Laurence Lecordier*, David Walgraffe*, Sara Devaux, Philippe Poelvoorde, Etienne Pays and Luc Vanhamme.
* The two first authors contributed equally to this work.
Laboratory of Molecular Parasitology,
IBMM (Institute for molecular biology and medicine), Free University of Brussels,
12 rue des Professeurs Jeener et Brachet, 6041 Gosselies, Belgium
Corresponding author:
Luc Vanhamme
Laboratory of Molecular Parasitology, IBMM (Institute for molecular biology and medicine), Free University of Brussels,
12 rue des Professeurs Jeener et Brachet, 6041 Gosselies, Belgium
Tel : 32-2-6509758 Telefax: 32-2-6509760
E-mail : Luc.vanhamme@ulb.ac.be
Abbreviations: dsRNA, double stranded RNA ; miRNA, micro RNA ; RNAi, RNA interference ; siRNA, small interfering RNA.
2 RNA interference abrogates gene expression through transcription of double stranded RNA (dsRNA). It is mediated by a natural biological mechanism which triggers the transformation of these dsRNAs into shorter versions of 21 to 23 nt. These siRNAs (small interfering RNAs) then drive gene silencing by sequence specific mRNA degradation or by inhibition of protein synthesis. Endogenous RNAi generates active siRNAs from long natural dsRNAs or from microRNA (miRNA) hairpin structures [1-5].
RNAi can theoretically be used as a tool in all cell types possessing this endogenous pathway. Trypanosoma brucei falls into this category. The feasibility of this technique in trypanosomes was originally demonstrated by Gull and colleagues [6] and by Tschudi and Ullu [7]. Several groups (Clayton, Cross [8,9] Englund [10], Donelson [11], Gull [12] and Tschudi and Ullu[13] ) have since engineered cell lines and plasmids allowing inducible dsRNA transcription from a fragment of interest [reviewed in 14, 15].
Trypanosoma brucei is a unicellular eukaryote belonging to the order kinetoplastidae. It is the agent of two major afflictions affecting the African continent, human sleeping sickness that kills an estimated 300,000 persons a year and nagana, which is the major obstacle to efficient cattle rearing throughout large regions of the African continent [www.who.org].
Trypanosomes are a privileged model for the study of mechanisms involved in host-parasite interactions. These parasites escape their mammalian hosts defences through
immunosuppressive activity [16] as well as antigenic variation [17,18], a regular change of their major surface antigen, the variant surface glycoprotein or VSG. In addition, some subspecies have also developed resistance to an innate trypanolytic factor [19]. While some aspects of these host-parasite interactions can be covered by studies in vitro, others, such as the analysis of trypanosome components on immunosuppressive activity are better suited to in vivo studies ..These studies would greatly benefit from the possibility of inducing RNAi in vivo. Two steps are required in order to obtain such a system .
which have lost the ability to differentiate into stumpy forms. They kill laboratory rodents within a few days after injection. On the contrary, pleomorphic strains differentiate from proliferative slender forms into quiescent stumpy forms in the bloodstream, a process which allows the development of chronic infections. The first step towards in vivo RNAi would therefore be the creation of an equivalent pleomorphic cell line..
In this paper, we address the second necessary step, the induction of RNAi in transgenic trypanosomes inside the mammalian host. To do so, systemic administration of tetracycline or an analogue is required. Tetracycline and its analogues have been successfully used in
animals, in order to induce the cre recombinase in conditional knockout mice [20], and to induce protein over-expression in trypanosomes [21].
We performed RNAi-mediated knock down of two genes coding for general transcription factors. TbTFIIS1 (GeneDB accession number Tb11.02.2600) and TbXPD (GeneDB accession number Tb08.11J15.890). The former is one of two TFIIS homologues found in the T. brucei genome database (Walgraffe et al., in preparation), the latter is the T. brucei homologue of a subunit of the TFIIH transcription factor (Lecordier et al., submitted). PCR products (nt 1-642 and nt 762-2160 of the TbTFIIS1 and TbXPD ORFs, respectively) were cloned in the pZJM vector [10] using the XhoI and HindIII restriction sites located between the two head-to-head inducible hybrid promoters. After linearization by NotI within the ribosomal spacer sequence, these DNA constructs were electroporated into the 13-90 bloodstream transgenic cell line expressing the T7 polymerase and the tetracycline sensitive repressor [9]. Transfected trypanosomes were selected in HMI-9 medium containing
phleomycin. Selected populations were cloned on agar plates [22]. The expression of dsRNA was induced in culture by adding the tetracycline analogue doxycycline to 1 µg/ml into the
4 culture medium. The genomic integration of the constructs was checked by Southern blot analysis (not shown), and the efficiency of induction was verified by Northern blot analysis (Fig. 1, panels A1 and B1). In both cases, the presence of doxycycline led to enhanced expression of dsRNA and concomitant decrease of the endogenous mRNA levels. This induction also led to reduction of protein expression to undetectable levels after two days (Fig. 1, panels A2 and B2). The growth of several clones of each transgenic cell line was monitored for several days in induced and control non-induced conditions. Similar results were obtained for all clones of 13-90pZJM TbTFIIS1 and of 13-90pZJM TbXPD. While the knock down of TFIIS1 only very mildly affected the cells, the TbXPD knock down led to cell death after 3 days (Fig. 1, panels A3 and B3, respectively).
As a first approach to the use of RNAi in vivo, sets of 6 outbred NMRI mice (Charles River) were injected with 5 x 106 transgenic trypanosomes from two independent clones of 13- 90pZJM TbTFIIS1 and of 13-90pZJM TbXPD. Half of the mice sets were given drinking water containing 1 mg/ml doxycycline that was changed every other day. The two clones of each transgenic line showed the same behaviour. In agreement with the results obtained in vitro, the parasitemia of mice injected with 13-90pZJM TbTFIIS1 was unaffected by the administration of doxycycline, killing the mice on day 3 after injection, , (Fig. 2A, B). In contrast, the 13-90pZJM TbXPD clones exhibited doxycycline dependant infection patterns While untreated trypanosomes killed the mice on day 3 after injection (Fig. 2C), the
doxycycline treated parasitemia grew to very mild levels on day 1 before falling to
undetectable levels on the following days (Fig. 2D). However, they relapsed between days 11 and 16 and killed the mice within 4 days (Fig. 2D). These results were reproduced twice. This suggests that administration of doxycycline in drinking water is sufficient to induce RNAi in vivo. The mild parasitemia observed on day 1 could be related to a lag period necessary either for the RNAi to affect growth or for doxycycline to reach its effective concentration in the
clones. This suggests that some trypanosomes are able to escape the selective pressure of gene silencing imposed by RNAi. To address this question, we repeated the experiment with mice that were watered with doxycycline 48 hours before injection (Fig. 2E). In this case, a mild parasitemia was still detected on day 1, but the mice remained negative thereafter for 25 days, and for and an additional 15 days after stopping doxycycline treatment. This result was reproduced in a second independent experiment. This time lapse necessary for doxycycline to induce RNAi is also observed in culture. The period without selective pressure, occurring during the transfer of trypanosomes from culture where they are maintained under
hygromycin/neomycin/phleomycin selection to the moment doxycycline reaches effective concentrations in the host’s blood, could delay the kinetics of induction enough to allow RNAi revertants to appear. We addressed this possibility by physiological and genetic analysis of antibiotic resistance of two of the relapses. These trypanosomes were harvested and put in HMI-9 culture medium. They were found to have lost resistance to the antibiotics used for transgene selection. Southern blot analysis confirmed that these trypanosomes had lost part or all of the pZJM plasmid since both TbXPD and phleoR transgenes were
undetectable (results not shown). They further suggested that the relapsed populations were clonal.
These observations emphasize the need to keep trypanosomes under permanent selective pressure in order to obtain a homogenous phenotype. Indeed, caution is recommended because in some cases RNAi induction imposes a negative pressure on cells. The ability of trypanosomes to perform genetic recombination could quickly lead to the appearance of relapsed trypanosomes and hide the effect of RNAi at the population level. Our results show that, provided that mice are prewatered with doxycycline, the system is strong enough to work in vivo. However, an ideal experimental protocol would involve a permanent selection for the
6 3 transgenes necessary for induction of RNAi. This would require watering the mice with geneticin, phleomycin and hygromycin. Although antitumoral activity [23] or selection [24,25] using 2 of these eukaryotic antibiotics in mice has been reported, the treatment was very short and the window of concentrations allowing selection without toxicity for mice was very narrow. Because of this toxicity, our attempts to select transgenic trypanosomes in mice, with a combination of these three antibiotics has so far failed (unpublished results).
The rearrangement events observed in the relapsed trypanosomes occur with a probability in the order of 10-7, as only 7 mice out of 12 injected with 5 x 106 13-90 pZJM TbXPD
trypanosomes became positive in the presence of doxycycline. This conclusion was supported by the results of additional experiments in which 2 sets of 4 mice were injected respectively with 5 x 105 and 5 x 104 of these trypanosomes,in the presence of doxycycline. None of these mice exhibited parasites within the 30 days of monitoring.
While RNAi emerges as a powerful tool in molecular biology, its application in vivo has been advocated. Pioneer experiments have demonstrated its effectiveness in antiviral therapy [26- 28]. We have shown that an in vivo RNAi system could be used for studying host-parasite interactions, since our data establishes the proof-of-principle for RNAi induction in
trypanosomes grown in their hosts. This method should allow the study of genes/factors involved in mechanisms such as the rate of antigenic variation, resistance or sensitivity to lysis by human serum, or immunosuppression. These applications would also benefit from the development of pleomorphic cell strains suitable for RNAi, allowing the analysis of the immune response on a scale of weeks rather than days. On a longer term, it theoretically opens the door to treatment of trypanosomiasis by systemic delivery of miRNAs.
Acknowledgements
– Belgian Science Policy and a “crédit aux chercheurs” given to L.V. by the Belgian National Fund for Scientific Research (FNRS). L.V. is Research Associate at the FNRS and D.W. and S.D. were supported by a FRIA fellowship. We thank Cécile Felu and Dr. Pierrick Uzureau for proofreading and valuable comments on the manuscript
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Trypanosoma brucei by heritable and inducible double-stranded RNA. RNA 2000;6(7):1069- 76.
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[17] Barry JD, McCulloch R. Antigenic variation in trypanosomes: enhanced phenotypic variation in a eukaryotic parasite. Adv Parasitol. 2001;49:1-70.
[18] Pays E, Vanhamme L, Perez-Morga D. Antigenic variation in Trypanosoma brucei:
facts, challenges and mysteries. Curr Opin Microbiol. 2004;7:369-74.
[19] Vanhamme L, Pays E. The trypanosome lytic factor of human serum and the molecular basis of sleeping sickness. Int J Parasitol. 2004;34:887-98.
[20] Manickan E, Satoi J, Wang TC, Liang TJ. Conditional liver-specific expression of simian virus 40 T antigen leads to regulatable development of hepatic neoplasm in transgenic mice. J Biol Chem. 2001;276(17):13989-94.
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Legends to figures.
Figure 1. Knock down of TbTFIIS1 and TbXPD by RNAi. The 13-90 bloodstream cell line was transfected by pZJM TbTFIIS1 (panels A) or pZJM TbXPD (panels B) and cloned after selection. A1 and B1: Northern blot analysis of RNA extracted from the cell lines during the growth curves before doxycycline addition (D0) or 1, 2 and 3 days (D1, D2 and D3) after doxycycline addition. The thin and open arrow respectively point at the mRNA and dsRNA.
The upper panel shows the hybridization with the relevant probe while the lower one shows hybridization with a control histone H2B probe. A2 and B2: Western blot analysis of total protein extracts from the cell lines during the growth curves before doxycycline addition (D0) and 1, 2 and 3 days (D1, D2 and D3) after doxycycline addition. The arrowhead points at the specific band while the asterisk indicates a non-specific band of unknown nature inadvertently recognised by the antiserum and used as loading control. A3 and B3: Growth curves of a clone from each cell line are shown in the absence (∆) or presence (■) of doxycycline.
Figure 2. Parasitemia curves in mice watered with or without doxycycline, given on day 0 or two days before injection. The results are shown for one representative experiment out of 4 that were performed. Each curve and symbol represents an individual mouse. Large crosses
12 indicate death. 5 x 106 bloodstream trypanosomes (13-90 strain) transfected with pZJMTFIIS (panels A,B) or pZJMXPD (panels C,D,E) were injected in each mouse. Mice were separated in groups of 3, watered either with regular water (panels A and C), or water supplemented with doxycycline given on day 0 (panels B and D) or -2 (panel E).
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A TFIIH homologue controls the mutually exclusive expression of the major stage- specific antigens of Trypanosoma brucei
Running title: Transcriptional control by TFIIH in T. brucei
Laurence Lecordier, Sara Devaux, David Walgraffe, Philippe Poelvoorde, Luc Vanhamme 1 and Etienne Pays 1,2
Laboratory of Molecular Parasitology, Institute of Molecular Biology and Medicine,
University of Brussels, 12, rue des Professeurs Jeener et Brachet, B-6041 Gosselies, Belgium.
1 L.V. and E.P. share last authorship of this paper
2 corresponding author epays@ulb.ac.be; tel 32 2 6509759; fax 32 2 6509750
subject categories: chromatin and transcription / microbiology and pathogens
character count: 54,779
2 Abstract
Bloodstream forms (BF) of Trypanosoma brucei continuously change their variant surface glycoprotein (VSG) to develop chronic infection in mammals. In insect-specific procyclic forms (PF), the VSG is replaced by another major surface protein termed procyclin. These antigens are strictly stage-specific because transcription of their genes is mutually exclusive.
In PF, conditional RNAi of any of the homologues of TFIIH subunits Tbp44, TbXPD or TbXPB, or over-expression of a TbXPD mutant, led to reduction of procyclin transcription linked to stimulation of RNA elongation and mRNA production from the silent VSG expression sites (ESs). These effects occurred irrespective of the relative severity of the cellular phenotypes, some of which were lethal. Similar effects were obtained following cell treatment with the DNA damaging agent MMS. In BF, TbTFIIH subunit knock down led to inhibition of the active ES linked to stimulation of procyclin transcription. Thus, a TFIIH-like DNA repair/ transcription factor is involved in the coordinated control of the VSG and
procyclin transcription units, both of which recruit RNA Pol I.
Keywords: Trypanosoma brucei/ surface antigens/ parasite life-cycle/ transcriptional control/
RNA elongation
2
Introduction
African trypanosomes, the prototype of which is Trypanosoma brucei, are protozoan parasites responsible for chronic infections in various mammals including man. These organisms are transmitted between hosts by tsetse (Glossina sp.) flies. Adaptation to their mammalian hosts involves a spectacular process of antigenic variation. Bloodstream forms (BF) are entirely covered with a dense antigenic coat containing 10 million molecules of a single component, the Variant Surface Glycoprotein or VSG. This antigen is repeatedly changed to avoid the antibody response, but as a strict rule a single VSG is ever expressed at a time (for recent reviews, see McCulloch, 2004; Pays et al, 2004). When transmitted by the insect vector, the trypanosomes differentiate and divide as procyclic forms (PF). In these forms the VSG is replaced by another major surface component termed procyclin (Roditi et al, 1998). Although VSG and procyclin are the most abundant proteins of their respective developmental forms, they are absolutely stage-specific and constitute therefore typical markers of the parasite life cycle.
VSG synthesis is controlled at the transcriptional level, but the mechanisms involved are not understood. In BF a single VSG is transcribed from a repertoire of several hundred genes, and in PF none of these genes are expressed. However, at both developmental stages a series of telomeric loci containing VSGs and termed VSG expression sites (ESs) are transcribed in their promoter-proximal region, although their VSGs are not expressed. These sites, called silent ESs, share the same architecture as the active ES of BF. They contain polycistronic
transcription units where the VSG is the most distal gene. The other genes are termed ESAGs, for ES-Associated Genes (Pays et al, 2001; Berriman et al, 2002). The promoters of these units, as well as those of the procyclin transcription units, resemble rDNA promoters and recruit RNA Pol I (Kooter and Borst, 1984; Janz and Clayton, 1994; Vanhamme et al, 1995b;
Lee and Van der Ploeg, 1997; Günzl et al, 2003). Despite the use of this polymerase, the transcripts are trans-spliced and polyadenylated exactly as mRNAs synthesized by RNA Pol II. Transcription of the active ES occurs in a discrete subnuclear location called the ES body (Navarro and Gull, 2001). This body is distinct from the nucleolus, and so far no such structure was detected for the silent ESs, whether from BF or PF, or for the procyclin units.
Although a model for the control of the ESs proposed promoter regulation by telomeric silencing (Horn and Cross, 1997), some evidence rather pointed to the crucial role of RNA elongation and processing. First, the silent ESs from both BF and PF are transcribed in their promoter-proximal region (Pays et al, 1989, 1990; Ansorge et al, 1999), giving rise to transcripts that are not polyadenylated and are retained within the nucleus (Vanhamme et al,
4 2000). Second, experimental treatments mimicking the environmental conditions that trigger the cellular differentiation of BF into PF showed striking opposite effects on RNA elongation over the ESs and procyclin units (Vanhamme et al, 1995a). Finally, the silencing of the active ES that occurs during the transformation of BF from proliferative long slender into non- dividing short stumpy forms involved progressive in situ stalling of RNA polymerase on the ES, also pointing to regulation at the RNA elongation level (Amiguet-Vercher et al, 2004).
These findings were in agreement with studies showing that the ES promoters are very simple and apparently not regulated (Jefferies et al, 1991; Rudenko et al, 1995; Vanhamme et al, 1995b; Navarro and Cross, 1998), and they were consistent with the general strategy for gene expression control in kinetoplastids, which does not seem to act on transcription initiation, but rather at co- and post-transcriptional levels (Vanhamme and Pays, 1995; Clayton, 2002).
Put together, the evidences that (i) the regulation of the ESs mainly occurs at the RNA elongation/processing level and (ii) mRNA production from these sites must involve a
concerted action between RNA Pol I and a “RNA factory” normally linked to RNA Pol II, led to our proposal that the control of the ESs would involve a limiting component bridging RNA Pol I and a elongation/processing complex normally associated with RNA Pol II (Vanhamme et al, 2000; Vanhamme et al, 2001).
A candidate for such a complex is TFIIH. In eukaryotes from yeast to man, TFIIH is a basal transcription factor essential not only for transcription by RNA Pol II, but also by RNA Pol I (Iben et al, 2002; Hoogstraten et al, 2002). This factor is involved in both RNA elongation and DNA repair, and contains several subunits with different enzymatic activities, which have been highly conserved during evolution (Zurita and Merino, 2003). TFIIH is composed of two subcomplexes that can exhibit independent functions. The first subcomplex, or core, is
involved in DNA repair and contains two DNA helicases, XPD and XPB, together with their respective regulatory subunits P44 and P52 and three other subunits of unknown function (Zurita and Merino, 2003; Ranish et al, 2004; Giglia-Mari et al, 2004). The XPD helicase bridges the core to the second subcomplex, a trimer termed CAK and involved in cell cycle control via the kinase cdk7 and cyclin H. Cdk7 is responsible for phosphorylation of the C- terminal domain (CTD) of RNA Pol II, which activates RNA elongation by this polymerase.
We identified T. brucei homologues of human p44, XPD and XPB, and provide evidence that these proteins are involved in the coordinated transcriptional control of the ESs and procyclin units, pointing to the involvement of a TFIIH-like factor in stage-specificity of the major parasite antigens.
4
Results
Characterization of T. brucei homologues of TFIIH subunits.
Analysis of the T. brucei genome database led to the identification of four putative
homologues of TFIIH subunits. Significant sequence alignments were obtained for subunits p44, XPD, XPB and p52 (Fig. 1A). The trypanosome homologue of p44 (Tbp44) shared 25%
sequence identity (41% similarity) with its human counterpart, and the C-terminal C4 zinc and RING fingers were conserved (Fig. 1B). The TbXPD and TbXPB homologues
respectively showed 41% and 42% sequence identity with their human counterparts (Fig. 1A), and in both cases all domains known to be involved in DNA helicase activity were conserved (Fig. 1B). Finally, an homologue of p52 was also discovered (25% identity, 38% similarity) (Fig. 1A).
We subcloned Tbp44, TbXPD and TbXPB after PCR amplification from T. brucei genomic DNA. The genes for these proteins were all found to be unique (data not shown). Whereas the TbXPD mRNA was present in similar amounts in BF and PF, the Tbp44 and TbXPB mRNAs were more abundant in BF than in PF (Fig. 2A). Rabbit polyclonal antibodies were raised against recombinant Tbp44 and TbXPD. Using these antibodies a similar level of TbXPD was detected in the two developmental forms (Fig. 2A). In the case of Tbp44, the pattern of bands suggested a differential processing and/or modification of this protein between the two forms (Fig. 2A). Immunofluorescence experiments localized Tbp44 and TbXPD to the nucleus of both BF and PF (data not shown).
Evidence for a complex between Tbp44 and TbXPD.
The possible interaction between TbP44 and TbXPD was assayed by two-hybrid experiments using the Gal4 system in yeast (Chevray and Nathans, 1992). An interaction was monitored between Gal4BD-TbXPD and Gal4AD-Tbp44 (Fig. 2B). No interaction was observed in any other combination tested, including Gal4BD-Tbp44-Gal4AD-TbXPD and homo-
combinations.
We constructed transgenic T. brucei lines expressing TAP-tagged versions of Tbp44 or TbXPD, and affinity-purified the components bound to these proteins. Silver nitrate staining revealed several bands absent from the control (Fig. 2C). Among those retained by Tbp44-tag, specific antibodies detected TbXPD (Fig. 2C). Reciprocally, Tbp44 was detected in the
fraction bound to TbXPD-tag (Fig. 2C). In order to evaluate the specificity of this interaction,
6 we used site-directed mutagenesis to change K732 of TbXPD-tag into W, a mutation known to prevent the interaction of XPD with p44 in man (Coin et al., 1998). Significantly, Tbp44 was no longer detected in the fraction bound to this mutant version of TbXPD-tag (Fig. 2C).
Altogether, these data suggested that TbXPD and Tbp44 directly interact in vivo.
TbXPD and TbXPB are essential in both PF and BF, but Tbp44 is dispensable in PF.
We constructed transgenic T. brucei lines to evaluate the phenotypic consequences of gene inactivation by conditional RNAi. Knock down of TbXPD and TbXPB rapidly resulted in cell death in both forms (Fig. 3A). The cellular morphology was severely altered, and the cell cycle was perturbed. In normal PF and BF, the mitochondrial genome, or kinetoplast (K), replicates and divides before nuclear (N) DNA division and cytokinesis (Fig. 3B, control cells). Ablation of either TbXPD or TbXPB resulted in a progressive decrease of the number of dividing cells and accumulation of cells with aberrant kinetoplast and nuclear DNA content, indicating a blockade in SK and SN phases of the cell cycle. While in BF, essentially 1K*2N cells were observed, PF seemed to be affected in a greater number of processes.
Indeed, in addition to 1K*2N cells, TbXPD and TbXPB RNAi cell populations also contained zoids (1K 0N cells) as well as large numbers of 1K*1N cells in the early phase of kinetoplast replication (Fig. 3B). In PF, Tbp44 appeared to be dispensable, since within 4 days no
significant effect was observed on cellular growth and morphology despite strong reduction of mRNA levels (Fig. 3A). However, after 4 days the growth slightly decreased (Figure 3C). As this could possibly be due to a defect in DNA repair, the Tbp44 RNAi cell line was submitted to the mutagenic agent MMS, and its growth rate was compared with control lines. MMS induced growth retardation after 4 days, and an additive effect was observed when combining MMS and TbP44 RNAi induction (Figure 3C). Thus, in PF ablation of Tbp44 led to increased sensitivity to DNA damage, indicating a role of Tbp44 in DNA repair. In BF, we could not manage to obtain Tbp44 RNAi or KO trypanosomes despite the use of different strategies and vectors.
Despite the strong perturbation of cell cycle and viability, TbXPD RNAi stimulated transcription of the ESs in PF, and procyclin in BF.
Run-on transcription assays were carried out in isolated nuclei to evaluate the effect of TbXPD RNAi in both PF and BF. Fig. 4 shows representative results of three independent experiments conducted after 3 days of RNAi induction in PF, and 1 day in BF. In PF transcription of rDNA was slightly enhanced (average + 30%). In contrast, transcription by
6
RNA Pol II was significantly inhibited, as observed for the tubulin, histone H2B and H4 genes (average inhibition + 60%). Transcription inhibition was also observed for the
procyclin locus (average inhibition + 30%). Strikingly, transcription of the first genes of the ESs, namely ESAG7/6, was strongly stimulated, with a reproducible increase of nearly 10- fold. This effect was not linked to these genes only, as stimulation was also observed for other genes located downstream in the same units, such as ESAG5 and ESAG8. However, the stimulation was progressively reduced with the distance from the promoter, and it was not detectable in the VSG region.
In BF, slight effects were observed for the rRNA and housekeeping genes (stimulation and inhibition, respectively). Transcription of procyclin was strongly stimulated (about 5-fold), whereas transcription of the ES exhibited a complex pattern, with stimulation (up to 50%) in the promoter-proximal region and inhibition (40 %) in the promoter-distal region that includes the VSG.
Thus, ablation of TbXPD led to contrasting transcriptional effects on the ES between the two developmental stages, and opposite effects were observed between the VSG and procyclin transcription units.
In PF the transcriptional stimulation of the ESs is specifically linked to disruption of TFIIH subunits, and leads to de novo production of fully processed mRNAs.
We extended the run-on study by analysing the steady-state levels of transcripts in TbXPD RNAi, and we also performed this analysis in Tbp44 and TbXPB RNAi. As shown by ethidium bromide staining in Fig. 5A, RNAi on any of these genes did not lead to major differences regarding the rRNA. In all three cases the amount of different mRNAs
synthesized by RNA Pol II, such as those of histone H2B (Fig 5A) and tubulin (not shown), decreased. The extent of this inhibition was higher for the TbXPB RNAi. The procyclin mRNA level decreased in Tbp44, TbXPD and TbXPB RNAi cells, with again a stronger effect in the latter case. Finally, a clear increase of the ESAG7/6 mRNA level was detectable in all cases. The induced ESAG7/6 transcripts exhibited the same size as the regular ESAG7/6 mRNAs from BF, and they were polyadenylated as revealed by their binding to oligo(dT) (not shown). These data were compared to those obtained in a control RNAi cell line, for the RNA Pol I subunit RPA12 (to be described elsewhere). In this case no ESAG7/6 mRNA could be detected despite RNAi-mediated mRNA knock down (not shown). Worth mentioning is the observation, already reported earlier (Pays et al, 1989; Coquelet et al, 1989), that no ESAG7/6
8 mRNA is normally visible in PF. Thus, inactivation of Tbp44, TbXPD or TbXPB led to de novo production of ESAG7/6 mRNA.
Increase of procyclin mRNA in BF.
In BF, Northern blot analysis of TbXPD and TbXPB RNAi cells revealed in both cases a strong increase of procyclin mRNA, although the kinetics of this effect was reproducibly different between these cells (Fig. 5B). No significant effect was observed on the other genes tested, apart from a decrease of VSG mRNA and a reproducible transient reduction of
ESAG7/6 mRNA at day 1 of induction, before moderate to strong increase of this mRNA (Fig.
5B).
In PF and BF, TbXPD and TbXPB RNAi did not change the allelic pattern of ES transcripts.
In order to see if the stimulation of ES transcription in PF concerned different sites or was restricted to a single one, we used RT-PCR to amplify the transcripts from the region between nucleotides 1 and 200 of the ESs, and we sequenced pools of 30 products after subcloning (Vanhamme et al, 2000; Amiguet-Vercher et al, 2004). In TbXPB RNAi cells these transcripts showed the typical heterogeneity of PF (12 variants in 30 sequences), indicating that
transcription was stimulated in different ESs.
In BF, neither TbXPD nor TbXPB knock down influenced the level of heterogeneity of ES transcripts, which remained homogeneous in both the 1-200 and ESAG 7 (nt 1700-1900) regions. Thus, disruption of TbTFIIH subunits did not affect the mono-allelic control of the ESs in BF.
Overexpression of TbTFIIH subunits mimics transcriptional effects of knock down As an alternative to RNAi, we attempted TbTFIIH disruption by dominant negative interference. Transgenic cell lines overexpressing TbXPD K732W were obtained through ectopic transcription of this gene in the ribosomal locus. Cell lines overexpressing wild-type TbXPD or Tbp44 were generated in parallel. Since all these subunits were provided with a TAP tag, a cell line expressing the TAP tag alone was used as a control. As shown in Fig. 6A, all constructs were expressed in both PF and BF.
In PF, cells overexpressing TbXPD K732W reproducibly grew faster than the control cells, whereas the Tbp44 overexpressors showed reduced growth rate (Fig. 6B). In BF, all cell lines grew similarly except the TbXPD K732W overexpressors, which divided significantly faster,
8
like in PF (not shown). In PF, morphological alterations and cell cycle phenotypes were observed, with a higher frequency in TbXPD overexpressing cell lines. Some, as 1K2N cells, were reminiscent of RNAi phenotypes. In particular, a frequent phenotype consisted of dividing cells showing incomplete cytokinesis while engaging a new kinetoplast and nuclear division cycle (Fig. 6B).
In PF transcription of the procyclin and ESAG7/6 genes was unaffected in the Tbp44 overexpressors, but respective down-regulation of procyclin and up- regulation of ESAG7/6 were observed in both TbXPD overexpressors, with a clearly more pronounced effect in the case of the TbXPD K732W mutant (Fig. 6C). In BF, overexpression of TbXPD induced reduction of both ESAG7 and H2B mRNAs, in parallel with an increase of procyclin mRNA (Fig. 6C).
Linkage to DNA repair
Tbp44 being involved in DNA repair (Fig. 3C), we evaluated the possible effect of DNA damaging on transcription of the ES and procyclin transcription units. In Tbp44 RNAi cells, the DNA alkylating agent MMS induced effects similar to those obtained upon induction of Tbp44 dsRNA, namely down-regulation of procyclin mRNA and up-regulation of ESAG7/6 mRNA (Fig. 7). Moreover, the effects of dsRNA induction and MMS treatment were additive (Fig. 7). Thus, DNA damaging mimicked the effects seen following TbTFIIH disruption.
Discussion
We report the characterization of T. brucei homologues of the TFIIH subunits p44, XPD and XPB. Although both yeast two-hybrid assays and TAP-tag purification provided some evidence for interaction between Tbp44 and TbXPD, it cannot yet be concluded that a full TFIIH complex is present in T. brucei, since so far the only additional subunit that could be detected in the genome database was p52, and no other subunit candidate was identified by either TAP-tagging or immunoprecipitation.
In PF Tbp44 knock down only sligthly affected growth, which may seem unexpected given the involvement of this subunit in transcription initiation by RNA Pol II in man (Tremeau- Bravard et al, 2001), and its essential nature in yeast (Yoon et al, 1992, Wang et al, 1995).
Trypanosomes are organisms that rely on polycistronic transcription at the chromosome scale, and therefore, transcription initiation is not a key level of control of gene expression
(Vanhamme and Pays, 1995; Clayton, 2002). Thus, if as in man Tbp44 controls the escape of RNA polymerase from the promoter, it is possible that interfering with this process in PF
10 would only lead to minor effects on cell viability and proliferation. At least Tbp44 could play a role in DNA repair, since in PF its down-regulation increased the cell sensitivity to DNA damage. Moreover, in BF our systematic failures to obtain Tbp44 knock down cells suggest a vital function at this stage. This difference would be in agreement with the relative stage- specificity of Tbp44 expression. In the cases of TbXPD and TbXPB, induction of RNAi led to a clear phenotype in both PF and BF. In all four cases the cells stopped dividing and died, as expected from the involvement of TFIIH in transcription, DNA repair and cell cycle
progression. In BF, kDNA segregation and cytokinesis were inhibited. In PF, all phases of the cell division were affected (kDNA replication and segregation, mitosis and cytokinesis).
These phenotypes are reminiscent of those obtained with drugs affecting mitosis such as the anti-microtubule agent rhizoxin and the inhibitor of nuclear DNA synthesis aphidicolin (Ploubidou et al, 1999). Moreover, stage-specific differences were also observed following knock down of a mitotic cyclin (Hammarton et al, 2003). Finally, TbXPD K732W
overexpressor cells were perfectly viable, but cell division was affected in these cells since they grew significantly faster than wild type cells, while growth of cells overexpressing Tbp44 was delayed. Although these phenotypes could be indirectly due to a general
transcription defect, the direct deregulation of cdk7 activity could also be involved. Indeed, the CAK complex is connected to the core TFIIH via XPB and XPD helicases (Rossignol et al, 1997; Sandrock and Egly, 2001), the latter being involved in regulation of CAK activity (Sandrock and Egly, 2001; Chen et al, 2003). So far we have not identified homologues of the CAK components cyclin H, cdk7 and MAT1 in trypanosome databases or in proteins bound to either TbXPD-Tag or Tbp44-Tag, but the obvious effects of TbTFIIH knock down on the cell cycle suggest that proteins analogous to CAK exist. The sequence of these proteins might significantly differ from those of other eukaryotes given the high divergence of trypanosomal RNA Pol CTD’s and the evidence for co-evolution between CTD’s and CTD-directed cdk’s (Guo and Stiller, 2004). Cdk7 is able to phosphorylate the RNA Pol II CTD when associated to the core TFIIH and, upon mitotic down-regulation of XPD, the free CAK complex is targeted to other proteins involved in cell cycle progression (Akoulitchev and Reinberg, 1998). However, these observations could not apply to trypanosomes, since in these
organisms transcription is not down-regulated during mitosis (L. L. and David Pérez-Morga, unpublished data) and the cell cycle checkpoints are clearly different from those of other eukaryotes, cytokinesis being apparently linked to kinetoplast segregation rather than nuclear DNA division (Ploubidou et al, 1999; Hammarton et al, 2003).
10
Despite the variability of the phenotypes observed between the individual transgenic cells, inactivation of each of the three Tbp44, TbXPD and TbXPB genes, as well as overexpression of the TbXPD K732W mutant, led to up regulation of the ESs in PF, where these sites are normally silent (Fig. 8A). Different arguments strengthen the significance of this
transcriptional stimulation.
(i) It combined increased RNA elongation, as measured in run-on experiments, with full mRNA maturation and processing, as observed in Northern blot analysis of steady-state RNA. The detection of ESAG7/6 mRNAs is especially remarkable considering that in PF, no trace of these mature transcripts could be observed despite the evidence for transcription of the relevant genomic region (Pays et al, 1989). In particular, no ESAG7/6 mRNA was detected in PF even after UV irradiation, under conditions leading to strong enrichment of promoter-proximal transcripts following RNA stabilization (Pays et al, 1989; Coquelet et al 1989, 1991). In these studies, only aberrantly processed transcripts were detected. The absence of ESAG7/6 mRNAs in PF is consistent with the dispensable nature of their function at this stage, since these mRNAs encode the trypanosomal receptor for transferrin, and no transferrin uptake occurs in PF (Salmon et al, 1994).
Therefore, the correct mRNA maturation observed here reflects a significant qualitative switch that extends beyond quantitative stimulation.
(ii) This effect was not related to a specific gene, since it was observed not only for the first genes of the transcription units, but also for the genes located downstream such as ESAG5 and ESAG8, which encode unrelated proteins (Pays et al, 2001).
Furthermore, the stimulation decreased progressively with the distance from the promoter.
(iii) The effect was always important, reaching nearly 10-fold in the ESAG7/6 region.
(iv) The transcriptional stimulation of the ESs occurred in cells where transcription by RNA Pol II was reduced.
Moreover, this transcriptional stimulation appeared to be specifically linked to the RNA elongation activity of the TbTFIIH complex.
(i) This effect was observed independently of the cellular phenotype of the different transgenic cells. In two cases, in TbXPD and TbXPB RNAi cells, it was associated with a lethal phenotype linked to arrest of cell division, but in other cases, in the Tbp44 RNAi cells and the TbXPD K732W overexpressor, the cells were viable.
12 Therefore, the transcriptional stimulation cannot be ascribed to an indirect
consequence of a pleotropic effect such as a blockade of the cell cycle.
(ii) The increase of mRNAs from the ESs was observed not only in TbTFIIH knocked down cells, but it was also obtained in overexpressors of TbXPD, particularly with a mutant version of this protein that lost interaction with Tbp44. In contrast, no mRNA increase was seen in cells with RNAi for other transcriptional factors, or overexpressing irrelevant TAP-tagged proteins.
Taking all these observations into account, it seems reasonable to propose that the
transcriptional control of the ESs, at least in PF, involves TbTFIIH components. This would be the first clue about factors involved in the control of the ESs, as the only information available so far in the field is the evidence that RNA Pol I is the enzyme used for
transcription, and that the latter occurs in a discrete body of the nucleus (Navarro and Gull, 2001; Günzl et al, 2003). The possible involvement of TbTFIIH subunits in the control of the ESs is consistent with our previous suggestion that a limiting elongation/processing factor could be a key component (Vanhamme et al, 2000, 2001). Moreover, it is fully supported by the known characteristics of this transcription factor in other eukaryotes. Indeed, TFIIH interacts with RNA Pol I (Iben et al, 2002) and is present in limiting amounts, being
redistributed between different subnuclear compartments when needed (Borggrefe et al, 2001;
Hoogstraten et al, 2002).
It is interesting to compare these results with those recently reported by the group of Rudenko (Sheader et al, 2004). These authors observed that inhibition of DNA synthesis or induction of DNA damage led to specific transcriptional stimulation of a silent ES of BF, apparently with similar extent and sequence range as those reported here. In these cases the cells were blocked in S-phase, and the stimulation also concerned other RNA Pol I
transcription units such as those of the ribosomal and procyclin genes. One of the hypotheses invoked by these authors was the recruitment to damaged DNA, of repair factors that would also influence transcriptional elongation. Sheader et al even proposed that TFIIH could be such a factor. According to this idea, DNA damage in BF would trigger a de-sequestration of the DNA repair complex TFIIH to the silent ESs, increasing RNA elongation in these sites.
In PF, TbTFIIH knock down induced transcriptional stimulation in the promoter-proximal region of the silent VSG units, whereas the active procyclin units were inhibited. In BF, the active ES was inhibited in its distal part, whereas the inactive procyclin units were stimulated.
Thus, at both stages the disruption of TbTFIIH led to inhibition of RNA elongation in the active transcription units linked to parallel activation of the silent ones, mimicking the inverse
12
RNA elongation effects occurring on the VSG and procyclin units during experimental
treatment of BF (Vanhamme et al, 1995a). However, in BF the silent ESs did not appear to be activated. Despite an increase of both run-on transcription in the ESAG7/6 region and of the ESAG7/6 mRNA level, the ES transcripts remained homogeneous. Thus, a paradoxical transcriptional stimulation occurred at the beginning of the active ES, whose distal part was down regulated. This observation supports the view that TbTFIIH controls RNA elongation, and evokes the promoter-proximal accumulation of RNA polymerase that was observed together with the arrest of RNA elongation during the differentiation of proliferative long slender into quiescent short stumpy forms (Amiguet-Vercher et al, 2004). We conclude that TbTFIIH is not required for the mono-allelic control of the ESs in BF, but is involved in the mutually exclusive control of RNA elongation in the two major stage-specific transcription units of the parasite, both of which are RNA Pol I-driven (Fig. 8A). A working hypothesis is presented in Fig. 8B. TbTFIIH would be involved in the recruitment of a RNA elongation factor, possibly a cdk7 homologue, to stage-specific transcription units. Disruption of TbTFIIH, through knock down or overexpression of individual subunits, would ablate the specificity of this recruitment. Similarly, DNA damage could also alter this recruitment. So far it cannot be determined if DNA damage is involved in all transcriptional effects studied in this work.
Materials and Methods
Cloning of T. brucei homologues of TFIIH subunits
The accession numbers of the different genes in database TRYP19.10 were 194.t00088, 196.t00139 and Tb03.5L5, for Tbp44, TbXPD and TbXPB respectively. The PCR products of these genes were cloned in the PCR:2 vector (Invitrogen) and sequenced on both strands. The K732W mutant version of TbXPD was obtained by site-directed mutagenesis (Stratagene).
Production of anti-Tbp44 and anti-TbXPD antibodies
The complete Tbp44 open reading frame and the fragment of TbXPD encoding residues 1 to 254 were amplified by PCR with appropriate flanking sites and inserted in frame with the GST coding sequence in the pGEX expression vector (Amersham). The fusion proteins were
produced in E. coli BL21. The GST-XPD and GST-Tbp44 fusion proteins were purified by affinity chromatography on glutathion agarose and electroelution, respectively. Anti-Tbp44 and anti-TbXPD antibodies were raised in rabbits.
14
Overexpression and purification of TAP tagged Tbp44, TbXPD and TbXPD K732W The TAP tag sequence (C-terminal version: Puig et al., 2001) was inserted in the pTSARib vector designed to overexpress proteins in trypanosomes (Xong et al, 1998). Tbp44, TbXPD and TbXPD K732W were amplified by PCR with appropriate flanking sites and were inserted in frame upstream of the TAP tag coding sequence. Constructs were linearized and
transfected into PF. Hygromycin resistant populations were analyzed by Western blotting for expression of TAP tagged proteins with anti-peroxydase rabbit antibodies. Overexpressing BF cell lines were obtained by cyclical transmission of the PF overexpressors in tsetse flies and subsequent growth in mice. For the purification of tagged proteins, PF from exponentially growing cultures were collected by centrifugation, washed and resuspended at 3.108 cells/ml in lysis buffer (0.1 M KCl, 5 mM MgCl2, 0.5 mM DTT, 17% glycerol, 0.5% NP40, 10 mM Tris pH 8.0) with protease inhibitors, and incubated 1h at 4°C with gentle agitation. The lysate was centrifuged for 15 min at 20,000 g at 4°C, and the supernatant was adjusted to 0.15 M NaCl before loading on rabbit IgG Agarose equilibrated in 150 mM NaCl, 0.1% NP40, 10 mM Tris pH 8.0. The TAP tagged proteins were further purified in two steps as described in Puig et al (2001).
Two hybrid interactions
The Tbp44 and TbXPD coding sequences were amplified by PCR and cloned in frame with the sequence encoding the Gal4 DNA binding domain (pPC97 vector) or the Gal4 activator domain (pPC86 vector) (Chevray and Nathans, 1992). The yeast strain pJ69-4A (James at al, 1996) (MATa trp1-901 leu2-3, 112ura3-52 his3-200 gal4∆ gal80∆ LYS2::Gal1-HIS3 Gal2- ADE2 met2::GAL7-LacZ) was sequentially transformed with the pPC97 and pPC86
constructs. Transformants were selected onto minimal medium lacking tryptophane and leucine. Protein interactions were assayed onto minimal medium lacking tryptophane, leucine and histidine.
Trypanosomes
PF from the EATRO 1125 strain were cultivated at 27°C in SDM79 medium (Brun and Jenni, 1977) supplemented with 10% fœtal bovine serum. In case of the RNAi PF strain 29-13 (Wirtz et al, 1999) the medium contained 15% foetal bovine serum, 25 µg/ml hygromycin and 15 µg/ml geneticin. BF RNAi was performed in strains 13.90 and 328.114 (Wirtz et al, 1999;
14
Morris et al, 2001). Parasites were cultured at 37°C in 5% CO2 in HMI9 medium supplemented with 10% foetal bovine serum and 10% Serum Plus (Hirumi and Hirumi, 1989). Transfection of PF was performed by electroporation of 10 µg of linearized plasmid DNA in 2.107 cells in Zimmerman postfusion medium (Bellofatto and Cross, 1989) using 0.4 cm cuvettes in a Biorad Gene Pulser II (two pulses of 1.5 kV, 25 µF capacitance). Parasites were transferred to 10 ml of medium for 16 h before selection with 25 µg/ml hygromycin (pTSARib constructs) or 2.5 µg/ml phleomycin (pZJM constructs). Cloning of phleomycin resistant 29.13 populations was performed by limiting dilution in 96-wells plates in the presence of non-recombinant 29.13 cells to maintain cell density. Transfection of BF was performed by electroporation of 10 µg of linearized plasmid DNA in 2.106 cells in 0.6 x Cytomix (van den Hoff et al, 1992) using 0.2 cm cuvettes in a Electro Square Porator (BTX) (one pulse of 1190 V, 252 µs pulse). Parasites were incubated in 10ml of medium for 16h at 37°C before selection with 1 µg/ml phleomycin (pZJM constructs) or 1 µg/ml hygromycin (p2T7 construct). Clones were obtained by plating resistant populations on agarose
(Carruthers and Cross, 1992). After amplification of PF or BF clones, correct integration of the constructs was verified by Southern blot analysis following restriction endonuclease digestion for pZJM constructs and pulsed field gel electrophoresis for p2T7 constructs.
RNAi experiments
Tbp44 and TbXPB fragments were obtained by PCR with primers creating appropriate flanking sites (HindIII and XhoI for pJZM, BamHI and XhoI for p2T7-177). The TbXPD DNA fragment was obtained by HindIII + SalI digestion of the gene. The tetracycline-inducible vectors were pZJM (Wirtz et al, 1999) and p2T7-TiTA177 (Wickstead et al, 2002). The different constructs and the trypanosomes cell lines transfected with these constructs are listed as follows.
construct ORF position trypanosomes
pZJM-Tbp44 30-834 29.13 PF
pZJM-TbXPD 762-2160 29.13 PF and 13.90 BF pZJM-TbXPB 555-2373 29.13 PF
p2T7-177-TbXPB 666-1731 328-114 BF
For RNAi analysis, trypanosomes were diluted at 2.5.105 cells/ml (PF) or 105 cells/ml (BF) in medium containing appropriate selection drugs. DsRNA synthesis was induced by the addition of 1 µg/ml of the tetracycline analogue doxycycline (Duchefa). Cultures were not allowed to
16 reach 2.106 cells/ml for PF or 106 cells/ml for BF, and doxycycline was added every day to maintain dsRNA induction. Methanesulfonic acid methyl ester (MMS) treatment of the TbP44 RNAi cell line was performed by addition of 0.0003% MMS to the culture.
Microscopy
The cells were washed in phosphate saline glucose buffer (36.5mM NaCl, 1.5% glucose, 50mM sodium phosphate, pH 8.0), fixed for 10 min at room temperature in 3.5%
formaldehyde, then washed twice in 0.15 M NaCl, 10 mM Tris-HCl (pH 8.0) and deposited onto poly L-lysine coated slides. DNA was further stained with DAPI.
Transcription studies
Run on experiments were performed as described in Murphy et al (1987). Three independent experiments including duplicates were conducted on PF harvested at day 3, and BF harvested at day 1 following dsRNA induction. Different clones of each RNAi produced similar results.
Acknowledgements
We acknowledge D. Nolan, D. Pérez-Morga, P. Uzureau, K. Winters, E. Dupont and A. Pays for help, and D. Lafontaine for helpful suggestions. This work was supported by the Belgian Fund for Scientific Research (FRSM and Crédit aux Chercheurs) and by the Interuniversity Attraction Poles Programme – Belgian Science Policy. L. V. is Research Associate at the Belgian Fund for Scientific Research, and S. D. and D. W. are financed by FRIA.
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24 Figure legends
Fig. 1. Sequence analysis of T. brucei homologues of TFIIH subunits. Panel A summarizes the results of sequence comparison between human and yeast p44, XPD, XPB and p52, and the T. brucei genome database. The numbers in each box indicate the percentages of sequence identity/similarity, as well as the E-value (between brackets). Panel B shows a schematic view of the sequence alignment between the human, S. cerevisiae and T. brucei homologues (top to bottom respectively) of p44, XPD and XPB, presented in full in the Supplementary
Information. The significant alignments are detailed in the different blow-ups. For XPD, domains I to VI are conserved regions known to be involved in DNA-RNA helicase activity.
For XPB, the most conserved domains are detailed.
Fig. 2. Characterization of T. brucei homologues of TFIIH subunits. Panel A: expression of Tbp44, TbXPD and TbXPB in PF and BF. Northern blots of 10 µg of poly(A+) RNA were hybridized with full-length probes from the relevant genes. Western blots of total protein extracts were incubated with specific rabbit polyclonal antibodies. Panel B: double hybrid evidence of interaction between Tbp44 and TbXPD. Interactions were monitored in yeast with the indicated pairs of constructs. Growth in the absence of histidine reveals interaction
between the two constructs. Panel C: TAP-tag evidence of in vivo interaction between Tbp44 and TbXPD. Extracts from PF expressing TAP-tagged versions of Tbp44, TbXPD or TbXPD K732W were purified by tandem affinity chromatography, and analyzed by silver nitrate staining and Western blotting with the indicated antibodies. Arrowheads designate the relevant proteins.
Fig. 3. Phenotype of Tbp44, TbXPD and TbXPB RNAi cells. Panel A shows the growth curves of PF and BF incubated in the absence (triangles) or presence (squares) of 1 µg/ml tetracycline (D = days). The kinetics of induction of dsRNAs are shown by Northern blot hybridization of 6 µg total RNA. Black and open arrows indicate the mRNAs and dsRNAs respectively. Panel B shows the morphology of the RNAi cells. In normal PF (control cells), kinetoplast DNA (K, arrowhead) migrates close to the nucleus (N), replicates and segregates before nuclear DNA mitosis (open arrow). Examples of TbXPD RNAi cells include a 1K*2N cell and a zoid (Z) generated by a 2K1N cell in which cytokinesis occurs before nuclear division. TbXPB RNAi examples include a 1K*1N cell where the kinetoplast is duplicated but not divided. As in PF, in normal BF the kinetoplast is replicated before nuclear DNA.
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