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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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ChIP analysis of genomic PfH2A.Z distribution shows enrichment near the transcription start site (TSS).ChIP was performed in ring, trophozoite and schizont stage parasites with antibodies against PfH2A.Z, H2A, H3 and non-immune control antibodies. Real time qPCR was performed targeting sequences near the TSS and in the open reading frame of various genes characterized by different expression profiles. Enrichment was calculated and the data are presented as ratio over H3 to correct for differences in nucleosome density in the inter- and intra-genic regions. Error bars represent standard deviation from two technical replicates. One representative experiment out of three biological replicates is shown. Amplified regions are depicted in the gene models under each graph. Arrows depict the TSS, and boxes the protein coding sequence. (A) Enrichment of PfH2A.Z/H3 is observed near the TSS in all investigated genes in all three stages. (B) Enrichment of H2A/H3 is equivalent near the TSS and in the coding regions of genes in all three stages.
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ppat-1001292-g003: ChIP analysis of genomic PfH2A.Z distribution shows enrichment near the transcription start site (TSS).ChIP was performed in ring, trophozoite and schizont stage parasites with antibodies against PfH2A.Z, H2A, H3 and non-immune control antibodies. Real time qPCR was performed targeting sequences near the TSS and in the open reading frame of various genes characterized by different expression profiles. Enrichment was calculated and the data are presented as ratio over H3 to correct for differences in nucleosome density in the inter- and intra-genic regions. Error bars represent standard deviation from two technical replicates. One representative experiment out of three biological replicates is shown. Amplified regions are depicted in the gene models under each graph. Arrows depict the TSS, and boxes the protein coding sequence. (A) Enrichment of PfH2A.Z/H3 is observed near the TSS in all investigated genes in all three stages. (B) Enrichment of H2A/H3 is equivalent near the TSS and in the coding regions of genes in all three stages.

Mentions: Our results show that PfH2A.Z is enriched in all three stages in the promoter regions of candidate genes that are differentially regulated throughout the life cycle (Figure 3A, S3A). In contrast to PfH2A.Z, H2A showed equal distribution in the promoter and the open reading frame of the investigated genes (Figure 3B). In rings, promoter enrichment of PfH2A.Z was highest in genes that are constitutively expressed (e.g. HSP70 and casein kinase) or induced during asexual intra-erythrocytic differentiation (e.g. schizont genes MSP2 and Eba175), but also clearly apparent in silent genes (e.g. sporozoite genes CSP and SSP2). In trophozoites and schizonts, PfH2A.Z enrichment in the promoter showed similar levels across all genes.


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)

ChIP analysis of genomic PfH2A.Z distribution shows enrichment near the transcription start site (TSS).ChIP was performed in ring, trophozoite and schizont stage parasites with antibodies against PfH2A.Z, H2A, H3 and non-immune control antibodies. Real time qPCR was performed targeting sequences near the TSS and in the open reading frame of various genes characterized by different expression profiles. Enrichment was calculated and the data are presented as ratio over H3 to correct for differences in nucleosome density in the inter- and intra-genic regions. Error bars represent standard deviation from two technical replicates. One representative experiment out of three biological replicates is shown. Amplified regions are depicted in the gene models under each graph. Arrows depict the TSS, and boxes the protein coding sequence. (A) Enrichment of PfH2A.Z/H3 is observed near the TSS in all investigated genes in all three stages. (B) Enrichment of H2A/H3 is equivalent near the TSS and in the coding regions of genes in all three stages.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3040674&req=5

ppat-1001292-g003: ChIP analysis of genomic PfH2A.Z distribution shows enrichment near the transcription start site (TSS).ChIP was performed in ring, trophozoite and schizont stage parasites with antibodies against PfH2A.Z, H2A, H3 and non-immune control antibodies. Real time qPCR was performed targeting sequences near the TSS and in the open reading frame of various genes characterized by different expression profiles. Enrichment was calculated and the data are presented as ratio over H3 to correct for differences in nucleosome density in the inter- and intra-genic regions. Error bars represent standard deviation from two technical replicates. One representative experiment out of three biological replicates is shown. Amplified regions are depicted in the gene models under each graph. Arrows depict the TSS, and boxes the protein coding sequence. (A) Enrichment of PfH2A.Z/H3 is observed near the TSS in all investigated genes in all three stages. (B) Enrichment of H2A/H3 is equivalent near the TSS and in the coding regions of genes in all three stages.
Mentions: Our results show that PfH2A.Z is enriched in all three stages in the promoter regions of candidate genes that are differentially regulated throughout the life cycle (Figure 3A, S3A). In contrast to PfH2A.Z, H2A showed equal distribution in the promoter and the open reading frame of the investigated genes (Figure 3B). In rings, promoter enrichment of PfH2A.Z was highest in genes that are constitutively expressed (e.g. HSP70 and casein kinase) or induced during asexual intra-erythrocytic differentiation (e.g. schizont genes MSP2 and Eba175), but also clearly apparent in silent genes (e.g. sporozoite genes CSP and SSP2). In trophozoites and schizonts, PfH2A.Z enrichment in the promoter showed similar levels across all genes.

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