Limits...
The Epigenome of Schistosoma mansoni Provides Insight about How Cercariae Poise Transcription until Infection.

Roquis D, Lepesant JM, Picard MA, Freitag M, Parrinello H, Groth M, Emans R, Cosseau C, Grunau C - PLoS Negl Trop Dis (2015)

Bottom Line: We demonstrate that in cercariae no transcription occurs, and we provide evidences that cercariae do not possess large numbers of canonical stem cells.We conclude that specific H3 modifications are a phylogenetically older and probably more general mechanism, i.e. not restricted to stem cells, to poise transcription.Since adult couples must form to cause the disease symptoms, changes in histone modifications appear to be crucial for pathogenesis and represent therefore a therapeutic target.

View Article: PubMed Central - PubMed

Affiliation: Université de Perpignan Via Domitia, Perpignan, France; CNRS, UMR 5244, Interactions Hôtes-Pathogènes-Environnements (IHPE), Perpignan, France.

ABSTRACT

Background: Chromatin structure can control gene expression and can define specific transcription states. For example, bivalent methylation of histone H3K4 and H3K27 is linked to poised transcription in vertebrate embryonic stem cells (ESC). It allows them to rapidly engage specific developmental pathways. We reasoned that non-vertebrate metazoans that encounter a similar developmental constraint (i.e. to quickly start development into a new phenotype) might use a similar system. Schistosomes are parasitic platyhelminthes that are characterized by passage through two hosts: a mollusk as intermediate host and humans or rodents as definitive host. During its development, the parasite undergoes drastic changes, most notable immediately after infection of the definitive host, i.e. during the transition from the free-swimming cercariae into adult worms.

Methodology/principal findings: We used Chromatin Immunoprecipitation followed by massive parallel sequencing (ChIP-Seq) to analyze genome-wide chromatin structure of S. mansoni on the level of histone modifications (H3K4me3, H3K27me3, H3K9me3, and H3K9ac) in cercariae, schistosomula and adults (available at http://genome.univ-perp.fr). We saw striking differences in chromatin structure between the developmental stages, but most importantly we found that cercariae possess a specific combination of marks at the transcription start sites (TSS) that has similarities to a structure found in ESC. We demonstrate that in cercariae no transcription occurs, and we provide evidences that cercariae do not possess large numbers of canonical stem cells.

Conclusions/significance: We describe here a broad view on the epigenome of a metazoan parasite. Most notably, we find bivalent histone H3 methylation in cercariae. Methylation of H3K27 is removed during transformation into schistosomula (and stays absent in adults) and transcription is activated. In addition, shifts of H3K9 methylation and acetylation occur towards upstream and downstream of the transcriptional start site (TSS). We conclude that specific H3 modifications are a phylogenetically older and probably more general mechanism, i.e. not restricted to stem cells, to poise transcription. Since adult couples must form to cause the disease symptoms, changes in histone modifications appear to be crucial for pathogenesis and represent therefore a therapeutic target.

No MeSH data available.


Related in: MedlinePlus

Comparison of EpiChIP profiles 2kb upstream and 4kb downstream of the TSS (average for 12,871 genes located on linkage groups that are assembled on chromosome level) for cercariae (light color) and adults (dark color).TSS and direction of transcription indicated by an arrowhead. X-axis in bp, y-axis in relative enrichment (% of all mapped reads). (A) H3K9me3, (B) H3K9ac, (C) H3K27me3, (D) H3K9me3, and (E) unbound fractions UB of mock ChIP without antibody that corresponds to input that shows no difference between cercariae and adults and no enrichment at the TSS.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4549315&req=5

pntd.0003853.g001: Comparison of EpiChIP profiles 2kb upstream and 4kb downstream of the TSS (average for 12,871 genes located on linkage groups that are assembled on chromosome level) for cercariae (light color) and adults (dark color).TSS and direction of transcription indicated by an arrowhead. X-axis in bp, y-axis in relative enrichment (% of all mapped reads). (A) H3K9me3, (B) H3K9ac, (C) H3K27me3, (D) H3K9me3, and (E) unbound fractions UB of mock ChIP without antibody that corresponds to input that shows no difference between cercariae and adults and no enrichment at the TSS.

Mentions: To link transcription and chromatin structure, we considered the correct identification of the TSS as crucial. Anecdotal evidences had suggested that the available automatic annotation had missed a certain proportion of first exons and we decided to perform RNA-Seq with RNA extracted from cercariae. Indeed, our data showed that in about 20% of the genes, TSS had to be corrected. We then performed ChIP-Seq. For H3K4me3, we found relatively sharp peaks with a mean peak maximum located 250 bp (or 1–2 nucleosomes) downstream of the transcription start site (TSS) of genes (Fig 1A). We detected on average 7,746 peaks in cercariae and 8,868 peaks in adults (Table 1). For about ~24% of genes, EpiChIP identified transcripts but no significant H3K4me3 peaks (FDR = 0.01). Examples for genes that are free of H3K4me3 peaks are all known micro exon genes (MEGs) supplementary table S5.4 in [41] (Fig 2A), with the exception of Smp_163710 that shows small peaks in adults. MEGs contain unusually small exons of 3–36 bp that are repeated in tandem [42]. Their function remains elusive but it is thought that hypervariable proteins can be generated by MEGs through a ‘pick and mix’ splicing strategy. It is conceivable that such types of genes have a specific control mechanism. Other H3K4me3-peak free genes are polycistronic genes that are located downstream of the trans-splicing site. Protasio et al. estimated trans-splicing events to occur in 11% of S. mansoni genes [18] and had compiled a list of putative polycistronic transcripts. We used this list and show that in 143 (89.9%) genes, the H3K4me3 peak is located exclusively in the gene upstream of the trans-splicing acceptor site (Fig 2B), in 2 (1.2%) cases peaks are present in both genes (upstream and downstream), and in 13 (8.2%) genes we did not find any peak, although in 3 cases we suspect that an upstream gene with a peak may be the first of the polycistronic unit (S2 Table). Our data are in line with the view that these genes are indeed transcribed as one unit from a single TSS (that has the H3K4me3 signal) and fragmented by trans-splicing. For H3K27me3 we found with Diagenode antibody cat# pAb-069-050 lot# A29900242 enrichment at the TSS (-500 to +1,000 bp), while it was reduced to background level in adults. Since there was anecdotic evidence for cross-reactivity of this antibody with H3K4me3, we repeated the experiment with cat# C15410069 (that replaces pAb-069-050) lot# A1821D. With this antibody we also observe enrichment of H3K27me3 in cercariae compared to adults, but shifted towards the body of the gene (Fig 1C and Fig 2C). Methylation and acetylation in H3K9 are mutually exclusive at the same histone and H3K9me3 is considered a repressive mark. H3K9me3 shows a peak about 200 bp upstream of the TSS in cercariae, which is reduced to an upstream background in adults (Fig 1D and Fig 2C). H3K9ac is a hallmark of active transcription. In cercariae we detected this modification primarily around the TSS (-500 to +1,000 bp) while in adults acetylation spans the body of the genes (Fig 1B and Fig 2C). Taken together, our results indicate that all four studied histone modifications show an unusual enrichment around the TSS in cercariae, while in adults their distribution is similar to that found in other metazoans. We saw no differences between the input fractions (negative controls used for normalization) of cercariae and adults (Fig 1E).


The Epigenome of Schistosoma mansoni Provides Insight about How Cercariae Poise Transcription until Infection.

Roquis D, Lepesant JM, Picard MA, Freitag M, Parrinello H, Groth M, Emans R, Cosseau C, Grunau C - PLoS Negl Trop Dis (2015)

Comparison of EpiChIP profiles 2kb upstream and 4kb downstream of the TSS (average for 12,871 genes located on linkage groups that are assembled on chromosome level) for cercariae (light color) and adults (dark color).TSS and direction of transcription indicated by an arrowhead. X-axis in bp, y-axis in relative enrichment (% of all mapped reads). (A) H3K9me3, (B) H3K9ac, (C) H3K27me3, (D) H3K9me3, and (E) unbound fractions UB of mock ChIP without antibody that corresponds to input that shows no difference between cercariae and adults and no enrichment at the TSS.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4549315&req=5

pntd.0003853.g001: Comparison of EpiChIP profiles 2kb upstream and 4kb downstream of the TSS (average for 12,871 genes located on linkage groups that are assembled on chromosome level) for cercariae (light color) and adults (dark color).TSS and direction of transcription indicated by an arrowhead. X-axis in bp, y-axis in relative enrichment (% of all mapped reads). (A) H3K9me3, (B) H3K9ac, (C) H3K27me3, (D) H3K9me3, and (E) unbound fractions UB of mock ChIP without antibody that corresponds to input that shows no difference between cercariae and adults and no enrichment at the TSS.
Mentions: To link transcription and chromatin structure, we considered the correct identification of the TSS as crucial. Anecdotal evidences had suggested that the available automatic annotation had missed a certain proportion of first exons and we decided to perform RNA-Seq with RNA extracted from cercariae. Indeed, our data showed that in about 20% of the genes, TSS had to be corrected. We then performed ChIP-Seq. For H3K4me3, we found relatively sharp peaks with a mean peak maximum located 250 bp (or 1–2 nucleosomes) downstream of the transcription start site (TSS) of genes (Fig 1A). We detected on average 7,746 peaks in cercariae and 8,868 peaks in adults (Table 1). For about ~24% of genes, EpiChIP identified transcripts but no significant H3K4me3 peaks (FDR = 0.01). Examples for genes that are free of H3K4me3 peaks are all known micro exon genes (MEGs) supplementary table S5.4 in [41] (Fig 2A), with the exception of Smp_163710 that shows small peaks in adults. MEGs contain unusually small exons of 3–36 bp that are repeated in tandem [42]. Their function remains elusive but it is thought that hypervariable proteins can be generated by MEGs through a ‘pick and mix’ splicing strategy. It is conceivable that such types of genes have a specific control mechanism. Other H3K4me3-peak free genes are polycistronic genes that are located downstream of the trans-splicing site. Protasio et al. estimated trans-splicing events to occur in 11% of S. mansoni genes [18] and had compiled a list of putative polycistronic transcripts. We used this list and show that in 143 (89.9%) genes, the H3K4me3 peak is located exclusively in the gene upstream of the trans-splicing acceptor site (Fig 2B), in 2 (1.2%) cases peaks are present in both genes (upstream and downstream), and in 13 (8.2%) genes we did not find any peak, although in 3 cases we suspect that an upstream gene with a peak may be the first of the polycistronic unit (S2 Table). Our data are in line with the view that these genes are indeed transcribed as one unit from a single TSS (that has the H3K4me3 signal) and fragmented by trans-splicing. For H3K27me3 we found with Diagenode antibody cat# pAb-069-050 lot# A29900242 enrichment at the TSS (-500 to +1,000 bp), while it was reduced to background level in adults. Since there was anecdotic evidence for cross-reactivity of this antibody with H3K4me3, we repeated the experiment with cat# C15410069 (that replaces pAb-069-050) lot# A1821D. With this antibody we also observe enrichment of H3K27me3 in cercariae compared to adults, but shifted towards the body of the gene (Fig 1C and Fig 2C). Methylation and acetylation in H3K9 are mutually exclusive at the same histone and H3K9me3 is considered a repressive mark. H3K9me3 shows a peak about 200 bp upstream of the TSS in cercariae, which is reduced to an upstream background in adults (Fig 1D and Fig 2C). H3K9ac is a hallmark of active transcription. In cercariae we detected this modification primarily around the TSS (-500 to +1,000 bp) while in adults acetylation spans the body of the genes (Fig 1B and Fig 2C). Taken together, our results indicate that all four studied histone modifications show an unusual enrichment around the TSS in cercariae, while in adults their distribution is similar to that found in other metazoans. We saw no differences between the input fractions (negative controls used for normalization) of cercariae and adults (Fig 1E).

Bottom Line: We demonstrate that in cercariae no transcription occurs, and we provide evidences that cercariae do not possess large numbers of canonical stem cells.We conclude that specific H3 modifications are a phylogenetically older and probably more general mechanism, i.e. not restricted to stem cells, to poise transcription.Since adult couples must form to cause the disease symptoms, changes in histone modifications appear to be crucial for pathogenesis and represent therefore a therapeutic target.

View Article: PubMed Central - PubMed

Affiliation: Université de Perpignan Via Domitia, Perpignan, France; CNRS, UMR 5244, Interactions Hôtes-Pathogènes-Environnements (IHPE), Perpignan, France.

ABSTRACT

Background: Chromatin structure can control gene expression and can define specific transcription states. For example, bivalent methylation of histone H3K4 and H3K27 is linked to poised transcription in vertebrate embryonic stem cells (ESC). It allows them to rapidly engage specific developmental pathways. We reasoned that non-vertebrate metazoans that encounter a similar developmental constraint (i.e. to quickly start development into a new phenotype) might use a similar system. Schistosomes are parasitic platyhelminthes that are characterized by passage through two hosts: a mollusk as intermediate host and humans or rodents as definitive host. During its development, the parasite undergoes drastic changes, most notable immediately after infection of the definitive host, i.e. during the transition from the free-swimming cercariae into adult worms.

Methodology/principal findings: We used Chromatin Immunoprecipitation followed by massive parallel sequencing (ChIP-Seq) to analyze genome-wide chromatin structure of S. mansoni on the level of histone modifications (H3K4me3, H3K27me3, H3K9me3, and H3K9ac) in cercariae, schistosomula and adults (available at http://genome.univ-perp.fr). We saw striking differences in chromatin structure between the developmental stages, but most importantly we found that cercariae possess a specific combination of marks at the transcription start sites (TSS) that has similarities to a structure found in ESC. We demonstrate that in cercariae no transcription occurs, and we provide evidences that cercariae do not possess large numbers of canonical stem cells.

Conclusions/significance: We describe here a broad view on the epigenome of a metazoan parasite. Most notably, we find bivalent histone H3 methylation in cercariae. Methylation of H3K27 is removed during transformation into schistosomula (and stays absent in adults) and transcription is activated. In addition, shifts of H3K9 methylation and acetylation occur towards upstream and downstream of the transcriptional start site (TSS). We conclude that specific H3 modifications are a phylogenetically older and probably more general mechanism, i.e. not restricted to stem cells, to poise transcription. Since adult couples must form to cause the disease symptoms, changes in histone modifications appear to be crucial for pathogenesis and represent therefore a therapeutic target.

No MeSH data available.


Related in: MedlinePlus