Trypanosoma brucei Orc1 is essential for nuclear DNA replication and affects both VSG silencing and VSG switching

Abstract

Binding of the Origin Recognition Complex (ORC) to replication origins is essential for initiation of DNA replication, but ORC has non-essential functions outside of DNA replication, including in heterochromatic gene silencing and telomere maintenance. Trypanosoma brucei, a protozoan parasite that causes human African trypanosomiasis, uses antigenic variation as a major virulence mechanism to evade the host's immune attack by expressing its major surface antigen, the Variant Surface Glycoprotein (VSG), in a monoallelic manner. An Orc1/Cdc6 homologue has been identified in T. brucei, but its role in DNA replication has not been directly confirmed and its potential involvement in VSG repression or switching has not been thoroughly investigated. In this study, we show that TbOrc1 is essential for nuclear DNA replication in mammalian-infectious bloodstream and tsetse procyclic forms (BF and PF). Depletion of TbOrc1 resulted in derepression of telomere-linked silent VSGs in both BF and PF, and increased VSG switching particularly through the in situ transcriptional switching mechanism. TbOrc1 associates with telomere repeats but appears to do so independently of two known T. brucei telomere proteins, TbRAP1 and TbTRF. We conclude that TbOrc1 has conserved functions in DNA replication and is also required to control telomere-linked VSG expression and VSG switching.

Figure 1

TbOrc1 is essential for BF cell growth. (A) Depletion of TbOrc1 in RNAi cells. Whole cell lysates from two independent cell lines with FLAG-HA-HA (F2H)-tagged TbOrc1, B4 and B5, were analyzed by western blotting using anti-HA antibody at the indicated time points. Tubulin is shown as a loading control. (B) Growth curves for WT, TbORC1 RNAi cell lines in wild-type or in TbORC1^−/+ (SKO) heterozygous background. TbORC1 RNAi was induced using 0.1 μg/ml doxycyclin (DOX). The average population doublings (PDs) versus days of induction and standard deviations were calculated from three independent experiments. (C) Depletion of TbOrc1 led to a cell cycle profile change. Top, gating schematic of uninduced TbORC1 RNAi cells in flow cytometry analysis. PI, propidium iodide staining intensity. Bottom, quantification of populations of cells at different cell cycle stages before (day 0) and after (days 1, 2, and 3) depletion of TbOrc1. Unpaired t-tests were done to compare Day 1, 2, and 3 values with the Day 0 value. One asterisk, 0.009<P≤0.05; two asterisks, 0.0001<P≤ 0.009; three asterisks, P≤0.0001.

Figure 2

TbOrc1 is required for PF and BF nuclear DNA replication. DNA replication was analyzed by comparing BrdU incorporation in TbORC1 RNAi uninduced and induced cells. Immunostaining of BrdU-incorporated cells with anti-BrdU antibody and bulk DNA that was counterstained with DAPI (A, C). DAPI fluorescence indicates cells at different stages of the cell cycle. Representative images are shown. Asterisk, kDNA only BrdU-positive; Arrow, nucleus and kDNA BrdU-positive; Arrowhead, nucleus only BrdU-positive. Percentage of BrdU-positive and BrdU-negative cells in control and RNAi induced samples (B, D). Data are presented as the mean percent of 200 cells (for PF experiments) and 150 cells (for BF experiments) counted per time point from two independent experiments. Error bars represent the variation range. Only 1N1K and 1N2K cells are included in the analysis of replicating cells. The scale bar in (A) and (C) is 10 μm.

Figure 3

TbOrc1 is required for complete silencing of subtelomeric BES-linked VSGs. (A) Depletion of TbOrc1 in BF cells leads to derepression of BES-linked silent VSG genes. Steady state mRNA levels of several silent VSGs, the active VSG2, and control genes were analyzed by Q-RT-PCR, using tubulin gene as an internal loading control. The fold changes in mRNA level after RNAi induction are plotted. Day 0 values were set to 1. rDNA, TbTERT, TbPGI, and TbRPS15 genes were analyzed as controls. Fold changes were averaged from at least three independent experiments. Standard deviations are shown as error bars. (B) Expression levels of silent VSG proteins and telomere proteins in TbOrc1-depleted BF cells. (C) BES-linked silent VSGs are derepressed in TbOrc1-depleted PF cells. The analysis was performed as described in (A). (D) Two silent VSG proteins can be expressed simultaneously on the surface of a single TbOrc1-depleted BF cell. VSG2-expressing BF cells were examined by immunofluorescence at indicated time points after TbORC1 RNAi induction. Cells co-expressing the silent VSG3 and VSG13 were visible four days post TbORC1 RNAi induction. A single layer of the z-stack images was shown for each time point to better illustrate the expression of derepressed VSGs and their localization.

Figure 4

TbOrc1 controls VSG switching mechanisms. (A) VSG switching mechanisms. Diagram exhibits four major switching events; in-situ (transcriptional inactivation of the active and activation of a silent BES promoter), ES GC + ES loss (recombination near BES promoters, or active BES loss associated with in-situ switching), VSG GC (gene conversion near VSGs), and crossover (XO) (exchanges of chromosome ends containing the active and a silent VSG). (B) Expression levels of TbOrc1-HA, histone H3, and tubulin in the TbORC1 RNAi cells. (C) VSG switching frequency increased about 3-fold in the TbOrc1-depleted cells. Switchers were enriched and selected after two days of induction followed by one day of recovery (see text for details). Three independent cultures each were examined. Standard deviations are shown as error bars. Unpaired T tests were done and means of switching frequencies were significantly different (P=0.0124). (D) TbOrc1 suppresses VSG switching at the BES promoter. Fifty-eight and 135 switchers from three uninduced and three induced cultures, respectively, were analyzed for their switching mechanisms (see text for details). Percentage of individual mechanisms is indicated in the sector. Approximately 1% of clones appeared to have undergone in-situ and homologous recombination because VSG2 is lost but BSD does not appear to be expressed from the active ES even though the BSD gene is still present.

Figure 5

TbOrc1 is located at telomeres. Quantification of TbOrc1 ChIP results in BF (A) or PF (C) cells. Probes used in different hybridizations were indicated at the top. Average enrichments were calculated from 15 (BF) or 4 (PF) independent experiments, and standard variations are shown as error bars. P values of unpaired t-tests are shown for indicated pairs of data. Representative Southern analyses of the TbOrc1 ChIP in BF (B) or PF (D) cells are shown. DNA precipitated with HA antibody (HA), TbTRF antibody (TbTRF), or no antibody (No AB) from BF cells, and DNA precipitated with IgG beads or protein G beads from PF cells, and the input DNA sample (20%) were hybridized with either a TTAGGG-repeat probe, a 177-bp repeat probe (BF), or a 50 bp repeat probe as indicated.