Limits...
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.

Show MeSH

Related in: MedlinePlus

Simultaneous activation of two var gene promoters in cis. A. Activity of the upsC and upsB promoter in 3D7/upsCBR parasites before (–WR) and after (+WR) WR selection. Northern blots showing episomal hdhfr and bsd transcription across the intra-erythrocytic cycle. Transcription of the endogenous cam gene serves as a stage-specific loading control. The vector map is shown on top. (1) 0–12 hpi; (2) 12–24 hpi; (3) 24–36 hpi; (4) 32–44 hpi. B. Growth assay. Blasticidin-S-selected 3D7/upsCBR and 3D7/upsBR parasites were challenged with WR at day 0. Parasite growth in the presence of WR was monitored over the following generations.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2169929&req=5

fig04: Simultaneous activation of two var gene promoters in cis. A. Activity of the upsC and upsB promoter in 3D7/upsCBR parasites before (–WR) and after (+WR) WR selection. Northern blots showing episomal hdhfr and bsd transcription across the intra-erythrocytic cycle. Transcription of the endogenous cam gene serves as a stage-specific loading control. The vector map is shown on top. (1) 0–12 hpi; (2) 12–24 hpi; (3) 24–36 hpi; (4) 32–44 hpi. B. Growth assay. Blasticidin-S-selected 3D7/upsCBR and 3D7/upsBR parasites were challenged with WR at day 0. Parasite growth in the presence of WR was monitored over the following generations.

Mentions: Because P. falciparum episomes are maintained as concatamers of tandemly repeated plasmids (O'Donnell et al., 2001), it was impossible in previous studies to determine whether only a single episomal var promoter was activated at any time, or whether instead multiple var promoters were active simultaneously on the same DNA molecule. To test this, we designed construct pHBupsCBR, where two var gene promoters, upsC and upsB, control transcription of the bsd and hdhfr reporter genes respectively (Fig. 4A). Transfection of pHBupsCBR followed by selection on blasticidin-S selected for the transgenic line 3D7/upsCBR Northern analysis revealed that upsC promoted transcription of the bsd gene in ring-stage and early trophozoite parasites as expected. Strikingly, in this context the downstream upsB promoter was fully activated by default, demonstrating that local var promoter activation dominates over silencing (Figs 2 and 4A). Consistent with this finding, WR-unselected 3D7/upsCBR parasites were resistant to WR challenge unlike 3D7/upsBR parasites, where the upsB-hdhfr cassette was silenced (Fig. 1A and 4B). Together, these results prove that multiple episomal var promoters in cis are active simultaneously. We hypothesize that this is related to the absence of boundary/insulator elements on the plasmids, which are naturally required to prevent simultaneous activation of neighbouring cis-linked var genes. These findings furthermore suggest that the local chromatin environment is important to determine the transcriptional state of var gene 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)

Simultaneous activation of two var gene promoters in cis. A. Activity of the upsC and upsB promoter in 3D7/upsCBR parasites before (–WR) and after (+WR) WR selection. Northern blots showing episomal hdhfr and bsd transcription across the intra-erythrocytic cycle. Transcription of the endogenous cam gene serves as a stage-specific loading control. The vector map is shown on top. (1) 0–12 hpi; (2) 12–24 hpi; (3) 24–36 hpi; (4) 32–44 hpi. B. Growth assay. Blasticidin-S-selected 3D7/upsCBR and 3D7/upsBR parasites were challenged with WR at day 0. Parasite growth in the presence of WR was monitored over the following generations.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2169929&req=5

fig04: Simultaneous activation of two var gene promoters in cis. A. Activity of the upsC and upsB promoter in 3D7/upsCBR parasites before (–WR) and after (+WR) WR selection. Northern blots showing episomal hdhfr and bsd transcription across the intra-erythrocytic cycle. Transcription of the endogenous cam gene serves as a stage-specific loading control. The vector map is shown on top. (1) 0–12 hpi; (2) 12–24 hpi; (3) 24–36 hpi; (4) 32–44 hpi. B. Growth assay. Blasticidin-S-selected 3D7/upsCBR and 3D7/upsBR parasites were challenged with WR at day 0. Parasite growth in the presence of WR was monitored over the following generations.
Mentions: Because P. falciparum episomes are maintained as concatamers of tandemly repeated plasmids (O'Donnell et al., 2001), it was impossible in previous studies to determine whether only a single episomal var promoter was activated at any time, or whether instead multiple var promoters were active simultaneously on the same DNA molecule. To test this, we designed construct pHBupsCBR, where two var gene promoters, upsC and upsB, control transcription of the bsd and hdhfr reporter genes respectively (Fig. 4A). Transfection of pHBupsCBR followed by selection on blasticidin-S selected for the transgenic line 3D7/upsCBR Northern analysis revealed that upsC promoted transcription of the bsd gene in ring-stage and early trophozoite parasites as expected. Strikingly, in this context the downstream upsB promoter was fully activated by default, demonstrating that local var promoter activation dominates over silencing (Figs 2 and 4A). Consistent with this finding, WR-unselected 3D7/upsCBR parasites were resistant to WR challenge unlike 3D7/upsBR parasites, where the upsB-hdhfr cassette was silenced (Fig. 1A and 4B). Together, these results prove that multiple episomal var promoters in cis are active simultaneously. We hypothesize that this is related to the absence of boundary/insulator elements on the plasmids, which are naturally required to prevent simultaneous activation of neighbouring cis-linked var genes. These findings furthermore suggest that the local chromatin environment is important to determine the transcriptional state of var gene 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