DNA helicase RecQ1 regulates mutually exclusive expression of virulence genes in Plasmodium falciparum via heterochromatin alteration

Abstract

The Plasmodium falciparum var gene family encodes ∼60 surface antigens by which parasites escape the host immune responses via clonal expression of var genes. However, the mechanism controlling this mutual exclusivity, associated with alterations in chromatin assembly, is not understood. Here, we determined how expression of the var gene family is regulated by two RecQ DNA helicase family members, PfRecQ1 and PfWRN, in P. falciparum Through genetic manipulation, we found that the complete var repertoire was silenced on PfRecQ1 knockout, whereas their expression did not show noticeable changes when PfWRN was knocked out. More important, mutually exclusive expression of var genes could be rescued by complementation of PfRecQ1. In addition, knocking out either of these two helicase genes changed the perinuclear cluster distribution of subtelomeres and subtelomeric var genes. Whereas deletion of PfRecQ1 increased the heterochromatin mark trimethylated (H3K9me3) at the transcription start site (TSS) of the var gene upsC1, that deletion had no effect on the global distribution of H3K9me3 over gene bodies, including those for the var genes. ChIP-seq assay showed that PfRecQ1 was enriched globally at the TSSs of all genes, whereas PfWRN-enriched regions occurred at the gene bodies of the var gene family, but not of other genes or at TSSs of all genes. On PfRecQ1 deletion, the upsC1 var gene moved from the active perinuclear transcription region to a silenced region of the upsC type. These findings imply that PfRecQ1, but not PfWRN, is essential for maintaining the clonal expression of var genes.

Keywords: DNA helicases; Plasmodium falciparum; heterochromatin; mutually exclusive expression; virulence gene.

Fig. 1.

Expression status of var genes on knock-out of PfRecQ1 or PfWRN. Var genes expression level (A) in WT 3D7C8, PfRecQ1∆ (strain D3), and PfWRN∆ were detected by RT-qPCR with five repeats, and (B) in different strains of PfRecQ1∆ with at least two repeats. PfRecQ1∆D4 and PfRecQ1∆D3 were clones from first transfection into 3D7C8 (PF3D7_1240600, dominantly expressed); PfRecQ1∆B2 and PfRecQ1∆B3 were clones from second transfection into 3D7C8. 3D7C8 is the WT parasite strain. (C) In PfRecQ1∆B2, PfRecQ1∆B2/vec, PfRecQ1∆B2/EX-1, and PfRecQ1∆B2/EX-2. PfRecQ1∆B2/vec was PfRecQ1∆B2 transfected with PCC4-G418 vector, as control of complementation transfection. PfRecQ1∆B2/EX-1 and PfRecQ1∆B2/EX-2 were PfRecQ1∆B2 transfected with PCC4-^TY1-FlagRecQ1-G418 plasmid to episomally express ^TY1-FlagPfRecQ1 to rescue the functions of PfRecQ1. PfRecQ1∆B2/EX-1 and PfRecQ1∆B2/EX-2 were results from twice-independent complementation transfections. Experiments were repeated three times. The y axis gives the relative abundance of var genes mRNA; var genes IDs are shown below the x axis. Arginyl-tRNA synthetase (PF3D7_0913900), a housekeeping gene, is used as internal control. Error bars represent SEM.

Fig. 2.

H3K9me3 modification and DNA-FISH of subtelomeric var genes on PfRecQ1∆ or PfWRN∆. (A) Rep20 DNA-FISH in WT 3D7C8, PfRecQ1∆D3, and PfWRN∆. Rep20 probe was labeled with Biotin and then incubated with streptavidin (Alexa Fluor 568 conjugate, Thermo Fisher, red); chromosomal DNA was stained with DAPI (blue), representing the nuclear area. Parasites synchronized at ring stage were used for this DNA-FISH. Resolution of A is 600 dpi. (B) Graphic representation of the statistical analysis of Rep20 DNA-FISH result. The experiment was repeated three times, each with a count of 100 nuclei at ring stage. Nuclei were classified as type 1 (1–3 foci/nucleus), type 2 (4–5 foci/nucleus), or type 3 (≥6 foci/nucleus). The percentage of each type per 100 nuclei was calculated. (Scale bars of all pictures of DNA-FISH, 1 µm.) Here all error bars were SEM, *P < 0.05; **P < 0.01; ***P < 0.001. (C) Integrative genomic view of ChIP-seq result of H3K9me3 at ring stage in P. falciparum. In parasites of WT-3D7C8 (black), RecQ1∆D3 (red), and WRN∆ (blue) at ring phase, H3K9me3 bound specially to sequences of telomeres and var genes. Var genes are categorized as being in the forward orientation (unfilled arrows) or reverse orientation (filled arrows). Telomere sequences were at the end of chromosomes, which were not labeled. ChIP was performed with the anti-H3K9me3 antibody twice, and the average was used for this IGV. Each read was normalized by the total number of uniquely mapped ChIP-seq reads. Values on the y axis were calculated by ChIP reads divided by input reads, representing the net signal of ChIP. Chromosomal numbers are shown to the left. Two var genes in the boxed region are amplified for a detailed view in SI Appendix, Fig. S4C. A scale bar representing 100 kilobases (kb) is shown to the right of chromosome 1. The data range on the y axis used for IGV here was 0–40. (D) ChIP-qPCR of H3K9me3. The primers at the TSS of four var genes were used for this assay. PF3D7_0400400 is a upsA type, also used as A1 var gene for paired DNA-FISH. PF3D7_0300100 is a upsB type var gene. PF3D7_0617400 is a upsC type, named as C3. PF3D7_1240600, is another upsC type, also used as C1 var genes for paired DNA-FISH. Var A1, B, C3 genes are silent in WT, PfRecQ1∆, and PfWRN∆; Var C1 is dominantly transcribed in WT or PfWRN∆, but silent in PfRecQ1∆. Primers for ChIP-qPCR are shown in SI Appendix, Table S3. The experiment was repeated five times. The P value of upsC1 between WT and PfRecQ1∆ was 0.013, whereas the others were >0.05 (no significant difference).

Fig. 3.

Localization of PfRecQ1 and PfWRN proteins to P. falciparum chromatins. (A) Integrative genomic view of ChIP-seq result of ^HA-FlagRecQ1 (red) and ^GFPWRN (blue). Anti-Flag and anti-GFP were used for ChIP of PfRecQ1 and PfWRN, respectively. y axis values were calculated using each ChIP signal as the numerator and the respective signal input as the denominator. Sixty var genes distributed along P. falciparum chromosomes 1–13 are indicated by forward orientation (unfilled) and reverse orientation (filled) arrows. The data range of values on the y axis used for RecQ1 and WRN was 0–15 and 0–3, respectively. (B and C) Average reads coverage profile of ^HA-FlagRecQ1 (red line) and ^GFPWRN (blue line) at gene bodies of (B) var gene family and (C) other genes (nonvar genes). The curves were normalized per million mapped reads. y axis values were calculated as each ChIP signal divided by its respective input signals.

Fig. 4.

Var gene DNA redistribution correlates with expression status on RecQ1∆. (A) Localizations of four picked var genes for paired DNA-FISH in chromosomes. (B) Representative image of paired DNA-FISH of upsA1 (PF3D7_0400400, green) with upsC1 (PF3D7_1240600, red). In 3D7C8 and RecQ1∆, upsA1 was labeled with fluorescein, whereas upsC1 was labeled with biotin. In WRN∆, upsA1 was labeled with biotin, whereas upsC1 was labeled with fluorescein. (C) Representative image of paired DNA-FISH of upsB1 (PF3D7_ 0115700, green) with upsC1 (red). In all strains, upsB1 was labeled with biotin, whereas upsC1 was labeled with fluorescein. (D) Representative image of paired DNA-FISH of upsC2 (PF3D7_0412400, green) with upsC1 (red). In 3D7C8 and RecQ1∆, upsC2 was labeled with fluorescein and upsC1 was labeled with biotin. In WRN∆, upsC2 was labeled with biotin and upsC1 was labeled with fluorescein. Parasites synchronized at ring stage were used for this DNA-FISH. Biotin labeling was visualized by incubating with streptavidin-Alexa Fluor 568. DAPI staining (blue) was used to locate nuclear area. Graphs at right are statistical analyses of paired DNA-FISH results. Experiments were repeated three times, each with a count of ≥50 nuclei. The percentage of colocalization or no colocalization were calculated. Error bars were SEM. Scale bars of all pictures of DNA-FISH were 1 µm. In DNA-FISH of upsC1 and upsC2, the P value of RecQ1∆ to 3D7C8 was 0.006. Resolution of B, C, and D is 600 dpi.