Transcriptomic, proteomic, and functional consequences of codon usage bias in human cells during heterologous gene expression.

Differences in codon frequency between genomes, genes, or positions along a gene, modulate transcription and translation efficiency, leading to phenotypic and functional differences. Here, we present a multiscale analysis of the effects of synonymous codon recoding during heterologous gene expression in human cells, quantifying the phenotypic consequences of codon usage bias at different molecular and cellular levels, with an emphasis on translation elongation. Six synonymous versions of an antibiotic resistance gene were generated, fused to a fluorescent reporter, and independently expressed in HEK293 cells. Multiscale phenotype was analyzed by means of quantitative transcriptome and proteome assessment, as proxies for gene expression; cellular fluorescence, as a proxy for single-cell level expression; and real-time cell proliferation in absence or presence of antibiotic, as a proxy for the cell fitness. We show that differences in codon usage bias strongly impact the molecular and cellular phenotype: (i) they result in large differences in mRNA levels and protein levels, leading to differences of over 15 times in translation efficiency; (ii) they introduce unpredicted splicing events; (iii) they lead to reproducible phenotypic heterogeneity; and (iv) they lead to a trade-off between the benefit of antibiotic resistance and the burden of heterologous expression. In human cells in culture, codon usage bias modulates gene expression by modifying mRNA availability and suitability for translation, leading to differences in protein levels and eventually eliciting functional phenotypic changes.

Schistosome W-Linked Genes Inform Temporal Dynamics of Sex Chromosome Evolution and Suggest Candidate for Sex Determination.

Schistosomes, the human parasites responsible for snail fever, are female-heterogametic. Different parts of their ZW sex chromosomes have stopped recombining in distinct lineages, creating "evolutionary strata" of various ages. Although the Z-chromosome is well characterized at the genomic and molecular level, the W-chromosome has remained largely unstudied from an evolutionary perspective, as only a few W-linked genes have been detected outside of the model species Schistosoma mansoni. Here, we characterize the gene content and evolution of the W-chromosomes of S. mansoni and of the divergent species S. japonicum. We use a combined RNA/DNA k-mer based pipeline to assemble around 100 candidate W-specific transcripts in each of the species. About half of them map to known protein coding genes, the majority homologous to S. mansoni Z-linked genes. We perform an extended analysis of the evolutionary strata present in the two species (including characterizing a previously undetected young stratum in S. japonicum) to infer patterns of sequence and expression evolution of W-linked genes at different time points after recombination was lost. W-linked genes show evidence of degeneration, including high rates of protein evolution and reduced expression. Most are found in young lineage-specific strata, with only a few high expression ancestral W-genes remaining, consistent with the progressive erosion of nonrecombining regions. Among these, the splicing factor u2af2 stands out as a promising candidate for primary sex determination, opening new avenues for understanding the molecular basis of the reproductive biology of this group. Keywords: sex chromosomes, evolutionary strata, W-linked gene, sex determining gene, schistosome parasites.

Dosage Compensation throughout the Schistosoma mansoni Lifecycle: Specific Chromatin Landscape of the Z Chromosome.

Differentiated sex chromosomes are accompanied by a difference in gene dose between X/Z-specific and autosomal genes. At the transcriptomic level, these sex-linked genes can lead to expression imbalance, or gene dosage can be compensated by epigenetic mechanisms and results into expression level equalization. Schistosoma mansoni has been previously described as a ZW species (i.e., female heterogamety, in opposition to XY male heterogametic species) with a partial dosage compensation, but underlying mechanisms are still unexplored. Here, we combine transcriptomic (RNA-Seq) and epigenetic data (ChIP-Seq against H3K4me3, H3K27me3, and H4K20me1 histone marks) in free larval cercariae and intravertebrate parasitic stages. For the first time, we describe differences in dosage compensation status in ZW females, depending on the parasitic status: free cercariae display global dosage compensation, whereas intravertebrate stages show a partial dosage compensation. We also highlight regional differences of gene expression along the Z chromosome in cercariae, but not in the intravertebrate stages. Finally, we feature a consistent permissive chromatin landscape of the Z chromosome in both sexes and stages. We argue that dosage compensation in schistosomes is characterized by chromatin remodeling mechanisms in the Z-specific region.

Evolution of gene dosage on the Z-chromosome of schistosome parasites.

XY systems usually show chromosome-wide compensation of X-linked genes, while in many ZW systems, compensation is restricted to a minority of dosage-sensitive genes. Why such differences arose is still unclear. Here, we combine comparative genomics, transcriptomics and proteomics to obtain a complete overview of the evolution of gene dosage on the Z-chromosome of Schistosoma parasites. We compare the Z-chromosome gene content of African (Schistosoma mansoni and S. haematobium) and Asian (S. japonicum) schistosomes and describe lineage-specific evolutionary strata. We use these to assess gene expression evolution following sex-linkage. The resulting patterns suggest a reduction in expression of Z-linked genes in females, combined with upregulation of the Z in both sexes, in line with the first step of Ohno's classic model of dosage compensation evolution. Quantitative proteomics suggest that post-transcriptional mechanisms do not play a major role in balancing the expression of Z-linked genes.

Parent-of-Origin-Dependent Gene Expression in Male and Female Schistosome Parasites.

Schistosomes are the causative agents of schistosomiasis, a neglected tropical disease affecting over 230 million people worldwide. Additionally to their major impact on human health, they are also models of choice in evolutionary biology. These parasitic flatworms are unique among the common hermaphroditic trematodes as they have separate sexes. This so-called "evolutionary scandal" displays a female heterogametic genetic sex-determination system (ZZ males and ZW females), as well as a pronounced adult sexual dimorphism. These phenotypic differences are determined by a shared set of genes in both sexes, potentially leading to intralocus sexual conflicts. To resolve these conflicts in sexually selected traits, molecular mechanisms such as sex-biased gene expression could occur, but parent-of-origin gene expression also provides an alternative. In this work we investigated the latter mechanism, that is, genes expressed preferentially from either the maternal or the paternal allele, in Schistosoma mansoni species. To this end, transcriptomes from male and female hybrid adults obtained by strain crosses were sequenced. Strain-specific single nucleotide polymorphism (SNP) markers allowed us to discriminate the parental origin, while reciprocal crosses helped to differentiate parental expression from strain-specific expression. We identified genes containing SNPs expressed in a parent-of-origin manner consistent with paternal and maternal imprints. Although the majority of the SNPs was identified in mitochondrial and Z-specific loci, the remaining SNPs found in male and female transcriptomes were situated in genes that have the potential to explain sexual differences in schistosome parasites. Furthermore, we identified and validated four new Z-specific scaffolds.

The deep conservation of the Lepidoptera Z chromosome suggests a non-canonical origin of the W.

Moths and butterflies (Lepidoptera) usually have a pair of differentiated WZ sex chromosomes. However, in most lineages outside of the division Ditrysia, as well as in the sister order Trichoptera, females lack a W chromosome. The W is therefore thought to have been acquired secondarily. Here we compare the genomes of three Lepidoptera species (one Dytrisia and two non-Dytrisia) to test three models accounting for the origin of the W: (1) a Z-autosome fusion; (2) a sex chromosome turnover; and (3) a non-canonical mechanism (e.g., through the recruitment of a B chromosome). We show that the gene content of the Z is highly conserved across Lepidoptera (rejecting a sex chromosome turnover) and that very few genes moved onto the Z in the common ancestor of the Ditrysia (arguing against a Z-autosome fusion). Our comparative genomics analysis therefore supports the secondary acquisition of the Lepidoptera W by a non-canonical mechanism, and it confirms the extreme stability of well-differentiated sex chromosomes.

Sex-Biased Transcriptome of Schistosoma mansoni: Host-Parasite Interaction, Genetic Determinants and Epigenetic Regulators Are Associated with Sexual Differentiation.

BACKGROUND: Among more than 20,000 species of hermaphroditic trematodes, Schistosomatidae are unusual since they have evolved gonochorism. In schistosomes, sex is determined by a female heterogametic system, but phenotypic sexual dimorphism appears only after infection of the vertebrate definitive host. The completion of gonad maturation occurs even later, after pairing. To date, the molecular mechanisms that trigger the sexual differentiation in these species remain unknown, and in vivo studies on the developing schistosomulum stages are lacking. To study the molecular basis of sex determination and sexual differentiation in schistosomes, we investigated the whole transcriptome of the human parasite Schistosoma mansoni in a stage- and sex-comparative manner. METHODOLOGY/ PRINCIPAL FINDINGS: We performed a RNA-seq on males and females for five developmental stages: cercariae larvae, three in vivo schistosomulum stages and adults. We detected 7,168 genes differentially expressed between sexes in at least one of the developmental stages, and 4,065 of them were functionally annotated. Transcriptome data were completed with H3K27me3 histone modification analysis using ChIP-Seq before (in cercariae) and after (in adults) the phenotypic sexual dimorphism appearance. In this paper we present (i) candidate determinants of the sexual differentiation, (ii) sex-biased players of the interaction with the vertebrate host, and (iii) different dynamic of the H3K27me3 histone mark between sexes as an illustration of sex-biased epigenetic landscapes. CONCLUSIONS/ SIGNIFICANCE: Our work presents evidence that sexual differentiation in S. mansoni is accompanied by distinct male and female transcriptional landscapes of known players of the host-parasite crosstalk, genetic determinants and epigenetic regulators. Our results suggest that such combination could lead to the optimized sexual dimorphism of this parasitic species. As S. mansoni is pathogenic for humans, this study represents a promising source of therapeutic targets, providing not only data on the parasite development in interaction with its vertebrate host, but also new insights on its reproductive function.

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

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.