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 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

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

Outbred (pigmented) <i>B</i>. <i>glabrata</i> snails contain higher DNA methyltransferase activity, MBD-binding capacity and genome 5mC compared to inbred (NMRI) snails.

<p>DNA methyltransferase and MBD-binding activity was fluorometrically measured in nuclear protein isolated from the head/foot tissue of adult snails. <b>A) DNMT activity was measured in 7</b> μ<b>g of <i>B</i>. <i>glabrata</i> (NMRI and pigmented hybrid strains) nuclear protein extract (n = 2) using EpiQuik DNA Methyltransferase Activity/Inhibition Assay Kit (Epigentek).</b> Relative fluorescence units (RFU) were obtained at 530_EX/590_EM nm and subsequently normalised to the blank negative control (assay buffer only) and positive control (Dnmt1). Error bars represent standard deviation (SD) of the normalised means. <b>B) MBD-binding activity within 10</b> μ<b>g of <i>B</i>. <i>glabrata</i> (NMRI and pigmented hybrid strains) nuclear protein extract (n = 2) was measured with the EpiQuik MBD2 Binding Activity/Inhibition Assay Kit (Epigentek).</b> 10 μg of BSA was used as a negative control. Fluorescence was read at 530_EX/590_EM nm and readings subsequently normalised to the blank negative control (assay buffer only). Error bars represent ± standard deviation (SD) of the normalised means. <b>C) 5mC was detected in <i>B</i>. <i>glabrata</i> gDNA (100ng) derived from both albino NMRI and pigmented hybrid strains (n = 2) using the MethylFlash methylated DNA Quantification Kit (Epigentek).</b> The level of 5mC was measured in relative fluorescence units (RFU) at 530_EX/590_EM nm and normalised to the negative (synthetic unmethylated DNA with 50% cytosine content) and positive control (synthetic methylated DNA with 50% 5mC content). * indicates a significant difference (Student’s two-tailed t test; <i>p<0</i>.<i>05</i>) between the 5mC level of NMRI and Pigmented snails. Readings are shown as means and error bars represent ± standard deviation (SD). 5mC abundance (%), displayed above bars, was calculated based on the <i>B</i>. <i>glabrata</i> genome GC content (35%) as described in the Materials and Methods.</p

<i>B</i>. <i>glabrata</i> DNMT homologs are novel members of the DNMT enzyme family.

<p>Phylogenetic relationships based on Bayesian (Mr Bayes v3.1.2) and Maximum Likelihood (MEGA v5.2.2) approaches, were inferred from a multiple sequence alignment of the six highly conserved motifs within the catalytic domain (PF00145) from 29 taxa using MUSCLE [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005246#pntd.0005246.ref031" target="_blank">31</a>]. BgDNMT1 and BgDNMT2 are indicated by red boxes. Abbreviations Bg, Ac, Lg, Ct, Hr, Ci, Mm, Smd, Em, Sm, Fh, Am and Cq relate to <i>B</i>. <i>glabrata</i>, <i>A</i>. <i>californica</i>, <i>L</i>. <i>gigantea</i>, <i>C</i>. <i>gigas</i>, <i>C</i>. <i>teleta</i>, <i>H</i>. <i>robusta</i>, <i>C</i>. <i>intestinalis</i>, <i>M</i>. <i>musculus</i>, <i>S</i>. <i>mediterranea</i>, <i>E</i>. <i>multilocularis</i>, <i>S</i>. <i>mansoni</i>, <i>F</i>. <i>hepatica</i>, <i>A</i>. <i>mellifera</i> and <i>C</i>. <i>quinquefasciatus</i>. The Bayesian analysis consensus tree is illustrated (Figtree v1.3.1, [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005246#pntd.0005246.ref036" target="_blank">36</a>]) with branch lengths signifying distance between taxa. Node labels within parentheses represent percentage bootstrap support values from Maximum Likelihood analysis (500 bootstrap replicates performed using the JTT model), while those outside parentheses represent Bayesian posterior probability support values (based on performing four independent Markov Chain Monte Carlo runs for 1,000,000 generations using the WAG model).</p

The <i>B</i>. <i>glabrata</i> 14-3-3 (BGLB005695) single exon gene contains methylated cytosines within its coding region.

<p><b>A) PCR amplification of bisulfite converted DNA reveals four 5mC sites within Bg14-3-3.</b> Bisulfite conversion of gDNA followed by PCR (BS-PCR) and sequencing was employed to investigate the methylation status of cytosines within the exon of <i>Bg14-3-3</i> (Scaffold1582:42425–42875). Following bisulfite conversion of gDNA, target sequences were amplified, subcloned, and individual clones subsequently sequenced. A filled circle indicates the presence of 5mC; an empty circle indicates the absence of 5mC. Each circle represents a cytosine in a CpG context. Numbers above the circles correspond to base pair positions. Percentages of 5mC detected at each cytosine are indicated. Grey box represents CDS of <i>Bg14-3-</i>3 and the dashed lines indicate the amplified region. B) <b>Confirmation of methylated CpGs within <i>Bg14-3-3</i> by WGBS.</b> An IGV v2.3 [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005246#pntd.0005246.ref057" target="_blank">57</a>] genome browser screenshot of the <i>Bg14-3-3</i> gene. Black bars indicate a methylated CpG position as determined by WGBS (Genome Publication, under review) and y-axis represents degree of methylation (between 0–1) as estimated by BSMAP v1.0.0 [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005246#pntd.0005246.ref100" target="_blank">100</a>].</p

The <i>B</i>. <i>glabrata</i> DNA methylation machinery is abundantly expressed in sex tissues and haemocytes.

<p><b>A) RNA-Seq analysis of the <i>B</i>. <i>glabrata</i> DNA methylation machinery in twelve snail tissues.</b> The normalised sequencing counts [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005246#pntd.0005246.ref051" target="_blank">51</a>] for each gene of interest (i.e. <i>Bgmbd2/3</i>, <i>Bgdnmt1</i> and <i>Bgdnmt2</i>) across the twelve tissues were used to estimate sample parameters for that gene i.e. the mean and standard deviation. The twelve observations for each gene were scaled to a standardised t-distribution. These standardised counts for the three genes were plotted (y-axis) against the twelve tissues (x-axis)—the continuous the red line on the y-axis at 1.79 represents <i>p</i> < 0.05 on a t-distribution with 11 degrees of freedom. The samples were divided into three groups, i.e Group 2: ovotestes (OVO), Group 3: terminal genitalia (TRG) and Group 1: salivary glands (SAL), digestive gland/hepatopancreas (DG/HP), central nervous system (CNS), buccal mass (BUC), albumin gland <b>(</b>AG), mantle edge (MAN), head/foot (FOOT), stomach (STO), heart/APO (HAPO) and kidney (KID). Differential expression analysis, using DESeq2 [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005246#pntd.0005246.ref051" target="_blank">51</a>], indicates the Group 1 vs. Group 2 and Group 1 vs. Group 3 comparisons of <i>Bgmbd2</i>/<i>3</i>, <i>Bgdnmt1</i> and <i>Bgdnmt2</i> abundance (i.e. tissue samples with data above the red line) are statistically significant for that gene of interest. <b>B) qRT-PCR data confirms the tissue-enriched expression of the <i>B</i>. <i>glabrata</i> DNA methylation machinery.</b> qRT-PCR was employed to verify the transcript abundance of <i>Bgdnmt1</i>, <i>Bgdnmt2</i> and <i>Bgmbd2/3</i> across five tissues previously analysed by RNAseq. In addition to albumin gland <b>(</b>AG), head/foot (FOOT), stomach (STO), ovotestes (OVO) and digestive gland/hepatopancreas (DG/HP), transcript abundance was also determined in haemocytes (HAEMO). Error bars represent standard deviation of the mean (SD). The Ct values of target genes were normalised to the reference gene S19 [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005246#pntd.0005246.ref077" target="_blank">77</a>]. Biological duplicates were used for each tissue and technical triplicates performed for every qRT-PCR reaction. For haemocytes, only one biological sample was available. <b>C) 5-AzaC treatment inhibits <i>B</i>. <i>glabrata</i> oviposition.</b> Adult NMRI snails (10–12 individuals/condition) were incubated in the presence or absence of 491μM 5-AzaC for a total of eight days. The bar chart represents mean eggs laid/condition at day eight + standard deviation (SD). The Student’s two-tailed <i>t</i> test was performed to identify significant differences between the treatments. Images are representative of egg sacs obtained from control and 5-AzaC conditions and were taken 7 days after deposition. <b>D) A heat map representation of genes within the neighbourhood of <i>Bgdnmt1</i> and <i>Bgmbd2/3</i> that are significantly over or under-expressed in OVO (ovotestes).</b> The genes are clustered in two directions i.e. across samples and across genes. Uniprot assigned short names to these genes based on sequence homology (full name included in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005246#pntd.0005246.s002" target="_blank">S2 Table</a>) are indicated.</p