A MECHANISTIC INVESTIGATION OF FCγR SIGNALING DURING ANTIBODY-ENHANCED DENGUE INFECTION

Ph.DDOCTOR OF PHILOSOPH

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

STAGEs: A web-based tool that integrates data visualization and pathway enrichment analysis for gene expression studies

Abstract Gene expression profiling has helped tremendously in the understanding of biological processes and diseases. However, interpreting processed data to gain insights into biological mechanisms remain challenging, especially to the non-bioinformaticians, as many of these data visualization and pathway analysis tools require extensive data formatting. To circumvent these challenges, we developed STAGEs (Static and Temporal Analysis of Gene Expression studies) that provides an interactive visualisation of omics analysis outputs. Users can directly upload data created from Excel spreadsheets and use STAGEs to render volcano plots, differentially expressed genes stacked bar charts, pathway enrichment analysis by Enrichr and Gene Set Enrichment Analysis (GSEA) against established pathway databases or customized gene sets, clustergrams and correlation matrices. Moreover, STAGEs takes care of Excel gene to date misconversions, ensuring that every gene is considered for pathway analysis. Output data tables and graphs can be exported, and users can easily customize individual graphs using widgets such as sliders, drop-down menus, text boxes and radio buttons. Collectively, STAGEs is an integrative platform for data analysis, data visualisation and pathway analysis, and is freely available at https://kuanrongchan-stages-stages-vpgh46.streamlitapp.com/ . In addition, developers can customise or modify the web tool locally based on our existing codes, which is publicly available at https://github.com/kuanrongchan/STAGES

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

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

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

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

Hypoxia enhances antibody-dependent dengue virus infection

10.15252/embj.201695642EMBO JOURNAL36101348-136