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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 occupancy in the active var gene upstream region is maintained at schizont stage in ΔSir2A but not ΔSir2B parasites.ChIP was performed in ring (A, C) and schizont stage (B, D) parasites of 3D7, 3D7ΔSir2A and 3D7ΔSir2B lines. One out of two biological replicates is shown. Real-time qPCR was performed in upstream and coding regions of different sets of five var genes that were expressed at high or low levels in 3D7ΔSir2A or in 3D7ΔSir2B, respectively. The ratio between PfH2A.Z enrichment in the upstream and coding sequence was determined (ups/orf ratio). Knock out parasites are presented in light grey, wild type 3D7 parasites in dark grey. Shown is the median boxed with 25th and 75th percentile and minimum/maximum values as whiskers. Non-parametric Mann-Whitney test was performed and significant differences are indicated. Analysed var genes were PF13_0003, MAL7P1.50, PF07_0050, PFD1005c and PFL1960w (ΔSir2A high); PF08_0141, PFD0020c, PFL0020w, PF08_0106 and PFD0995c (ΔSir2A low); PFL2665c, PFA0005w, PF13_0364, PFD0615c and PF07_0049 (ΔSir2B high); PFE0005w, PF07_0139, PFD1245c, PFD0020c and PFD0005w (ΔSir2B low).
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ppat-1001292-g005: PfH2A.Z occupancy in the active var gene upstream region is maintained at schizont stage in ΔSir2A but not ΔSir2B parasites.ChIP was performed in ring (A, C) and schizont stage (B, D) parasites of 3D7, 3D7ΔSir2A and 3D7ΔSir2B lines. One out of two biological replicates is shown. Real-time qPCR was performed in upstream and coding regions of different sets of five var genes that were expressed at high or low levels in 3D7ΔSir2A or in 3D7ΔSir2B, respectively. The ratio between PfH2A.Z enrichment in the upstream and coding sequence was determined (ups/orf ratio). Knock out parasites are presented in light grey, wild type 3D7 parasites in dark grey. Shown is the median boxed with 25th and 75th percentile and minimum/maximum values as whiskers. Non-parametric Mann-Whitney test was performed and significant differences are indicated. Analysed var genes were PF13_0003, MAL7P1.50, PF07_0050, PFD1005c and PFL1960w (ΔSir2A high); PF08_0141, PFD0020c, PFL0020w, PF08_0106 and PFD0995c (ΔSir2A low); PFL2665c, PFA0005w, PF13_0364, PFD0615c and PF07_0049 (ΔSir2B high); PFE0005w, PF07_0139, PFD1245c, PFD0020c and PFD0005w (ΔSir2B low).

Mentions: Because H2A.Z may function as a barrier to prevent the spread of Sir2-mediated silencing in yeast [53] we further investigated the relationship between Sir2 and PfH2A.Z in the control of var gene expression in P. falciparum. ChIP and expression analyses were performed on ring and schizont stage parasites in which Sir2A or Sir2B had been disrupted (3D7Δsir2A and 3D7Δsir2B) [41], [48]. By q-RT-PCR we first monitored the expression profiles of all var genes in ring stages of both 3D7Δsir2 parasite lines (Figure S8). With the aim to understand how each Sir2 paralogue influences PfH2A.Z deposition and how this correlates with var transcription, we selected five var genes that were highly expressed and five var genes that were lowly expressed in 3D7Δsir2A parasites for further analysis, all of which had previously been shown to be regulated by Sir2A [41], [48]. We used the same strategy to choose ten var genes previously shown to be regulated by Sir2B [48] for analysis in 3D7Δsir2B. We then analysed PfH2A.Z deposition by ChIP and qPCR in upstream and coding regions of these var genes in knock out and wild type parasites. The ups/orf ratios were determined and compared between 3D7Δsir2A or 3D7Δsir2B and 3D7 parasites, respectively (Figure 5).


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 occupancy in the active var gene upstream region is maintained at schizont stage in ΔSir2A but not ΔSir2B parasites.ChIP was performed in ring (A, C) and schizont stage (B, D) parasites of 3D7, 3D7ΔSir2A and 3D7ΔSir2B lines. One out of two biological replicates is shown. Real-time qPCR was performed in upstream and coding regions of different sets of five var genes that were expressed at high or low levels in 3D7ΔSir2A or in 3D7ΔSir2B, respectively. The ratio between PfH2A.Z enrichment in the upstream and coding sequence was determined (ups/orf ratio). Knock out parasites are presented in light grey, wild type 3D7 parasites in dark grey. Shown is the median boxed with 25th and 75th percentile and minimum/maximum values as whiskers. Non-parametric Mann-Whitney test was performed and significant differences are indicated. Analysed var genes were PF13_0003, MAL7P1.50, PF07_0050, PFD1005c and PFL1960w (ΔSir2A high); PF08_0141, PFD0020c, PFL0020w, PF08_0106 and PFD0995c (ΔSir2A low); PFL2665c, PFA0005w, PF13_0364, PFD0615c and PF07_0049 (ΔSir2B high); PFE0005w, PF07_0139, PFD1245c, PFD0020c and PFD0005w (ΔSir2B low).
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ppat-1001292-g005: PfH2A.Z occupancy in the active var gene upstream region is maintained at schizont stage in ΔSir2A but not ΔSir2B parasites.ChIP was performed in ring (A, C) and schizont stage (B, D) parasites of 3D7, 3D7ΔSir2A and 3D7ΔSir2B lines. One out of two biological replicates is shown. Real-time qPCR was performed in upstream and coding regions of different sets of five var genes that were expressed at high or low levels in 3D7ΔSir2A or in 3D7ΔSir2B, respectively. The ratio between PfH2A.Z enrichment in the upstream and coding sequence was determined (ups/orf ratio). Knock out parasites are presented in light grey, wild type 3D7 parasites in dark grey. Shown is the median boxed with 25th and 75th percentile and minimum/maximum values as whiskers. Non-parametric Mann-Whitney test was performed and significant differences are indicated. Analysed var genes were PF13_0003, MAL7P1.50, PF07_0050, PFD1005c and PFL1960w (ΔSir2A high); PF08_0141, PFD0020c, PFL0020w, PF08_0106 and PFD0995c (ΔSir2A low); PFL2665c, PFA0005w, PF13_0364, PFD0615c and PF07_0049 (ΔSir2B high); PFE0005w, PF07_0139, PFD1245c, PFD0020c and PFD0005w (ΔSir2B low).
Mentions: Because H2A.Z may function as a barrier to prevent the spread of Sir2-mediated silencing in yeast [53] we further investigated the relationship between Sir2 and PfH2A.Z in the control of var gene expression in P. falciparum. ChIP and expression analyses were performed on ring and schizont stage parasites in which Sir2A or Sir2B had been disrupted (3D7Δsir2A and 3D7Δsir2B) [41], [48]. By q-RT-PCR we first monitored the expression profiles of all var genes in ring stages of both 3D7Δsir2 parasite lines (Figure S8). With the aim to understand how each Sir2 paralogue influences PfH2A.Z deposition and how this correlates with var transcription, we selected five var genes that were highly expressed and five var genes that were lowly expressed in 3D7Δsir2A parasites for further analysis, all of which had previously been shown to be regulated by Sir2A [41], [48]. We used the same strategy to choose ten var genes previously shown to be regulated by Sir2B [48] for analysis in 3D7Δsir2B. We then analysed PfH2A.Z deposition by ChIP and qPCR in upstream and coding regions of these var genes in knock out and wild type parasites. The ups/orf ratios were determined and compared between 3D7Δsir2A or 3D7Δsir2B and 3D7 parasites, respectively (Figure 5).

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