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

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.

Fig. 1

Epigenetic regulation of upsB promoters. A. Silencing and activation of upsB-regulated transcription. Northern blots showing episomal hdhfr and bsd transcription across the intra-erythrocytic asexual cycle in 3D7/upsB, 3D7/upsB^R and 3D7/upsB^RI before (–WR) and after (+WR) WR selection. Transcription of the endogenous cam gene serves as a stage-specific loading control. Vector maps are shown above each set of autoradiographs. hsp86, heat-shock protein 86 promoter; Pb T, P. berghei dhfr-thymidilate synthase terminator; upsB, upsB upstream sequence; Pf T, P. falciparum hrp2 3′ terminator; rep20, 0.5 kb rep20 repeats; intron, 0.6 kb var intron sequence. (1) 0–12 hpi (hours post-invasion); (2) 12–24 hpi; (3) 24–36 hpi; (4) 32–44 hpi. B. Growth assay. Blasticidin-S-selected parasites were challenged with WR at day 0. Parasite growth in the presence of WR was monitored over the following generations. Assays were repeated twice with the same result. C. WR sensitivities of 3D7/upsB, 3D7/upsB^R and 3D7/upsB^RI before (open) and after (filled) WR selection, and of 3D7 wild-type parasites.

Fig. 2

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.

Fig. 3

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.

Fig. 4

Simultaneous activation of two var gene promoters in cis. A. Activity of the upsC and upsB promoter in 3D7/upsCB^R 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/upsCB^R and 3D7/upsB^R parasites were challenged with WR at day 0. Parasite growth in the presence of WR was monitored over the following generations.

Fig. 5

Low-resolution chromatin analysis of episomal upsB promoters. Native chromatin was digested with MNase followed by indirect end-labelling. Southern blots were hybridized with a 257 bp hdhfr probe to detect MNase-sensitive sites in the silenced and activated upsB promoters on pHBupsB^R. A map of the 4172 bp XbaI/SphI fragment of pHBupsB^R containing the upsB promoter is shown on the right. MNase-sensitive sites are highlighted with respect to the hdhfr start codon. The positions of the cis-acting elements SPE1 and SPE2 and the rep20 repeat region are indicated. The arrow depicts the MNase-resistant region (−1760 to −2320) containing five direct SPE2 repeats (−2093 to −2231) (Voss et al., 2003). Asterisks identify sites that are also preferentially cut in naked plasmid DNA (Fig. S3). Circles represent a proposed nucleosomal organization.

Fig. 6

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.