Chemical modulation of Schistosoma mansoni lysine specific demethylase 1 (SmLSD1) induces wide-scale biological and epigenomic changes - Supplementary tables and movies

Extended data This project contains the following extended data: Table S1. Chemical structures of the 39 compounds included in this study. Table S2. Z´ values for both phenotype and motility of the Roboworm screens performed on the 39 compounds. Table S3. Z´ values for both phenotype and motility of the Roboworm screens performed on the titration of the five selected compounds (compounds 15, 16, 33, 35 and 36). Table S4A. List of the 71,859 ATAC-Seq peaks found in the female dataset Table S4B. List of corresponding 844 genomic loci associated to statistically significant ATAC-seq differences (adjusted p-value ≤0.05) following compound 33 treatment (female dataset) Table S4C. List of the 84,997 ATAC-Seq peaks found in male dataset Table S4D. List of corresponding 2,107 genomic loci associated to statistically significant ATAC-seq differences (adjusted p-value ≤0.05) following compound 33 treatment (male dataset) Table S4E. List of female and male genes associated to statistically significant ATAC-seq differences between compound 33 and mock treatments. Table S5A. List of the 844 genomic loci associated to statistically significant ATAC-seq differences (female subset). The table contains the gene ID, chromosome position, start and end of the ATAC-seq peak position, ATAC-seq peak identifier, value of log2-foldchange (log2FC).  Table S5B. Occurrence of ATAC-seq peaks (accessible chromatin) per genes (female subset). The table contains the gene ID and the associated number of ATAC-seq peaks Table S5C. List of ATAC-seq positive signals (accessible chromatin) found in the mock female samples (filtered from S5A Table with log2FC>0) Table S5D. List of ATAC-seq positive signals (accessible chromatin) found in the compound 33-treated female samples (filtered from S5A Table with log2FC Table S5E. List of female genes containing higher ATAC-seq positive signals (accessible chromatin) in compound 33-treated samples. The table contains the gene ID, the number of associated ATAC-seq peaks and the average log2FC across all the peaks associated with each gene (log2FC33 treated samples).  Table S5F. List of female genes containing higher ATAC-seq positive signals (accessible chromatin) in mock samples. The table contains the gene ID, the number of associated ATAC-seq peaks and the average log2FC value across all the peaks associated to each gene (log2FC>0 corresponds to higher ATAC-seq positive signals found in the control treatment).  Table S6A. List of corresponding 2,107 genomic loci associated with statistically significant ATAC-seq differences (male subset). The table contains the gene ID, chromosome position, start and end of the ATAC-seq peak position, ATAC-seq peak identifier, value of log2-foldchange (log2FC).  Table S6B. Occurrence of ATAC-seq peaks per genes (male subset). The table contains the gene ID and the associated number of ATAC-seq peaks Table S6C. List of ATAC-seq positive signals (accessible chromatin) found in the mock male samples (filtered from S6A Table with log2FC>0) Table S6D. List of ATAC-seq positive signals (accessible chromatin) found in the compound 33-treated male samples (filtered from S6A Table with log2FC Table S6E. List of male genes associated with higher ATAC-seq positive signals (accessible chromatin) in compound 33-treated samples. The table contains the gene ID, the number of associated ATAC-seq peaks and the average log2FC across all the peaks associated to each gene (log2FC33 treated samples).  Table S6F. List of male genes associated with higher ATAC-seq positive signals (accessible chromatin) in mock samples. The table contains the gene ID, the number of associated ATAC-seq peaks and the average log2FC value across all the peaks associated with each gene (log2FC>0 corresponds to higher ATAC-seq positive signals found in the control treatment). Table S7. List of the structure, the docking score and EC50 values (on schistosomula and adult worms) of the five most active compounds. Table S8. List of 24 adult worm ATAC-seq sample IDs, primer sequences and additional PCR cycles used for preparation of ATAC-seq libraries Table S9. Excel workbook used for the hypergeometric test for overrepresentation of ATAC-Seq signal within the available scRNA-Seq data derived from adult worms [70].  Movie S1. Video of S. mansoni compound 33-treated (3.13 µM, right-hand side) and control (DMSO, left-hand side) worm pairs after 72 h incubation in tissue culture wells. Notice the lack of parasite attachment and the presence of cellular material within the compound treated well.  Movie S2. Serial optical sections of DAPI-stained, S. mansoni egg. Comparison between the compound 33-treated (3.13 µM, right-hand side) and the negative control (DMSO, left-hand side) egg is provided. Movie S3. Serial optical sections of S. mansoni adult female worm stained with DAPI and EdU. In these series of optical sections, three different anatomical regions (anterior region, gonadal system and posterior region, from left to right) of the worm were observed. Comparison between the negative control (DMSO, first row) and the compound 33-treated (3.13 µM, second row) parasites is provided. Movie S4. Serial optical sections of S. mansoni adult male worm stained with DAPI and EdU. In these series of optical sections, three different anatomical regions (anterior region, gonadal system and posterior region, from left to right) of the worm were observed. Comparison between the negative control (DMSO, first row) and the compound 33-treated (3.13 µM, second row) parasites is provided.</p

Chemical modulation of Schistosoma mansoni lysine specific demethylase 1 (SmLSD1) induces wide-scale biological and epigenomic changes - Supplementary Figures

Extended data This project contains the following extended data:      - Fig S1. Dose response titrations of compounds 15, 16, 33, 35 and 36 against schistosomula.  - Fig S2. Compound 33 treatment induces the release of oocytes, spermatozoa and vitelline cells from adult worms.  - Fig S3. Fast Blue BB stain (orange-red labelling) showing loss of mature vitellocytes in compound 33-treated female worms compared to the control ones.  - Fig S4. Compound 33 treatment reduces stem cell proliferation in adult male worms. - Fig S5. SmLSD1 inhibition causes changes in chromatin structure in S. mansoni male adult worms. - Fig S6. Visualisation of ATAC-seq samples.  - Fig S7. Computational preparation of the covalent adduct derived from the interaction of compound 33 with the FAD cofactor.  - Fig S8. Chemical space covered by the library of 39 HsLSD1 inhibitors.  - Fig S9. Compound 33 treatment inhibits H3K4me2 demethylation in adult male worms. Fig S1. Dose response titrations of compounds 15, 16, 33, 35 and 36 against schistosomula. The five hit compounds were screened against mechanically-transformed schistosomula at 10 µM and lower concentrations (5, 2.50, 1.25 and 0.625 µM). Three independent dose response titrations were performed and each compound concentration was evaluated in duplicate. Each concentration point defines the average mean of the three biological replicates (each of them with two technical replicates). Dose response curves for S. mansoni schistosomula phenotype (P) are presented here using GraphPad Prism (mean +/- SE of mean is indicated for each compound concentration). Estimated EC50s (and corresponding 95% confidence interval) calculated from these dose response curves are summarised in the table underneath. Z´ scores for motility and phenotype for the three screens are reported in Table S3. Fig S2. Compound 33 treatment induces the release of oocytes, spermatozoa and vitelline cells from adult worms. After 72 h incubation, representative images of eggs, oocytes (oc), spermatozoa (sp), mature spermatozoa (ms) and vitelline cells (vc) in tissue culture medium of worm pairs treated with DMSO (A and C) and a sublethal dose of compound 33 (3.13 μM, panels B, D, E and F) were taken. Images were acquired with Olympus microscope (4x for panels A and B), 10x for panels C and D and 20x for panels E and F)). Fig S3. Fast Blue BB stain (orange-red labelling) showing loss of mature vitellocytes in compound 33-treated female worms compared to the control ones. Two representative full-body images of a compound 33-treated female worm vs an untreated female. Fig S4. Compound 33 treatment reduces stem cell proliferation in adult male worms. Male schistosomes were treated with 3.13 μM of compound 33 (n = 6) or DMSO (n = 6) for 48 h and then were labelled with EdU for an additional 24 h. (A) – Schematic of a S. mansoni adult male with representative anterior region (yellow box), gonadal (magenta) and posterior region (brown) of untreated (top row, grey) compared to the compound-treated (bottom row, light brown) worms. Fluorescent microscopic images (6 males per treatment) were acquired on a Leica TCS SP8 super resolution laser confocal microscope fitted with a 40X objective (water immersion, 1,00 zoom factor, Z stack of 60 steps) using the Leica Application Suite X. DAPI stain = blue; EdU+ cells = green. Scale bar represents either 1 mm or 40 μm. Scatter plots illustrate the percentage of proliferative stem cells present in control (DMSO treated worms, n = 6) versus compound 33-treated males (3.13 µM of compound 33, n = 6) in the head region (B), the testes (C) and the tail region (D). Standard errors are shown and a Mann-Whitney test (with ** corresponding to p Fig S5. SmLSD1 inhibition causes changes in chromatin structure in S. mansoni male adult worms. (A) - Combined metagene ATAC profiles derived from compound 33 – (green) and mock (DMSO, blue) treated S. mansoni adult male worm libraries. The bold lines represent average values (from the 12 replicates), with the standard errors in shades (light green for compound 33, light blue for DMSO, light grey for the overlapping area of the first two). X-axis in kilobase (kb). TSS = Transcription start site, TES = transcription end site. −2.0 represents 2 kb upstream of the TSS, and 2.0 represents 2 kb downstream of the TES. Y-axis represents the averaged intensity value of the ATAC-Seq enrichment, which is based on the number of aligned reads but also the average distance between reverse and forward reads that were converted into ‘peak scores’ by MACS and a background correction with Poisson Pvalue (-log10(pvalue) using control as lambda and treatment as observation. (B) - Pie chart illustration of genomic distribution for the ATAC-seq peaks differentially found between mock and compound treated male worms. The percentage of ATAC-seq peaks is provided for each genomic feature: exonic (1st exon or other exons), intronic (1st intron or other introns), distal intergenic (regions located between genes - more than 300 bp downstream the end gene and more than 3 kb upstream, promoter region, downstream region (up to 300 bp downstream the end gene). (C) - Heatmap of differentially accessible ATAC-seq peaks (p-value 33-treated male worms (n = 12).  Clustering method: average linkage; Distance Measurement Method: Manhattan; clustering applied to rows and columns. Heatmap represents row-based z-scores of DESeq2 normalized Tn5 insertion counts for each differentially accessible ATAC-seq peak. Every line represents a peak, 2,107 peaks in total (peaks were not labelled for readability reasons). High (ATAC-seq up) and low (ATAC-seq down) chromatin accessibility is indicated in yellow and blue, respectively. Fig S6. Visualisation of ATAC-seq samples. Representative IGV screenshot of the DMSO (depicted in purple) and compound 33 (depicted in pink) ATAC-seq peaks derived from the female libraries. Differential ATAC-seq peaks tracks are represented with solid dark red boxes. (A) - Snapshot of the IGV browser showing smp_312640 (Locus: SM_V7_4:10754393-10755036), which showed an ATAC-seq positive signal (accessible chromatin) found in the compound 33-treated female samples. This gene was found differentially expressed in the late female germ cell cluster. (B) - Snapshot of the IGV browser showing smp_200410 (Locus: SM_V7_1:31057921-31059724), which showed an ATAC-seq positive signal (accessible chromatin) found in the DMSO female samples. This gene was found differentially expressed in the GSC cluster.  Fig S7. Computational preparation of the covalent adduct derived from the interaction of compound 33 with the FAD cofactor. (A) - Chemical structure of compound 33. (B) - Stick representation of the chemical structure of the cofactor FAD. (C) - Stick representation of the covalent adduct of compound 33 with the flavin ring of the cofactor. Fig S8. Chemical space covered by the library of 39 HsLSD1 inhibitors. The scatter plot shows the distribution of the calculated logP (cLogP) vs the molecular weight (MW, g/mol) of the 39 compounds under study in this investigation. Each compound is shown as orange dot where the five most active compounds are shown in green. The reference compound (Tranylcypromine, compound 1) of this family of LSD1 inhibitors and the two more closely related derivatives (GSK-LSD1 and ORY-1001 - compounds 2 and 3, respectively) are labelled for comparison to the five most active anti-schistosomal compounds presented in this study (in green). The anti-schistosomal controls used in the ex vivo assays (praziquantel and auranofin) are also indicated in purple.  Fig S9. Compound 33 treatment inhibits H3K4me2 demethylation in adult male worms. Visualisation of H3/H3K4me2 marks in adult male worm histone extracts (derived from 20 individuals, three biological replicates) after 72 h incubation with 3.13 µM of compound 33. Western blots of each biological replicate are reported here showing the H3 (loading controls) and H3K4me2 abundances of each experimental replicate.</p