Toxoplasma gondii chromodomain protein 1 binds to heterochromatin and colocalises with centromeres and telomeres at the nuclear periphery

Abstract

Background: Apicomplexan parasites are responsible for some of the most deadly parasitic diseases afflicting humans, including malaria and toxoplasmosis. These obligate intracellular parasites exhibit a complex life cycle and a coordinated cell cycle-dependant expression program. Their cell division is a coordinated multistep process. How this complex mechanism is organised remains poorly understood.

Methods and findings: In this study, we provide evidence for a link between heterochromatin, cell division and the compartmentalisation of the nucleus in Toxoplasma gondii. We characterised a T. gondii chromodomain containing protein (named TgChromo1) that specifically binds to heterochromatin. Using ChIP-on-chip on a genome-wide scale, we report TgChromo1 enrichment at the peri-centromeric chromatin. In addition, we demonstrate that TgChromo1 is cell-cycle regulated and co-localised with markers of the centrocone. Through the loci-specific FISH technique for T. gondii, we confirmed that TgChromo1 occupies the same nuclear localisation as the peri-centromeric sequences.

Conclusion: We propose that TgChromo1 may play a role in the sequestration of chromosomes at the nuclear periphery and in the process of T. gondii cell division.

Figure 1. TgChromo1 is a chromodomain containing protein.

A: TgChromo1 specifically binds to H3K9me3. TgChromo1 protein was bound to H3K9me3 peptides fixed on Agarose-beads. The binding was then competed by excess amount of different H3 peptides. A fraction of the input protein sample was loaded along with elution with an unmodified H3 (Beads+H3), H3K9ac (Beads+H3K9ac), H3K9me3 (Beads+H3K9me3) or H3K27me3 (Beads+H3K27me3) peptides. Molecular weight markers (kDa) are indicated on the right. B: Native TgChromo1-HA protein binds to H3K9me3 peptides. Extracts from the TgChromo1-HA RH ΔKu80 strain were purified using either H3K9me3 peptide bound beads or unmodified H3 bound beads. Eluates were subjected to Western-blot using anti-HA antibody to reveal the presence of TgChromo1-HA. Molecular weight markers (kDa) are indicated on the left side of the panel. C: The majority of TgChromo1 is co-purified with insoluble material. Cytoplasmic, nuclear and insoluble extracts from the TgChromo1-HA RHΔKu80 strain were subjected to a Western-blot using the anti-HA specific antibody. A control, a cytoplasmic resident (TgLDH1) and a nuclear resident (TgHistone4) were also identified in the same extracts.

Figure 2. TgChromo1 expression is cell cycle regulated.

A: IFA of TgChromo1-HA (green) and IMC1 (red), a marker of the inner membrane complex, throughout the cell cycle. Parasites representative of interphase (G1) and mitosis are presented. Parasite nuclei are labelled with DAPI. B: Comparison of the intensity of the signal produced by IFA of TgChromo1-HA in interphase (G1) and during budding (Budding, B). IMC1, a marker of the inner membrane complex, is used to identify emerging daughter cells during the budding. Parasites during budding (B) are arrowed. Parasite nuclei are labelled with DAPI. C: TgChromo1 is concentrated in foci of different intensity. IFA was performed using an anti-HA antibody and the IMC1 antibody. Parasites nuclei are labelled with DAPI. Foci of lesser intensity are arrowed.

Figure 3. TgChromo1 is maintained near the centrosome and the centrocone throughout the cell cycle.

A: IFA of TgChromo1-HA (green) and centrin1 (red), a marker of the centrosome. Parasite nuclei are labelled with DAPI (blue) at the interphase (G1), mitosis and the beginning of budding. B: IFA of TgChromo1-HA (green) and MORN1 (red), a marker of the centrocone. Parasite nuclei are labelled with DAPI (blue) at the interphase (G1), mitosis and the beginning of budding.

Figure 4. TgChromo1 binds to peri-centromeric heterochromatin.

ChIP on chip was performed with the TgChromo1 antibody (anti-CHD1, red) or the anti-HA antibody (HA, black) and hybridized on a genome-wide tiling microarray. The regions of enrichment for H3K9me3 are represented in blue. A snapshot of the 12 chromosomes where a centromere was identified is presented. ChIP on chip signals are represented as a log2 ratio of the signal given by the immunoprecipitated DNA over the input and plotted according to the genomic position of the oligonucleotide.

Figure 5. Colocalisation of TgChromo1 and peri-centromeric sequences and identification of a missing centromere on chromosome IV.

A: Genomic localisation of the FISH probes. Fish probes (red rectangles) approximate locations are plotted against the signal of the TgChromo1-HA ChIP on chip. B: FISH/IFA of TgChromo1-HA (green) and chromosome IX peri-centromeric repeats (red). Parasite nuclei are labelled with DAPI (blue). C: FISH/IFA of TgChromo1-HA (green) and chromosome IV putative peri-centromeric repeats (red). Parasites nuclei are labelled with DAPI (blue). D: Quantification of the overlap coefficient between the signals of FISH and IFA. The coefficient is expressed as a ratio of the number of pixel overlapping in IFA over FISH in a given area.

Figure 6. Subtelomeric repeats occupy the same nucleus territory as TgChromo1.

FISH/IFA of TgChromo1-HA (green) and chromosome IX telomeric repeats (red). Parasite nuclei are labelled with DAPI (blue). Colocalising signals from FISH and IFA are arrowed.

Figure 7. TgChromo1 participates to the nuclear organisation of the nucleus.

TgChromo1 participates in the functional organisation of the nucleus. The schematic represents the chromosomes in the nucleus with the centromere and telomere clusters occupied by TgChromo1 and their position at the periphery of the nucleus.