18 research outputs found
A Moonlighting Function of Plasmodium falciparum Histone 3, Mono-Methylated at Lysine 9?
BACKGROUND: In the human malaria parasites Plasmodium falciparum, histone modifications have been implicated in the transcriptional regulation. The acetylation and methylation status of the histones have been linked with transcriptional regulation of the parasite surface virulence factors as well as other genes with stage specific expression. In P. falciparum as well as other eukaryotes, different histone modifications were found to be compartmentalized to distinct regions in the nuclei. This compartmentalization is believed to be one of the main prerequisites for their function in epigenetic regulation of gene expression. METHODOLOGY/PRINCIPAL FINDINGS: Here we investigate intracellular distributions of five previously uncharacterized histone modifications including histone 4 acetylation on lysine residue 5 (H4K5Ac), H4K8Ac, H3K9Ac, H4Ac4 and H3K9Me1 during the asexual developmental stages. With the exception of H3K9Me1, the modified histones were localized to the nuclear periphery. This provides a strong indication that the P. falciparum nuclear periphery is one of the most active regions in epigenetic regulation of gene expression. Interestingly, H3K9Me1 is not associated with the nuclei but instead resides in the parasitophorous vacuole (PV), the double membrane compartments surrounding the parasite cell within the host erythrocyte. In this compartment, H3K9Me1 partially co-localizes with Etramp proteins. The localization of H3K9Me1 in the PV is conserved in the other species including P. yoelii and P. vivax. CONCLUSIONS: Similar to other eukaryotes, the periphery of the P. falciparum nuclei is likely one of the most active areas in epigenetic regulation of gene expression involving multiple histone modifications. On the other hand, H3K9Me1 evolved a new function that is linked with the PV. This functional role appears to be evolutionarily conserved in Plasmodium species
Histone Deacetylases Play a Major Role in the Transcriptional Regulation of the Plasmodium falciparum Life Cycle
The apparent paucity of molecular factors of transcriptional control in the genomes of Plasmodium parasites raises many questions about the mechanisms of life cycle regulation in these malaria parasites. Epigenetic regulation has been suggested to play a major role in the stage specific gene expression during the Plasmodium life cycle. To address some of these questions, we analyzed global transcriptional responses of Plasmodium falciparum to a potent inhibitor of histone deacetylase activities (HDAC). The inhibitor apicidin induced profound transcriptional changes in multiple stages of the P. falciparum intraerythrocytic developmental cycle (IDC) that were characterized by rapid activation and repression of a large percentage of the genome. A major component of this response was induction of genes that are otherwise suppressed during that particular stage of the IDC or specific for the exo-erythrocytic stages. In the schizont stage, apicidin induced hyperacetylation of histone lysine residues H3K9, H4K8 and the tetra-acetyl H4 (H4Ac4) and demethylation of H3K4me3. Interestingly, we observed overlapping patterns of chromosomal distributions between H4K8Ac and H3K4me3 and between H3K9Ac and H4Ac4. There was a significant but partial association between the apicidin-induced gene expression and histone modifications, which included a number of stage specific transcription factors. Taken together, inhibition of HDAC activities leads to dramatic de-regulation of the IDC transcriptional cascade, which is a result of both disruption of histone modifications and up-regulation of stage specific transcription factors. These findings suggest an important role of histone modification and chromatin remodeling in transcriptional regulation of the Plasmodium life cycle. This also emphasizes the potential of P. falciparum HDACs as drug targets for malaria chemotherapy
Characterization of transcription factors in plasmodium falciparum
Malaria is an important human infectious disease affecting millions of people every year. Despite years of research, transcriptional regulatory mechanisms in Plasmodium are still poorly understood. The presence of a large number of zinc finger proteins amongst the potential specific transcription factors suggests the importance of these proteins. In this thesis, we characterized PF10_0083, a putative transcription factor that belongs to the CCCH type zinc finger family. Using over-expression and knock-out cell lines, we carried out molecular characterization of this protein. PF10_0083 protein level was found to peak during the schizont stage, and the peak protein levels coincided with its nuclear localization. PF10_0083 was found to be expressed in only a sub-population of schizonts. We demonstrated that the over-expression of PF10_0083 resulted in enhanced gametocyte production, while the knock-out of PF10_0083 caused decreased gametocyte production. In addition, we showed that PF10_0083 is capable of associating with DNA. We demonstrated that PF10_0083 preferentially binds to the IGRs on the genome, and that this binding positively correlated with the transcriptional up-regulation of the gametocyte genes. As such, we conclude that PF10_0083 promotes the differentiation of asexual parasites to sexual forms under stress conditions.DOCTOR OF PHILOSOPHY (SBS
H3K9Me1 localized to the parasitophorous vacuole during the ring stage.
<p>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.</p
Loss of H3K9Me1 with disruption of the parasitophorous vacuole.
<p><i>P. falciparum</i> 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.</p
Immunodetection of Etramp 2, H3K9Me1 and H4K5Ac under different concentrations of saponin treatment.
<p>Ring stage <i>P. falciparum</i> 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.</p
Immunofluorescence analysis of H3K9Me1 localization in <i>P. yoelii</i> and <i>P. vivax</i>.
<p>In both <i>P. yoelii and P. vivax</i> H3K9Me1 (red) co-localized with the PV marker Etramp 4 (green) whereas H4K5Ac (red) co-localized with the DAPI stained nuclear DNA (blue).</p
Immunofluorescence analysis of histone modifications in <i>P. falciparum</i>.
<p>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.</p
Histone modification patterns on ectopically integrated promoters.
<p>(A) <i>Cloning strategy for ectopic integration of promoter regions</i>. Four promoter regions (1.5–2 kb upstream of the ATG) corresponding to upstream regions of MAL13P1.122, PF14_0705, PFD0240c and PFC0210c were cloned upstream of the luciferase reporter gene pLN-luc (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003170#s4" target="_blank">Materials and Methods</a>). <i>P. falciparum</i> strain Dd2<sup>attB</sup> was transfected with the above vectors to achieve integration at the <i>cg6</i> locus and the transgenic cell lines were selected on blasticidin. Primer pair P2/P4 was used to confirm integration (data not shown). (B) <i>H4K8ac occupancy at the four ectopically integrated promoter regions</i>. The graphs represent real time PCR results carried out on H4K8ac-immunoprecipitated DNA from rings (R), trophozoites (T) and schizonts (S). In order to distinguish between the endogenous and luciferase tagged promoter, specific primers were designed to amplify regions spanning the 3′ end of the promoter and either the start of the endogenous gene or the start of the luciferase gene. The positions of forward (F) and reverse (R) primers are shown in panel A. Grey lines refer to the ChIP enrichment of the native <i>cg6</i> locus in the untransfected parasites. Orange and green lines represent the ChIP enrichment of native promoters and integrated promoters, respectively, in the transfectants. The error bars give the standard deviation from triplicate experiments.</p