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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 present in the euchromatin compartment.(A) Colocalization analysis shows PfH2A.Z overlap with the euchromatin marks H3K9ac and H3K4me3 but not with the subtelomeric heterochromatin marks H3K9me3 and HP1. PfH2A.Z, H3K4me3, H3K9ac and H3K9me3 were detected with specific antibodies. (B) Immunoelectronmicroscopy with anti-PfH2A.Z antibodies indicates nuclear compartmentalization of PfH2A.Z. N  =  nucleus, C  =  cytoplasm. (C) Immunoprecipitation (IP) of mononucleosomes with anti-PfH2A.Z (H2A.Z) co-precipitates PfH2A.Z and H3K4me3. IP with pre-immune serum (pI) in parallel verified the specificity of the precipitation. Precipitated material was separated by SDS-PAGE and analysed by western blot. 3D7 whole parasite lysate was used as a positive control (Input). Western blots were probed with anti-PfH2A.Z and anti-H3K4me3 antibodies, respectively.
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ppat-1001292-g002: PfH2A.Z is present in the euchromatin compartment.(A) Colocalization analysis shows PfH2A.Z overlap with the euchromatin marks H3K9ac and H3K4me3 but not with the subtelomeric heterochromatin marks H3K9me3 and HP1. PfH2A.Z, H3K4me3, H3K9ac and H3K9me3 were detected with specific antibodies. (B) Immunoelectronmicroscopy with anti-PfH2A.Z antibodies indicates nuclear compartmentalization of PfH2A.Z. N  =  nucleus, C  =  cytoplasm. (C) Immunoprecipitation (IP) of mononucleosomes with anti-PfH2A.Z (H2A.Z) co-precipitates PfH2A.Z and H3K4me3. IP with pre-immune serum (pI) in parallel verified the specificity of the precipitation. Precipitated material was separated by SDS-PAGE and analysed by western blot. 3D7 whole parasite lysate was used as a positive control (Input). Western blots were probed with anti-PfH2A.Z and anti-H3K4me3 antibodies, respectively.

Mentions: H2A.Z has been shown to contribute to diverse biological processes associated with different chromatin compartments, such as gene activation and poising [63], [64], [65], [66], chromosome segregation [67], [68] and heterochromatin structure [69], [70], [71], [72], [73]. To investigate the chromatin association of PfH2A.Z we performed co-localization experiments with well-characterized chromatin marks. Double staining showed good overlap between PfH2A.Z and the euchromatin marks H3K4me3 and H3K9ac (Figure 2A), which are enriched across the P. falciparum genome [22]. In contrast, PfH2A.Z staining was distinct from the subtelomeric heterochromatin marks H3K9me3 and HP1 (Figure 2A). Consistent with these results, immunoelectron microscopy indicated that PfH2A.Z was not restricted to the nuclear periphery, where inactive subtelomeric and internal var genes cluster. However, its distribution appeared concentrated in certain subnuclear compartments, frequently at the border of the electron lucent and electron dense nuclear material that is presumed to represent euchromatin and heterochromatin, respectively (Figure 2B) [38].


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 present in the euchromatin compartment.(A) Colocalization analysis shows PfH2A.Z overlap with the euchromatin marks H3K9ac and H3K4me3 but not with the subtelomeric heterochromatin marks H3K9me3 and HP1. PfH2A.Z, H3K4me3, H3K9ac and H3K9me3 were detected with specific antibodies. (B) Immunoelectronmicroscopy with anti-PfH2A.Z antibodies indicates nuclear compartmentalization of PfH2A.Z. N  =  nucleus, C  =  cytoplasm. (C) Immunoprecipitation (IP) of mononucleosomes with anti-PfH2A.Z (H2A.Z) co-precipitates PfH2A.Z and H3K4me3. IP with pre-immune serum (pI) in parallel verified the specificity of the precipitation. Precipitated material was separated by SDS-PAGE and analysed by western blot. 3D7 whole parasite lysate was used as a positive control (Input). Western blots were probed with anti-PfH2A.Z and anti-H3K4me3 antibodies, respectively.
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ppat-1001292-g002: PfH2A.Z is present in the euchromatin compartment.(A) Colocalization analysis shows PfH2A.Z overlap with the euchromatin marks H3K9ac and H3K4me3 but not with the subtelomeric heterochromatin marks H3K9me3 and HP1. PfH2A.Z, H3K4me3, H3K9ac and H3K9me3 were detected with specific antibodies. (B) Immunoelectronmicroscopy with anti-PfH2A.Z antibodies indicates nuclear compartmentalization of PfH2A.Z. N  =  nucleus, C  =  cytoplasm. (C) Immunoprecipitation (IP) of mononucleosomes with anti-PfH2A.Z (H2A.Z) co-precipitates PfH2A.Z and H3K4me3. IP with pre-immune serum (pI) in parallel verified the specificity of the precipitation. Precipitated material was separated by SDS-PAGE and analysed by western blot. 3D7 whole parasite lysate was used as a positive control (Input). Western blots were probed with anti-PfH2A.Z and anti-H3K4me3 antibodies, respectively.
Mentions: H2A.Z has been shown to contribute to diverse biological processes associated with different chromatin compartments, such as gene activation and poising [63], [64], [65], [66], chromosome segregation [67], [68] and heterochromatin structure [69], [70], [71], [72], [73]. To investigate the chromatin association of PfH2A.Z we performed co-localization experiments with well-characterized chromatin marks. Double staining showed good overlap between PfH2A.Z and the euchromatin marks H3K4me3 and H3K9ac (Figure 2A), which are enriched across the P. falciparum genome [22]. In contrast, PfH2A.Z staining was distinct from the subtelomeric heterochromatin marks H3K9me3 and HP1 (Figure 2A). Consistent with these results, immunoelectron microscopy indicated that PfH2A.Z was not restricted to the nuclear periphery, where inactive subtelomeric and internal var genes cluster. However, its distribution appeared concentrated in certain subnuclear compartments, frequently at the border of the electron lucent and electron dense nuclear material that is presumed to represent euchromatin and heterochromatin, respectively (Figure 2B) [38].

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