The transcription of bradyzoite genes in Toxoplasma gondii is controlled by autonomous promoter elements

Abstract

Experimental evidence suggests that apicomplexan parasites possess bipartite promoters with basal and regulated cis-elements similar to other eukaryotes. Using a dual luciferase model adapted for recombinational cloning and use in Toxoplasma gondii, we show that genomic regions flanking 16 parasite genes, which encompass examples of constitutive and tachyzoite- and bradyzoite-specific genes, are able to reproduce the appropriate developmental stage expression in a transient luciferase assay. Mapping of cis-acting elements in several bradyzoite promoters led to the identification of short sequence spans that are involved in control of bradyzoite gene expression in multiple strains and under different bradyzoite induction conditions. Promoters that regulate the heat shock protein BAG1 and a novel bradyzoite-specific NTPase during bradyzoite development were fine mapped to a 6-8 bp resolution and these minimal cis-elements were capable of converting a constitutive promoter to one that is induced by bradyzoite conditions. Gel-shift experiments show that mapped cis-elements are bound by parasite protein factors with the appropriate functional sequence specificity. These studies are the first to identify the minimal sequence elements that are required and sufficient for bradyzoite gene expression and to show that bradyzoite promoters are maintained in a 'poised' chromatin state throughout the intermediate host life cycle in low passage strains. Together, these data demonstrate that conventional eukaryotic promoter mechanisms work with epigenetic processes to regulate developmental gene expression during tissue cyst formation.

Fig. 1

Regulation of mRNA expression during bradyzoite differentiation in low-passage Type III-CTG, Type I-GT1 and Type II-Me49B7 strains. A. The expression of 267 genes that were up- or downregulated by Compound 1 treatment in the Type-II and III strains (3 μM drug for 48 h) are shown (for a list of these genes see Fig. S1). Each line represents one gene in the gene list allowing one to trace the expression of the genes across the experiment. Colouring of the lines (fold scale to the right) is fixed across the graph by the expression level of just one sample, Type III Compound 1 (CMPD1) (yellow-to-red = upregulated; yellow-to-blue = downregulated). Genes induced by Compound 1 include all known bradyzoite genes. BAG1, LDH2, ENO1, SAG4.2 and B-NTPase mRNAs are among the highest expressed mRNAs detected the Type III-CTG and Type II-Me49B7 strains induced by Compound 1 (indicated by labels and with line graphs highlighted in black). Note that the group of bradyzoite genes that are coloured in red or highlighted in black based on the mRNA expression in the Type III-CTG strain are minimally increased in Type I-GT1 parasites exposed to Compound 1. Each data point shown represents RMA-normalized values from two independent biological replicates. B. RMA-normalized fluorescent values for five known bradyzoite genes are listed for three strains maintained as tachyzoites or induced by Compound 1.

Fig. 2

Histone modifications on bradyzoite promoters are poised for activation in low-passage strains. A. ChIP results for the BAG1 (B), SAG2A (S) and β-tubulin (T) promoter regions in three major genetic lineages. The BAG1 promoter was associated with acetylated histones (H3 or H4) in low-passage tachyzoites (left panels) of all major strains (Type I-GT1, Type II-Me49B7 and Type III-CTG) and the levels detected were similar to acetylated H3 levels in the tachyzoite-specific gene SAG2A or the β-tubulin gene, which is constitutively expressed in both tachyzoite and bradyzoites. The levels of H3 or H4 acetylation in these strains was largely sustained in parasites stimulated to differentiate with 3 μM Compound 1 (CMPD1) for 48 h (right panels). Acetylated H3 associated with the BAG1 promoter in the lab-adapted, Type I-RH strain was minimal in tachyzoites or parasites exposed to Compound 1. AcH3, AcH4 = ChIP with antibody specific for acetylated histone H3 or H4; No Ab = no antibody control ChIP; Input = input DNA collected prior to immunoprecipitation. B. ChIP of the LDH2 promoter region in different strains. LDH2 promoter has complex H3 acetylation patterns in different strains. In tachyzoites, we observed strong H3 acetylation associated with the LDH2 promoter in Type I and III strains, while Type II strain tachyzoites the promoter was hypoacetylated (left panels). Following induction by Compound 1, hyperacetylated H3 was detected in the LDH2 promoter from Type II-Me49B7 and III-CTG parasites, while this promoter became hypoacetylated in the Type I-GT1 strain (right panels). In the cell culture adapted Type I-RH strain, the LDH2 promoter was hypoacetylated regardless of stage or culture condition. C. ChIP of the B-NTPase promoter regions in different strains. B-NTPase promoter is associated with substantial H3 acetylation in both tachyzoite and Compound 1-induced extracts in all three low-passage strains with the level of H3 acetylated in Type II-Me49B7 tachyzoites and bradyzoites lower than the Type I-GT1 and Type III-CTG strains.

Fig. 3

Intergenic genomic regions control developmental gene expression. A. Promoter regions from bradyzoite genes are strongly induced by 3 μM Compound 1 treatment. Several bradyzoite promoters engineered to drive the expression of firefly luciferase were tested under tachyzoite (open bars) and bradyzoite induction (solid bars) conditions. Type III-VEG_msj tachyzoites were electroporated with the appropriate plasmid combination (see Experimental procedures), inoculated into HFF monolayers (T25cm² flasks), allowed to grow overnight, and then experimental flasks were placed under 3 μM Compound 1 for 48 h. Four readings were collected for firefly and renilla luciferase activity for each promoter construct. Fold increase in luciferase activity in comparison to α-tubulin-renilla controls are plotted for each promoter construct tested under tachyzoite or bradyzoite-induction conditions. All promoters exhibited strong induction following Compound 1 induction with the exception of BSR4, which has been shown to be equally expressed in tachyzoites and bradyzoites (Van et al., 2007). B. Integrated promoter constructs in transgenic parasites display natural developmental expression. Clone Type II-Prugniaud-IC2 expresses an integrated copy of the firefly luciferase controlled by the BAG1 promoter (−1197 bp region). Type II-Prugniaud-IC2 parasites were inoculated into HFF monolayers, allowed to grow overnight and then placed under 3 μM Compound 1. Infected monolayers were assessed for luciferase activity (average of four readings) at the indicated time points. Luciferase induction is first observed between 24 and 36 h and increases dramatically in parallel to native BAG1 protein (Radke et al., 2006). C. Differentiation conditions vary significantly in their strength of induction. Type II-Prugniaud-IC2 parasites (2 million) were inoculated into HFF monolayers, allowed to grow overnight and then placed under six different induction conditions (see Experimental procedures). Drug concentrations: Compound 1 = 3 μM or pyrolidine dithiocarbamate = 100 μM. Infected monolayers were assayed for luciferase activity (4-readings/condition) using standard protocols (Promega manual #TM058) at 72 h post-treatment. Results were calculated as ratios of induced/untreated control (e.g. Compound 1 = 20 970 normalized light units, pH 8.2 = 4838 units, and PDTC = 2900 units) and then graphed in comparison to the pH 8.2 values set to 100%. The order of strength of induction: Compound 1 > pH 8.2 > PDTC > CO₂ and arginine depletion > high temperature.

Fig. 4

Sequential and internal deletion of the BAG1 and B-NTPase promoters. Transient transfections were performed in duplicate and firefly and renilla luciferase activity was assayed sequentially in each of the samples. Firefly luciferase results were normalized by α-tubulin-renilla levels and graphed as the induced level of expression as compared with the full-length promoter construct (relative response ratio). For each sequential deletion construct, fold change was also determined with respect to the level of luciferase expression in the α-tubulin-renilla control (fold change values and standard deviations in parentheses are listed adjacent each deletion construct). Nucleotide positions in these deletion studies are referenced with respect to the start of translation (+1) in each construct. A. Results of sequential deletion of the BAG1 promoter compared with the full-length 1195 bp promoter construct. B. Results of internal deletion of the BAG1 promoter. Note the regions identified by sequence deletion are referenced by arrow in this internal deletion series. C. Results of sequential deletion of the B-NTPase promoter (fold change values also included) with respect to the 1495 bp full-length promoter. D. A series of overlapping 10 bp internal deletions further refine the required sequence elements first identified by the sequential deletion between −611–403 bp in this promoter (see arrow).

Fig. 5

BAG1, B-NTPase and LDH2 promoter induction in Type I, II and III parasites under two different induction conditions. A. Type III-VEG_msj, Type II-Me49B7 and Type I-RH strains were transiently transfected in duplicate and induced by 3 μM Compound 1 (CMPD1) or pH 8.2 media conditions (48 h). Infected monolayers were harvested at the appropriate times (parasite + HFF cells), lysates prepared and luciferase activity was determined (4-readings/condition). Firefly luciferase results are normalized by α-tubulin-renilla levels and reported as the relative induced expression levels in reference to the corresponding full-length promoter (relative response ratio; fold change values for these experiments are listed in Table S1). Three promoters were tested, BAG1, B-NTPase and LDH2, with two internal deletion constructs compared with three promoter constructs that show maximum inductive activity (BAG1 −1195, B-NTPase −801 and LDH2 −708). Internal deletion in the BAG1 −457–365 bp, B-NTPase −477–441 bp and LDH2 −426–377 bp promoters of the critical cis-elements ablate induction of these promoters in all three strains regardless of the induction condition, while neighbouring deletions in each promoter retained at least 50% of the activity of the fully active promoter construct. Each control internal deletion behaved similarly in each strain including the strong induction observed when a putative repressor element that lies between −340 and 277 bp of the LDH2 promoter was deleted.

Fig. 6

BAG1 and Brady-NTPase cis-elements are autonomous in Toxoplasma. Copies (1–3×) of the BAG1 or B-NTPase cis-element (see Table 1) were introduced into the DHFR promoter (see diagram). The cis-elements were placed 100 bp upstream of the major start of transcription at −369 bp (Matrajt et al., 2004). The Type III-VEG_msj strain was transiently transfected in duplicate and induced by 3 μM Compound 1. Infected monolayers were harvested at the appropriate times (parasite + HFF cells), lysates prepared, and luciferase activity was determined (4-readings/condition). Firefly luciferase results are normalized by α-tubulin-renilla levels and reported as the relative induced expression levels in reference to the corresponding full-length promoter (relative response ratio). No change in luciferase expression was observed under tachyzoite conditions with any construct (data not shown). However, 48 h following 3 μM Compound 1 addition there was a stepwise increase in luciferase activity compared with the WT-DHFR promoter construct based on the number of cis-element copies.

Fig. 7

B-NTPase EMSA assay. The B-NTPase cis- element forms sequence specific protein–DNA complexes. Incubation of [³²P]-end-labelled DNA encoding B-NTPase cis-element (−467–438 bp, see Table S2 for all primer designs) with nuclear extracts from Type III-CTG parasites induced for 48 h with 3 μM Compound 1 (CMPD1). Note the major complex (complex 2) formed with labelled Brady-NTPase cis-element fragment was not diminished by unlabelled B-NTPase mutant #3 sequence (Table 1) or with unlabelled BAG1 cis-element. Lane 1, probe alone; lane 2, nuclear extract + probe; lanes 3–7, probe + extract + cold competitor. Lanes 3 and 4, unlabelled wild-type B-NTPase competitor at 5- and 25-fold excess respectively. Lanes 5 and 6, unlabelled B-NTPase mutant #3 (Table 1 and Table S2) at 5- and 25-fold excess. Lane 7, 25-fold excess unlabelled BAG1 cis-element.

Fig. 8

BAG1 EMSA assay. Identification of sequence specific protein–DNA interactions using the BAG1 cis- element. Incubation of [³²P]-labelled BAG1 cis-element (−386–355 bp, Table S2 primer designs) with nuclear extracts from Type III-CTG parasites induced for 48 h with Compound 1 (CMPD1). Lane 1, probe alone; lane 2, nuclear extract + probe; lanes 3–7, probe + extract + cold competitor. Lanes 3 and 4, unlabelled wild-type BAG1 competitor at 5- and 25-fold excess respectively. Lanes 5 and 6, unlabelled BAG1 mutant competitor at 5-, 25-fold excess. Lane 7, fivefold excess unlabelled B-NTPase competitor.