A novel ISWI is involved in VSG expression site downregulation in African trypanosomes
2007; Springer Nature; Volume: 26; Issue: 9 Linguagem: Inglês
10.1038/sj.emboj.7601678
ISSN1460-2075
AutoresKatie R. Hughes, Matthew E. Wand, Lucy Foulston, Rosanna Young, Kate Harley, Stephen J. Terry, Klaus Ersfeld, Gloria Rudenko,
Tópico(s)Insect symbiosis and bacterial influences
ResumoArticle12 April 2007free access A novel ISWI is involved in VSG expression site downregulation in African trypanosomes Katie Hughes Katie Hughes Peter Medawar Building for Pathogen Research, University of Oxford, Oxford, UK Search for more papers by this author Matthew Wand Matthew Wand Peter Medawar Building for Pathogen Research, University of Oxford, Oxford, UK Search for more papers by this author Lucy Foulston Lucy Foulston Peter Medawar Building for Pathogen Research, University of Oxford, Oxford, UK Search for more papers by this author Rosanna Young Rosanna Young Peter Medawar Building for Pathogen Research, University of Oxford, Oxford, UK Search for more papers by this author Kate Harley Kate Harley Peter Medawar Building for Pathogen Research, University of Oxford, Oxford, UK Search for more papers by this author Stephen Terry Stephen Terry Peter Medawar Building for Pathogen Research, University of Oxford, Oxford, UK Search for more papers by this author Klaus Ersfeld Klaus Ersfeld Department of Biological Sciences, University of Hull, Hull, UK Search for more papers by this author Gloria Rudenko Corresponding Author Gloria Rudenko Peter Medawar Building for Pathogen Research, University of Oxford, Oxford, UK Search for more papers by this author Katie Hughes Katie Hughes Peter Medawar Building for Pathogen Research, University of Oxford, Oxford, UK Search for more papers by this author Matthew Wand Matthew Wand Peter Medawar Building for Pathogen Research, University of Oxford, Oxford, UK Search for more papers by this author Lucy Foulston Lucy Foulston Peter Medawar Building for Pathogen Research, University of Oxford, Oxford, UK Search for more papers by this author Rosanna Young Rosanna Young Peter Medawar Building for Pathogen Research, University of Oxford, Oxford, UK Search for more papers by this author Kate Harley Kate Harley Peter Medawar Building for Pathogen Research, University of Oxford, Oxford, UK Search for more papers by this author Stephen Terry Stephen Terry Peter Medawar Building for Pathogen Research, University of Oxford, Oxford, UK Search for more papers by this author Klaus Ersfeld Klaus Ersfeld Department of Biological Sciences, University of Hull, Hull, UK Search for more papers by this author Gloria Rudenko Corresponding Author Gloria Rudenko Peter Medawar Building for Pathogen Research, University of Oxford, Oxford, UK Search for more papers by this author Author Information Katie Hughes1, Matthew Wand1, Lucy Foulston1, Rosanna Young1, Kate Harley1, Stephen Terry1, Klaus Ersfeld2 and Gloria Rudenko 1 1Peter Medawar Building for Pathogen Research, University of Oxford, Oxford, UK 2Department of Biological Sciences, University of Hull, Hull, UK *Corresponding author. The Peter Medawar Building for Pathogen Research, University of Oxford, South Parks Road, Oxford OX1 3SY, UK. Tel.: +44 1865 281 548; Fax: +44 1865 281 894; E-mail: [email protected] The EMBO Journal (2007)26:2400-2410https://doi.org/10.1038/sj.emboj.7601678 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info African trypanosomes show monoallelic expression of one of about 20 telomeric variant surface glycoprotein (VSG) gene-expression sites (ESs) while multiplying in the mammalian bloodstream. We screened for genes involved in ES silencing using flow cytometry and RNA interference (RNAi). We show that a novel member of the ISWI family of SWI2/SNF2-related chromatin-remodelling proteins (TbISWI) is involved in ES downregulation in Trypanosoma brucei. TbISWI has an atypical protein architecture for an ISWI, as it lacks characteristic SANT domains. Depletion of TbISWI by RNAi leads to 30–60-fold derepression of ESs in bloodstream-form T. brucei, and 10–17-fold derepression in insect form T. brucei. We show that although blocking synthesis of TbISWI leads to derepression of silent VSG ES promoters, this does not lead to fully processive transcription of silent ESs, or an increase in ES-activation rates. VSG ES activation in African trypanosomes therefore appears to be a multistep process, whereby an increase in transcription from a silent ES promoter is necessary but not sufficient for full ES activation. Introduction Monoallelic transcription of one out of a large family of related genes is a little understood phenomenon in a variety of experimental systems. Examples of this include control of the olfactory receptor genes, whereby only one out of more than 1500 olfactory receptor genes is activated in a mutually exclusive manner in each olfactory neuron (Serizawa et al, 2004; Lomvardas et al, 2006). Likewise, in the malaria parasite Plasmodium falciparum, only one of about 50 VAR gene transcription units is activated in stringently monoallelic fashion (Scherf, 2006; Voss et al, 2006). Similarly, African trypanosomes show monoallelic expression of one of many highly similar telomeric variant surface glycoprotein (VSG) expression sites (ESs) while multiplying in the bloodstream of the mammalian host. Very little is known about how the counting machinery behind this stringent control operates. The African trypanosome Trypanosoma brucei is a protozoan parasite causing African sleeping sickness, transmitted by tsetse flies. Bloodstream-form T. brucei is covered with a homogeneous coat of a single VSG. Although trypanosomes have more than 1200 VSG genes and pseudogenes (Berriman et al, 2005), the active VSG is transcribed in a tightly regulated fashion from one of about 20 telomeric VSG ES transcription units (Borst and Ulbert, 2001; Becker et al, 2004). During a chronic infection, bloodstream-form T. brucei successively switches to new VSG types, allowing escape from antibodies against the old VSG. VSG switching can be mediated by activating different ESs or via DNA rearrangements inserting one of many silent VSG genes or pseudogenes into an active ES (Barry and McCulloch, 2001). In contrast, in insect-form T. brucei, all of the ESs are downregulated, as this life-cycle stage expresses an invariant procyclin coat on its surface rather than VSG (Roditi and Liniger, 2002). Unusually for a eukaryote, transcription of ESs, as well as of the procyclin transcription units, is mediated by RNA polymerase I (pol I) rather than RNA polymerase II (pol II) (Günzl et al, 2003). In bloodstream-form T. brucei, the active ES is located in an extranucleolar pol I transcriptional body (expression-site body or ESB), which is hypothesised to contain the transcription and RNA-processing factories necessary for high levels of expression of fully processed transcripts (Vanhamme et al, 2000; Navarro and Gull, 2001). Only a single ES can be stably activated at a time, and selection for simultaneous activation of two different ESs gives rise to trypanosomes which appear to be rapidly switching between the two (Chaves et al, 1999). ESs are controlled as regulated domains and activation of exogenous promoters, integrated at the chromosome end, cannot be uncoupled from activation of the endogenous ES promoter many tens of kilobases upstream (Horn and Cross, 1995). Downregulation of ESs differs in a number of characteristics in insect-form compared with bloodstream-form T. brucei. In insect-form T. brucei, all ESs are downregulated. There are low levels of transcription from silent ESs in both life-cycle stages, but these levels are significantly higher in the insect-form (Rudenko et al, 1994). Second, ES downregulation is promoter sequence specific in insect-form T. brucei, whereas in bloodstream-form T. brucei, both ES and rDNA promoters located within an ES are silenced essentially equally effectively (Rudenko et al, 1994, 1995; Horn and Cross, 1995). Last, it has been postulated that there is chromatin-mediated silencing of ESs in insect-form, but not in bloodstream-form T. brucei, as assessed using exogenous T7 RNA polymerase to probe for ES chromatin accessibility in both of these life-cycle stages (Navarro et al, 1999). It is possible that ES downregulation is mediated primarily at the level of transcription elongation rather than transcription initiation (discussed in Pays et al, 2004). We have developed an experimental approach allowing us to screen candidate genes for their role in ES downregulation. Constructs containing fluorescent reporter genes (DsRed or GFP) were inserted downstream of downregulated ES promoters in both insect and bloodstream-form T. brucei. Tetracycline-inducible RNA interference (RNAi) was induced against candidate genes, and ES derepression was monitored by flow cytometry. We identify the first gene shown to play a role in ES control in T. brucei, and show that it is a new member of the ISWI family of chromatin-remodelling proteins. Results Monitoring expression from downregulated VSG ESs by flow cytometry We attempted to identify proteins, which, if depleted by RNAi, cause ES derepression. Our approach was to integrate a construct containing DsRed (encoding red fluorescent protein) 216 bp downstream of different endogenous ES promoters into insect-form T. brucei 29–13 (Wirtz et al, 1999) (construct ESDsB in Figure 1A). This T. brucei cell line contains genes encoding T7 RNA polymerase and the tetracycline repressor allowing tetracycline-inducible expression. Clonal T. brucei transformants were isolated with the ESDsB construct integrated into chromosomal bands containing either the VSG121 or VSG221 ESs (Figure 1A). Using PCR, the constructs were shown to be linked to ES-associated genes (ESAG)7, the most upstream of the ESAGs. Figure 1.Monitoring ES derepression using flow cytometry in insect-form T. brucei. (A) The schematic shows the ESDsB construct integrating downstream of a VSG ES promoter. Various genes present within ESs are indicated with black boxes, and characteristic repeat arrays with striped boxes. The construct contains DsRed (DRed) and blasticidin resistance (Blast) genes flanked by tubulin (white boxes) or actin (black box) RNA-processing signals. Below: pulsed field gel analysis of insect-form T. brucei ESDsB transformants, where DNA from the parental T. brucei 29–13 cell line (lane P) is compared with that from T. brucei 29–13 transformants D1–D4 (lanes D1–D4). An ethidium stain of the gel (Eth.) is shown with size markers indicated in megabases and the slot indicated with an arrow. The gel was blotted and hybridised with a probe for DsRed (panel DsRed). Chromosomal bands containing either the VSG221 or VSG121 ESs are indicated with arrows. (B) Levels of expression of the DsRed gene in inactive ESs in insect-form T. brucei as measured by flow cytometry. Above: representative flow cytometry traces are shown, with DsRed expression monitored in the FL-2 channel (x axis). The T. brucei 29–13 TBT cell line (TBT) does not contain DsRed. The T. brucei 29–13 D1-D4 cell lines contain DsRed integrated behind different ES promoters. In the T. brucei 29–13 Dr1 and Dr2 cell lines, a DsRed containing construct was integrated behind an rDNA promoter in T. brucei 29–13. Quantitation of DsRed expression from different DsRed containing T. brucei lines was measured as total mean fluorescence (arbitrary units). Results are the mean of six experiments, with standard deviation indicated by error bars. Download figure Download PowerPoint Expression of DsRed in T. brucei can be monitored by flow cytometry (Figure 1B). Insect-form T. brucei not containing DsRed (TBT) did not fluoresce, whereas T. brucei with DsRed inserted downstream of a highly active rDNA promoter showed high levels of DsRed expression (Dr1 and Dr2 in Figure 1B). This did not lead to a growth arrest, indicating that DsRed is not significantly toxic in insect-form T. brucei. In contrast, trypanosomes containing the DsRed construct integrated behind inactive ES promoters (D1–D4) only showed levels of fluorescence that were marginally higher than in trypanosomes not containing a DsRed gene (Figure 1B). TbISWI is a member of the ISWI family of SWI2/SNF2-related chromatin-remodelling complexes We next tested if proteins previously found to bind DNA sequences present in transcriptionally silent regions of the T. brucei genome play a role in ES downregulation. TbISWI was originally identified in a screen for DNA-binding proteins interacting with the T. brucei 177 bp simple sequence repeats, which constitute the bulk of the transcriptionally inactive T. brucei minichromosomes (Wickstead et al, 2004). T. brucei proteins binding 177 bp repeat containing sequences were isolated and identified by mass spectrometry. These results will be presented elsewhere (Tilston V and K Ersfeld, manuscript in preparation). One of the proteins isolated (provisionally called TbISWI) was found to have a highly conserved SNF2 N-terminal domain (Eisen et al, 1995) (e value of e−110), a conserved helicase domain (5e−32) and a region resembling a myb-like DNA-binding domain (6e−9) (Figure 2A). SNF2 domains have DNA-dependent ATPase activity, and are present in the SWI2/SNF2-related class of proteins involved in chromatin remodelling (Mohrmann and Verrijzer, 2005). One of the subclasses of the SWI2/SNF2 family comprises members of the ISWI family (Corona and Tamkun, 2004; Mellor and Morillon, 2004). Sequence database interrogation with the TbISWI sequence preferentially identified ISWI family members from other species. High sequence similarity was found over the SNF2-domain-containing region, particularly over seven regions containing highly conserved ATPase/helicase motifs (Figure 2B). Figure 2.T. brucei TbISWI is a member of the ISWI subclass of SWI2/SNF2-related chromatin-remodelling complexes. (A) Schematic of TbISWI with protein domains identified using 3D-JIGSAW (version 2.0). A conserved SNF2 family N-terminal domain is indicated with a green oval, conserved helicase C-terminal domain with an orange hexagon, and a Myb-like DNA-binding domain with a red oval. (B) Alignment of TbISWI over the SNF2 and helicase domains with other ISWI family members: Saccharomyces cerevisiae ISWI2p, S. cerevisiae ISW1p, Caenorhabditis elegans ISWI homologue protein 1, Drosophila melanogaster ISWI isoform C, Xenopus laevis ISWI, Mus musculus DNA-dependent ATPase SNF2 H, and Homo sapiens SNF2 H (accession numbers in the Materials and methods). Seven conserved regions characteristic of ATPase/helicase motifs are indicated with numbered boxes, with particularly essential amino acids indicated by asterisks (Richmond and Peterson, 1996). Identical amino acids are highlighted in yellow, with conservative or similar amino-acid changes highlighted with blue or green blocks respectively. (C) TbISWI transcript is expressed in both insect and bloodstream-form T. brucei. Northern blot analysis of RNA from wild-type insect-form T. brucei (Lane 1), insect-form T. brucei 29–13 (Lane 2), or bloodstream-form T. brucei HNIR1(221+) (Lane 3). The blot was hybridised with a probe for TbISWI or tubulin (Tub) as a loading control. (D) TbISWI is expressed at comparable levels in both insect-form (PF) or bloodstream-form (BF) T. brucei. The Western blot was reacted with an antibody against TbISWI, and as a loading control with an antibody against BiP. Download figure Download PowerPoint Members of the ISWI family are recognisable by the presence of both an SNF2 domain and a SANT/SLIDE domain, which is an ISWI-specific subclass of myb domain with DNA-binding activity (Boyer et al, 2004). TbISWI has a myb domain, which could play a role in contact of TbISWI with DNA, but does not appear to have a clear SANT/SLIDE domain. We have nonetheless categorised our protein as an ISWI on the basis of the high homology with other ISWIs, and the lack of other ISWI candidates in T. brucei. TbISWI is expressed in both insect and bloodstream-form T. brucei at comparable levels (Figure 2C and D) as detected using a rabbit polyclonal antibody raised against the C-terminal 207 aa of TbISWI. Inactivation of TbISWI by RNAi in insect-form T. brucei leads to a growth arrest and VSG expression site upregulation We first tested the role of TbISWI in the T. brucei D1 cell line containing DsRed integrated behind an ES promoter located on a chromosomal band containing the VSG121 ES. Induction of RNAi against TbISWI using a tetracycline-inducible system led to a growth reduction in insect-form T. brucei D1-SA1 and D1-SA2 after about 6 days (Figure 3A). This phenotype was also observed using another nonoverlapping TbISWI RNAi fragment (result not shown). Using Western blot analysis, there was almost complete depletion of a band, which appeared to correspond to the TbISWI protein after 2 days induction of TbISWI RNAi (Figure 3B). This lag between the reduction in TbISWI protein to undetectable levels, and the appearance of the growth arrest could indicate that very low levels of TbISWI can still rescue the cell. Alternatively, the growth arrest could be an indirect consequence of TbISWI knock down. Figure 3.Blocking synthesis of TbISWI results in ES derepression in insect-form T. brucei. (A) Growth curves in insect-form T. brucei after induction of RNAi against TbISWI with tetracycline. Growth of the parental T. brucei 29–13 D1 cell line (D1) is compared with two independent T. brucei TbISWI RNAi transformants D1-SA1 and DA-SA2 in the presence (+) or absence (−) of tetracycline. The cumulative cell density is indicated on the y axis as cells/ml multiplied by 105. Time is indicated in days. (B) Disappearance of TbISWI protein after the induction of TbISWI RNAi in insect-form T. brucei. Protein lysates from the parental T. brucei 29–13 D1 cell line (D1) were compared with those from T. brucei D1-SA1 or D1-SA2. Lysates were isolated from cells after the induction of TbISWI RNAi with tetracycline for 0, 2, 4, 6 or 8 days. TbISWI as well as a cross-reacting band are indicated by arrows. As a loading control the blot was reacted with an antibody against BiP (BiP). (C) Derepression of DsRed marked ES promoters after induction of RNAi against TbISWI. The T. brucei 29–13 D1 cell lines contain a DsRed containing construct inserted behind a promoter on the VSG121 ES containing chromosome. DsRed expression was monitored by flow cytometry in the FL-2 channel in the T. brucei D1-SA1 or D1-SA2 cell lines grown in the presence (+) or absence (−) of tetracycline (Tet) to induce TbISWI RNAi. The total mean fluorescence is plotted over time. Examples of FACS traces from an uninduced population or one with TbISWI RNAi induced for 10 days are shown above. Results are presented as the mean of three experiments, with standard deviation indicated with error bars. (D) Promoters from different ESs are derepressed after the induction of TbISWI RNAi. TbISWI RNAi was induced in the T. brucei 29–13 D3 cell line, where DsRed is inserted behind an ES promoter on the VSG221 ES containing chromosome. The parental T. brucei 29–13 D3 cell line (D3) does not contain a TbISWI RNAi construct, and is compared with T. brucei transformants D3-SA1 and D3-SA2 grown in the presence (+) or absence (−) of tetracycline to induce TbISWI RNAi. Mean fluorescence in the FL2 channel is indicated on the Y-axis. Representative flow cytometry traces are shown for cells grown for 0 or 9 days in the presence of tetracycline. Download figure Download PowerPoint We next used flow cytometry to monitor DsRed expression from the ES promoter in this cell line after the induction of TbISWI RNAi. The T. brucei D1-SA1 and D1-SA2 cell lines started to show derepression of DsRed 2–4 days after induction of TbISWI RNAi, which reached 10–17-fold background after 6–10 days induction (Figure 3C). Comparable ES derepression was also seen with the T. brucei D3-SA1 cell line, where DsRed was integrated behind an ES promoter on the VSG221 ES-containing chromosome (parental T. brucei D3 cell line) (Figure 3D). ES derepression was not observed when RNAi was induced against other unrelated essential genes, indicating that derepression of DsRed was not simply a stress response caused by lack of an essential protein. Genes tested included NUP1 (Rout and Field, 2001), DAC1 (Ingram and Horn, 2002) and TDP-1 (Erondu and Donelson 1992), whereby induction of RNAi resulted in a growth reduction within 3 days; however, no significant VSG expression-site derepression was observed over a period extending up to 9 days (results not shown). ES promoters are flanked downstream by different families of ESAGs. After induction of TbISWI RNAi, transcription from the normally downregulated ES promoters extended through the adjacent ESAG7, ESAG6 and ESAG5 (Figure 4A and B). These derepressed transcripts were also present as variants with nucleotide lengths longer than expected for mature transcripts, possibly indicating inefficient trans-splicing or polyadenylation. Alternatively, these larger ESAG5 transcripts could be of varying sizes as they are derived from polymorphic ESAG5 genes present in multiple derepressed ESs. No increase in transcripts was observed from the ESAG4 or ESAG8 genes located downstream of ESAG5 (result not shown). We did not see evidence for upregulated transcripts derived from the 177 bp repeat sequences present on transcriptionally silent minichromosomes after the induction of TbISWI RNAi (result not shown). However, as the 177 bp repeat arrays do not contain RNA-processing signals, fortuitous transcription initiation in these areas of the genome would not necessarily give rise to stable transcripts. Figure 4.Transcription of derepressed ESs after the induction of TbISWI RNAi in insect-form T. brucei. (A) A schematic of a typical ES is shown with the promoter indicated by a flag, the integrated ESDsB construct indicated as per Figure 1A, and adjacent ES associated genes (ESAGs) with numbered boxes. Northern blot analysis shows RNA from bloodstream-form T. brucei 90–13 (B), insect-form T. brucei 29–13 (P) and the parental T. brucei 29–13 D1 cell line (D), which has a DsRed containing construct integrated behind an ES promoter. These lanes are compared with RNA from the T. brucei D1-SA1 or D1-SA2 cell lines after the induction of TbISWI RNAi for the time in days indicated above. The blot was hybridised with probes for TbISWI, DsRed, ESAG6/7, and ESAG5. The ethidium stained gel is shown below (Eth). (B) Quantitation of the increase in DsRed, ESAG6/7 and ESAG5 transcript in the T. brucei D1-SA1 and D1-SA2 cell lines after the induction of TbISWI RNAi for the number of days shown below. Radioactive signal is indicated as counts per mm2 in arbitrary units. Download figure Download PowerPoint TbISWI is localised in the nucleus and is present in the chromatin fraction of both insect and bloodstream-form T. brucei The cellular localisation of TbISWI was determined by expressing TbISWI-GFP fusion protein from a tetracycline-inducible T7 promoter. TbISWI-GFP protein localised to the nucleus of both insect and bloodstream-form T. brucei (Figure 5A). Next, we determined if TbISWI was present in chromatin-enriched cell fractions. Cells can be fractionated into a pellet fraction containing histone H3 as a marker for chromatin, and a supernatant fraction containing nonchromatin-associated proteins, including the nuclear RNA-binding protein La (Arhin et al, 2005; DiPaolo et al, 2005). At low salt concentrations, TbISWI was present in the pellet fraction together with histone H3, indicating that it is associated with chromatin (Figure 5B). In contrast, the RNA-binding protein La was present in the supernatant. Performing this fractionation procedure in the presence of increasing concentrations of NaCl showed that TbISWI was released into the supernatant at a concentration between 200 and 300 mM NaCl. This indicates that TbISWI binds DNA with a lower affinity than histone H3, which remained associated with the chromatin fraction in up to 500 mM NaCl (Stunnenberg and Birnstiel, 1982). Equivalent results were obtained using fractionated lysates from insect-form T. brucei (results not shown). These results are all compatible with TbISWI being a chromatin-associated protein critical for ES downregulation in both insect and bloodstream-form T. brucei. Figure 5.TbISWI has a nuclear localisation and is present in the chromatin fraction. (A) TbISWI-GFP fusion protein localises in the nucleus of both insect-form (PF) and bloodstream-form (BF) T. brucei. Results are shown after 8 h induction of TbISWI-GFP in insect-form T. brucei (PF) or bloodstream-form T. brucei (BF). Panels show trypanosomes imaged using differential interference contrast (DIC). DNA is stained with DAPI, and TbISWI-GFP is visualised in the FITC channel. (B) TbISWI colocalises with histone H3 in the chromatin fraction of BF T. brucei. Cell lysates were fractionated into a pellet (P) and supernatant (S) fraction in the presence of increasing NaCl (lanes 1–7 respectively 0, 50, 100, 200, 300, 400 and 500 mM NaCl). Panels were reacted with an antibody against TbIWSI (TbISWI), histone H3 (H3) or the RNA-binding protein La (La). Download figure Download PowerPoint TbISWI is essential in bloodstream-form T. brucei and is involved in downregulation of silent VSG ESs We next developed an experimental approach allowing us to investigate the role of TbISWI in ES control in bloodstream-form T. brucei (Figure 6A). A construct containing GFP was integrated downstream of the promoter of the VSG221 ES in the bloodstream-form T. brucei 'single-marker' line containing the T7 RNA polymerase and tetracycline repressor genes allowing tetracycline-inducible expression (Wirtz et al, 1999). A construct allowing tetracycline-inducible VSG221 RNAi was introduced into these VSG221-expressing cells (Sheader et al, 2005). Induction of VSG221 RNAi allows the selection of cells, which have switched to the expression of different VSGs (Aitcheson et al, 2005). After screening for cells that had activated the VSGT3 ES, we integrated a construct containing a blasticidin-resistance gene immediately behind the VSGT3 ES promoter. This allowed us to maintain cultures of trypanosomes, which were homogeneous for expression of the active VSGT3 ES. Subsequently, a TbISWI RNAi construct (MC177 TbISWI-A) was integrated into this cell line, allowing for monitoring for derepression of GFP integrated behind the silent VSG221 ES promoter after the induction of TbISWI RNAi. Figure 6.TbISWI is involved in VSG expression site downregulation in bloodstream-form T. brucei. (A) The bloodstream-form T. brucei T3-SA1 cell line used for investigating VSG expression site control using RNAi and flow cytometry. The large box indicates a trypanosome expressing VSGT3. ESs are shown with flags for the promoters, and relevant genes including GFP, blasticidin resistance (Blast) and VSG221, VSGT3 and VSG121 with filled boxes. The T. brucei T3-SA1 cell line contains genes encoding T7 RNA polymerase and the tetracycline repressor (TetR) allowing tetracycline-inducible transcription from modified T7 promoters (black flags). A TbISWI RNAi construct is integrated into these cells. Derepression of the GFP marked VSG221 ES can be monitored by flow cytometry. (B) Growth reduction after the induction of TbISWI RNAi in VSGT3 expressing T. brucei. T. brucei T3SA1 and T3SA2 cell lines were grown in the presence (+) or absence (−) of tetracycline to induce TbISWI RNAi. In comparison, the parental VSGT3 expressing cell line T. brucei VSGT3-SM (T3) did not contain an RNAi construct. The cell density is indicated as a cumulative amount multiplied by 105, and time is indicated in hours. (C) Disappearance of TbISWI protein after induction of TbISWI RNAi in bloodstream-form T. brucei expressing VSGT3. Protein lysate from bloodstream-form T. brucei expressing a TbISWI-GFP fusion protein (SG) was compared with lysate from the parental bloodstream-form T. brucei VSGT3-SM cell line (T3) or T. brucei TbISWI RNAi transformant T3-SA1, where TbISWI RNAi had been induced for 0, 6, 12, 24 or 48 h. The top panels show blots reacted with a polyclonal anti-TbISWI antiserum, with the bands containing TbISWI and the TbISWI-GFP fusion protein indicated with arrows. The bottom panels show an internal cross-reactive band which functions as a loading control. Comparable results were found with the T. brucei T3-SA2 cell line. (D) Derepression of the GFP marked VSG221 ES after the induction of TbISWI RNAi in VSGT3 expressing T. brucei. The parental T. brucei VSGT3-SM cell line (T3) is compared with the T. brucei T3SA1 and T3SA2 cell lines containing the TbISWI RNAi construct. The upper panel shows the representative flow cytometry traces with fluorescence in the FL1 channel indicated on the x axis. The bottom panel shows the quantitation of a representative experiment through time in the presence (+) or absence (−) of tetracycline to induce TbISWI RNAi. (E) GFP is derepressed after the induction of TbISWI RNAi in VSG121 expressing T. brucei. The T. brucei VSG121-SM cell line (121) is compared with the T. brucei 121SA1 and 121SA2 cell lines containing the TbISWI RNAi construct. Flow cytometry traces and quantitation of an experiment performed in the presence (+) or absence (−) of tetracycline are as indicated above (C). Download figure Download PowerPoint Induction of TbISWI RNAi in bloodstream-form T. brucei resulted in a reduction in growth rate after about 48 h (Figure 6B). The induction of TbISWI RNAi resulted in depletion of TbISWI protein, which was undetectable by 24 h, as monitored by Western blot analysis in T. brucei T3-SA1 (Figure 6C). Comparable depletion of TbISWI protein was seen in T. brucei T3-SA2 (result not shown). Induction of TbISWI RNAi in VSGT3 expressors led to an average 61-fo
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