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Alterations in local chromatin environment are involved in silencing and activation of subtelomeric var genes in Plasmodium falciparum.

Voss TS, Tonkin CJ, Marty AJ, Thompson JK, Healer J, Crabb BS, Cowman AF - Mol. Microbiol. (2007)

Bottom Line: However, they are active by default when placed downstream of a second active var promoter, underscoring the significance of local chromatin environment and nuclear compartmentalization in var promoter regulation.Native chromatin covering the SPE2-repeat array in upsB promoters is resistant to nuclease digestion, and insertion of these regulatory elements into a heterologous promoter causes local alterations in nucleosomal organization and promoter repression.Our findings suggest a common logic underlying the transcriptional control of all var genes, and have important implications for our understanding of the epigenetic processes involved in the regulation of this major virulence gene family.

View Article: PubMed Central - PubMed

Affiliation: Division of Infection and Immunity, The Walter and Eliza Hall Institute of Medical Research, Parkville 3050, Australia.

ABSTRACT
Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), encoded by the var gene family, undergoes antigenic variation and plays an important role in chronic infection and severe malaria. Only a single var gene is transcribed per parasite, and epigenetic control mechanisms are fundamental in this strategy of mutually exclusive transcription. We show that subtelomeric upsB var gene promoters carried on episomes are silenced by default, and that promoter activation is sufficient to silence all other family members. However, they are active by default when placed downstream of a second active var promoter, underscoring the significance of local chromatin environment and nuclear compartmentalization in var promoter regulation. Native chromatin covering the SPE2-repeat array in upsB promoters is resistant to nuclease digestion, and insertion of these regulatory elements into a heterologous promoter causes local alterations in nucleosomal organization and promoter repression. Our findings suggest a common logic underlying the transcriptional control of all var genes, and have important implications for our understanding of the epigenetic processes involved in the regulation of this major virulence gene family.

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Insertion of SPE2 elements into the heterologous cam promoter. A. The effect of SPE2 insertion on cam promoter timing. Plasmid maps are shown on the left. Stage-specific Northern blots to detect hdhfr and cam (control) transcription is shown on the right. (2) 12–24 hpi; (3) 24–36 hpi; (4) 32–44 hpi. B. Comparison of the chromatin structure of the episomal cam and camSPE2 promoters. MNase-sensitive sites are indicated with respect to the hdhfr start codon. The position of the inserted SPE2 elements (−829 to −911) in camSPE2 is shown. SPE2 insertion causes local protection from MNAse digestion (asterisk), and the two MNAse-sensitive sites in the cam wild-type promoter disappear (arrows). C. Comparison of the chromatin structure of the episomal camSPE2 and camSPE2m promoters. The positions of the inserted SPE2 (−829 to −911) and SPE2m (−829 to −889) elements are shown. Note that the two MNase-sensitive sites in the cam wild-type promoter (−890 and −1050) are retained after insertion of the mutated SPE2m.
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fig06: Insertion of SPE2 elements into the heterologous cam promoter. A. The effect of SPE2 insertion on cam promoter timing. Plasmid maps are shown on the left. Stage-specific Northern blots to detect hdhfr and cam (control) transcription is shown on the right. (2) 12–24 hpi; (3) 24–36 hpi; (4) 32–44 hpi. B. Comparison of the chromatin structure of the episomal cam and camSPE2 promoters. MNase-sensitive sites are indicated with respect to the hdhfr start codon. The position of the inserted SPE2 elements (−829 to −911) in camSPE2 is shown. SPE2 insertion causes local protection from MNAse digestion (asterisk), and the two MNAse-sensitive sites in the cam wild-type promoter disappear (arrows). C. Comparison of the chromatin structure of the episomal camSPE2 and camSPE2m promoters. The positions of the inserted SPE2 (−829 to −911) and SPE2m (−829 to −889) elements are shown. Note that the two MNase-sensitive sites in the cam wild-type promoter (−890 and −1050) are retained after insertion of the mutated SPE2m.

Mentions: We were interested in testing the potential role of SPE2 elements in chromatin organization and transcriptional regulation. We generated construct pHBcamSPE2R with three tandem copies of SPE2 inserted into the heterologous P. falciparum calmodulin (cam) promoter (Fig. 6A). pHBcamR (Voss et al., 2006) and pHBcamSPE2mR, where point mutations in the SPE2 sequence prevent the interaction with the SPE2 binding protein (Voss et al., 2003), were used as controls. The three blasticidin-S-resistant transgenic lines 3D7/cam, 3D7/camSPE2R and 3D7/camSPE2mR were resistant to WR challenge, indicating that the cam promoter was not silenced by insertion of SPE2 elements (data not shown). However, insertion of SPE2 strongly affected the temporal activity profile of the cam promoter. The activity of the camSPE2 promoter was restricted to schizont-stage parasites [32–44 h post invasion (hpi)]. In contrast, the wild-type cam promoter was active across much of the intra-erythrocytic cycle, and insertion of mutated SPE2 elements had no effect (Fig. 6A). To obtain evidence that this observation was due to the actual interaction of the SPE2 binding activity with its cognate binding site, we analysed the chromatin structure in these promoters by indirect end-labelling. The pattern of MNase-sensitive sites in the episomal wild-type cam promoter was similar to the pattern observed in a cam promoter at a subtelomeric transgene locus (Duraisingh et al., 2005). However, after insertion of the SPE2 elements at position −833, the two sensitive sites at −890 and −1050 were now protected from MNase digestion (Fig. 6B). Furthermore, these alterations were specific to the SPE2 elements as insertion of the mutated SPE2 repeats had no effect (Fig. 6C). These findings are consistent with the MNase resistance of the SPE2 array in upsB promoters, and indicate that the change in temporal cam promoter activity is a direct result of SPE2 binding factor recruitment to the introduced SPE2 elements.


Alterations in local chromatin environment are involved in silencing and activation of subtelomeric var genes in Plasmodium falciparum.

Voss TS, Tonkin CJ, Marty AJ, Thompson JK, Healer J, Crabb BS, Cowman AF - Mol. Microbiol. (2007)

Insertion of SPE2 elements into the heterologous cam promoter. A. The effect of SPE2 insertion on cam promoter timing. Plasmid maps are shown on the left. Stage-specific Northern blots to detect hdhfr and cam (control) transcription is shown on the right. (2) 12–24 hpi; (3) 24–36 hpi; (4) 32–44 hpi. B. Comparison of the chromatin structure of the episomal cam and camSPE2 promoters. MNase-sensitive sites are indicated with respect to the hdhfr start codon. The position of the inserted SPE2 elements (−829 to −911) in camSPE2 is shown. SPE2 insertion causes local protection from MNAse digestion (asterisk), and the two MNAse-sensitive sites in the cam wild-type promoter disappear (arrows). C. Comparison of the chromatin structure of the episomal camSPE2 and camSPE2m promoters. The positions of the inserted SPE2 (−829 to −911) and SPE2m (−829 to −889) elements are shown. Note that the two MNase-sensitive sites in the cam wild-type promoter (−890 and −1050) are retained after insertion of the mutated SPE2m.
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Related In: Results  -  Collection

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fig06: Insertion of SPE2 elements into the heterologous cam promoter. A. The effect of SPE2 insertion on cam promoter timing. Plasmid maps are shown on the left. Stage-specific Northern blots to detect hdhfr and cam (control) transcription is shown on the right. (2) 12–24 hpi; (3) 24–36 hpi; (4) 32–44 hpi. B. Comparison of the chromatin structure of the episomal cam and camSPE2 promoters. MNase-sensitive sites are indicated with respect to the hdhfr start codon. The position of the inserted SPE2 elements (−829 to −911) in camSPE2 is shown. SPE2 insertion causes local protection from MNAse digestion (asterisk), and the two MNAse-sensitive sites in the cam wild-type promoter disappear (arrows). C. Comparison of the chromatin structure of the episomal camSPE2 and camSPE2m promoters. The positions of the inserted SPE2 (−829 to −911) and SPE2m (−829 to −889) elements are shown. Note that the two MNase-sensitive sites in the cam wild-type promoter (−890 and −1050) are retained after insertion of the mutated SPE2m.
Mentions: We were interested in testing the potential role of SPE2 elements in chromatin organization and transcriptional regulation. We generated construct pHBcamSPE2R with three tandem copies of SPE2 inserted into the heterologous P. falciparum calmodulin (cam) promoter (Fig. 6A). pHBcamR (Voss et al., 2006) and pHBcamSPE2mR, where point mutations in the SPE2 sequence prevent the interaction with the SPE2 binding protein (Voss et al., 2003), were used as controls. The three blasticidin-S-resistant transgenic lines 3D7/cam, 3D7/camSPE2R and 3D7/camSPE2mR were resistant to WR challenge, indicating that the cam promoter was not silenced by insertion of SPE2 elements (data not shown). However, insertion of SPE2 strongly affected the temporal activity profile of the cam promoter. The activity of the camSPE2 promoter was restricted to schizont-stage parasites [32–44 h post invasion (hpi)]. In contrast, the wild-type cam promoter was active across much of the intra-erythrocytic cycle, and insertion of mutated SPE2 elements had no effect (Fig. 6A). To obtain evidence that this observation was due to the actual interaction of the SPE2 binding activity with its cognate binding site, we analysed the chromatin structure in these promoters by indirect end-labelling. The pattern of MNase-sensitive sites in the episomal wild-type cam promoter was similar to the pattern observed in a cam promoter at a subtelomeric transgene locus (Duraisingh et al., 2005). However, after insertion of the SPE2 elements at position −833, the two sensitive sites at −890 and −1050 were now protected from MNase digestion (Fig. 6B). Furthermore, these alterations were specific to the SPE2 elements as insertion of the mutated SPE2 repeats had no effect (Fig. 6C). These findings are consistent with the MNase resistance of the SPE2 array in upsB promoters, and indicate that the change in temporal cam promoter activity is a direct result of SPE2 binding factor recruitment to the introduced SPE2 elements.

Bottom Line: However, they are active by default when placed downstream of a second active var promoter, underscoring the significance of local chromatin environment and nuclear compartmentalization in var promoter regulation.Native chromatin covering the SPE2-repeat array in upsB promoters is resistant to nuclease digestion, and insertion of these regulatory elements into a heterologous promoter causes local alterations in nucleosomal organization and promoter repression.Our findings suggest a common logic underlying the transcriptional control of all var genes, and have important implications for our understanding of the epigenetic processes involved in the regulation of this major virulence gene family.

View Article: PubMed Central - PubMed

Affiliation: Division of Infection and Immunity, The Walter and Eliza Hall Institute of Medical Research, Parkville 3050, Australia.

ABSTRACT
Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), encoded by the var gene family, undergoes antigenic variation and plays an important role in chronic infection and severe malaria. Only a single var gene is transcribed per parasite, and epigenetic control mechanisms are fundamental in this strategy of mutually exclusive transcription. We show that subtelomeric upsB var gene promoters carried on episomes are silenced by default, and that promoter activation is sufficient to silence all other family members. However, they are active by default when placed downstream of a second active var promoter, underscoring the significance of local chromatin environment and nuclear compartmentalization in var promoter regulation. Native chromatin covering the SPE2-repeat array in upsB promoters is resistant to nuclease digestion, and insertion of these regulatory elements into a heterologous promoter causes local alterations in nucleosomal organization and promoter repression. Our findings suggest a common logic underlying the transcriptional control of all var genes, and have important implications for our understanding of the epigenetic processes involved in the regulation of this major virulence gene family.

Show MeSH
Related in: MedlinePlus