Trypanosoma brucei TIF2 suppresses VSG switching by maintaining subtelomere integrity

Abstract

Subtelomeres consist of sequences adjacent to telomeres and contain genes involved in important cellular functions, as subtelomere instability is associated with several human diseases. Balancing between subtelomere stability and plasticity is particularly important for Trypanosoma brucei, a protozoan parasite that causes human African trypanosomiasis. T. brucei regularly switches its major variant surface antigen, variant surface glycoprotein (VSG), to evade the host immune response, and VSGs are expressed exclusively from subtelomeres in a strictly monoallelic fashion. Telomere proteins are important for protecting chromosome ends from illegitimate DNA processes. However, whether they contribute to subtelomere integrity and stability has not been well studied. We have identified a novel T. brucei telomere protein, T. brucei TRF-Interacting Factor 2 (TbTIF2), as a functional homolog of mammalian TIN2. A transient depletion of TbTIF2 led to an elevated VSG switching frequency and an increased amount of DNA double-strand breaks (DSBs) in both active and silent subtelomeric bloodstream form expression sites (BESs). Therefore, TbTIF2 plays an important role in VSG switching regulation and is important for subtelomere integrity and stability. TbTIF2 depletion increased the association of TbRAD51 with the telomeric and subtelomeric chromatin, and TbRAD51 deletion further increased subtelomeric DSBs in TbTIF2-depleted cells, suggesting that TbRAD51-mediated DSB repair is the underlying mechanism of subsequent VSG switching. Surprisingly, significantly more TbRAD51 associated with the active BES than with the silent BESs upon TbTIF2 depletion, and TbRAD51 deletion induced much more DSBs in the active BES than in the silent BESs in TbTIF2-depleted cells, suggesting that TbRAD51 preferentially repairs DSBs in the active BES.

Figure 1

TbTIF2 interacts with TbTRF and associates with telomeres. (A) TbTIF2 and TbTRF interact in yeast two-hybrid analysis. Top, different Gal4 Activation Domain (GAD)-fused TbTIF2 fragments (starting and ending amino acids of each fragment are listed in the parentheses) were tested for their interaction with LexA-fused full-length TbTRF or just LexA (empty). The expression of GAD-TbTIF2 fragments was examined by western blotting (Supplementary information, Figure S1C). Middle, different LexA-fused TbTRF fragments were tested for their interaction with the GAD-fused full-length TbTIF2. Control experiments testing the interaction between LexA-TbTRF and GAD alone were performed previously. β-galactosidase activities, shown as average (calculated from at lease four independent tests) ± standard deviations, reflect the expression of the reporter LacZ gene resulting from the interaction between the LexA- and GAD-fused proteins. A summary of the interaction between TbTIF2 and TbTRF is shown at the bottom. (B) TbTIF2 and TbTRF interact in vivo. Whole Cell Extract (WCE) was prepared from SM/TbTIF2-F2H. The soluble fraction of the lysate (soluble lysate) was cleared with protein G beads (IP input) and Immunoprecipitated with TbTRF antibody 1261, HA antibody 12CA5, or no antibody. IP supernatant (sup) and IP product (pellet) were subsequently analyzed by western blotting using HA antibody F-7 (Santa Cruz Biotechnology, Inc.) or chicken anti-TbTRF antibody 606. (C) Immunofluorescence analysis of SM/TbTIF-F2H cells using 12CA5 and 1261. DAPI stains DNA in both the nucleus (larger blue circle) and the kinetoplast (small blue dot). (D) ChIP analysis in SM/TbTIF2-F2H cells using F-7, 1261, or mouse IgG (M-IgG). ChIP products were hybridized with a TTAGGG or a 50 bp repeat probe. Representative slot blots are shown in Supplementary information, Figure S1F. The blots were exposed to a phosphorimager and results were quantified by ImageQuant. Average was calculated from at least three independent experiments. In this and following figures, error bars represent standard deviation. Numbers next to the brackets indicate P-values (unpaired t-tests) between different experiments. P < 0.05 is considered to be significant.

Figure 2

TbTIF2 is essential for cell survival. (A) Induction of TbTIF2 RNAi led to a decreased TbTIF2 protein level. Whole cell extracts were prepared from 2/TIF2i clone B9 cells at different time points after induction of TbTIF2 RNAi. HA antibody F-7 and EF-2 antibody (as a loading control, Santa Cruz Biotechnologies) were used in western blotting analyses. (B) Depletion of TbTIF2 by RNAi led to a growth arrest in T. brucei cells. Growth curves for SM/TbTIF2-F2H and 2/TIF2i cells in the presence (+) or the absence (−) of doxycycline (Dox). Average population doublings were calculated from three independent cultures. (C) TbTIF2 depletion had mild effects on subtelomeric VSG silencing. mRNA levels for several BES-linked VSGs and mVSGs were estimated by qRT-PCR at different time points after induction of TbTIF2 RNAi. The fold changes in mRNA levels were calculated from three independent experiments. 0 h values are all equal to “1” but not shown. Changes significantly different from that of rRNA are indicated by asterisks. ***P < 0.001 (unpaired t-tests).

Figure 3

Transient depletion of TbTIF2 led to an elevated VSG switching frequency. (A) Induction of TbTIF2 for 30 h led to a growth arrest for ∼24 h. S/TIF2i clone A14 and S/v cells were cultured without doxycycline, with doxycycline for 30 h, or with doxycycline throughout the experiment. Average population doublings were calculated from four independent experiments to plot the growth curve. (B) The TbTIF2 protein level decreased for ∼24 h when TbTIF2 RNAi was induced for 30 h in S/TIF2i cells. Western blotting analyses were performed using HA antibody F-7 and EF-2 antibody. S/v cells were treated the same way as a control. (C) VSG switching frequencies in S/TIF2i and control cells. Cells were cultured with doxycycline for 30 h or without doxycycline as indicated. Average switching frequencies were calculated from 4-12 independent assays. P-values (unpaired t-tests) between S/v and other cells are shown as numbers on top of the corresponding columns. (D) VSG switching pathways in S/TIF2i and control cells. Percent of each VSG switching pathway of total events was plotted. Listed on top of each column is the number of switchers analyzed. Detailed switcher characterization results are listed in Supplementary information, Tables S3-S6.

Figure 4

Depletion of TbTIF2 led to increased amount of DSBs at subtelomeric BES regions. (A) The principle of LMPCR. LMPCR analyses were performed in S/TIF2i clone A14 (B-D) and S/v (E) cells. The LMPCR products were hybridized with VSG2 (B), VSG21 (C), Tb427.9.9970 (which encodes a putative small nuclear RNA gene activation protein 50, SNAP50) (D), and 70 bp repeat (E) probes. In each panel of this figure and Figure 6, the Ethidium bromide (EtBr)-stained LMPCR products are shown at the top, the Southern blot result is shown in the middle, and the PCR products using primers specific to TbRAP1 are shown at the bottom as a loading control. The amounts of input genomic DNA, either treated (+) or untreated (−) with T4 DNA polymerase, were marked on top of each lane. Molecular weight markers (in kb unless otherwise indicated) are labeled on the left. (F) The signal intensities of LMPCR products (treated with T4 DNA polymerase) were quantified by ImageQuant from Southern blots exposed to phosphorimagers. The fold changes were calculated by dividing post TbTIF2 depletion value by preinduction value. The average was calculated from at least three independent experiments. Error bars represent standard deviation. P-values (unaired t-tests) were calculated using Graphpad Prism between values at each subtelomeric locus and that at Tb427.9.9970. Asterisks indicate significant difference. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 5

Depletion of TbTIF2 led to increased TbRAD51 association with telomeric and subtelomeric chromatin. (A) Quantification of cells with punctate nuclear TbRAD51 foci at various time points after induction of TbTIF2 RNAi in S/TIF2i A14 cells. Number of cells counted was indicated in the parentheses. Averages were calculated from three independent experiments. Numbers on columns indicate P-values (unpaired t-tests) compared to the 0 h value. (B, C) ChIP analysis using the TbRAD51 antibody or rabbit IgG. ChIP products and input DNA were hybridized with either a TTAGGG or a 50 bp repeat probe in Southern blotting, which were exposed to a phosphorimager. The hybridization signal intensity was quantified by ImageQuant, and averages were calculated from four independent experiments (B). ChIP products were analyzed by qPCR using primer pairs specific to various loci (Supplementary information, Table S1). Averages were calculated from 5-10 independent experiments (C). In both B and C, numbers on top of brackets indicate P-values (unpaired t-tests) comparing TbRAD51 ChIP values before and after TbTIF2 depletion.

Figure 6

More subtelomeric DSBs persisted when TbRAD51 was deleted in TbTIF2-depleted cells. The LMPCR products from S/TIF2i A14 and S/TIF2i/TbRAD51Δ C6 cells were hybridized with the Ψ_BES1 (A) or Ψ_BES11 (B) probe. (C) The signal intensities of LMPCR products (treated with T4 DNA polymerase) were quantified by ImageQuant from Southern blots exposed to phosphorimagers. The fold changes were calculated by dividing post TbTIF2 depletion value in S/TIF2i/ΔTbRAD51 cells by that in S/TIF2i cells on the same gel. The average was calculated from at least three independent experiments. Error bars represent standard deviation. P-values (unpaired t-tests) between pairs of loci were indicated.