Fig 1.
Life cycle of the human parasite Schistosoma mansoni including the five developmental stages presented in this work.
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 Biomphalaria 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.
Table 1.
Details of the antibodies used for ChIP-Seq.
Table 2.
Parameters used in chromstaR for the detection of ‘peaks’ (300 bp to 10 kb wide).
Bin size: Size (in bp) in which the genome was fragmented to analyze the histone mark distribution. Differential score: value generated by chromstaR which provide an estimation on how divergent two bins are (0 = no difference, 1 = extremely different). Minimum read count: 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. False discovery rate = minimum value to eliminate false positives. Gap = 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 S. mansoni genome. The reason for that is only uniquely mapped reads are used, but 47.73% of the genome is repetitive [36].
Table 3.
Parameters used in chromstaR for the detection of ‘ranges’ (10 kb to 100 kb).
Bin size: Size (in bp) in which the genome was fragmented to analyze the histone mark distribution. Differential score: value generated by chromstaR which provide an estimation on how divergent two bins are (0 = no difference, 1 = extremely different). Minimum read count: 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. False discovery rate = minimum value to eliminate false positives. Gap = 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 S. mansoni genome. The reason for that is only uniquely mapped reads are used, but 47.73% of the genome is repetitive [36].
Fig 2.
Sample clustering and genome-wide frequency of combinatorial states during the life cycle of Schistosoma mansoni.
(A) ChIP-seq data heatmap between five parasite developmental stages for H3K4me3 and H3K27me3. Typical example of chromatin profile for H3K4me3 (B) and H3K27me3 (C). (D) Genome-wide frequency of all combinatorial states in all stages. Two waves of repressive H3K27me3 (red line) with a maximum of methylation can be seen in Sp1 and adults, while a constant line of H3K4me3 (green line) is seen throughout the whole cycle (left Y-axis). A single wave of bivalent chromatin (H3K4me3 and H3K27me3 co-localizing, purple line, secondary axis) has its maximum in cercariae (right Y-axis). (E) Frequency methylation at TSS. Intense repressive methylation inside intermediate and definitive hosts (H3K27me3, red line, left Y-axis), intense bivalency state in cercariae (H3K4me3+H3K27me3 at same locus, purple line, secondary axis, right Y-axis) with loss of single active H3K4me3 histone mark (H3K4me3, green line, left Y-axis). X-axis: Five developmental stage of S. mansoni; Y- axis: Genome-wide methylation frequency (%).
Fig 3.
Heatmap and enrichment of chromatin states around transcription start sites (TSS) of five developmental stages of the Schistosoma mansoni life cycle.
(A) Heatmap of miracidia stage for H3K4me3, H3K27me3 and H3K4me3+H3K27me3. (B) Heatmap of sporocyst stage for H3K4me3, H3K27me3 and H3K4me3+H3K27me3. (C) Heatmap of cercaria stage for H3K4me3, H3K27me3 and H3K4me3+H3K27me3. (D) Heatmap of miracidia stage for H3K4me3, H3K27me3 and H3K4me3+H3K27me3. (E) Heatmap of miracidia stage for H3K4me3, H3K27me3 and H3K4me3+H3K27me3. Logarithms (observed/ expected) for each histone modification were plotted along S. mansoni genome and 4 kb upstream and downstream for each stage (A-E). Colors: Blue = H3k4me3; Yellow = H3K27me3; Purple = H3K4me3+H3K27me3.
Table 4.
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).
Fig 4.
Histone methyltransferase inhibitors block miracidium to primary sprorocyst transformation.
Each treatment was set up in triplicate and parasites were cultured in CBSS with 1% DMSO at a controlled temperature of 26°C (in the dark). An ANOVA followed by post hoc analysis with Tukey’s multiple comparison test was performed to infer statistical significance; *p<0.005. A. Effect of 0.4 μM of A366, 2 μM of A366 and DMSO (negative control). B. Effect of 0.4 μM of GSK343, 0.4 μM of GSK343 and DMSO (negative control). C. Photomicrographs of miracidia to sporocyst transition in the presence of histone methyltransferase inhibitors. Miracidia transformed in the presence of DMSO (negative control) show normal transformation into sporocysts, but fail to lose their ciliated plates and do not develop into primary sporocysts in the presence of of A366 and GSK343 (10 μM). Representative images were acquired at low power (10X objective).
Table 5.
Description of the distributions of peaks (0.3–10 kb) between cercariae and adults for each histone mark.
TSS = Transcription Start Site of genes. Multiple = H3K4me3 + H3K27me3 + H4K40me1 at the same locus. * In the case of H4K20me1, most of the marks were wide and often covering a large part of the gene, if not the whole gene.
Table 6.
Description of the large differentially enriched regions (ranges, 10–100 kb) between cercariae and adults for each histone mark combination, as well as the number of annotated genes present in these regions and the number of regions without any coding genes (and their cumulated length).
tRNAs genes were excluded from the gene counts. We also calculated the total length of these regions for each mark.
Table 7.
Gene ontology overrepresentation depending on the type of chromatin enrichment.
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.
Fig 5.
Principal component analysis (PCA) of ChIP enrichment for H3K4me3 (left) and H3K27me3 (right) over repetitive sequences.
All life cycle stages segregate in the PCA but miracida and Sp1 (separated by 48 hours) are very close and could only be differentiated by H3K4me3 methylation. Arrows indicate developmental direction and are there to guide the eye.
Table 8.
Details for the mixed sexes ChIP-Seq reads available at the NCBI-SRA.
Table 9.
Details for the female ChIP-Seq reads available at the NCBI-SRA.