Figure 1.
Immunofluorescence analysis of histone modifications in P. falciparum.
Localization of histone modifications were analyzed with the ring, trophozoite, schizont, late schizont and merozoite stages of the IDC. IFAs were carried out with antibodies against specific histone 3 and 4 lysine residue acetylations: H3K9Ac, H4K5Ac, H4K8Ac, and H4Ac4, as well as methylations: H3K4Me3, H3K9Me3 and H3K9Me1, and unmodified histone H3. Nuclear DNA was stained with DAPI (blue). All modifications, with the exception of H3K9Me1 and H3 (antibody raised against H3 C terminal), showed specific and distinct localization in the nucleus in all the stages. In contrast, H3K9Me1 was localized mainly outside the nucleus with very low levels detected inside the nucleus.
Figure 2.
H3K9Me1 localized to the parasitophorous vacuole during the ring stage.
Co-localization of H3K9Me1 with Etramp 2 (A) and 4 (B) was performed in ring and schizont stage parasites respectively. Similarly, co-localization of H4K5Ac with Etramp 2 (C) and 4 (D) was performed in ring and schizont stage parasites respectively. H3K9Me1/H4K5Ac and Etramp 2/4 were stained red and green respectively. DAPI stained nuclear DNA blue. Yellow and white arrows indicate foci of more intense fluorescence produced by H3K9Me1 and Etramp labeling respectively. In ring stage parasites, compared to schizonts, H3K9Me1 partially co-localized with Etramp 2 indicating localization to different compartments of the PV. H4K5Ac was localized solely to the nucleus and did not co-localize with either Etramp 2 or 4.
Figure 3.
Immunodetection of Etramp 2, H3K9Me1 and H4K5Ac under different concentrations of saponin treatment.
Ring stage P. falciparum infected red blood cells were subjected to different concentrations of saponin treatment, ranging from 0.06% to 0.14%. A strong signal corresponding to the expected molecular weights for Etramp 2 and H3K9Me1 was detected with 0.06%, 0.08% and 0.1% saponin. At higher saponin concentrations (0.12% and 0.14%), disruption of the PVM resulted in significant reductions in Etramp 2 and H3K9Me1 signal. In comparison, levels of H4K5Ac were unaffected by the saponin treatment. A faint band appeared at approximately 34 kDa due to non-specific reaction of the secondary antibody. Molecular weights are shown in kDa.
Figure 4.
Loss of H3K9Me1 with disruption of the parasitophorous vacuole.
P. falciparum infected red blood cells were subjected to various concentrations of saponin treatment and analyzed by IFA. H4K5 acetylation (red) remained co-localized with the DAPI stained nuclear DNA (blue) regardless of increasing concentrations of saponin. Etramp 2 (green) and H3K9Me1 (red) were gradually lost, resulting from the disruption of the PVM.
Figure 5.
Immunofluorescence analysis of H3K9Me1 localization in P. yoelii and P. vivax.
In both P. yoelii and P. vivax H3K9Me1 (red) co-localized with the PV marker Etramp 4 (green) whereas H4K5Ac (red) co-localized with the DAPI stained nuclear DNA (blue).
Figure 6.
Immunofluorescence analysis of Nup 100.
H3K9Me1, H4K5Ac and Nup 100 were stained red and green respectively. DAPI stained DNA blue. Yellow arrow indicates the co-localization of the H3K5Ac and Nup-100 in the ring stage parasites. White arrows indicate the co-localization of H3K9Me1 and Nup 100 outside the DAPI stained area, in the ring and schizont stage parasites.