Native chromatin immunoprecipitation (N-ChIP) and ChIP-Seq of Schistosoma mansoni: Critical experimental parameters

Abstract

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.

The Cytoplasm Affects the Epigenome in Drosophila melanogaster

“…A standard native ChIP protocol [67] was adapted to D. melanogaster adults. We used an anti-H3K4me3 monoclonal antibody (Millipore cat.…”

Towards an understanding of the epigenetics of schistosomes: a comparative epigenomic study

“…Posttranslational modifications of histones (e.g., methylation, acetylation and phosphorylation) are widely studied and have been shown to play a role in chromatin compaction and control of gene transcription (Lee et al 2010). Orthologues of all histones apart from MacroH2A are predicted in the current genome assembly of Schistosoma mansoni and our laboratory and others have confirmed the presence of histone H3 and H4 and the isoforms H3K4Me3, H3S10P (unpublished observations), H3K4Me2, H3K9Ac, H3K9Me3, H3K27Me1 and H4Ac (Souza et al 2002, Caby & Pierce 2009, Cosseau et al 2009). An analysis of data available in SchistoDB indicates that genes for all canonical histone modifying enzymes can be found.…”

“…Chromatin status analysis by ChIP -N-ChIP was performed as previously described (Cosseau et al 2009) (methdb.univ-perp.fr/cgrunau/methods/native_chip_ sm.html). Briefly, chromatin was extracted from both C and IC miracidia and fragmented with micrococcal nuclease.…”

“…The N-terminal ends of Schistosoma histones H3 and H4 are conserved with other model species and, therefore, commercial antibodies can be used to immunoprecipitate modified histones and identify DNA to which they are bound. Cross-linked and native ChIP (N-ChIP) have been successfully applied to schistosomes (Caby & Pierce 2009, Cosseau et al 2009). Massive sequencing analysis of the immunoprecipitated DNA (ChIP-Seq) and alignment against the S. mansoni genome has afforded a whole genome approach to visualise enrichment of specific epigenetic marks at specific Schistosoma loci.…”

“…ChIP-Seq analysis can be performed using different antibodies that recognise different epigenetic marks responsible for euchromatic or heterochromatic structures. This technique allows the comparison of chromatin structure between life cycle stages (Cosseau et al 2009), male or female adults (unpublished observations) or different strains. This comparative epigenomic approach has highlighted differences in chromatin structure that may impact the physiology of S. mansoni, such as development, sexual dimorphism and adaptation to environmental change.…”