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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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Analysis of var promoter activity by densitometry. A. Analysis of hdhfr transcript abundance in transfected 3D7 ring-stage parasites. Vector maps for each transfected line are shown on the left. The relative hdhfr transcript production per promoter before and after selection on WR is displayed (see also Experimental procedures and Fig. S1). B. Analysis of bsd transcript abundance in 3D7 trophozoites. The cam promoter construct at the bottom of the panel was used as control. The relative bsd transcript production per promoter before and after selection on WR is displayed. Numbers behind open bars (WR-selected) indicate the fold increase in steady-state transcripts compared with the default state (WR-unselected) (black bars). epis, episomal; chr, chromosomal.
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fig02: Analysis of var promoter activity by densitometry. A. Analysis of hdhfr transcript abundance in transfected 3D7 ring-stage parasites. Vector maps for each transfected line are shown on the left. The relative hdhfr transcript production per promoter before and after selection on WR is displayed (see also Experimental procedures and Fig. S1). B. Analysis of bsd transcript abundance in 3D7 trophozoites. The cam promoter construct at the bottom of the panel was used as control. The relative bsd transcript production per promoter before and after selection on WR is displayed. Numbers behind open bars (WR-selected) indicate the fold increase in steady-state transcripts compared with the default state (WR-unselected) (black bars). epis, episomal; chr, chromosomal.

Mentions: To derive relative activity values for upsB promoters, we determined hdhfr transcript levels by densitometry (Figs 2 and S1). UpsB promoters produced 61-fold (pHBupsBR), 37-fold (pHBupsB) and 28-fold (pHBupsBRI) more transcripts after activation compared with the silenced state, similar to the level of upsC activation (44-fold). A comparable increase in activity (54-fold) was also measured after integration of pHBupsBR into the subtelomeric var locus PFL0005w (Fig. S2). The silencing emanating from upsB promoters spread in cis, which was evident from the reduced hsp86 promoter activity on silenced episomes (Fig. 2). While the intron is not required for upsB silencing, it appears to decrease upsB activity (Fig. 2), a tendency we also observed with upsC promoters (Voss et al., 2006). In contrast to upsC promoters, however, upsB promoter activation did not occur less frequently in the presence of the intron as demonstrated by the similar sensitivities of 3D7/upsB, 3D7/upsBR and 3D7/upsBRI parasites to WR challenge (Fig. 1B), suggesting that the var intron does not augment silencing of upsB 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)

Analysis of var promoter activity by densitometry. A. Analysis of hdhfr transcript abundance in transfected 3D7 ring-stage parasites. Vector maps for each transfected line are shown on the left. The relative hdhfr transcript production per promoter before and after selection on WR is displayed (see also Experimental procedures and Fig. S1). B. Analysis of bsd transcript abundance in 3D7 trophozoites. The cam promoter construct at the bottom of the panel was used as control. The relative bsd transcript production per promoter before and after selection on WR is displayed. Numbers behind open bars (WR-selected) indicate the fold increase in steady-state transcripts compared with the default state (WR-unselected) (black bars). epis, episomal; chr, chromosomal.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2169929&req=5

fig02: Analysis of var promoter activity by densitometry. A. Analysis of hdhfr transcript abundance in transfected 3D7 ring-stage parasites. Vector maps for each transfected line are shown on the left. The relative hdhfr transcript production per promoter before and after selection on WR is displayed (see also Experimental procedures and Fig. S1). B. Analysis of bsd transcript abundance in 3D7 trophozoites. The cam promoter construct at the bottom of the panel was used as control. The relative bsd transcript production per promoter before and after selection on WR is displayed. Numbers behind open bars (WR-selected) indicate the fold increase in steady-state transcripts compared with the default state (WR-unselected) (black bars). epis, episomal; chr, chromosomal.
Mentions: To derive relative activity values for upsB promoters, we determined hdhfr transcript levels by densitometry (Figs 2 and S1). UpsB promoters produced 61-fold (pHBupsBR), 37-fold (pHBupsB) and 28-fold (pHBupsBRI) more transcripts after activation compared with the silenced state, similar to the level of upsC activation (44-fold). A comparable increase in activity (54-fold) was also measured after integration of pHBupsBR into the subtelomeric var locus PFL0005w (Fig. S2). The silencing emanating from upsB promoters spread in cis, which was evident from the reduced hsp86 promoter activity on silenced episomes (Fig. 2). While the intron is not required for upsB silencing, it appears to decrease upsB activity (Fig. 2), a tendency we also observed with upsC promoters (Voss et al., 2006). In contrast to upsC promoters, however, upsB promoter activation did not occur less frequently in the presence of the intron as demonstrated by the similar sensitivities of 3D7/upsB, 3D7/upsBR and 3D7/upsBRI parasites to WR challenge (Fig. 1B), suggesting that the var intron does not augment silencing of upsB 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