Schistosome nuclear protein extracts and recombinant SmMBD2/3 both contain 5mC binding activities.

<p>(A) The 5mC binding capacity of nuclear protein extracts derived from adult male and female worms were quantified using the Epigentek MBD2 binding activity/inhibition assay. NIH-3T3 nuclear protein extracts and BSA were included as positive and negative controls, respectively. A significant difference in 5mC binding (in the CpG context) amongst protein samples was found. (B) IPTG-induced rSmMBD2/3-His₆ protein (arrowhead; 36.5 kDa) was produced in <i>E</i>. <i>coli</i>, purified by Ni²⁺-NTA column chromatography and subjected to MALDI-TOF MS (<i>Materials and Methods</i>; 22 peptides covering 67% of full length SmMBD2/3 identified). An un-induced sample was also produced and similarly processed. (C) The 5mC binding activity (within a CpG context) of purified rSmMBD2/3-His₆ was measured using the Epigentek MBD2 binding activity/inhibition assay and compared to un-induced bacterial and BSA protein samples. Significant differences in 5mC binding were observed between rSmMBD2/3-His₆ and both the BSA and un-induced samples.</p

SmMBD2/3 interacts with the nuclear chromobox protein SmCBX (Smp_179650).

<p>(A) A truncated version of Smp_179650 (delta 1–160) was repeatedly (5/14 times or 36% of all hits; <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007107#ppat.1007107.s002" target="_blank">S2 Table</a>) identified as an interacting partner of SmMBD2/3 in Y2H assays. This truncated version of Smp_179650 contained the chromo shadow domain (CSD; blue oval, PF01393), a region associated with protein-protein interactions [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007107#ppat.1007107.ref050" target="_blank">50</a>]. Full-length Smp_179650 also contains the chromodomain (CD; yellow rectangle, PF00385) and a monopartite nuclear localisation signal (NLS, ¹⁰⁹VPEPAKKKRTS¹¹⁹). Amino acid positions are indicated (bold numbers). (B) The SmMBD2/3 –SmCBX (Δ1–160) interaction strength was quantified using the X-β-gal based (PXG) assay [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007107#ppat.1007107.ref035" target="_blank">35</a>]. Experimental controls included: p53 + SV40 large T antigen (positive) and SmMBD2/3 + pGADT7 (empty prey vector), pGBKT7 (empty bait vector) + SmCBX/ Δ1–160, pGBKT7 + pGADT7 (all negative). (C) DNA microarray analysis of <i>Smcbx</i> expression throughout 15 lifecycle stages. Bar chart represents normalised mean fluorescent intensities + standard deviation (n = 3 replicates/lifecycle stage except adult female, where n = 2) of <i>Smcbx</i> transcript abundance derived from oligonucleotide CONTIG6649 as described previously [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007107#ppat.1007107.ref036" target="_blank">36</a>]. Inset drawing represents SmCBX (Smp_179650) gene organisation (4 exons–yellow boxes; 3 introns–black lines) and localisation of oligonucleotide CONTIG6649 to exon 3 (SchistoGeneDB v5.2).</p

SmMBD2/3 and SmCBX are both required for schistosome oviposition.

<p>(A) Seven-week old adult male and female schistosome pairs were electroporated with 5 μg siRNA duplexes targeting luciferase (si<i>Luc</i>), <i>Smcbx</i> (si<i>Smcbx</i>) or <i>Smmbd2/3</i> (si<i>Smmbd2/3</i>). At day 7 post treatment, eggs were collected from wells (5 worm pairs/well; n = 3) and counted. A one-way ANOVA followed by Tukey HSD test was performed to identify statistically significant treatments. (B) Percentage of eggs (n = 14 for si<i>Luc</i>, n = 20 for si<i>Smcbx</i>, n = 15 for si<i>Smmbd2/3</i>) demonstrating abnormal (grey) versus normal (black) phenotypes. Normal = oval eggs with lateral spine, containing regular surface autofluorescence. (C) Representative fluorescent images of eggs collected from wells of si<i>Luc</i>, si<i>Smcbx</i> and si<i>Smmbd2/3</i> treated worm pairs. Green = eggshell autofluorescence; blue = DAPI⁺ cells. Bar = 20 μm.</p

<i>Smmbd2/3</i> and <i>Smcbx</i> are broadly expressed in male and female schistosomes.

<p>(A) Expression of <i>Smmbd2/3</i> and <i>Smcbx</i> in male somatic tissues, (B) testes and (C) ovaries relative to <i>Smhistone H2B</i>. Both genes are broadly expressed in somatic tissues, including the <i>histone H2B</i>⁺ neoblasts and in most cell types (<i>histone H2B</i>^<i>-</i>) within the male and female germ line. Scale bars = 20 μm. Blue = DAPI. Magenta and green = pseudocoloured antisense RNA probes for <i>Smmbd2/3</i>, <i>Smcbx</i> and <i>Smhistone H2B</i>. White = co-localisation of two RNA probes. Where illustrated, delineated areas (dashed white boxes) are magnified in the insets (solid white boxes).</p

RNAi-mediated knockdown of <i>Smmbd2/3</i> and <i>Smcbx</i> affects the proliferation of schistosome stem cells.

<p>(A) Seven-week old adult male and female schistosomes were electroporated with 5 μg siRNA duplexes targeting luciferase (si<i>Luc</i>), <i>Smcbx</i> (si<i>Smcbx</i>) or <i>Smmbd2/3</i> (si<i>Smmbd2/3</i>). Following 48 hr, total RNA was harvested and subjected to qRT-PCR. Percent knockdown (KD) and statistical significance (Student’s <i>t</i> test, two tailed, unequal variance) is indicated. All siRNA and qRT-PCR DNA sequences are included in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007107#ppat.1007107.s001" target="_blank">S1 Table</a>. (B) Representative anterior ends and ovaries of female schistosomes treated with siRNA duplexes at day seven post treatment. Blue = DAPI; Green = EdU⁺ cells. Bar = 50 μM. Column scatter plot (horizontal bars = mean and +/- StDev of mean) represents the percentage of proliferating cells remaining in female worms treated with siRNA duplexes for seven days (si<i>Luc</i>, n = 11; si<i>Smcbx</i>, n = 11; si<i>Smmbd2/3</i> = 12). The percentage of proliferating cells affected by knockdown (in comparison to <i>siLuc</i> control worms) is indicated where significant (one-way ANOVA followed by Tukey HSD test).</p

SmMBD2/3 contains amino acid residues critical for binding to 5mC templates.

<p><b>(A)</b> Diagrammatic representation of the 314 amino acid SmMBD2/3 (encoded by Smp_138180) illustrating the methyl-CpG (mCpG) binding domain (PF01429), two putative predicted bipartite nuclear localisation signals (NLS), a coiled-coil domain (CC) and a C-terminal domain of methyl CpG binding protein 2 and 3 (PF140489). Amino acid positions are indicated in bold numbering. <b>(B)</b> A multiple sequence alignment of the methyl-CpG (mCpG) binding domains (PF01429) collected from SmMBD2/3 (italics and contained in a blue box) and MBD homologs was generated. MBDs unable to bind 5mC are indicated in red. Highly conserved residues are highlighted in turquoise and moderately conserved residues are shaded grey. A ‘*’ indicates amino acid residues that contribute to 5mC binding as assessed by mutational studies (summarised in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007107#ppat.1007107.t001" target="_blank">Table 1</a>). A ‘#’ signifies additional amino acid residues that directly interact with 5mC and a ‘:’ indicates amino acid residues that interact with the DNA phosphate backbone [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007107#ppat.1007107.ref021" target="_blank">21</a>]. Amino acid insertions in AmMBD1 and DmMBD2/3 are indicated in black boxes above and below the alignment, respectively. AmMBD1: XP_003250634.1, HsMBD1: NP_002375.1, MmMBD1: NP_038622.2, HsMeCP2: NP_001104262.1, MmMeCP2: NP_001075448.1, XlMeCP2: NP_001081854.1, HsMBD4: NP_001263201.1, MmMBD4: NP_034904.2, HsMBD2: NP_003918.1, MmMBD2 NP_034903.2, GgMBD2: NP_001012403.1, HsMBD3: NP_001268382.1, MmMBD3: NP_038623.1, XlMBD3: AAD55389.1, BmMBD2/3: XP_004929675.1, ApMBD2/3: ACF05483.1, HpMBD2/3: ACF05485.1, AdMBD2/3: [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007107#ppat.1007107.ref025" target="_blank">25</a>], SmMBD2/3: CCD59176.1, DmMBD2/3: NP_731370.1. <b>(C)</b> Ribbon representation of SmMBD2/3 homology model (green) depicts 5mC-interacting residues (stick representation: amino acids in red, 5mC in orange) found within PF01429. The SmMD2/3 model was generated using the solved crystal structure of <i>G</i>. <i>gallus</i> MBD2 co-complexed with methylated DNA as detailed in the <i>Materials and Methods</i>. The tetra-amino acid archetypal binding pocket is indicated (red).</p

Description of frequency and covered chromatin state (kb) in miracidia, Sp1, cercariae, schistosomula and adults for H3K4me3, H3K27me3 or both (bivalent state), genome-wide and at TSS (Transcription Start Site of genes).

<p>Description of frequency and covered chromatin state (kb) in miracidia, Sp1, cercariae, schistosomula and adults for H3K4me3, H3K27me3 or both (bivalent state), genome-wide and at TSS (Transcription Start Site of genes).</p

Parameters used in chromstaR for the detection of ‘peaks’ (300 bp to 10 kb wide).

<p><i>Bin size</i>: Size (in bp) in which the genome was fragmented to analyze the histone mark distribution. <i>Differential score</i>: value generated by chromstaR which provide an estimation on how divergent two bins are (0 = no difference, 1 = extremely different). <i>Minimum read count</i>: minimum number of reads which must be mapped inside a bin in order to take it into consideration. Minimum region length: minimum size (in bp) of consecutive adjacent bins with a different chromatin profile between samples. <i>False discovery rate</i> = minimum value to eliminate false positives. <i>Gap</i> = size of gaps which are allowed between two bins or group of bins with a different chromatin profile between samples. This is important, as gaps (where no reads are present) are frequent on <i>S</i>. <i>mansoni</i> genome. The reason for that is only uniquely mapped reads are used, but 47.73% of the genome is repetitive [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007066#ppat.1007066.ref036" target="_blank">36</a>].</p

Gene ontology overrepresentation depending on the type of chromatin enrichment.

<p>In short regions, we removed genes that had also been identified in the long region analysis. Symbol code: *Panther Pathways, ** = Biological Process, *** = Molecular Function, **** = Protein Class. Short regions: 300 bp– 10 kb. Long regions: 10 kb– 100 kb.</p

Life cycle of the human parasite <i>Schistosoma mansoni</i> including the five developmental stages presented in this work.

<p>The life cycle starts when eggs are in contact with freshwater and release a free-swimming larva, the miracidium. Miracidia seek out an intermediate host, a freshwater snail of the <i>Biomphalaria</i> genus, penetrate the tegument and transform into primary sporocysts. Sporocysts multiply asexually for approximately ten days and then mature into secondary sporocysts, which generate hundreds of cercariae, a second type of free-swimming larva. Cercariae actively seek a definitive mammalian host (rodent, primate or human) and penetrate the dermis of the host, reaching the vascular system. Schistosomula follow a complex maturation process, ultimately leading to adult worms. Male and female worms form pairs and migrate toward mesenteric veins, where a single female can lay approximately one-hundred eggs per day.</p