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Expression of P. falciparum var genes involves exchange of the histone variant H2A.Z at the promoter.

Petter M, Lee CC, Byrne TJ, Boysen KE, Volz J, Ralph SA, Cowman AF, Brown GV, Duffy MF - PLoS Pathog. (2011)

Bottom Line: Throughout the asexual, intraerythrocytic lifecycle of P. falciparum we found that the P. falciparum ortholog of H2A.Z (PfH2A.Z) colocalizes with histone modifications that are characteristic of transcriptionally-permissive euchromatin, but not with markers of heterochromatin.This result indicates that PfH2A.Z occupancy at the active var promoter is antagonized by PfSir2A during the intraerythrocytic life cycle.We conclude that PfH2A.Z contributes to the nucleosome architecture at promoters and is regulated dynamically in active var genes.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Royal Melbourne Hospital, University of Melbourne, Melbourne, Australia.

ABSTRACT
Plasmodium falciparum employs antigenic variation to evade the human immune response by switching the expression of different variant surface antigens encoded by the var gene family. Epigenetic mechanisms including histone modifications and sub-nuclear compartmentalization contribute to transcriptional regulation in the malaria parasite, in particular to control antigenic variation. Another mechanism of epigenetic control is the exchange of canonical histones with alternative variants to generate functionally specialized chromatin domains. Here we demonstrate that the alternative histone PfH2A.Z is associated with the epigenetic regulation of var genes. In many eukaryotic organisms the histone variant H2A.Z mediates an open chromatin structure at promoters and facilitates diverse levels of regulation, including transcriptional activation. Throughout the asexual, intraerythrocytic lifecycle of P. falciparum we found that the P. falciparum ortholog of H2A.Z (PfH2A.Z) colocalizes with histone modifications that are characteristic of transcriptionally-permissive euchromatin, but not with markers of heterochromatin. Consistent with this finding, antibodies to PfH2A.Z co-precipitate the permissive modification H3K4me3. By chromatin-immunoprecipitation we show that PfH2A.Z is enriched in nucleosomes around the transcription start site (TSS) in both transcriptionally active and silent stage-specific genes. In var genes, however, PfH2A.Z is enriched at the TSS only during active transcription in ring stage parasites. Thus, in contrast to other genes, temporal var gene regulation involves histone variant exchange at promoter nucleosomes. Sir2 histone deacetylases are important for var gene silencing and their yeast ortholog antagonises H2A.Z function in subtelomeric yeast genes. In immature P. falciparum parasites lacking Sir2A or Sir2B high var transcription levels correlate with enrichment of PfH2A.Z at the TSS. As Sir2A knock out parasites mature the var genes are silenced, but PfH2A.Z remains enriched at the TSS of var genes; in contrast, PfH2A.Z is lost from the TSS of de-repressed var genes in mature Sir2B knock out parasites. This result indicates that PfH2A.Z occupancy at the active var promoter is antagonized by PfSir2A during the intraerythrocytic life cycle. We conclude that PfH2A.Z contributes to the nucleosome architecture at promoters and is regulated dynamically in active var genes.

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PfH2A.Z is expressed in the nucleus throughout asexual differentiation.Full length PfH2A.Z was expressed as a GST-fusion protein in E. coli and used to immunize rabbits. (A) Specificity of antisera. Parasite extracts were separated by SDS-PAGE and analysed by western blot. Anti-PfH2A.Z antiserum specifically reacted with PfH2A.Z at 18 kDa in parasite extracts and did not cross-react with H2A at 15 kDa (1st panel). Pre-immune serum (pI) does not show any reactivity (2nd panel). Anti-H2A antiserum specifically detects H2A migrating at 15 kDa (3rd panel). Anti-PfH2A.Z detects human H2A.Z in BeWo cell lysate (4th panel). (B) Anti-PfH2A.Z immunoprecipitates acetylated PfH2A.Z. Upper panel: Anti-H4K12ac antibody labels immunoprecipitated PfH2A.Z (lane 3). Anti-H4K12ac recognises an acetylated epitope present in both H4 and PfH2A.Z. Anti-H4K12ac IP (lane 1) was performed as a positive control and shows precipitation of a band corresponding to H4. No bands are apparent after IP with pI serum (lane 2). The IgG light chain (IgGLC) from the precipitating antibodies is also detected by the secondary antibody. Lower Panel: western blot reprobed with anti-PfH2A.Z confirms specificity of the immunoprecipitation. (C) Western blot analysis across the asexual life cycle demonstrates expression of PfH2A.Z and H3 in all stages. In comparison to H3, PfH2A.Z protein expression peaks in parasites 34–40 hours post-invasion which corresponds to late trophozoites/early schizonts. The ratio of H2A.Z/H3 signal in the western blot was determined by densitometry and is presented in a bar graph. (D) Nuclear localization of PfH2A.Z is shown by indirect immunofluorescence analysis and confocal microscopy of fixed 3D7 parasites using anti-PfH2A.Z antibodies. DNA was visualized with DAPI. R  =  ring stage, T  =  trophozoite stage, S  =  schizont stage. DIC  =  differential interference contrast.
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ppat-1001292-g001: PfH2A.Z is expressed in the nucleus throughout asexual differentiation.Full length PfH2A.Z was expressed as a GST-fusion protein in E. coli and used to immunize rabbits. (A) Specificity of antisera. Parasite extracts were separated by SDS-PAGE and analysed by western blot. Anti-PfH2A.Z antiserum specifically reacted with PfH2A.Z at 18 kDa in parasite extracts and did not cross-react with H2A at 15 kDa (1st panel). Pre-immune serum (pI) does not show any reactivity (2nd panel). Anti-H2A antiserum specifically detects H2A migrating at 15 kDa (3rd panel). Anti-PfH2A.Z detects human H2A.Z in BeWo cell lysate (4th panel). (B) Anti-PfH2A.Z immunoprecipitates acetylated PfH2A.Z. Upper panel: Anti-H4K12ac antibody labels immunoprecipitated PfH2A.Z (lane 3). Anti-H4K12ac recognises an acetylated epitope present in both H4 and PfH2A.Z. Anti-H4K12ac IP (lane 1) was performed as a positive control and shows precipitation of a band corresponding to H4. No bands are apparent after IP with pI serum (lane 2). The IgG light chain (IgGLC) from the precipitating antibodies is also detected by the secondary antibody. Lower Panel: western blot reprobed with anti-PfH2A.Z confirms specificity of the immunoprecipitation. (C) Western blot analysis across the asexual life cycle demonstrates expression of PfH2A.Z and H3 in all stages. In comparison to H3, PfH2A.Z protein expression peaks in parasites 34–40 hours post-invasion which corresponds to late trophozoites/early schizonts. The ratio of H2A.Z/H3 signal in the western blot was determined by densitometry and is presented in a bar graph. (D) Nuclear localization of PfH2A.Z is shown by indirect immunofluorescence analysis and confocal microscopy of fixed 3D7 parasites using anti-PfH2A.Z antibodies. DNA was visualized with DAPI. R  =  ring stage, T  =  trophozoite stage, S  =  schizont stage. DIC  =  differential interference contrast.

Mentions: To analyse PfH2A.Z, antiserum against recombinant full length PfH2A.Z was generated. The serum specifically recognizes PfH2A.Z but not H2A and cross-reacts with human H2A.Z, which has a different N terminal acetylation pattern, indicating reactivity of the antibody with the non-acetylated, conserved C-terminal domains (Figure 1A). We further showed that anti-PfH2A.Z immunoprecipitates both acetylated and non-acetylated forms of PfH2A.Z because antibodies specific for an acetylated peptide present in the PfH2A.Z and H4 N-termini [61] labels immunoprecipitated PfH2A.Z by Western Blot (Figure 1B).


Expression of P. falciparum var genes involves exchange of the histone variant H2A.Z at the promoter.

Petter M, Lee CC, Byrne TJ, Boysen KE, Volz J, Ralph SA, Cowman AF, Brown GV, Duffy MF - PLoS Pathog. (2011)

PfH2A.Z is expressed in the nucleus throughout asexual differentiation.Full length PfH2A.Z was expressed as a GST-fusion protein in E. coli and used to immunize rabbits. (A) Specificity of antisera. Parasite extracts were separated by SDS-PAGE and analysed by western blot. Anti-PfH2A.Z antiserum specifically reacted with PfH2A.Z at 18 kDa in parasite extracts and did not cross-react with H2A at 15 kDa (1st panel). Pre-immune serum (pI) does not show any reactivity (2nd panel). Anti-H2A antiserum specifically detects H2A migrating at 15 kDa (3rd panel). Anti-PfH2A.Z detects human H2A.Z in BeWo cell lysate (4th panel). (B) Anti-PfH2A.Z immunoprecipitates acetylated PfH2A.Z. Upper panel: Anti-H4K12ac antibody labels immunoprecipitated PfH2A.Z (lane 3). Anti-H4K12ac recognises an acetylated epitope present in both H4 and PfH2A.Z. Anti-H4K12ac IP (lane 1) was performed as a positive control and shows precipitation of a band corresponding to H4. No bands are apparent after IP with pI serum (lane 2). The IgG light chain (IgGLC) from the precipitating antibodies is also detected by the secondary antibody. Lower Panel: western blot reprobed with anti-PfH2A.Z confirms specificity of the immunoprecipitation. (C) Western blot analysis across the asexual life cycle demonstrates expression of PfH2A.Z and H3 in all stages. In comparison to H3, PfH2A.Z protein expression peaks in parasites 34–40 hours post-invasion which corresponds to late trophozoites/early schizonts. The ratio of H2A.Z/H3 signal in the western blot was determined by densitometry and is presented in a bar graph. (D) Nuclear localization of PfH2A.Z is shown by indirect immunofluorescence analysis and confocal microscopy of fixed 3D7 parasites using anti-PfH2A.Z antibodies. DNA was visualized with DAPI. R  =  ring stage, T  =  trophozoite stage, S  =  schizont stage. DIC  =  differential interference contrast.
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ppat-1001292-g001: PfH2A.Z is expressed in the nucleus throughout asexual differentiation.Full length PfH2A.Z was expressed as a GST-fusion protein in E. coli and used to immunize rabbits. (A) Specificity of antisera. Parasite extracts were separated by SDS-PAGE and analysed by western blot. Anti-PfH2A.Z antiserum specifically reacted with PfH2A.Z at 18 kDa in parasite extracts and did not cross-react with H2A at 15 kDa (1st panel). Pre-immune serum (pI) does not show any reactivity (2nd panel). Anti-H2A antiserum specifically detects H2A migrating at 15 kDa (3rd panel). Anti-PfH2A.Z detects human H2A.Z in BeWo cell lysate (4th panel). (B) Anti-PfH2A.Z immunoprecipitates acetylated PfH2A.Z. Upper panel: Anti-H4K12ac antibody labels immunoprecipitated PfH2A.Z (lane 3). Anti-H4K12ac recognises an acetylated epitope present in both H4 and PfH2A.Z. Anti-H4K12ac IP (lane 1) was performed as a positive control and shows precipitation of a band corresponding to H4. No bands are apparent after IP with pI serum (lane 2). The IgG light chain (IgGLC) from the precipitating antibodies is also detected by the secondary antibody. Lower Panel: western blot reprobed with anti-PfH2A.Z confirms specificity of the immunoprecipitation. (C) Western blot analysis across the asexual life cycle demonstrates expression of PfH2A.Z and H3 in all stages. In comparison to H3, PfH2A.Z protein expression peaks in parasites 34–40 hours post-invasion which corresponds to late trophozoites/early schizonts. The ratio of H2A.Z/H3 signal in the western blot was determined by densitometry and is presented in a bar graph. (D) Nuclear localization of PfH2A.Z is shown by indirect immunofluorescence analysis and confocal microscopy of fixed 3D7 parasites using anti-PfH2A.Z antibodies. DNA was visualized with DAPI. R  =  ring stage, T  =  trophozoite stage, S  =  schizont stage. DIC  =  differential interference contrast.
Mentions: To analyse PfH2A.Z, antiserum against recombinant full length PfH2A.Z was generated. The serum specifically recognizes PfH2A.Z but not H2A and cross-reacts with human H2A.Z, which has a different N terminal acetylation pattern, indicating reactivity of the antibody with the non-acetylated, conserved C-terminal domains (Figure 1A). We further showed that anti-PfH2A.Z immunoprecipitates both acetylated and non-acetylated forms of PfH2A.Z because antibodies specific for an acetylated peptide present in the PfH2A.Z and H4 N-termini [61] labels immunoprecipitated PfH2A.Z by Western Blot (Figure 1B).

Bottom Line: Throughout the asexual, intraerythrocytic lifecycle of P. falciparum we found that the P. falciparum ortholog of H2A.Z (PfH2A.Z) colocalizes with histone modifications that are characteristic of transcriptionally-permissive euchromatin, but not with markers of heterochromatin.This result indicates that PfH2A.Z occupancy at the active var promoter is antagonized by PfSir2A during the intraerythrocytic life cycle.We conclude that PfH2A.Z contributes to the nucleosome architecture at promoters and is regulated dynamically in active var genes.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Royal Melbourne Hospital, University of Melbourne, Melbourne, Australia.

ABSTRACT
Plasmodium falciparum employs antigenic variation to evade the human immune response by switching the expression of different variant surface antigens encoded by the var gene family. Epigenetic mechanisms including histone modifications and sub-nuclear compartmentalization contribute to transcriptional regulation in the malaria parasite, in particular to control antigenic variation. Another mechanism of epigenetic control is the exchange of canonical histones with alternative variants to generate functionally specialized chromatin domains. Here we demonstrate that the alternative histone PfH2A.Z is associated with the epigenetic regulation of var genes. In many eukaryotic organisms the histone variant H2A.Z mediates an open chromatin structure at promoters and facilitates diverse levels of regulation, including transcriptional activation. Throughout the asexual, intraerythrocytic lifecycle of P. falciparum we found that the P. falciparum ortholog of H2A.Z (PfH2A.Z) colocalizes with histone modifications that are characteristic of transcriptionally-permissive euchromatin, but not with markers of heterochromatin. Consistent with this finding, antibodies to PfH2A.Z co-precipitate the permissive modification H3K4me3. By chromatin-immunoprecipitation we show that PfH2A.Z is enriched in nucleosomes around the transcription start site (TSS) in both transcriptionally active and silent stage-specific genes. In var genes, however, PfH2A.Z is enriched at the TSS only during active transcription in ring stage parasites. Thus, in contrast to other genes, temporal var gene regulation involves histone variant exchange at promoter nucleosomes. Sir2 histone deacetylases are important for var gene silencing and their yeast ortholog antagonises H2A.Z function in subtelomeric yeast genes. In immature P. falciparum parasites lacking Sir2A or Sir2B high var transcription levels correlate with enrichment of PfH2A.Z at the TSS. As Sir2A knock out parasites mature the var genes are silenced, but PfH2A.Z remains enriched at the TSS of var genes; in contrast, PfH2A.Z is lost from the TSS of de-repressed var genes in mature Sir2B knock out parasites. This result indicates that PfH2A.Z occupancy at the active var promoter is antagonized by PfSir2A during the intraerythrocytic life cycle. We conclude that PfH2A.Z contributes to the nucleosome architecture at promoters and is regulated dynamically in active var genes.

Show MeSH
Related in: MedlinePlus