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• W2131249903 abstract "The African trypanosome Trypanosoma brucei transcribes the active variant surface glycoprotein (VSG) gene from one of about 20 VSG expression sites (ESs). In order to study ES control, we made reporter lines with a green fluorescent protein gene inserted behind the promoter of different ESs. We attempted to disrupt the silencing machinery, and we used fluorescence-activated cell sorter analysis for the rapid and sensitive detection of ES up-regulation. We find that a range of treatments that either block nuclear DNA synthesis, like aphidicolin, or modify DNA-like cisplatin and 1-methyl-3-nitro-1-nitrosoguanidine results in up-regulation of silent ESs. Aphidicolin treatment was the most effective, with almost 80% of the cells expressing green fluorescent protein from a silent ES. All of these treatments blocked the cells in S phase. In contrast, a range of toxic chemicals had little or no effect on expression. These included berenil and pentamidine, which selectively cleave the mitochondrial kinetoplast DNA, the metabolic inhibitors suramin and difluoromethylornithine, and the mitotic inhibitor rhizoxin. Up-regulation also affected other RNA polymerase I (pol I) transcription units, as procyclin genes were also up-regulated after cells were treated with either aphidicolin or DNA-modifying agents. Strikingly, this up-regulation of silent pol I transcription units was bloodstream form-specific and was not observed in insect form T. brucei. We postulate that the redistribution of a limiting bloodstream form-specific factor involved in both silencing and DNA repair results in the derepression of normally silenced pol I transcription units after DNA damage. The African trypanosome Trypanosoma brucei transcribes the active variant surface glycoprotein (VSG) gene from one of about 20 VSG expression sites (ESs). In order to study ES control, we made reporter lines with a green fluorescent protein gene inserted behind the promoter of different ESs. We attempted to disrupt the silencing machinery, and we used fluorescence-activated cell sorter analysis for the rapid and sensitive detection of ES up-regulation. We find that a range of treatments that either block nuclear DNA synthesis, like aphidicolin, or modify DNA-like cisplatin and 1-methyl-3-nitro-1-nitrosoguanidine results in up-regulation of silent ESs. Aphidicolin treatment was the most effective, with almost 80% of the cells expressing green fluorescent protein from a silent ES. All of these treatments blocked the cells in S phase. In contrast, a range of toxic chemicals had little or no effect on expression. These included berenil and pentamidine, which selectively cleave the mitochondrial kinetoplast DNA, the metabolic inhibitors suramin and difluoromethylornithine, and the mitotic inhibitor rhizoxin. Up-regulation also affected other RNA polymerase I (pol I) transcription units, as procyclin genes were also up-regulated after cells were treated with either aphidicolin or DNA-modifying agents. Strikingly, this up-regulation of silent pol I transcription units was bloodstream form-specific and was not observed in insect form T. brucei. We postulate that the redistribution of a limiting bloodstream form-specific factor involved in both silencing and DNA repair results in the derepression of normally silenced pol I transcription units after DNA damage. African trypanosomes including Trypanosoma brucei evade immune attack during chronic infections by periodically switching a variant surface glycoprotein (VSG) 1The abbreviations used are: VSG, variant surface glycoprotein; BAC, bacterial artificial chromosome; BrdUrd, 5-bromo-2′-deoxyuridine; DFMO, difluoromethylornithine; ES, expression site; ESAG, expression site associated gene; ESB, expression site body; FACS, fluorescence-activated cell sorter; GFP, green fluorescent protein; eGFP, enhanced GFP; MNNG, 1-methyl-3-nitro-1-nitrosoguanidine; pol I, polymerase I. 1The abbreviations used are: VSG, variant surface glycoprotein; BAC, bacterial artificial chromosome; BrdUrd, 5-bromo-2′-deoxyuridine; DFMO, difluoromethylornithine; ES, expression site; ESAG, expression site associated gene; ESB, expression site body; FACS, fluorescence-activated cell sorter; GFP, green fluorescent protein; eGFP, enhanced GFP; MNNG, 1-methyl-3-nitro-1-nitrosoguanidine; pol I, polymerase I. coat (1Barry J.D. McCulloch R. Adv. Parasitol. 2001; 49: 1-70Google Scholar, 2Borst P. Ulbert S. Mol. Biochem. Parasitol. 2001; 114: 17-27Google Scholar, 3Donelson J.E. Acta Trop. 2003; 85: 391-404Google Scholar, 4Vanhamme L. Lecordier L. Pays E. Int. J. Parasitol. 2001; 31: 523-531Google Scholar). The predominant VSG is encoded by a gene transcribed from 1 of about 20 telomeric VSG expression sites (ES). Switching VSG coats is mediated by DNA rearrangements moving a new VSG into the active ES from a repertoire of hundreds of silent VSG genes and pseudogenes in chromosome internal tandem arrays or at telomeres. Alternatively, a switch can be mediated by a transcriptional switch between ESs. The large polycistronic ES transcription units contain an assortment of expression site-associated genes (ESAGs) in addition to the telomeric VSG (reviewed in Ref. 5Pays E. Lips S. Nolan D. Vanhamme L. Perez-Morga D. Mol. Biochem. Parasitol. 2001; 114: 1-16Google Scholar). Although the basic ES structure is conserved, there is variation in exactly which ESAGs are present, as well as their number and order (6Berriman M. Hall N. Sheader K. Bringaud F. Tiwari B. Isobe T. Bowman S. Corton C. Clark L. Cross G.A. Hoek M. Zanders T. Berberof M. Borst P. Rudenko G. Mol. Biochem. Parasitol. 2002; 122: 131-140Google Scholar). Switching between the polymorphic ESAGs present in different ESs can allow the trypanosome to adapt to life in different hosts. This has been best investigated for the polymorphic ESAG6 and -7 genes encoding transferrin receptor subunits (7Bitter W. Gerrits H. Kieft R. Borst P. Nature. 1998; 391: 499-502Google Scholar, 8Gerrits H. MuBmann R. Bitter W. Kieft R. Borst P. Mol. Biochem. Parasitol. 2002; 119: 237-247Google Scholar, 9Isobe T. Holmes E.C. Rudenko G. J. Mol. Evol. 2003; 56: 377-386Google Scholar) and the serum resistance-associated gene conferring human serum resistance (10Xong H.V. Vanhamme L. Chamekh M. Chimfwembe C.E. Van Den Abbeele J. Pays A. Van Meirvenne N. Hamers R. De Baetselier P. Pays E. Cell. 1998; 95: 839-846Google Scholar, 11Vanhamme L. Paturiaux-Hanocq F. Poelvoorde P. Nolan D.P. Lins L. Van Den Abbeele J. Pays A. Tebabi P. Van Xong H. Jacquet A. Moguilevsky N. Dieu M. Kane J.P. De Baetselier P. Brasseur R. Pays E. Nature. 2003; 422: 83-87Google Scholar). The polycistronic ESs are regulated as domains flanked upstream by extensive arrays of 50-bp repeats (12Horn D. Cross G.A. Cell. 1995; 83: 555-561Google Scholar). Exogenous promoters integrated upstream of the 50-bp repeat arrays escape the transcriptional control operating on the downstream ES (13Sheader K. Berberof M. Isobe T. Borst P. Rudenko G. Mol. Biochem. Parasitol. 2003; 128: 147-156Google Scholar). In bloodstream form T. brucei, ES control is not sequence-specific (12Horn D. Cross G.A. Cell. 1995; 83: 555-561Google Scholar, 14Rudenko G. Blundell P.A. Dirks-Mulder A. Kieft R. Borst P. Cell. 1995; 83: 547-553Google Scholar), does not appear to involve a repressed chromatin state (15Navarro M. Cross G.A. Wirtz E. EMBO J. 1999; 18: 2265-2272Google Scholar, 16Navarro M. Cross G.A. Mol. Biochem. Parasitol. 1998; 94: 53-66Google Scholar), or require homologues of genes involved in yeast telomere position effect, despite having a superficial resemblance to this phenomenon (17Conway C. McCulloch R. Ginger M.L. Robinson N.P. Browitt A. Barry J.D. J. Biol. Chem. 2002; 277: 21269-21277Google Scholar). The multiple ESs are transcribed in a mutually exclusive fashion (reviewed in Ref. 18Borst P. Cell. 2002; 109: 5-8Google Scholar). Stable maximal activation of two ESs does not appear possible (19Chaves I. Rudenko G. Dirks-Mulder A. Cross M. Borst P. EMBO J. 1999; 18: 4846-4855Google Scholar). Selection for double ES expressors using selectable markers inserted immediately downstream of different ES promoters results in trypanosomes that have one ES maximally active and another ES partially up-regulated, or that rapidly switch between two different ESs (19Chaves I. Rudenko G. Dirks-Mulder A. Cross M. Borst P. EMBO J. 1999; 18: 4846-4855Google Scholar, 20Ulbert S. Chaves I. Borst P. Mol. Biochem. Parasitol. 2002; 120: 225-235Google Scholar). Possibly these rapidly switching trypanosomes alternate between a privileged subnuclear location that has been called an expression site body (ESB), a pol I transcriptional body specific to bloodstream form T. brucei (21Navarro M. Gull K. Nature. 2001; 414: 759-763Google Scholar). Location in an ESB transcription/RNA processing factory may give the active ES access to limiting factors necessary for transcription elongation and polyadenylation (22Vanhamme L. Poelvoorde P. Pays A. Tebabi P. Van Xong H. Pays E. Mol. Microbiol. 2000; 36: 328-340Google Scholar). In contrast, in insect form T. brucei all ESs appear to be down-regulated to a great extent, although not as tightly as in the bloodstream form (23Rudenko G. Blundell P.A. Taylor M.C. Kieft R. Borst P. EMBO J. 1994; 13: 5470-5482Google Scholar). Silencing is mechanistically different, as it is promoter sequence-specific and appears to involve repressed chromatin (15Navarro M. Cross G.A. Wirtz E. EMBO J. 1999; 18: 2265-2272Google Scholar). However, it is unlikely that the telomeric location of ESs is critical for silencing in this life cycle stage, as ES promoters on circular bacterial artificial chromosomes (BACs) are as effectively silenced as those in the genome (24te Vruchte D. Aitcheson N. Rudenko G. Mol. Biochem. Parasitol. 2003; 128: 123-133Google Scholar). In order to investigate the machinery mediating ES down-regulation in bloodstream form T. brucei, we constructed reporter T. brucei strains with GFP inserted immediately behind the promoter of silenced ESs. We attempted to disrupt the silencing machinery using various chemical treatments. Fluorescence-activated cell sorting (FACS) analysis allowed rapid and sensitive detection of ES derepression. We find that treatments inhibiting DNA synthesis or causing DNA damage result in a block in S phase and a concurrent up-regulation of silent ESs. In addition, up-regulation of procyclin transcript was also observed. Procyclin is transcribed from RNA polymerase I transcription units that are normally down-regulated in the bloodstream form. Surprisingly, this up-regulation of transcripts from normally silent sites is life cycle-specific, as there was no evidence for up-regulation of silenced ESs in insect form T. brucei after comparable treatments. We postulate the presence of a limiting bloodstream form-specific factor involved in both silencing and DNA repair. Redistribution after DNA damage could result in the derepression of normally silenced pol I transcription units. T. brucei Transformants and Culturing Conditions—All trypanosomes used were T. brucei 427, and bloodstream form variants were all derived from T. brucei 427 VSG221a (25Bernards A. de Lange T. Michels P.A. Liu A.Y. Huisman M.J. Borst P. Cell. 1984; 36: 163-170Google Scholar). Bloodstream form T. brucei was maintained at 37 °C in HMI-9 medium with the addition of 10% fetal calf serum and 10% Serum Plus (JRH Biosciences) (26Hirumi H. Hirumi K. J. Parasitol. 1989; 75: 985-989Google Scholar). In order to ensure population homogeneity of the VSG coat expressed, trypanosome transformants with drug markers in the active VSG ES were continuously maintained on the appropriate drug selection pressure to prevent ES switching. The T. brucei RP2X-1 transformant was described previously (14Rudenko G. Blundell P.A. Dirks-Mulder A. Kieft R. Borst P. Cell. 1995; 83: 547-553Google Scholar). T. brucei HNI has a hygromycin gene inserted immediately behind the promoter of the 221 ES and a neomycin gene inserted behind the promoter of the VO2 ES and was described previously (27Rudenko G. Chaves I. Dirks-Mulder A. Borst P. Mol. Biochem. Parasitol. 1998; 95: 97-109Google Scholar). T. brucei 221GP1(221+) has a GFP gene in the active 221 ES and was made by replacing the hygromycin gene in the active 221 ES of T. brucei HNI(221+) with the 221GP1 construct containing GFP and the puromycin resistance gene. These trypanosomes were subsequently selected on G418 to select for trypanosomes that had activated the neomycin gene marked VO2 ES and silenced the GFP, resulting in T. brucei 221GP1(VO2+). T. brucei VO2GP1(VO2+) has GFP in the active VO2 ES and was made by modifying T. brucei HNI(VO2+) by replacing the neomycin gene in the active VO2 ES with the VO2GP1 construct containing the GFP and blasticidin genes. These trypanosomes were subsequently selected on hygromycin to select for trypanosomes that had activated the hygromycin gene marked 221 ES and silenced the VO2 ES producing T. brucei VO2GP1(221+). The hygromycin resistance gene was subsequently replaced by a gene encoding puromycin resistance to create T. brucei VO2GP2(221+). Insect form T. brucei 427 was maintained at 27 °C in SDM-79 medium with the addition of 10% fetal calf serum (28Brun R. Schonenberger M. Acta Trop. 1979; 36: 289-292Google Scholar). The insect form T. brucei transformants (RPX1-1, ESX1-1, ESX1-2, and rDES1-1) were described previously (23Rudenko G. Blundell P.A. Taylor M.C. Kieft R. Borst P. EMBO J. 1994; 13: 5470-5482Google Scholar) and were maintained on 25 μg ml–1 hygromycin. Transfection Constructs—The 221GP1 construct has a puromycin resistance gene excised from construct pBS-Pur (gift of Isabel Roditi, University of Berne) (29Ruepp S. Furger A. Kurath U. Renggli C.K. Hemphill A. Brun R. Roditi I. J. Cell Biol. 1997; 137: 1369-1379Google Scholar) and inserted between tubulin intergenic regions containing splice and polyadenylation sites (23Rudenko G. Blundell P.A. Taylor M.C. Kieft R. Borst P. EMBO J. 1994; 13: 5470-5482Google Scholar). The GFP gene is eGFP (Clontech) flanked upstream by the tubulin 3′ splice site present on a tubulin intergenic region, and downstream by the 221 VSG untranslated region and polyadenylation signal. The downstream flanking sequences are on a 612-bp fragment amplified by PCR using Pwo polymerase (Roche Applied Science) from a genomic clone of the 221 VSG 2T. Isobe and G. Rudenko, unpublished data. using the following primers: 221VSG3445s, 5′-TTTCCCCCCTCAAATTTCCCC-3′, and 221VSG4057as, 5′-CGAAAAATTAAGATTCAAAACCACGG-3′. The 1.3-kb 5′ 221 ES target fragment was amplified from the H25N7 BAC containing the 221 VSG ES (GenBank™ account number AL671259) (6Berriman M. Hall N. Sheader K. Bringaud F. Tiwari B. Isobe T. Bowman S. Corton C. Clark L. Cross G.A. Hoek M. Zanders T. Berberof M. Borst P. Rudenko G. Mol. Biochem. Parasitol. 2002; 122: 131-140Google Scholar) using PCR with Pwo polymerase and primers as follows: HNES481s, 5′-TTAAGCTTTCTAACACTTCCTTTTTG-3′, and VO228551as, 5′-AACCTCAATGGACGAAGGAG-3′. Integration of the construct was 216 bp downstream of the transcription start site. The 714-bp 3′ 221 ES target fragment was isolated from the H25N7 BAC using primers 221–73592s 5′-ACGAAGAGCAGGGGTGCAAC-3′ and HNES4244as 5′-GCTTCATCTGC-TGGTCGTCTTC-3′. The VO2GP1 construct was analogous to the 221GP1 construct, and only the puromycin resistance gene was replaced by a blasticidin resistance gene from construct tubBSRtub (gift of P. Borst, Netherlands Cancer Institute, Amsterdam), and the target fragments were amplified by PCR from the N19B2 BAC containing part of the VO2 ES (GenBank™ account number AL671256) (6Berriman M. Hall N. Sheader K. Bringaud F. Tiwari B. Isobe T. Bowman S. Corton C. Clark L. Cross G.A. Hoek M. Zanders T. Berberof M. Borst P. Rudenko G. Mol. Biochem. Parasitol. 2002; 122: 131-140Google Scholar). The 5′ target fragment was amplified using the HNES481s and VO228551as primers listed above. The 3′ target fragment was amplified using primers VO2–118336s 5′-CTCTAGTGAGCGTATTTTAGAGG-3′ and HNES 4244as. T. brucei Transformation—Transfection was performed using mid-log phase bloodstream form trypanosomes, which were washed and then resuspended at 5 × 107 ml–1 in cytomix without glutathione or ATP (30van den Hoff M.J. Moorman A.F. Lamers W.H. Nucleic Acids Res. 1992; 20: 2902Google Scholar). 2.5 × 107 cells were electroporated with 10 μg of linearized DNA using a Bio-Rad Gene Pulser II with a single pulse of 1.5-kV and 25-microfarad capacitance in 0.2-cm cuvettes (Bio-Rad). After ∼6 h recovery in HMI-9 medium, cells were distributed over 24-wells plates at densities between 7 × 104 and 3 × 105 cells ml–1. Selection was with 0.2 μg ml–1 puromycin (Sigma) or 2 μg ml–1 blasticidin (Invitrogen). After 5–7 days on selection transformants were expanded for further analysis. ES Switching—ES switching was performed using drug selection on agarose plates (31Carruthers V.B. Cross G.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8818-8821Google Scholar). T. brucei 221GP1(221+) with the puromycin gene in the active 221 ES was maintained in the presence of 0.2 μg of puromycin ml–1 (Sigma). To switch to the VO2 ES, 1 × 105 to 1 × 107 trypanosomes were spread on agarose plates containing 5 μg of G418 (Invitrogen) ml–1. Colonies were counted after 9–10 days to determine the switching frequency (average of 4.4 × 10–6 determined on three plates) and analyzed by PCR to check for retention of the previously active 221 ES (27Rudenko G. Chaves I. Dirks-Mulder A. Borst P. Mol. Biochem. Parasitol. 1998; 95: 97-109Google Scholar). T. brucei VO2GP1(VO2+) was switched in an analogous fashion; only these trypanosomes were maintained on 2 μgof blasticidin ml–1 and selected on 5 μg of hygromycin ml–1 to select for activation of the 221 ES. The frequency of 221 ES activation was 3 × 10–6 (average of four plates). Again PCR was used to monitor for retention of the VO2 ES. T. brucei Chemical or DNA Damage Treatments—Mid-log phase cultures were pelleted and resuspended in fresh medium to remove drug selection pressure. Bloodstream form T. brucei was resuspended at 5 × 105 cells ml–1, and insect form T. brucei was resuspended at 5 × 106 cells ml–1. Cultures were treated with the appropriate chemical for 24 h for bloodstream form T. brucei unless stated otherwise in the figure legends or 48 h for insect form T. brucei. Alternatively, bloodstream form T. brucei was exposed to UV or γ-radiation and then recovered for 24 h. Aphidicolin, MNNG, berenil, pentamidine, suramin, trichostatin A, difluoromethylornithine (DFMO), and pCPT-cAMP were all from Sigma, and ethidium bromide from BDH. Cisplatin was a gift of Adrian Begg and Ben Floot (Netherlands Cancer Institute, Amsterdam) and rhizoxin a gift of Keith Gull. Growth curves were initially performed with each of the different substances, and concentrations were chosen that flattened cell growth in a comparable fashion. Concentrations used are as follows: aphidicolin (30 μm) (32Ploubidou A. Robinson D.R. Docherty R.C. Ogbadoyi E.O. Gull K. J. Cell Sci. 1999; 112: 4641-4650Google Scholar), MNNG (2 μgml–1) (20Ulbert S. Chaves I. Borst P. Mol. Biochem. Parasitol. 2002; 120: 225-235Google Scholar, 33King D.L. Turco S.J. Mol. Biochem. Parasitol. 1988; 28: 285-293Google Scholar), berenil (1 μm) (34Shapiro T.A. Englund P.T. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 950-954Google Scholar), pentamidine (1 μm) (34Shapiro T.A. Englund P.T. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 950-954Google Scholar), suramin (400 nm), trichostatin A (2 μg ml–1), DFMO (difluoromethylornithine) (200 μm) (35Zweygarth E. Kaminsky R. Acta Trop. 1991; 48: 223-232Google Scholar), pCPT-cAMP (1 mm) (36Breidbach T. Ngazoa E. Steverding D. Exp. Parasitol. 2002; 101: 223-230Google Scholar), ethidium bromide (8 μm) (37Boibessot I. Turner C.M. Watson D.G. Goldie E. Connel G. McIntosh A. Grant M.H. Skellern G.G. Acta Trop. 2002; 84: 219-228Google Scholar), cisplatin (4 μm) (38Balber A.E. Gonias S.L. Pizzo S.V. Exp. Parasitol. 1985; 59: 74-80Google Scholar), and rhizoxin (10 nm) (32Ploubidou A. Robinson D.R. Docherty R.C. Ogbadoyi E.O. Gull K. J. Cell Sci. 1999; 112: 4641-4650Google Scholar). UV irradiation was performed with 3840 J/m2 UV light (254 nm) using a UV cross-linker (Stratagene). Cells were in 25 ml of tissue culture dishes with the lid removed during exposure (adapted from Ref. 39Johnson P.J. Kooter J.M. Borst P. Cell. 1987; 51: 273-281Google Scholar). γ-Irradiation was performed using a 137Cs source (Gravitron RX30/55M, Graviner Manufacturing, Gosport, Hampshire, UK). Trypanosomes in HMI-9 medium were placed at a proximity to the cesium source so that the dose rate was 4.4 gray/min for a total dose of 160 gray. After removal from the exposure to UV or γ-radiation, cells were recovered for 24 h. The number of double-strand DNA breaks introduced into the T. brucei genome was estimated using agarose blocks containing T. brucei transfected with BACs of ∼150 kb (24te Vruchte D. Aitcheson N. Rudenko G. Mol. Biochem. Parasitol. 2003; 128: 123-133Google Scholar). Introduction of a single double-strand break into these large circles results in linearization. We estimated that 160 gray irradiation resulted in the introduction of ∼370 double-strand breaks into the T. brucei genome. FACS Analysis—Derepression of GFP was analyzed by FACS using a BD Biosciences FACSCalibur with an excitation wavelength of 488 nm. After treatment and recovery, cells were washed once in PSG (60 mm Na2HPO4, 3 mm NaH2PO4, 44 mm NaCl, 56 mm glucose) and resuspended at 106 cells ml–1 for analysis. Data were analyzed using CellQuest software version 3.3. To correct for fluorescence of the chemical used, T. brucei HNI, which lacks GFP, was also treated and analyzed by FACS, and any fluorescence in the FL-1 channel was subtracted from the final values. In addition, values were corrected by subtracting background fluorescence of untreated cells containing silenced GFP. For cell cycle analysis cells were stained with propidium iodide as described previously (40Hammarton T.C. Clark J. Douglas F. Boshart M. Mottram J.C. J. Biol. Chem. 2003; 278: 22877-22886Google Scholar). Briefly, after treatment and recovery, cells were fixed in 70% methanol (106 cells ml–1) and incubated at 4 °C overnight. Cells were washed in cold phosphate-buffered saline, resuspended in 1 ml of phosphate-buffered saline containing 10 μg ml–1 propidium iodide and 10 μg ml–1 RNase A, and incubated at 37 °C for 45 min. Detector FL2-A and an AmpGain value of 1.75 were used. RNA Analysis—Total RNA was isolated from bloodstream form T. brucei treated for 24 (bloodstream form) or 48 h (procyclic form) with various chemicals in the absence of drug selection pressure. RNA was isolated from ∼3 to 5 × 107 cells using an RNeasy RNA isolation kit (Qiagen). 5 μg of total RNA was electrophoresed in formaldehyde gels and blotted according to Ref. 41Sambrook J. Russell D.W. Molecular Cloning: A Laboratory Manual. 3rd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2001Google Scholar. Northern blots were hybridized with probes radiolabeled by random priming using the Megaprime DNA labeling system (Amersham Biosciences). The probe for eGFP is a 719-bp SalI/NotI fragment from the eGFP plasmid (Clontech). The probe for the 221 VSG is an 800-bp fragment of the 221 VSG corresponding to positions 122–925 in the sequence (GenBank™ account number X56762). The probe for the VO2 VSG is a 600-bp EcoRI/HindIII fragment from the VO2 VSG cDNA. 3K. Sheader and G. Rudenko, unpublished data. The tubulin probe is a 700-bp HindIII/EcoRI fragment. The procyclin probe is the entire CPT4 cDNA (42Rudenko G. Bishop D. Gottesdiener K. Van der Ploeg L.H. EMBO J. 1989; 8: 4259-4263Google Scholar). The procyclin probe can be expected to hybridize with transcript from both the EP and GPEET procyclin variants. Quantitation was performed with a Bio-Rad PhosphorImager. DNA Damage Results in the Up-regulation of GFP Marked Silent ESs—We constructed T. brucei strains with GFP immediately downstream of the promoter of the inactivated 221 or VO2 VSG ESs. As the ES promoters were silent, these strains allowed us to assay for disruption of the ES silencing machinery. We introduced a puromycin resistance gene linked to GFP into the active 221 VSG ES of T. brucei HNI(221+) (27Rudenko G. Chaves I. Dirks-Mulder A. Borst P. Mol. Biochem. Parasitol. 1998; 95: 97-109Google Scholar) (Fig. 1A). The resulting transformant T. brucei 221GP1(221+) had the hygromycin gene located downstream of the 221 VSG ES promoter of T. brucei HNI(221+) replaced by a puromycin gene. As this modified variant of T. brucei HNI contains a neomycin gene located downstream of the silent VO2 VSG ES promoter, selection with G418 allowed us to screen for reactivation of the VO2 VSG ES, producing the reporter strain T. brucei 221GP1(VO2+) (Fig. 1B). T. brucei expressing GFP from an active VSG ES is more than 1000-fold brighter than background, allowing it to be very easily detected (Fig. 1C). FACS analysis of the T. brucei 221GP1(VO2+) trypanosomes with a silenced GFP provided a very sensitive and rapid assay system for measuring a change in expression of the silent VSG ES. We incubated the T. brucei 221GP1(VO2+) reporter strain with a range of chemicals in order to screen for those resulting in the up-regulation of silent VSG ESs. First, we tested the histone deacetylase inhibitor trichostatin A, which generally causes derepression of silenced transcription units in a wide range of experimental systems due to disruption of repressed chromatin (reviewed in Ref. 43Yoshida M. Horinouchi S. Beppu T. BioEssays. 1995; 17: 423-430Google Scholar). Although trichostatin A suppressed growth of bloodstream form T. brucei, it had no detectable effect on derepression of the silent 221 VSG ES (result not shown). As there is no evidence that silenced ESs in bloodstream form T. brucei have a more repressed chromatin structure than active ESs (15Navarro M. Cross G.A. Wirtz E. EMBO J. 1999; 18: 2265-2272Google Scholar, 16Navarro M. Cross G.A. Mol. Biochem. Parasitol. 1998; 94: 53-66Google Scholar), this result is not surprising. Although trichostatin A did not cause up-regulation of silent ESs, a range of treatments resulting in DNA modification or damage did (Fig. 2). Effective treatments that resulted in high levels of up-regulation of normally silent VSG ESs included DNA-modifying agents. Cisplatin treatment results in the introduction of a bulky cisplatin adduct into DNA (44van de Vaart P.J. Belderbos J. de Jong D. Sneeuw K.C. Majoor D. Bartelink H. Begg A.C. Int. J. Cancer. 2000; 89: 160-166Google Scholar), and MNNG methylates DNA (45Bignami M. O'Driscoll M. Aquilina G. Karran P. Mutat. Res. 2000; 462: 71-82Google Scholar). Treatment of bloodstream form T. brucei with both of these chemicals resulted in up to 40% of the cells expressing GFP (Fig. 2). Treatment with UV light also resulted in DNA modification in the form of thymidine dimers. This treatment was also effective at producing the up-regulation of GFP, as was the intercalating agent ethidium bromide. DNA damage in the absence of DNA modification was also effective in inducing up-regulation of GFP transcript. The introduction of double-strand DNA breaks with a cesium source also resulted in very high levels of silent VSG ES up-regulation, with more than 50% of the cells expressing GFP (Fig. 2). In contrast, berenil and pentamidine selectively induced double-strand breaks in the kinetoplast DNA rather than nuclear DNA (34Shapiro T.A. Englund P.T. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 950-954Google Scholar). Treatment with these drugs did not result in significant ES derepression. Treatment with the metabolic inhibitor suramin (46Fairlamb A.H. Bowman I.B. Mol. Biochem. Parasitol. 1980; 1: 315-333Google Scholar) or the ornithine decarboxylase inhibitor DFMO (35Zweygarth E. Kaminsky R. Acta Trop. 1991; 48: 223-232Google Scholar, 47Phillips M.A. Coffino P. Wang C.C. J. Biol. Chem. 1987; 262: 8721-8727Google Scholar) also had no detectable effect, despite inhibiting T. brucei cell growth in a comparable fashion to the other treatments (see “Experimental Procedures” for details). It is therefore likely that modification or damage of nuclear DNA is the key factor in the up-regulation of silent ESs that we observed. Up-regulation of Silent ESs Correlates with a Block in S Phase—Treatment of T. brucei with DNA-modifying agents resulted in a block in DNA synthesis, as observed by measuring incorporation of BrdUrd in treated cells (result not shown) (48Woodward R. Gull K. J. Cell Sci. 1990; 95: 49-57Google Scholar). In order to block nuclear DNA synthesis without introducing DNA damage, we incubated bloodstream form T. brucei with aphidicolin, which selectively binds and inhibits nuclear DNA polymerase α (49Pedrali-Noy G. Spadari S. J. Virol. 1980; 36: 457-464Google Scholar, 50Sheaff R. Ilsley D. Kuchta R. Biochemistry. 1991; 30: 8590-8597Google Scholar). Aphidicolin has been used previously to stall trypanosomes at the G1/S phase transition (32Ploubidou A. Robinson D.R. Docherty R.C. Ogbadoyi E.O. Gull K. J. Cell Sci. 1999; 112: 4641-4650Google Scholar, 51Mutomba M.C. Wang C.C. Mol. Biochem. Parasitol. 1996; 80: 89-102Google Scholar). Treatment of bloodstream form T. brucei 221GP1(VO2+) with 30 μm aphidicolin resulted in very striking up-regulation of the silenced GFP (Fig. 2). Almost 80% of the cells expressed GFP, with almost 20% of the cells expressing GFP at maximal levels (dark bar in Fig. 2) (see Fig. 1C for description of the gates used). As up-regulation of silent ESs was observed after treatments that resulted in a block in nuclear DNA synthesis, we investigated if this could also be observed if T. brucei was stalled in the G1/G0 phase of the cell cycle. DNA synthesis is inhibited in stumpy form T. bruc" @default.
• W2131249903 created "2016-06-24" @default.
• W2131249903 creator A5041211806 @default.
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• W2131249903 creator A5084898942 @default.
• W2131249903 date "2004-04-01" @default.
• W2131249903 modified "2023-09-26" @default.
• W2131249903 title "Bloodstream Form-specific Up-regulation of Silent VSG Expression Sites and Procyclin in Trypanosoma brucei after Inhibition of DNA Synthesis or DNA Damage" @default.
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• W2131249903 doi "https://doi.org/10.1074/jbc.m312307200" @default.
• W2131249903 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/14726511" @default.
• W2131249903 hasPublicationYear "2004" @default.
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