.V alone. Our study reveals that gene expression depends on different epigenetic cues depending on chromosomal location and on the transcribing polymerase. This work provides insight into how these signals may have evolved into the more nuanced and fine-tuned gene regulatory mechanisms observed in other model systems.
References
[1]
Matthews KR (2005) The developmental cell biology of Trypanosoma brucei. J Cell Sci 118: 283–290. doi: 10.1242/jcs.01649. pmid:15654017
[2]
Matthews KR, Tschudi C, Ullu E (1994) A common pyrimidine-rich motif governs trans-splicing and polyadenylation of tubulin polycistronic pre-mRNA in trypanosomes. Genes Dev 8: 491–501. pmid:7907303 doi: 10.1101/gad.8.4.491
[3]
Lee JH, Nguyen TN, Schimanski B, Günzl A (2007) Spliced leader RNA gene transcription in Trypanosoma brucei requires transcription factor TFIIH. Eukaryotic Cell 6: 641–649. doi: 10.1128/EC.00411-06. pmid:17259543
[4]
Schimanski B, Nguyen TN, Günzl A (2005) Characterization of a multisubunit transcription factor complex essential for spliced-leader RNA gene transcription in Trypanosoma brucei. Mol Cell Biol 25: 7303–7313. doi: 10.1128/MCB.25.16.7303-7313.2005. pmid:16055738
[5]
Nguyen TN, Nguyen BN, Lee JH, Panigrahi AK, Günzl A (2012) Characterization of a novel CITFA subunit that is indispensable for transcription by Trypanosoma brucei's multifunctional RNA polymerase I. Eukaryotic Cell. doi: 10.1128/EC.00250-12.
[6]
Siegel TN, Hekstra DR, Kemp LE, Figueiredo LM, Lowell JE, et al. (2009) Four histone variants mark the boundaries of polycistronic transcription units in Trypanosoma brucei. Genes Dev 23: 1063–1076. doi: 10.1101/gad.1790409. pmid:19369410
[7]
Wright JR, Siegel TN, Cross GAM (2010) Histone H3 trimethylated at lysine 4 is enriched at probable transcription start sites in Trypanosoma brucei. Mol Biochem Parasitol 172: 141–144. doi: 10.1016/j.molbiopara.2010.03.013. pmid:20347883
[8]
Borst P, Sabatini R (2008) Base J: discovery, biosynthesis, and possible functions. Annu Rev Microbiol 62: 235–251. doi: 10.1146/annurev.micro.62.081307.162750. pmid:18729733
[9]
Cross M, Kieft R, Sabatini R, Dirks-Mulder A, Chaves I, et al. (2002) J-binding protein increases the level and retention of the unusual base J in trypanosome DNA. Mol Microbiol 46: 37–47. pmid:12366829 doi: 10.1046/j.1365-2958.2002.03144.x
[10]
Bullard W, Lopes da Rosa-Spiegler J, Liu S, Wang Y, Sabatini R (2014) Identification of the glucosyltransferase that converts hydroxymethyluracil to base J in the trypanosomatid genome. J Biol Chem 289: 20273–20282. doi: 10.1074/jbc.M114.579821. pmid:24891501
[11]
Cliffe LJ, Kieft R, Southern T, Birkeland SR, Marshall M, et al. (2009) JBP1 and JBP2 are two distinct thymidine hydroxylases involved in J biosynthesis in genomic DNA of African trypanosomes. Nucleic Acids Res 37: 1452–1462. doi: 10.1093/nar/gkn1067. pmid:19136460
[12]
Cliffe LJ, Siegel TN, Marshall M, Cross GAM, Sabatini R (2010) Two thymidine hydroxylases differentially regulate the formation of glucosylated DNA at regions flanking polymerase II polycistronic transcription units throughout the genome of Trypanosoma brucei. Nucleic Acids Res 38: 3923–3935. doi: 10.1093/nar/gkq146. pmid:20215442
[13]
Reynolds D, Cliffe L, F？rstner KU, Hon C-C, Siegel TN, et al. (2014) Regulation of transcription termination by glucosylated hydroxymethyluracil, base J, in Leishmania major and Trypanosoma brucei. Nucleic Acids Res 42: 9717–9729. doi: 10.1093/nar/gku714. pmid:25104019
[14]
van Luenen HGAM, Farris C, Jan S, Genest P-A, Tripathi P, et al. (2012) Glucosylated hydroxymethyluracil, DNA base j, prevents transcriptional readthrough in leishmania. Cell 150: 909–921. doi: 10.1016/j.cell.2012.07.030. pmid:22939620
[15]
Anderson BA, Wong ILK, Baugh L, Ramasamy G, Myler PJ, et al. (2013) Kinetoplastid-specific histone variant functions are conserved in Leishmania major. Mol Biochem Parasitol 191: 53–57. doi: 10.1016/j.molbiopara.2013.09.005. pmid:24080031
[16]
Hertz-Fowler C, Figueiredo LM, Quail MA, Becker M, Jackson A, et al. (2008) Telomeric expression sites are highly conserved in Trypanosoma brucei. PLoS ONE 3: e3527. doi: 10.1371/journal.pone.0003527. pmid:18953401
[17]
Cross GAM, Kim H-S, Wickstead B (2014) Capturing the variant surface glycoprotein repertoire (the VSGnome) of Trypanosoma brucei Lister 427. Mol Biochem Parasitol 195: 59–73. doi: 10.1016/j.molbiopara.2014.06.004. pmid:24992042
[18]
Kim H-S, Park SH, Günzl A, Cross GAM (2013) MCM-BP is required for repression of life-cycle specific genes transcribed by RNA polymerase I in the mammalian infectious form of Trypanosoma brucei. PLoS ONE 8: e57001. doi: 10.1371/journal.pone.0057001. pmid:23451133
[19]
Lowell JE, Cross GAM (2004) A variant histone H3 is enriched at telomeres in Trypanosoma brucei. J Cell Sci 117: 5937–5947. doi: 10.1242/jcs.01515. pmid:15522895
[20]
van Leeuwen F, Wijsman ER, Kuyl-Yeheskiely E, van der Marel GA, van Boom JH, et al. (1996) The telomeric GGGTTA repeats of Trypanosoma brucei contain the hypermodified base J in both strands. Nucleic Acids Res 24: 2476–2482. pmid:8692684 doi: 10.1093/nar/24.13.2476
[21]
van Leeuwen F, Wijsman ER, Kieft R, van der Marel GA, van Boom JH, et al. (1997) Localization of the modified base J in telomeric VSG gene expression sites of Trypanosoma brucei. Genes Dev 11: 3232–3241. pmid:9389654 doi: 10.1101/gad.11.23.3232
[22]
Kolev NG, Ramey-Butler K, Cross GAM, Ullu E, Tschudi C (2012) Developmental progression to infectivity in Trypanosoma brucei triggered by an RNA-binding protein. Science 338: 1352–1353. doi: 10.1126/science.1229641. pmid:23224556
Hovel-Miner GA, Boothroyd CE, Mugnier M, Dreesen O, Cross GAM, et al. (2012) Telomere length affects the frequency and mechanism of antigenic variation in Trypanosoma brucei. PLoS Pathog 8: e1002900. doi: 10.1371/journal.ppat.1002900. pmid:22952449
[25]
Benetti R, García-Cao M, Blasco MA (2007) Telomere length regulates the epigenetic status of mammalian telomeres and subtelomeres. Nat Genet 39: 243–250. doi: 10.1038/ng1952. pmid:17237781
[26]
Kolev NG, Franklin JB, Carmi S, Shi H, Michaeli S, et al. (2010) The transcriptome of the human pathogen Trypanosoma brucei at single-nucleotide resolution. PLoS Pathog 6. doi: 10.1371/journal.ppat.1001090.
[27]
Ekanayake DK, Minning T, Weatherly B, Gunasekera K, Nilsson D, et al. (2011) Epigenetic regulation of transcription and virulence in Trypanosoma cruzi by O-linked thymine glucosylation of DNA. Mol Cell Biol 31: 1690–1700. doi: 10.1128/MCB.01277-10. pmid:21321080
[28]
Delatte B, Fuks F (2013) TET proteins: on the frenetic hunt for new cytosine modifications. Brief Funct Genomics 12: 191–204. doi: 10.1093/bfgp/elt010. pmid:23625996
[29]
Pfaffeneder T, Spada F, Wagner M, Brandmayr C, Laube SK, et al. (2014) Tet oxidizes thymine to 5-hydroxymethyluracil in mouse embryonic stem cell DNA. Nat Chem Biol 10: 574–581. doi: 10.1038/nchembio.1532. pmid:24838012
[30]
Pastor WA, Aravind L, Rao A (2013) TETonic shift: biological roles of TET proteins in DNA demethylation and transcription. Nat Rev Mol Cell Biol 14: 341–356. doi: 10.1038/nrm3589. pmid:23698584
[31]
Delatte B, Deplus R, Fuks F (2014) Playing TETris with DNA modifications. EMBO J 33: 1198–1211. doi: 10.15252/embj.201488290. pmid:24825349
[32]
Wu H, D'Alessio AC, Ito S, Xia K, Wang Z, et al. (2011) Dual functions of Tet1 in transcriptional regulation in mouse embryonic stem cells. Nature 473: 389–393. doi: 10.1038/nature09934. pmid:21451524
[33]
Wirtz E, Leal S, Ochatt C, Cross GA (1999) A tightly regulated inducible expression system for conditional gene knock-outs and dominant-negative genetics in Trypanosoma brucei. Mol Biochem Parasitol 99: 89–101. pmid:10215027 doi: 10.1016/s0166-6851(99)00002-x
[34]
Hirumi H, Hirumi K (1989) Continuous cultivation of Trypanosoma brucei blood stream forms in a medium containing a low concentration of serum protein without feeder cell layers. J Parasitol 75: 985–989. pmid:2614608 doi: 10.2307/3282883
[35]
Boothroyd CE, Dreesen O, Leonova T, Ly KI, Figueiredo LM, et al. (2009) A yeast-endonuclease-generated DNA break induces antigenic switching in Trypanosoma brucei. Nature 459: 278–281. doi: 10.1038/nature07982. pmid:19369939
[36]
Kim H-S, Cross GAM (2010) TOPO3alpha influences antigenic variation by monitoring expression-site-associated VSG switching in Trypanosoma brucei. PLoS Pathog 6: e1000992. doi: 10.1371/journal.ppat.1000992. pmid:20628569
[37]
Langmead B, Trapnell C, Pop M, Salzberg SL (2009) Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol 10: R25. doi: 10.1186/gb-2009-10-3-r25. pmid:19261174
