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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 enriched in the active var upstream region and in var introns.(A) ChIP analysis of PfH2A.Z distribution along the var2csa gene throughout differentiation in 3D7 parasites selected for var2csa expression by CSA panning. PfH2A.Z is enriched at −1000 and −1500 bp upstream of the ATG in ring and trophozoite stages, but not in schizonts. These positions correspond to nucleosomes surrounding the TSS. One representative experiment out of four biological replicates is shown. (B) PfH2A.Z occupancy in ring stage parasites expressing var2csa (var2CSA ON) or not (var2CSA OFF). PfH2A.Z enrichment near the TSS is only observed when var2csa is expressed. (C) PfH2A.Z distribution along var2csa, PFL0020c (var20), PF08_0141 (var41) and caseine kinase (CK) as a control. The transcribed var2csa locus (var2CSA ON) shows enrichment of PfH2A.Z around the TSS and moderately in the intron. In silent var genes, PfH2A.Z is exclusively enriched in the intron. Amplified regions are depicted in the gene models under each graph. ups  =  upstream, orf  =  open reading frame, int  =  intron. Error bars represent standard deviation from two technical replicates. (D) PfH2A.Z is significantly enriched in the var intron as compared to the orf (Mann-Whittney, N = 10, p = 0.0052). Intron data are compiled from three different experiments of ten different var genes.
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ppat-1001292-g004: PfH2A.Z is enriched in the active var upstream region and in var introns.(A) ChIP analysis of PfH2A.Z distribution along the var2csa gene throughout differentiation in 3D7 parasites selected for var2csa expression by CSA panning. PfH2A.Z is enriched at −1000 and −1500 bp upstream of the ATG in ring and trophozoite stages, but not in schizonts. These positions correspond to nucleosomes surrounding the TSS. One representative experiment out of four biological replicates is shown. (B) PfH2A.Z occupancy in ring stage parasites expressing var2csa (var2CSA ON) or not (var2CSA OFF). PfH2A.Z enrichment near the TSS is only observed when var2csa is expressed. (C) PfH2A.Z distribution along var2csa, PFL0020c (var20), PF08_0141 (var41) and caseine kinase (CK) as a control. The transcribed var2csa locus (var2CSA ON) shows enrichment of PfH2A.Z around the TSS and moderately in the intron. In silent var genes, PfH2A.Z is exclusively enriched in the intron. Amplified regions are depicted in the gene models under each graph. ups  =  upstream, orf  =  open reading frame, int  =  intron. Error bars represent standard deviation from two technical replicates. (D) PfH2A.Z is significantly enriched in the var intron as compared to the orf (Mann-Whittney, N = 10, p = 0.0052). Intron data are compiled from three different experiments of ten different var genes.

Mentions: ChIP analysis showed that in ring stage parasites, when var2csa transcription peaks (Figure S4) [76], PfH2A.Z is strongly enriched in the areas flanking the predicted TSS (−1500 and −1000 bp), but not further downstream (−575 bp) or within the open reading frame (ATG, DBL3, DBL6) (Figure 4A, S3B). As the parasites progress through the trophozoite to the schizont stage var transcription declines and PfH2A.Z enrichment around the TSS decreases. No enrichment of PfH2A.Z was detectable around the TSS of the var2csa gene at schizont stage. This stage-specific deposition of PfH2A.Z in the var gene promoter contrasts with the continuous presence of PfH2A.Z in the promoters of the limited number of other genes we analysed (Figure 3A) indicating that PfH2A.Z deposition differs in its temporal regulation in var 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)

PfH2A.Z is enriched in the active var upstream region and in var introns.(A) ChIP analysis of PfH2A.Z distribution along the var2csa gene throughout differentiation in 3D7 parasites selected for var2csa expression by CSA panning. PfH2A.Z is enriched at −1000 and −1500 bp upstream of the ATG in ring and trophozoite stages, but not in schizonts. These positions correspond to nucleosomes surrounding the TSS. One representative experiment out of four biological replicates is shown. (B) PfH2A.Z occupancy in ring stage parasites expressing var2csa (var2CSA ON) or not (var2CSA OFF). PfH2A.Z enrichment near the TSS is only observed when var2csa is expressed. (C) PfH2A.Z distribution along var2csa, PFL0020c (var20), PF08_0141 (var41) and caseine kinase (CK) as a control. The transcribed var2csa locus (var2CSA ON) shows enrichment of PfH2A.Z around the TSS and moderately in the intron. In silent var genes, PfH2A.Z is exclusively enriched in the intron. Amplified regions are depicted in the gene models under each graph. ups  =  upstream, orf  =  open reading frame, int  =  intron. Error bars represent standard deviation from two technical replicates. (D) PfH2A.Z is significantly enriched in the var intron as compared to the orf (Mann-Whittney, N = 10, p = 0.0052). Intron data are compiled from three different experiments of ten different var genes.
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ppat-1001292-g004: PfH2A.Z is enriched in the active var upstream region and in var introns.(A) ChIP analysis of PfH2A.Z distribution along the var2csa gene throughout differentiation in 3D7 parasites selected for var2csa expression by CSA panning. PfH2A.Z is enriched at −1000 and −1500 bp upstream of the ATG in ring and trophozoite stages, but not in schizonts. These positions correspond to nucleosomes surrounding the TSS. One representative experiment out of four biological replicates is shown. (B) PfH2A.Z occupancy in ring stage parasites expressing var2csa (var2CSA ON) or not (var2CSA OFF). PfH2A.Z enrichment near the TSS is only observed when var2csa is expressed. (C) PfH2A.Z distribution along var2csa, PFL0020c (var20), PF08_0141 (var41) and caseine kinase (CK) as a control. The transcribed var2csa locus (var2CSA ON) shows enrichment of PfH2A.Z around the TSS and moderately in the intron. In silent var genes, PfH2A.Z is exclusively enriched in the intron. Amplified regions are depicted in the gene models under each graph. ups  =  upstream, orf  =  open reading frame, int  =  intron. Error bars represent standard deviation from two technical replicates. (D) PfH2A.Z is significantly enriched in the var intron as compared to the orf (Mann-Whittney, N = 10, p = 0.0052). Intron data are compiled from three different experiments of ten different var genes.
Mentions: ChIP analysis showed that in ring stage parasites, when var2csa transcription peaks (Figure S4) [76], PfH2A.Z is strongly enriched in the areas flanking the predicted TSS (−1500 and −1000 bp), but not further downstream (−575 bp) or within the open reading frame (ATG, DBL3, DBL6) (Figure 4A, S3B). As the parasites progress through the trophozoite to the schizont stage var transcription declines and PfH2A.Z enrichment around the TSS decreases. No enrichment of PfH2A.Z was detectable around the TSS of the var2csa gene at schizont stage. This stage-specific deposition of PfH2A.Z in the var gene promoter contrasts with the continuous presence of PfH2A.Z in the promoters of the limited number of other genes we analysed (Figure 3A) indicating that PfH2A.Z deposition differs in its temporal regulation in var 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