Trypanosome strain generation and culturing

Bloodstream form (BF) Trypanosoma brucei strain 427 expressing VSG221 from BES1 (12) was used for all experiments, and cultured in modified HMI-9 medium supplemented with 15% foetal calf serum. All cell lines used in this study are detailed in Supplementary Table S1, and are all based on the ‘single marker’ (SM) cell line (25) which is referred to here as SM221. The SM221pur cell line has a puromycin resistance gene inserted immediately behind the BES1 promoter allowing selection for maintenance of VSG221 expression (26). All genetically modified cell lines were validated using linking PCR.

To mark an individual MES in BF T. brucei, constructs were inserted downstream of the endogenous promoter of MES VSG653 (10). The T. brucei SM221 MES-pur cell line was generated by transfecting the pMES653PurBla plasmid into SM221 cells resulting in a puromycin resistance gene inserted downstream of the endogenous MES VSG653 promoter. The T. brucei SM221 MES-GFP cell line was generated through the transfection of the pMES653eGFPBla plasmid into T. brucei SM221 cells resulting in an eGFP gene inserted downstream of the endogenous MES VSG653 promoter. In both cell lines, construct integration was selected for using a blasticidin resistance driven by an ectopic rDNA promoter. Both plasmids were generated by Gibson assembly using the primers listed in Supplementary Table S2.

T. brucei SM221pur Cas9 cells constitutively expressing Cas9 endonuclease were generated by transfecting the pSMOx2 Cas9 plasmid (derived from a plasmid kindly provided by Jack Sunter, Oxford Brookes University, UK). This integrates a codon-optimised Cas9 gene upstream of the first β-tubulin gene in the tubulin array, which is expressed by endogenous Pol II transcription. Cas9 mediated gene tagging and gene knock-out were carried out as described in (27). pPOT plasmids for this purpose were kindly provided by Sam Dean, (University of Warwick, UK) and primers were designed using the LeishGEedit website (www.leishgedit.net). All plasmids and primers used for Cas9-mediated gene tagging and knock-out are listed in Supplementary Table S2.

Stem–loop RNAi constructs were generated by cloning a PEX11 stuffer fragment flanked upstream by HindIII/XbaI and downstream by BamHI/ AscI into pDexv4-mSt-KIN17 (from the Bill Wickstead lab). A PEX11 stuffer was cloned downstream of a tetracycline inducible T7 promoter. Complementary gene sequences were cloned flanking this PEX11 stuffer, and the construct was inserted into the VSGG4 locus (Tb427VSG-31, Tb427_00805900) on a T. brucei mini-chromosome (28) after digestion with NotI and transfection into SM221pur cells.

All RNAi cell lines were generated from SM221pur MES-GFP cells, with the exception of the α-tubulin RNAi cell line which was generated using T. brucei BNII V02+ pSMOx2. This cell line was generated using the BNII V02+ cell line (29) transfected with a derivative of the pSMOx plasmid containing codon optimised tetracycline repressor and T7 RNA polymerase (30) with alterations allowing construct integration upstream of the first β-tubulin gene of the tubulin array (kind gift of Mark Carrington, University of Cambridge, UK). PFR2 (Tb927.8.4970) was targeted for RNAi using the first 400 bp of its CDS, TbSAP was targeted for RNAi from position 130 to 634 of its CDS and α-tubulin was targeted for RNAi from position 629 to 1340 of its CDS.

RNAi library generation and selection for repressors of MES transcription

The T. brucei SM221MES-pur cell line was transfected with pRP^Sce*. This construct was generated following transfer of the Sce* cassette, containing the I-SceI meganuclease and its recognition site, from pRPa^Sce*(31) to the pRP construct via HindIII/ Bsp120I cloning (32). In contrast to pRPa^Sce*, which targets an ectopic rDNA spacer landing pad locus in 2T1 T. brucei (32), pRP^Sce* is able to integrate at any of the conserved rDNA spacer sequences in the SM221MES-pur T. brucei cell line. Transformants were selected with hygromycin.

pRPa^Sce* Hyg transfected cells were pooled for a total number of ∼3 × 10⁷ cells, and incubated for 24 h without hygromycin before transfection of the T. brucei whole genome RNAi plasmid library from (33). Transfection of the plasmid RNAi library was performed after 3 h of tetracycline induction to induce I-SceI expression and target site cleavage, enabling high efficiency integration of the RNAi library at the I-SceI cleaved rDNA spacer sequence (31). Phleomycin was added 5 h after transfection to select for RNAi library integration. T. brucei RNAi library cells were counted daily and expanded continuously until total cell number exceeded 1 × 10⁸. Screens for repressors of MES expression were initiated directly from expanded RNAi library cells using 2 × 10⁷ cells per screen. The RNAi library was induced with tetracycline for 24 h to ensure robust RNAi target depletion prior to addition of puromycin selection (50, 60 or 70 ng/ml). Genomic DNA for high throughput RIT-seq analysis was harvested (Blood and Tissue Kit, Qiagen) from the 50 ng or 60 ng/ml puromycin screens following ten days under selection, and from the 70 ng/ml screen after 23 days under selection. The genomic DNA from library cells grown in the absence of tetracycline or puromycin was harvested after either three or ten days of growth.

RIT-seq (RNA interference target sequencing) was carried out as previously described (31). Briefly, the remaining RNAi target fragments in each selected RNAi library were PCR amplified using LIB2f and LIB2r primers as described in (34). The amplified products were sequenced on an Illumina MiSeq platform (BGI Genomics). Sequence reads containing the RNAi construct-specific 14-base barcode were identified using a custom script (34). Mapping was carried out using Bowtie2 (35) set to ‘very sensitive local’ alignment, and output SAM files were processed using SAMtools (36). The resultant BAM files were viewed against the reference genome in the Artemis genome browser (37). RNAi target fragment read numbers were converted to RPKM (reads per kilobase per million reads mapped) to account for interlibrary read depth variations when comparing sequencing outputs from independently selected RNAi libraries.

Immunofluorescence microscopy

In order to visualise target proteins using antibodies, cells were washed twice in cold PSG buffer and fixed in 2% formaldehyde for 15 minutes at room temperature. Fixed cells were washed twice in PBS and settled on ColorFrost Plus microscopy slides (Shandon) for 30 min in a humidity chamber. Cells were then permeabilised using 0.1% NP-40 and then subsequently incubated with the following primary antibodies: A rabbit polyclonal anti-GFP antibody (Abcam, Ab290) and anti-Ty epitope mouse monoclonal BB2 (kind gift of the Keith Gull lab). The secondary antibodies used were: goat anti-rabbit IgG (H+L) conjugated with Dylight 488, or goat anti-mouse IgG (H+L) conjugated to Dylight 594 (both from ThermoFisher Scientific). Cells were then mounted in Vectashield with DAPI (Vector Laboratories) and imaged on a Zeiss M1 Imager microscope with an AxioCam MRm camera. Post-acquisition analyses were carried out using ImageJ with the BAR plug-in for quantitation of signal intensity (NIH, Bethesda, MD, USA). Sample preparation for visualisation of telomeres using DNA FISH was carried out as detailed above, although instead of antibodies, the DAKO Telomere PNA FISH kit/FITC (Agilent) was used according to the manufacturer's instructions.

Chromosome immunoprecipitation (ChIP) experiments

ChIP analysis of TbSAP and histone H3 was carried out as described in (38) and (22) with minor modifications. T. brucei TbSAP-eGFP/TbSAP KO or untagged SM221pur cells were cross-linked with 1% formaldehyde for 1 h at room temperature before the reaction was quenched by glycine addition to a final concentration of 125 mM. Cross-linked cells were sonicated (BioRuptor, Diagenode) to obtain an average DNA fragment size of 200 bp. Immunoprecipitation was carried out by incubation at 4°C for 18 h with anti-GFP (Ab290, Abcam) or anti-histone H3 (Ab1791, Abcam) antibodies bound to Protein G magnetic Dynabeads (ThermoFisher). Dynabeads without bound antibodies were included as controls for all ChIP experiments. ChIP material was analysed by qPCR (Applied Biosystems 7500 real time PCR machine) using Brilliant II SYBR low ROX master mix as previously described (38) and (22) using the primers listed in Supplementary Table S3. Quantitation of target sequence enrichment in the ChIP material was calculated from the percentage of input chromatin after normalisation by subtraction of the value obtained from the no-antibody controls.

Flow cytometry analysis

In order to quantitate levels of eGFP fluorescence following TbSAP RNAi, 1–3 × 10⁶T. brucei cells were washed twice in cold PSG and fixed with 2% formaldehyde. Fixed cells were then washed and resuspended in PBS. In order to determine the percentage of live or dead cells following either PFR2 or TbSAP RNAi, 0.5–1.0 × 10⁶ unfixed cells were washed and resuspended in cold PSG. Propidium iodide (PI) was added to samples to an end concentration of 10 μg/ ml with cells kept on ice to prevent internalisation of PI through endocytosis. Flow cytometry data was acquired using a BD LSR Fortessa cell analyser operated by BD FACS Diva software (BD Biosciences) with 10 000 events recorded per sample. Post-acquisition analysis of flow cytometry data was with FlowJo v10 software (BD Biosciences).

qPCR analyses of mRNA transcript levels

Total RNA was extracted using a Qiagen RNeasy Mini kit with genomic DNA depleted using Turbo DNase (Ambion) according to manufacturer's instructions. Reverse transcription with 100 ng of total RNA was carried out using a Qiagen Omniscript RT kit with random hexamers from Promega. Generated cDNA was diluted 10-fold and the equivalent of 0.5 ng of RNA was used as the template for the qPCR reactions. qPCR reactions were carried out using an Applied Biosystems 7500 real time PCR machine using Brilliant II SYBR low ROX master mix (Agilent) with primers listed in Supplementary Table S4.

Mass spectrometry analysis of cell surface GPI-anchored proteins

Approximately 4 × 10⁷T. brucei cells per sample were washed twice at 4°C in trypanosome dilution buffer (5 mM KCl, 80 mM NaCl, 1 mM MgSO₄, 20 mM Na₂HPO₄, 2 mM NaH₂PO₄, 20 mM glucose pH 7.6) and resuspended in 45 μl cold 10 mM sodium phosphate buffer (pH 8.0) with protease inhibitors. Samples were then incubated for 5 min at 37°C in order to activate endogenous GPI-specific phospholipase C (GPI-PLC) which cleaves GPI-anchored proteins on the cell surface (39). Samples were then cooled on ice and the supernatant was harvested and diluted with NuPAGE LDS sample buffer (ThermoFisher) and dithiothreitol to a final concentration of 1× sample buffer with 100 mM dithiothreitol. Harvested proteins were denatured at 70°C for 10 min and then frozen before processing for mass spectrometry using the protocol described in (40).

RNA-seq of bloodstream form T. brucei after induction of TbSAP RNAi

Total RNA was extracted using a Qiagen RNeasy Mini kit and genomic DNA was depleted using Turbo DNase (Ambion). Quality of extracted RNA was determined by agarose gel electrophoresis and TapeStation (Agilent) analysis. Generation of Illumina-indexed RNA-seq libraries and sequencing (HiSeq 4000, Illumina) was carried out at the BRC Genomics facility (Imperial College London). RNA-seq libraries were generated by polyA-enrichment for mRNA and strand selection. Paired-end sequencing was carried out to a read length of 75 bp at each end, resulting in approximately 13 million pairs of reads for each sample. Reads were processed using Trim Galore v0.4.4 (www.bioinformatics.babraham.ac.uk; -q28—illumina—stringency 3—length 50—max _n10—trim-n—paired–trim1) and aligned to reference sequence using bowtie2v2.3.4 (41) (–no-mixed—no-discordant-I50-X 500). Alignment was performed against the long-read based assembly of the T. brucei 427 genome (427_2018; v42). For comparative analysis to a previously published metacyclic transcriptome (42), reads from study SRP103532 (www.ebi.ac.uk/ena/) were remapped to the 427 long-read assembly with the parameters above.

Total transcript abundance was assessed using HTSeq v0.5.4 (43) considering only fragments mapping with MAPQ≥2 (to discriminate between ES copies). Differential expression was estimated using edgeR package (44), using the ‘RLE’ method for calculation of normalisation factors, an estimate of dispersion robustified against outliers, and fitting count data to a quasi-likelihood negative binomial generalised log-linear model [glmQLFit method]. To compare transcriptomic changes due to TbSAP RNAi to those in the metacyclic transcriptome, databases were remapped to the long-read assembly of the T. brucei 427 genome (v46 based on the work of (45)) as above. Read depth analysis was performed using bedtools v2.25.0 (46). Alternatively, the RNA-seq reads were mapped to the T. brucei 427 genome and expression comparison was carried out using DEseq.

A genome-scale RNAi library screen identifies a putative MES repressor protein

In order to identify novel repressors of the silent metacyclic VSG expression sites (MES) of bloodstream form T. brucei, we performed genome-scale RNAi library screens. These have previously successfully elucidated novel regulatory pathways in African trypanosomes (47–49). We first generated the bloodstream form T. brucei SM221 MES-Pur reporter strain, which expresses VSG221 from the VSG221 bloodstream form expression site (BES1) (Figure 1A, Supplementary Figure S1A). We inserted a reporter construct containing a puromycin resistance gene immediately downstream of the inactive metacyclic VSG expression site (MES) VSG653 promoter (10) (42). As we could not select for construct integration using the repressed MES VSG653 promoter, we included an ectopic ribosomal DNA (rDNA) promoter directing expression of a blasticidin resistance gene downstream of the silent puromycin resistance gene (Figure 1A).

Figure 1.

A whole genome RNAi library screen designed to identify repressors of metacyclic expression site (MES) transcription in bloodstream form T. brucei. (A) Schematic of the T. brucei SM221 MES-Pur cell line with a marked MES used for conducting the screen. A puromycin resistance gene (Pur) was inserted immediately downstream of the endogenous promoter (white flag) of the VSG653 (purple box) MES. A downstream blasticidin resistance gene (Blast) expressed from an ectopic rDNA promoter (black flag) allowed selection for construct integration. A putative MES repressor protein is indicated with a red dot, various genes with coloured boxes, telomere repeats with horizontal arrows and transcription with a dashed arrow. After transfection of a tetracycline inducible whole genome RNAi library, the cell line was placed on different levels of puromycin selection to select for derepression of the inactive MES promoter after the induction of RNAi with tetracycline. (B) Cumulative growth curve of T. brucei RNAi library cells in the presence (+) or absence (−) of tetracycline (Tet) to induce RNAi in the presence of different levels of puromycin (Pur) selection. Tetracycline was added at day 0, and puromycin selection at day 1 (arrow). The growth curve shows the cumulative number of cells through time in the presence (+) or absence (−) of tetracycline and puromycin. (C) Venn diagram of shared MES repressor candidates identified from high throughput RITseq (100 read equivalent RPKM cutoff) from the 50, 60 and 70 ng/ml puromycin (Pur) selection screens. (D) Correlation plot of mapped reads from RITseq data from tetracycline induced trypanosome libraries selected with either 50 ng/ ml or 60 ng/ml puromycin (pur). Each dot represents a single T. brucei gene. The minimum threshold of 100 reads is marked by red dotted lines, and genes with <100 reads in both screens shown in dark grey, and genes with at least 100 reads in both screens shown in green. Correlation between the reads obtained with either the 50 or 60 ng/ml screens is high (Pearson correlation coefficient R² = 0.985). TbSAP (Tb927.10.4440) is annotated with a red dot.

The MES repressor RNAi library screen involved introducing a plasmid library containing fragments of the entire T. brucei genome cloned between opposing tetracycline inducible T7 promoters into the T. brucei SM221 MES-Pur reporter strain (33). Use of the rare cutting homing endonuclease I-SceI, allowed high efficiency targeting of the plasmid library to rDNA spacers (50). Knockdown of a putative MES repressor through the induction of RNAi with tetracycline would result in derepression of the normally inactive MES VSG653 promoter, and increased puromycin resistance of the selected trypanosomes. Readthrough transcription from the MES located ectopic rDNA promoter in the SM221 MES-Pur strain resulted in increased expression of MES VSG653 transcript (Supplementary Figure S1B). However, introduction of the construct did not result in significantly increased transcription from the endogenous MES VSG653 promoter, as the relative puromycin resistance of the T. brucei SM221 MES-Pur strain was approximately equivalent to that of the parental T. brucei SM221 cells (50–100 ng/ml puromycin) (Supplementary Figure S1C).

We therefore performed the MES repressor RNAi library screen three times using three different concentrations of puromycin (50, 60 and 70 ng/ml) in order to select at different degrees of stringency and thereby increase chances of success (Figure 1B). First, we electroporated the RNAi library into SM221 MES-Pur cells, generating a T. brucei RNAi library with an estimated 6.3x coverage of the haploid T. brucei genome. RNAi was induced with tetracycline at time point 0, and cells were subsequently placed on selection with puromycin 24 h later. Cells in the absence of tetracycline and puromycin (–Tet, 0 ng/ml Pur) grew at a normal rate (Figure 1B). Cells in the presence of puromycin but no tetracycline to induce RNAi died.

RNAi library induction and selection in 50 or 60 ng/ml puromycin, led to reduced growth for seven days, followed by the emergence of a resistant population. In the presence of 70 ng/ml puromycin, growth retardation was more pronounced, with a resistant population only emerging at day 19. Genomic DNA was harvested when each library had achieved a consistent robust growth rate (Figure 1B), and the remaining RNAi target fragments were amplified, using RNAi construct-specific primers (Supplementary Figure S2A).

RIT-seq (RNA interference target sequencing) was carried out to identify the RNAi target fragments enriched after puromycin selection. The PCR-amplified RNAi target fragments were deep sequenced, generating >3 million sequence reads per selected RNAi library, of which 16–24% contained the RNAi construct barcode. Barcode-containing reads were mapped against the T. brucei 927 reference genome and read values were converted to reads per kilobase per million reads mapped (RPKM) to account for read depth variations between the sequenced RNAi libraries. High confidence hits were identified as those represented by RPKM equivalent to >99 reads containing the RNAi construct barcode. Selection in 50 or 60 ng/ml puromycin followed a similar growth profile and identified a highly similar set of genes (Pearson's correlation coefficient of R² = 0.985) (Figure 1C, D).

In contrast, selection in 70 ng/ml puromycin resulted in delayed emergence of a resistant population and a substantially greater number of apparent hits, which showed little concordance with those identified following selection in 50 or 60 ng/ml puromycin (Pearson's correlation coefficients of R² = 0.104 or R² = 0.102 respectively) (Supplementary Figure S2B, C). We initially assumed that selection in 70 ng/ml puromycin would enhance the stringency of our screen. However, the delayed recovery time and the large number of hits identified, suggests that secondary adaptations occurred in a founder population, enabling a proportion of cells to survive in the presence of puromycin, independent of specific RNAi library-mediated knockdown, thereby reducing the stringency of the screen. Hence, while selection in 70 ng/ml puromycin identified some hits also seen following selection in 50 and/or 60 ng/ml puromycin, none of the 364 unique hits were investigated further. Forty eight hits were identified following selection in 50 and 60 ng/ml puromycin, of which 14 were also identified following selection in 70 ng/ml puromycin (Figure 1C, Supplementary File S1). Remarkably, no genes were identified that had been previously documented to play a role in the repression of BESs (reviewed in (13)), suggesting that in bloodstream form T. brucei MES silencing is mechanistically fundamentally different to the silencing of BESs.

Most of the proteins identified following puromycin selection of our MES Pur RNAi library have no obvious connection with transcriptional regulation, suggesting that rather than derepressing the MES PUR gene expression, they may influence puromycin action (either directly or indirectly), with their depletion reducing T. brucei drug sensitivity. However, of the 14 hits identified in all three screens, Tb927.10.4440 stood out as the only protein with a known nuclear localisation (Tryptag.org; (51); (Supplementary File S1). This protein contains a predicted SAP DNA binding domain (InterPro) (Figure 2A), a 35 residue motif consisting of two parallel α-helices connected by an extended loop (52).

Figure 2.

TbSAP is a protein with a canonical SAP domain which is enriched at the nuclear periphery of bloodstream form T. brucei. (A) Schematic of TbSAP (Tb927.10.4440) with a canonical SAP domain indicated with a pink box and disordered domains with red boxes as determined using InterPro. The predicted SAP domain of TbSAP is aligned with SAP motifs from Homo sapiens (Hs) SAF-A, SAF-B, Acinus and PIAS1 proteins using T-coffee (http://tcoffee.crg.cat/apps/tcoffee/). Conserved amino acid residues are highlighted in green, and helical domains with pink cylinders as predicted using PSIPRED. (B) Enrichment of TbSAP at the nuclear periphery is shown in representative nuclei of bloodstream form T. brucei expressing a SAP-eGFP fusion protein from the endogenous TbSAP locus. DNA was visualised with DAPI and GFP using an anti-GFP antibody. Pixel intensity of each colour along a profile (white dashed line) was calculated using the ImageJ BAR multi-plot analysis tool, with the blue line indicating DAPI stained DNA and the green line SAP-GFP. The white scale bar indicates 5 μm. (C) TbSAP foci do not obviously colocalise with regions enriched for the telomere binding protein TbTRF. Representative nuclei (1K1N) of a T. brucei cell line containing one allele of TbSAP tagged with eGFP and one allele of TbTRF tagged with a 10X Ty1 epitope are shown. SAP-GFP is visualised with an anti-GFP antibody (green), TbTRF with an anti-Ty1 antibody (red) and DNA is stained with DAPI. Scale bar represents 2 μm.

The SAP motif was first identified on the scaffold attachment factors A and B (SAF-A and SAF-B), and was later found on a range of proteins involved in nucleic acid metabolism (53). It was postulated that this SAP motif could target proteins to specific chromosomal locations. In addition, both SAP as well as the related LEM motif, were proposed to be involved in tethering chromatin to the nuclear periphery, thereby impacting on chromosomal organisation within the nucleus (52). As this, and Tb10.v4.0034 (which is identical at the amino acid level) are the only proteins in the T. brucei genome matching a SAP domain profile (e value < 1e^–3), we named it TbSAP, and focused on it for further investigation.

TbSAP is a nuclear protein enriched at ES promoters and MESs

We first determined the cellular localisation of TbSAP by epitope tagging the N-terminus with eGFP in bloodstream form T. brucei using CRISPR-Cas9 (27,54). We validated correct epitope tagging of an endogenous TbSAP gene using PCR (data not shown). As fluorescence signal from the eGFP tagged TbSAP protein was weak, we performed immunofluorescence microscopy using anti-GFP antibodies (Figure 2B). TbSAP had a heterogeneous distribution in the nucleoplasm, but was particularly enriched in foci at the nuclear periphery. These results correspond with the cellular localisation of TbSAP in procyclic form T. brucei documented by the TrypTag initiative (TrypTag.org).

Telomeres in bloodstream form T. brucei are particularly enriched at the nuclear periphery (55,56). Our attempts at combining TbSAP localisation with DNA fluorescence in situ hybridisation (FISH) to visualise T. brucei telomeres were unsuccessful. We therefore tested for colocalisation of TbSAP with the T. brucei TTAGGG-binding factor (TRF). TbTRF protein binds telomeres, and plays an important role in telomere biology, including suppressing VSG switching mediated by DNA rearrangements (57–59). We therefore epitope tagged TbTRF at the N-terminus with the Ty epitope in cells containing an allele of TbSAP which was endogenously tagged with eGFP. Immunofluorescence microscopy experiments visualising both TbTRF and TbSAP at the same time showed that TbSAP foci were often adjacent to TbTRF foci, but we did not see obvious colocalisation of the two proteins (Figure 2C). We do not think that the N-terminal addition of eGFP to TbSAP in bloodstream form T. brucei compromised its function, as knocking out the second TbSAP allele in these cells using CRISPR/Cas9 was successful (Supplementary Figure S3A). This did not alter the cellular localisation of the epitope tagged TbSAP, and as expected, nuclear staining of TbSAP was enriched at the nuclear periphery (Supplementary Figure S3B). In addition, even though tagging one allele of SAP with eGFP led to a slight reduction in growth compared with the parental cell line, this was not further reduced after the knock-out of the second SAP allele (Supplementary Figure S3C). This argues that the SAP-GFP protein was not functionally impaired to a very significant extent.

As the SAP motif is a DNA binding domain, we determined the distribution of TbSAP-GFP on T. brucei DNA using chromatin immunoprecipitation (ChIP). As TbSAP-GFP was functional, we performed the ChIP experiments in the T. brucei TbSAP-GFP/ TbSAP KO cells. The ChIP experiments were also performed in the parental T. brucei SM221pur-Cas 9 cell line as a negative control (Figure 3). Unfortunately, we were never able to isolate enough anti-GFP-TbSAP ChIP enriched DNA to perform ChIP-seq. We therefore analysed the TbSAP-ChIP enriched DNA using qPCR. We first determined the distribution of TbSAP over MES VSG653 (Figure 3A). MES sequences are divergent from each other, and we were not able to use universal primer pairs to analyse TbSAP distribution over all MESs. However, a low but significant level of TbSAP was detected at the MES VSG653, with higher levels observed at the MES promoter (primer b) (*P < 0.05) (Figure 3B upper panel). TbSAP was also detected at the VSG gene in MES VSG653 (primer c), as well as the VSG in MES VSG397 (primer d). Histone H3 was also immunoprecipitated in parallel ChIP experiments as a control, and showed an approximately equivalent distribution in both cell lines (Figure 3B lower panel).

Figure 3.

TbSAP is enriched at the promoter regions of metacyclic and bloodstream form ESs. (A) Schematic of a telomeric metacyclic ES (MES) with the promoter indicated with a white flag and the metacyclic VSG653 gene with a purple box. Primer pairs used for analysis of chromatin immunoprecipitated (ChIP) material are indicated with lettered bars. Primer pair ‘c’ recognises VSG653 and primer pair ‘d’ recognises VSG397 present within a different MES. (B) The level of SAP-GFP present at MESs in the SAP-GFP/SAP KO line (green bars), which has one allele of TbSAP epitope tagged with eGFP, and the other TbSAP allele knocked out. In comparison, the same ChIP was performed with the parental cell line (T. brucei SM221 Pur-Cas9) (red bars) which does not contain epitope tagged TbSAP. In the top graph the ChIP was performed with an anti-GFP antibody. In the lower graph ChIP was performed using anti-histone H3 antibodies. Results are presented as the percentage of the total input immunoprecipitated after subtraction of a no antibody control. Data for the ChIP experiments is the mean of three biological replicates, with the standard deviation indicated with error bars. Statistical significance (pair-wise t-test against ChIP material from the untagged parental SM221pur cells) is indicated with asterisks (*P< 0.05). (C) Schematic of bloodstream form ES (BES1) with a puromycin resistance gene (blue box) inserted behind the active BES1 promoter (white flag). Expression site associated genes (ESAG) 7 and 6 are indicated with numbered boxes, and VSG221 with a red box. Simple sequence repeats (50 or 70 bp) are indicated with vertically striped boxes. An enlargement of the BES promoter region is indicated below. Primer pairs a–e (indicated with bars) are conserved across all BESs, while primer pairs f-h are specific for sequences present within either the active BES1 (VSG221, primer pair ‘f’ and puromycin, primer pair ‘g’) or the silent BES5 (VSG800 or VSG-18, primer pair ‘h’) respectively. (D) The ChIP experiments were performed as detailed in panel (B) only the distribution of SAP-GFP or histone H3 was determined across BESs in either the T. brucei SAP-GFP/ SAP KO line, or the untagged parental T. brucei SM221Pur-Cas9 cell line.

We next determined the distribution of TbSAP over the 15 BESs of T. brucei 427, which contain large stretches of highly similar DNA sequence (Figure 3C) (12). A significant enrichment of TbSAP was observed in the region immediately upstream of the BES promoters (*P < 0.05) (primers a–c in Figure 3D, upper panel), with negligible binding of TbSAP to BES downstream regions. More TbSAP appeared to be immunoprecipitated on DNA from the vicinity of BES compared with MES promoters. This is presumably as the BES promoter primers recognise ∼20 highly conserved BES promoters, compared with the MES VSG653 primers which identify single copy sequence. Again as a control, the distribution of histone H3 was determined in both T. brucei cell lines, which as expected, was enriched on silent BES5 sequences and depleted on active BES2 sequences (Figure 3D, lower panel). The distribution of TbSAP over the Pol I transcribed rDNA and Pol II transcribed actin loci was also determined (Supplementary Figure S4A). There was some TbSAP observed within the 20 rDNA transcription units, with reduced levels detected in the rDNA spacers presumably reflecting the relative divergence of these sequences compared with the conserved 18S and 28S rDNA genes. Negligible amounts of TbSAP were found at the constitutively transcribed actin Pol II loci (Supplementary Figure S4B). As a control, the distribution of histone H3 was determined, which is particularly enriched in nontranscribed regions (Supplementary Figure S4C) (60).

TbSAP knockdown leads to MES derepression in bloodstream form T. brucei

We next determined the essentiality of TbSAP, and reproducibility of MES derepression following TbSAP knockdown. We constructed a reporter cell line with an eGFP gene inserted immediately downstream of the inactive MES VSG653 promoter (Figure 4A). Construct integration was selected for using a downstream blasticidin resistance gene driven by an ectopic rDNA promoter. Tetracycline inducible TbSAP RNAi was expressed from a construct containing a stem–loop fragment for TbSAP inserted at a single copy VSGG4 (VSG-31) gene present at a T. brucei mini-chromosome (28). The induction of TbSAP RNAi with tetracycline resulted in a severe reduction in growth after about 24 h, but there was no evidence for a discrete cell cycle arrest (Figure 4B). There was a minor amount of cell death, but the majority of cells were alive after 96 h (Figure 4C), indicating that the knockdown of TbSAP resulted in reduced growth rate rather than acute lethality. Quantitation of TbSAP transcript using qPCR confirmed that the induction of TbSAP RNAi resulted in its reduction to ∼40% normal levels after 72 h (Figure 4D).

Figure 4.

RNAi mediated depletion of TbSAP results in a reduction in growth rate with minimal lethality in bloodstream form T. brucei. (A) Schematic of the SM221pur MES-eGFP reporter cell line which expresses VSG221 from BES1, and which is selected for with a puromycin resistance gene (Pur). An eGFP gene was inserted immediately downstream of the endogenous MES VSG653 promoter (white flag). The construct was selected for using a downstream blasticidin gene (Blast) driven by an ectopic rDNA promoter (black flag). Transcription is indicated with arrows. A TbSAP stem-loop RNAi construct containing a tetracycline inducible T7 promoter (grey flag) was inserted into a locus containing the single copy minichromosomal VSGG4 gene (28). (B) Cumulative growth curve for two MES-eGFP TbSAP RNAi clones (c1 and c2) in the presence (+) or absence (−) of tetracycline (Tet) to induce TbSAP RNAi. Time is indicated in hours (h). The mean of three biological replicates is shown with standard deviation indicated with error bars. (C) Determination of live/ dead bloodstream form T. brucei cells stained with propidium iodide and analysed by flow cytometry after the induction of TbSAP RNAi for the time indicated. Data for clone c1 are shown with error bars representing the standard deviation from three biological replicates. Tests for significance (t-test) show slightly increased lethality after induction of TbSAP RNAi compared with time 0, with statistically significant results indicated with **P< 0.01 and ***P< 0.001. (D) Quantitation of TbSAP transcript with qPCR after induction of TbSAP RNAi for the time indicated in hours (h). Data from clone c1 are shown with error bars representing the standard deviation from three biological replicates. Statistical significance for the knock-down was determined using t-tests comparing with time 0 h (***P< 0.001).

In order to determine the effect of TbSAP knockdown on the bloodstream form T. brucei transcriptome, we performed RNA-seq after the induction of TbSAP RNAi for 72 h. As TbSAP was identified in a screen for MES repressors, we first investigated if we had induced a global shift to a more ‘metacyclic-like’ cell. We compared the bloodstream form transcriptome after the induction of TbSAP RNAi for 72 h with the transcriptome from the parental MES-GFP strain (X-axis of Figure 5). In addition, we compared the available transcriptome from in vitro derived metacyclic T. brucei 427 cells with the parental MES-GFP strain, which was remapped to the same assembly (Y-axis of Figure 5) (42). Transcripts which were significantly changed specifically in metacyclic forms are indicated with red dots, and those which have specifically changed after the induction of TbSAP RNAi are indicated with blue dots. Transcripts which are significantly changed in both transcriptomes compared with the parental cells are indicated with pink dots.

Figure 5.

Comparison of the TbSAP RNAi transcriptome with a previously published metacyclic transcriptome reveals that although the induction of TbSAP RNAi results in the upregulation of metacyclic VSGs, this does not lead to ‘metacyclic-like’ cells. (A) Transcripts are indicated with dots, with changes in expression level after the induction of TbSAP RNAi for 72 h relative to uninduced parental cells (MES-GFP), shown on the X-axis. This is plotted against differential expression of transcripts in the metacyclic form (MCF) transcriptome described in (42) compared with parental MES-GFP cells on the Y-axis. Differentially expressed genes in either comparison (adjusted P-value ≤ 0.05) are highlighted. Transcripts which are only differentially expressed in the metacyclic form transcriptome are indicated in maroon. Transcripts which are only significantly changed after the induction of TbSAP RNAi for 72 h are indicated in blue. Transcripts which are significantly changed in both transcriptomes are indicated in pink, with MES VSGs indicated with green circles. Transcripts which have not significantly changed with respect to the transcriptome of the parental cells are indicated with grey circles. (B) Principal component analysis of sample to sample distances of the metacyclic form (MCF), parental (MES-GFP) or TbSAP RNAi (MES-GFP TbSAP) transcriptomes. The first two dimensions covering 96% of the variance are shown. Samples are coloured according to group.

As expected, eight metacyclic VSGs were significantly upregulated in the metacyclic transcriptome (green circles) (Figure 5A). The induction of TbSAP RNAi resulted in significant derepression of seven of these different metacyclic VSGs (green circles). The most significantly upregulated transcripts observed both after the induction of TbSAP RNAi for 72 h in bloodstream form T. brucei, as well as in the metacyclic transcriptome, were metacyclic VSGs. However, we did not find that that knocking down TbSAP with RNAi made the cell generally more ‘metacyclic-like’, and both induced and non-induced samples cluster together distinct from the metacyclic transcriptome in principal component analysis (Figure 5B). This argues that TbSAP knockdown results in derepression of MES telomeres within cells which still fundamentally have a bloodstream form transcriptome.

When looking at the changes in the transcriptome observed after the knock-down of TbSAP for 72 h, there is a strong bias towards transcript upregulation rather than downregulation (Figure 6). In total, 397 transcripts were upregulated using a cut-off value of a 1.4-fold increase and an adjusted P-value of ≤ 0.1 (Supplemental file S2). In contrast, only 25 transcripts were downregulated using the same threshold. Strikingly, the degree of dysregulation was also much stronger for the genes with increased transcript abundance (mean of 6.3-fold changed for genes upregulated at fdr ≤0.1, compared to 1.2-fold for downregulated genes). This is suggestive of a predominant role for TbSAP in silencing genomic regions in T. brucei.

Figure 6.

Knock-down of TbSAP leads to striking up-regulation of both VSGs and ESAGs with relatively few downregulated transcripts. (A) Volcano plot showing differential gene expression after induction of TbSAP RNAi for 72 h compared with the parental T. brucei MES-GFP cell line. Dots indicate individual transcripts with those corresponding to MES VSGs indicated in light orange. Results are plotted as log₂ (fold change) of read count values versus adjusted P-value. Thresholds at adjusted P-value ≤0.1 and fold change ≥1.4 are shown (dashed lines). Values were calculated from three biological replicates of each dataset. The other panels are as in (A), only in panel (B) VSGs located in BESs are highlighted with dark orange dots, panel (C), chromosome internal (CI) VSGs located within the silent VSG arrays are highlighted with purple, panel (D) VSGs located on minichromosomes are indicated with pink dots, panel (E) ESAGs are indicated in green dots and panel (F) the EP and procyclin transcripts are indicated with blue dots.

A striking number of VSGs in different genomic locations were upregulated, as were transcripts from Expression Site Associated Genes (ESAGs) found in BESs (Figures 6, 7A, Supplemental file S2). Of eight VSGs in T. brucei 427 genome assemblies sited <4 kb downstream of a putative MES promoter, seven were upregulated between 6.5- and 18.5-fold after TbSAP knockdown (Figures 6A, 7A, Supplementary Table S5). The eighth MES VSG was MES VSG653, which is the site of integration of the reporter construct, and which was upregulated to a lesser extent. A further putative metacyclic VSG, VSG-631, is a long distance from a possible MES promoter (8.9 kb), and was not differentially expressed in the RNA-seq experiments. In contrast to VSGs in MESs, none of the 14 VSGs located in BESs, were significantly differentially expressed (Figure 6B).

Figure 7.

Knockdown of TbSAP results in significant upregulation of metacyclic VSGs in bloodstream form T. brucei. (A) Relative abundance of VSG transcripts following RNAi against TbSAP for 72 h. Only transcripts other than VSG221 (VSG-2), present in the active BES1 are shown. The MES with mVSG-653 is the site of integration of the reporter construct. High levels of MES VSG-653 expression are due to the ectopic rDNA promoter which has been introduced in the construct. Abundance is shown as a percentage of the total VSG transcript reads. (B) Depletion of TbSAP leads to significant up-regulation of all eight MES VSGs. RT-qPCR was performed using RNA isolated from cells where TbSAP RNAi had been induced with tetracycline (Tet) for the time indicated in hours (h). Values were normalised against actin and shown as fold-change relative to the uninduced sample. Values plotted are the mean of three independent experiments with the standard deviation indicated with error bars. Statistical significance for upregulation was determined using Student's t-test (*P< 0.05, **P< 0.01, ***P< 0.001).

Significantly, knockdown of TbSAP causes a substantial upregulation of transcripts from silent subtelomeric VSG arrays (chromosome internal, or CI) VSGs (Figure 6C). In total, 121 of 2875 CI VSGs (4.2%) are significantly changed at the thresholds used, compared to 230 of 14 196 non-VSG, non-ES genes (1.6%, P <10^–15; proportions test). CI VSG transcripts increase from 4.6 to 5% of total VSG transcripts in the parental or uninduced cell lines to 6.9% after the induction of TbSAP RNAi for 72 h (Figure 7A). In contrast, none of the VSGs located on the T. brucei minichromosomes appeared to be significantly upregulated after the induction of TbSAP RNAi (Figure 7D). Although there was no significant upregulation of VSGs located in BESs, significant upregulation of ESAGs located in BESs was observed (Figure 6E).

However, a concern was that the derepression of both silent VSGs from the VSG arrays, and ESAGs was exclusively a secondary consequence of compromised transcription in sick cells. To resolve this, we determined the transcriptome of cells where RNAi against α-tubulin had been induced for 16 h as a lethality control. The induction of RNAi against α-tubulin results in an abrupt growth arrest, with disruption of cytoskeletal structures and cells unable to undergo cytokinesis (61). In spite of ablation of TbSAP having a much weaker effect on cell growth, more chromosomal internal VSGs from the VSG arrays (CI VSG) were upregulated after induction of TbSAP RNAi compared with after induction of α-tubulin RNAi (Supplementary Figure S5A). We did not find a correlation between which VSG transcripts were upregulated after both perturbations (Pearson's coefficient R² = 0.0017). A similar trend was found for the ESAG transcripts although the degree of upregulation was less (Supplementary Figure S5B).

To confirm the derepression of MES VSGs seen in the RNA-seq, we analysed RNA levels by quantitative RT-PCR (qPCR) (Figure 7B). We observed that all eight T. brucei 427 MES VSGs were significantly derepressed after 72 h of TbSAP RNAi. Highest were MES VSG636 with 25.6 ± 7.7-fold upregulation (**P ≤ 0.01), MES VSG1954 with 16.8 ± 3.2-fold (***P ≤ 0.001) and MES VSG397 with 16.2 ± 3.0-fold upregulation (***P ≤ 0.001) (Supplementary Table S5). The MES VSG653 which contained the reporter construct was only upregulated 2.1 ± 0.1 fold after knockdown of TbSAP. This SM221pur MES-GFP cell line expresses relatively high levels of VSG653 as a consequence of readthrough transcription from the exogenous rDNA promoter integrated upstream of the telomeric VSG653 (Supplementary Figure S6A, B). These relatively high background levels possibly influence the amount of derepression observed. In agreement with the qPCR quantitation of the VSG653 transcript, levels of fluorescence from the eGFP gene inserted upstream of the ectopic rDNA promoter within this MES also showed only 1.4–1.6-fold increase after SAP knockdown (Supplementary Figure S6C). It is likely that the genetic modification of this MES impacted its ability to be derepressed.

A concern with these MES derepression experiments, is that the upregulation of MES VSGs was a secondary consequence of cells responding to the reduction in cell growth rather than a phenotype specific to TbSAP. We therefore investigated if there was derepression of MES VSGs after knockdown of the essential gene T. brucei PFR2 (62). As expected, induction of RNAi against PFR2 led to a very abrupt growth arrest within 8 h induction of PFR2 RNAi, with PFR2 transcript reduced to ∼30% normal levels after induction of PFR2 RNAi for 16 h (Supplementary Figure S7A–C). Despite the rapid lethality observed after knockdown of the highly essential PFR2, there was little significant increase in metacyclic VSG transcripts, with the possible exception of MES VSG531 and MES VSG1954 where we observed only a very marginal increase in transcript (Supplementary Figure S7D). This argues that the increase in MES VSG transcripts observed after the induction of TbSAP RNAi is indeed a specific phenotype related to TbSAP rather than a secondary effect.

Derepression of BES promoters and chromosome internal VSGs after TbSAP knockdown

VSGs at the telomeres of the BESs were not significantly derepressed after knockdown of TbSAP. However, the BES ESAGs, particularly those closest to the BES promoters (ESAGs 10, 7 and 6), were derepressed to a greater extent (Figure 8A, B). This indicates that although knockdown of TbSAP leads to derepression of the BES promoters, this transcription is not fully processive. We next investigated the derepressed transcription observed after TbSAP knockdown at a whole genome level (Figure 9, Supplementary Figure S8). The T. brucei 427 genome contains 11 diploid megabase chromosomes with extensive polycistronic transcription units containing house-keeping genes (2). After SAP knockdown, we did not see an increase in transcripts either within or at the strand-switch regions of the polycistronic Pol II transcription units (Figure 9, Homo. core).

Figure 8.

Differential upregulation of promoter proximal expression site associated genes (ESAGs) in bloodstream form expression sites (BESs) after the induction of TbSAP RNAi. (A) Schematic of a typical telomeric BES with promoters indicated with white flags and relevant expression site associated genes (ESAGs) or pseudogenes (ψ) with numbered coloured boxes (12). 70 bp repeat arrays are indicated with a vertically hatched box. (B) Differential regulation of ESAGs in inactive BESs following induction of TbSAP RNAi for 72 h. ESAGs have been arranged according to their common conserved position in T. brucei 427 BESs (12). A Tukey boxplot is shown with median (bar), interquartile range (box) and inner fences (whiskers) for all transcripts resulting from individual ESAGs of each family, excluding those of the active BES1. Only transcripts directed with MAPQ ≥2 at ≥1 reads per million reads are included (one transcript each of inactive ESAG12 and ESAG8 copies pass this threshold). Asterisks indicate Bonferroni-corrected P-values for individual transcript sets being different from 0 (Student's t-test; *** P≤ 0.001, ** P≤ 0.01, * P≤ 0.05).

Figure 9.

Increased transcription of silent VSGs present within the silent VSG sub-telomeric arrays after the induction of TbSAP RNAi for 72 h in MES-eGFP TbSAP RNAi cells. Transcript levels (mean fragment count over three replicates) in the presence (+) of TbSAP RNAi (red line) or absence (−) of TbSAP RNAi (black line) were mapped over the T. brucei megabase chromosomes. Below the traces gene architecture is shown, with housekeeping genes indicated with grey bars, and silent VSGs with blue bars. Magnified detail is presented from regions within chromosomes (Chr.) 4, 6, 8 and 9. These include regions within the silent VSG arrays present at the heterozygous transcriptionally silent aneuploid chromosomal arms (Hetero. arm). Alternatively, these regions are within the homologous transcriptionally active diploid chromosomal cores (Homo.core). In order to display on the log scale, counts of 0 have been set to 0.2).

The silent VSG arrays comprise one of the few non-transcribed regions in African trypanosomes, and are present as extensive single copy stretches at the arms of the diploid megabase chromosomes (45,63). In several regions of the silent VSG arrays, SAP knockdown led to an increase in inappropriate transcription of silent VSG genes (Figure 9, Hetero. arm). There are not thought to be Pol II promoters in these regions. However, it is possible that a repressive chromatin structure prevents activation of cryptic Pol II promoters (45,64). The derepression of these silent VSG arrays after the induction of TbSAP RNAi indicates that TbSAP could be playing a role in maintenance of a repressed chromatin state in these silenced areas of the T. brucei genome.

TbSAP RNAi mediated MES derepression produces functional mVSG proteins

We next investigated if these upregulated VSGs were present on the trypanosome cell surface, indicating functionality. Bloodstream form T. brucei expresses an endogenous phospholipase C (PLC) which can be activated in trypanosomes in vitro, where it cleaves membrane bound VSG at the GPI anchor, releasing a soluble form of VSG (39). We performed quantitative proteomics to determine the relative amounts of different VSGs in soluble VSG fractions following the induction of TbSAP RNAi. In the T. brucei SM221 MES-Pur cell line expressing VSG221 from the active BES1, MES VSG653 comprises 1.3% total VSG protein (Figure 10A). As mentioned earlier, this high expression level of VSG653 is presumably a consequence of readthrough from the ectopic rDNA promoter (Supplementary Figure S1). If a TbSAP RNAi construct is introduced into this cell line, the amount of MES VSG653 increases about three fold (presumably as a consequence of leaky transcription of the RNAi fragment), and then further increases after the induction of TbSAP RNAi (Figure 10A).

Figure 10.

Multiple up-regulated VSGs are present on the cell surface following the induction of TbSAP RNAi (A) Quantitative proteomic analysis using mass spectrometry of surface VSGs cleaved by release of endogenous GPI-PLC after hypotonic lysis. Samples are from the parental VSG221 expressing SM221pur MES-GFP cells (MES-GFP) or cells where TbSAP RNAi has been induced with tetracycline (Tet) for the time indicated in hours (h). VSG221 expressed from the active BES1 is omitted from the BES VSGs dataset, as it displayed an abundance of 95–99% in each sample. In these cell lines MES VSG653 is expressed from readthrough transcription from an ectopic rDNA promoter introduced upstream of the VSG653 gene. The different classes of VSGs are indicated with coloured bars, with MES VSG653 indicated with a blue bar, other MES VSGs (purple), BES VSGs (green bar) or VSGs in chromosome internal (CI) silent VSG arrays (black). Data are the mean of three biological replicates. (B) As in panel (A) only both MES VSG653 and VSG221 are omitted from the VSG dataset, as together they made up >99% of the sample. Different types of VSGs (present in either MESs or BESs) are indicated with coloured bars, with VSGs not located in ESs indicated as CI VSGs (grey and black bars). Data are the mean of three biological replicates.

For the other MES VSGs, the proteomic analyses globally reflect what was observed at the transcript level. Metacyclic VSGs with the highest transcript reads (VSG397, VSG531, VSG636 and VSG1954) were all detected by mass spectrometry following phospholipase-C cleavage, indicating localisation on the cell surface (Figure 10B). Although metacyclic VSGs are not detectable on the surface of the parental VSG221 cells, after the induction of TbSAP RNAi for 72 or 96 h, they comprise 0.35% or 0.54% of the total VSG protein respectively. Less protein was detected from VSGs present in either the chromosome internal VSG arrays or BESs than would be expected from the transcript reads. These results show that if MESs are derepressed, they can produce functional VSG protein in bloodstream form T. brucei.