Quantitative chromatin immunoprecipitation (Q-ChIP) applied to Schistosoma mansoni (original) (raw)
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Molecular and Biochemical Parasitology, 2009
Histone modifications are important epigenetic marks that influence chromatin structure and consequently play a role in the control of eukaryotic transcription. Several histone modifying enzymes have been characterized in Schistosoma mansoni and it has been suggested that the regulation of gene transcription in schistosomes may require the action of these enzymes. However, the influence of chromatin structure on gene transcription in schistosomes has never been investigated. Chromatin immunoprecipitation (ChIP) is the technique of choice to study the relationship between histone modifications and gene expression. Although this technique has been widely used with cultured cells from model organisms and with many unicellular organisms, it remains challenging to apply this technique to non-conventional organisms that undergo complex life cycles. In this work, we describe a native ChIP procedure that is applicable to all the stages of the S. mansoni life cycle and does not require expensive equipment. Immunoprecipitated DNA was analysed on a whole-genome scale using massively parallel sequencing (ChIP-Sequencing or ChIP-Seq). We show that ChIP-Seq and conventional quantitative PCR deliver comparable results for a life-cycle regulated locus, smRHO, that encodes a guanine-protein coupled receptor. This is the first time that the ChIP-Seq procedure has been applied to a parasite. This technique opens new ways for analyzing epigenetic mechanisms in S. mansoni at a whole-genome scale and on the level of individual loci.
Measuring Histone Modifications in the Human Parasite Schistosoma mansoni
Methods in molecular biology, 2020
DNA-binding proteins play critical roles in many major processes such as development and sexual biology of Schistosoma mansoni and are important for the pathogenesis of schistosomiasis. Chromatin immunoprecipitation (ChIP) experiments followed by sequencing (ChIP-seq) are useful to characterize the association of genomic regions with posttranslational chemical modifications of histone proteins. Challenges in the standard ChIP protocol have motivated recent enhancements in this approach, such as reducing the number of cells required and increasing the resolution. In this chapter, we describe the latest advances made by our group in the ChIP methods to improve the standard ChIP protocol to reduce the number of input cells required and to increase the resolution and robustness of ChIP in S. mansoni.
Histone Methylome of the Human Parasite Schistosoma Mansoni
RNA Technologies
The trematode Schistosoma mansoni belongs to the group of digenetic parasites which need obligatory multiple hosts to develop. They transit between hosts as free-swimming stages in fresh water ecosystems. They generate phenotypically different developmental stages throughout their lifecycle and receive hugly heterogenous environmental cues. Each developmental stage is characterized by specific posttranslational histone modifications, in particular methylations. The combination of the different marks result in stage specific chromatin structure that is essential for development, sexual biology and pathogenesis. Histone methylation also responds to environmental changes and seems to be involved in an adaptive reponse or adjustment to the environment. Histone methylation thus represent promising source of therapeutic targets. In this chapter we will present the state-ofthe-art of how the dynamics of histone methylation are involved in multiple factors of the schistosome's development, as well as what is still lacking for better understanding it.
PLOS Pathogens
Epigenetic mechanisms and chromatin structure play an important role in development. Their impact is therefore expected to be strong in parasites with complex life cycles and multiple, strikingly different, developmental stages, i.e. developmental plasticity. Some studies have already described how the chromatin structure, through histone modifications, varies from a developmental stage to another in a few unicellular parasites. While H3K4me3 profiles remain relatively constant, H3K27 trimethylation and bivalent methylation show strong variation. Inhibitors (A366 and GSK343) of H3K27 histone methyltransferase activity in S. mansoni efficiently blocked miracidium to sporocyst transition indicating that H3K27 trimethylation is required for life cycle progression. As S. mansoni is a multicellular parasite that significantly affects both the health and economy of endemic areas, a better understanding of fluke developmental processes within the definitive host will likely highlight novel disease control strategies. Towards this goal, we also studied H4K20me1 in female cercariae and adults. In particular, we found that bivalent trimethylation of H3K4 and H3K27 at the transcription start site of genes is a landmark of the cercarial stage. In cercariae, H3K27me3 presence and strong enrichment in H4K20me1 over long regions (10-100 kb) is associated with development related genes. Here, we provide a broad overview of the chromatin structure of a metazoan parasite throughout its most important lifecycle stages. The five developmental stages studied here present distinct chromatin structures, indicating that histone methylation plays an important role during development. Hence, components of the histone methylation (and demethylation) machinery may provide suitable Schistosomiasis control targets.
The class I histone deacetylases of the platyhelminth parasite Schistosoma mansoni
Biochemical and Biophysical Research Communications, 2008
Histone deacetylases (HDAC) form a conserved enzyme family that control gene expression via the removal of acetyl residues from histones and other proteins and are under increasing investigation as therapeutic targets, notably in cancer and parasitic diseases. To investigate the conservation of these enzymes in the platyhelminth parasite Schistosoma mansoni, we cloned and characterized three class I HDACs, orthologues of mammalian HDAC1, 3 and 8, and confirmed their identities by phylogenetic analysis. The identification of an HDAC8 orthologue showed that it is not vertebrate-specific as previously thought and insertions in its catalytic domain suggest specific enzymatic properties. SmHDAC1, 3, and 8 mRNAs are expressed at all schistosome life-cycle stages. SmHDAC1 repressed transcriptional activity in a mammalian cell line and this activity was dependent on its catalytic activity since transcription was partially restored by treatment with trichostatin A and a catalytic site mutant failed to repress transcription.
Control of transcription in Schistosoma mansoni: Chromatin remodeling and other regulatory elements
Acta Tropica, 2008
The platyhelminth parasite Schistosoma mansoni, the causative agent of schistosomiasis, is a dioecious parasite with a complex life cycle that includes two different hosts and two free-living stages. Yet very little is known about the biochemical details connected to these different transitions. In the present work, results will be presented showing the most recent results in S. mansoni regarding the characterization of transcription factors and coactivators that act directly on the transcriptional machinery and those that are involved with chromatin remodeling. It is hoped that the information gathered here may contribute towards the understanding of crucial events in the parasite life cycle. Likewise, the development of new drugs that could interfere with oogenesis and sexual maturation may eventually profit from the information contained herein.
Genome Biology and Evolution, 2019
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.
PLoS Pathogens, 2014
Treatment and control of schistosomiasis relies on the only available drug, praziquantel, and the search for alternative chemotherapeutic agents is therefore urgent. Egg production is required for the transmission and immunopathology of schistosomiasis and females of S. mansoni lay 300 eggs daily. A large fraction of the total mRNA in the mature female worm encodes one eggshell protein, Smp14. We report that the nuclear receptors SmRXR1 and SmNR1 regulate Smp14 transcription through the recruitment of two histone acetyltransferases (HATs), SmGCN5 and SmCBP1. The treatment of HEK293 cells with histone deacetylase (HDAC) inhibitors (NaB or TSA) produced an 8-fold activation of the SmRXR1/SmNR1mediated Smp14 promoter activity. Incubation with synthetic HAT inhibitors, including PU139, significantly impaired the Smp14 promoter activity in these cells. Worm pairs cultivated in the presence of PU139 exhibited limited expression of Smp14 mRNA and protein. ChIP analysis demonstrated chromatin condensation at the Smp14 promoter site in worms treated with PU139. ChIP also revealed the presence of H3K27me3 and the absence of RNA Pol II at the Smp14 promoter region in the PU139-treated worms. Most significantly, the PU139-mediated inhibition of Smp14 expression resulted in a significant number of abnormal eggs as well as defective eggs within the ootype. In addition, scanning electron microscopy revealed structural defects and unformed eggshells, and vitelline cell leakage was apparent. The dsRNAi-targeting of SmGCN5 or SmCBP1 significantly decreased Smp14 transcription and protein synthesis, which compromised the reproductive system of mature female worms, egg-laying and egg morphology. Our data strongly suggest that the inhibition of Smp14 expression targeting SmGCN5 and/or SmCBP1 represents a novel and effective strategy to control S. mansoni egg development.
Epigenetic control of gene function in schistosomes: a source of therapeutic targets?
Frontiers in Genetics, 2014
The discovery of the epigenetic regulation of gene expression has revolutionized both our understanding of how genomes function and approaches to the therapy of numerous pathologies. Schistosomes are metazoan parasites and as such utilize most, if not all the epigenetic mechanisms in play in their vertebrate hosts: histone variants, histone tail modifications, non-coding RNA and, perhaps, DNA methylation. Moreover, we are acquiring an increasing understanding of the ways in which these mechanisms come into play during the complex schistosome developmental program. In turn, interest in the actors involved in epigenetic mechanisms, particularly the enzymes that carry out epigenetic modifications of histones or nucleic acid, as therapeutic targets has been stimulated by the finding that their inhibitors exert profound effects, not only on survival, but also on the reproductive function of Schistosoma mansoni. Here, we review our current knowledge, and what we can infer, about the role of epigenetic mechanisms in schistosome development, differentiation and survival. We will consider which epigenetic actors can be targeted for drug discovery and what strategies can be employed to develop potent, selective inhibitors as drugs to cure schistosomiasis.