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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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PfEMP1 is not expressed in var knock-down parasites. Detection of PfEMP1 by Western analysis of Triton X-100-insoluble/SDS-soluble membrane fractions from uninfected (control) and iRBCs. The two bands at approximately 250 kDa (asterisks) represent cross-reactive α- and β-spectrin (Cooke et al., 2006). sil., silenced var promoter; act., activated var promoter.
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fig03: PfEMP1 is not expressed in var knock-down parasites. Detection of PfEMP1 by Western analysis of Triton X-100-insoluble/SDS-soluble membrane fractions from uninfected (control) and iRBCs. The two bands at approximately 250 kDa (asterisks) represent cross-reactive α- and β-spectrin (Cooke et al., 2006). sil., silenced var promoter; act., activated var promoter.

Mentions: Our findings reveal striking similarities between the regulation of subtelomeric upsB and chromosome-central upsC promoters, suggesting that silencing of all var genes is generally induced by their cis-linked regulatory elements, independent of chromosomal location and/or promoter type. Moreover, activation of artificial var loci, both episomal and chromosomal, prevents transcription of endogenous var genes, emphasizing the central role of var promoters in mutually exclusive var gene transcription (Dzikowski et al., 2006; Voss et al., 2006). However, for upsB promoters this effect was only demonstrated for a chromosomal locus (Dzikowski et al., 2006). To test whether activation of artificial upsB loci carried on episomes also interferes with mutual exclusion, we investigated the ability of WR-selected 3D7/upsBRI parasites to express PfEMP1 and to adhere to the endothelial receptor CD36. Similar to WR-selected 3D7/upsCRI parasites (Voss et al., 2006), we find that 3D7/upsBRI expressing the hdhfr-resistance gene failed to express PfEMP1 (Fig. 3). Consequently, adherence to CD36 of red blood cells (RBCs) infected with WR-selected 3D7/upsBRI occurred at an average of 14.4% (13.8% and 15%) compared with unselected parasites. Moreover, fluorescent in situ hybridization (FISH) revealed a significant difference (P< 0.05) in the colocalization of pHBupsB with telomeric clusters in WR-unselected (39 ± 2.8%; mean n = 113) and WR-selected 3D7/upsB parasites (62 ± 3.5%; mean n = 102). This finding indicates that upsB promoter activation occurs in perinuclear chromosome-end clusters and provides a further parallel to the regulation of upsC (Voss et al., 2006) and upsE (Marty et al., 2006) promoters.


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)

PfEMP1 is not expressed in var knock-down parasites. Detection of PfEMP1 by Western analysis of Triton X-100-insoluble/SDS-soluble membrane fractions from uninfected (control) and iRBCs. The two bands at approximately 250 kDa (asterisks) represent cross-reactive α- and β-spectrin (Cooke et al., 2006). sil., silenced var promoter; act., activated var promoter.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2169929&req=5

fig03: PfEMP1 is not expressed in var knock-down parasites. Detection of PfEMP1 by Western analysis of Triton X-100-insoluble/SDS-soluble membrane fractions from uninfected (control) and iRBCs. The two bands at approximately 250 kDa (asterisks) represent cross-reactive α- and β-spectrin (Cooke et al., 2006). sil., silenced var promoter; act., activated var promoter.
Mentions: Our findings reveal striking similarities between the regulation of subtelomeric upsB and chromosome-central upsC promoters, suggesting that silencing of all var genes is generally induced by their cis-linked regulatory elements, independent of chromosomal location and/or promoter type. Moreover, activation of artificial var loci, both episomal and chromosomal, prevents transcription of endogenous var genes, emphasizing the central role of var promoters in mutually exclusive var gene transcription (Dzikowski et al., 2006; Voss et al., 2006). However, for upsB promoters this effect was only demonstrated for a chromosomal locus (Dzikowski et al., 2006). To test whether activation of artificial upsB loci carried on episomes also interferes with mutual exclusion, we investigated the ability of WR-selected 3D7/upsBRI parasites to express PfEMP1 and to adhere to the endothelial receptor CD36. Similar to WR-selected 3D7/upsCRI parasites (Voss et al., 2006), we find that 3D7/upsBRI expressing the hdhfr-resistance gene failed to express PfEMP1 (Fig. 3). Consequently, adherence to CD36 of red blood cells (RBCs) infected with WR-selected 3D7/upsBRI occurred at an average of 14.4% (13.8% and 15%) compared with unselected parasites. Moreover, fluorescent in situ hybridization (FISH) revealed a significant difference (P< 0.05) in the colocalization of pHBupsB with telomeric clusters in WR-unselected (39 ± 2.8%; mean n = 113) and WR-selected 3D7/upsB parasites (62 ± 3.5%; mean n = 102). This finding indicates that upsB promoter activation occurs in perinuclear chromosome-end clusters and provides a further parallel to the regulation of upsC (Voss et al., 2006) and upsE (Marty et al., 2006) promoters.

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