Schematic representation of transcriptional activity of episomal promoters in transfection experiments.

<p>The string of nucleosomes represents chromosomal genes, whereas the circle represents an episome. Colored lines or boxes represent a promoter. (<b>A</b>) Promoter sequences placed in an episome recapitulate correct temporal expression. Orange and blue represent a ring stage–specific promoter and a late stage–specific promoter, respectively. (<b>B</b>) The transcriptional status of clonally variant promoters often does not coincide with the state of the endogeneous promoter. Possible states for endogenous and episomal promoters are represented. See <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002943#ppat-1002943-box001" target="_blank">Box 1</a> for details.</p

Schematic representation of processes in parasite biology that involve chromatin changes.

<p>(<b>A</b>) CVGE. Expression dynamics of a representative clonally variant gene. In a clonal population, stochastic switches between the active and silenced states of the gene result in transcriptional heterogeneity upon long-term growth. The active state is associated with activating histone PTMs (e.g., H3K9ac, green marks), whereas the silenced state is associated with repressive histone PTMs (e.g., H3K9me3, red marks). Subcloning followed by short-term growth results in populations of parasites that predominantly maintain the same transcriptional state as the single parasite from which they are derived, demonstrating epigenetic inheritance. (<b>B</b>) Asexual blood cycle progression. The three stretches of chromatin represent a gene expressed only in ring stages, trophozoites, or schizonts, respectively. Hypothetical stage-specific transcription factors (represented by ovals) control expression of the genes by acting on regulatory elements in the promoter sequences (colored DNA). Some histone PTMs are associated with active transcription (green marks), and global changes in chromatin organization occur at specific stages (e.g., higher nucleosome density in schizonts). It is unlikely that these modifications are stably transmitted from one generation to the next because they change during each cycle. (<b>C</b>) Adaptation via directed transcriptional responses. A change in the environment is sensed and via signal transduction (colored circles) results in a directed transcriptional response, which can operate via changes in chromatin structure (e.g., deposition of activating histone PTMs). Only if the chromatin changes and transcriptional status are maintained after the external signal disappears, is there epigenetic transmission of information (red star). To date, there is no well-described pathway in <i>P. falciparum</i> that involves sensing external conditions followed by a directed transcriptional response, but here we propose a conceptual framework to determine whether there is epigenetic inheritance of transcriptional states if such a pathway is ever identified.</p

The MEE inhibits translation in the natural context of the <i>upsC</i> promoter.

<p>(A) Schematic depiction of <i>upsC var</i> promoter reporter constructs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100183#pone.0100183-Brancucci1" target="_blank">[54]</a>. Deletions are represented by dashed lines. Numbers refer to the nucleotide positions relative to the ATG start codon. The position of the MEE is highlighted. (B) Expression of hDHFR-GFP and GAPDH (loading control) in WR-selected parasites was analysed by semi-quantitative Western blot. (C) Top panel: Proportion of total steady-state h<i>dhfr-gfp</i> transcripts in WR-selected parasites carrying truncated upstream sequences relative to the control line 3D7/pBC. Values are derived from three independent experiments (mean +/− s.d.) (normalised to PF3D7_1331700 transcripts). Middle panel: Proportion of steady-state h<i>dhfr-gfp</i> transcripts produced by a single promoter in WR-selected parasites carrying truncated upstream sequences relative to the control line 3D7/pBC. Values represent the data displayed in the top panel divided by the average plasmid copy number determined from the same batch of parasites (bottom panel). (D) Mean increase in plasmid copy numbers (+/− s.d.) after WR selection in parasites transfected with constructs carrying MEE-positive upstream sequences (red) or MEE-negative upstream sequences (green). The increase in plasmid copy numbers in WR-selected 3D7/pBC4 is shown in black. Individual plasmid copy numbers determined for each population are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100183#pone.0100183.s003" target="_blank">Figure S3</a>. Asterisk, p = 0.0015 (Student’s t-test).</p

Integration of the <i>upsC</i> 5′ upstream sequence into a heterologous context at the <i>kahrp</i> locus.

<p>(A) Schematic map of the transfection construct pBK_minC. Single-crossover integration was guided by <i>kahrp</i> 5′ homology. The position of the <i>kahrp</i> TSS is indicated <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100183#pone.0100183-Lanzer1" target="_blank">[81]</a>. Numbers refer to the nucleotide positions relative to the ATG start codon. The <i>bsd</i> resistance cassette selects for stably transfected parasites. The <i>var</i> intron is indicated by a bold dashed line. hsp86 5′, <i>hsp86</i> promoter; Pb DT 3′, <i>P. berghei dhfr</i>-thymidylate synthase terminator; rep20, 0.5 kb TARE6 repeat element; hrp2 3′; histidine-rich protein 2 terminator. MEE, location of the 101 bp mutual exclusion element MEE <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100183#pone.0100183-Brancucci1" target="_blank">[54]</a>. (B) Genomic situation after integration of the pBK_minC concatamer into the endogenous <i>kahrp</i> locus. Restriction sites used in Southern analysis and fragment lengths are indicated and colour-coded. S, <i>Stu</i>I; B, <i>Bgl</i>II. The Southern blot on <i>Bgl</i>II/<i>Stu</i>I-digested gDNA shows integration of pBK_minC into the endogenous locus of <i>kahrp</i>. The membrane was hybridised with h<i>dhfr</i> (top) and <i>kahrp</i> (bottom). Fragments are colour-coded according to the integration map. wt, size of the <i>kahrp</i> fragment in 3D7 wild-type parasites. i, integration event; p, plasmid fragment. (C) The <i>upsC</i> 5′ UTR sequence represses <i>kahrp</i> promoter activity. The bars represent the ratio of relative h<i>dhfr-gfp</i> and <i>msp8</i> transcript levels in 3D7/pBK_minC parasites (open bars) compared to the 3D7/pBK_min control (black bars) cultured in absence of WR. Results are the mean +/− s.d. of three independent experiments. Values are normalised for PF3D7_1331700 transcripts.</p

The <i>upsC</i> 5′ UTR element inhibits translation.

<p>Semi-quantitative analysis of transcript and protein abundance in 3D7/pBK_min (control) and 3D7/pBK_minC ring stage parasites (6–14 hpi) cultured in absence of WR (−WR). Top panels: h<i>dhfr-gfp</i> and <i>hsp86</i> (loading control) transcripts were detected by Northern blot. Ethidium bromide-stained 18S and 28S rRNAs serve as second loading control. Bottom panels: expression of hDHFR-GFP and GAPDH (loading control) in the same parasite samples were analysed by Western blot.</p

A gene conversion event revokes translational inhibition of h<i>dhfr-gfp</i> transcripts.

<p>(A) Southern analysis on digested gDNA from unselected and WR-selected 3D7/pBK_minC parasites. Additional h<i>dhfr</i>-containing fragments detected in WR-selected parasites only are highlighted by pink arrows. S, <i>Stu</i>I; B, <i>Bgl</i>II; K, <i>Kpn</i>I; i, integration event; p, plasmid fragment. (B) The ends of chromosome 2 and 4 in unselected and 4/2 in WR-selected parasites are schematically depicted. Gene IDs (<a href="http://www.plasmoDB.org" target="_blank">www.plasmoDB.org</a>) are indicated for a subset of genes as reference. The dashed arrow highlights the site of gene conversion. The blue box represents the duplicated region of chromosome 2. The green box represents the region of chromosome 4 that was deleted. The brown box displays a zoom-in view of the gene conversion event and the resulting recombined locus. Detailed mapping and identification of the recombination site is presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100183#pone.0100183.s001" target="_blank">Figures S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100183#pone.0100183.s002" target="_blank">S2</a>. (C) h<i>dhfr-gfp</i> transcripts are produced from the <i>var</i> gene intron on chromosome 4 in WR-selected 3D7/pBK_minC parasites. Values represent relative <i>var</i> intron-derived h<i>dhfr-gfp</i> (grey bars) and ring stage-specific <i>msp8</i> (open bars, control) transcript levels at three consecutive time points in WR-selected 3D7/pBK_minC parasites (normalised to PF3D7_1331700 transcripts). hpi, hours post invasion. (D) Semi-quantitative analysis of transcript and protein abundance in 3D7/pBK_min (control) and 3D7/pBK_minC ring stage parasites (6–14 hpi) cultured in presence of WR99210 (+WR). Top panels: h<i>dhfr-gfp</i> and <i>hsp86</i> (loading control) transcripts were detected by Northern blot. Ethidium bromide-stained 18S and 28S rRNAs serve as second loading control. Bottom panels: expression of hDHFR-GFP and GAPDH (loading control) in the same parasite samples were analysed by Western blot.</p