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Plant Methods  2010 

Protocol: fine-tuning of a Chromatin Immunoprecipitation (ChIP) protocol in tomato

DOI: 10.1186/1746-4811-6-11

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Abstract:

We developed a chromatin immunoprecipitation protocol suitable for tomato by adjusting the parameters to optimise in vivo crosslinking, purification of nuclei, chromatin extraction, DNA shearing and precipitate analysis using real-time PCR. Results were obtained with two different antibodies, five control loci and two normalisation criteria.Here we provide a chromatin immunoprecipitation procedure for tomato leaves that could be combined with high-throughput sequencing to generate a detailed map of epigenetic modifications or genome-wide nucleosome positioning data.Emerging high-throughput methods and bioinformatics technologies have great potential to accelerate the discovery of specific DNA regulatory elements that interact with transcription factors (TFs). However, the vast majority of plant cis-elements corresponding to the majority of TFs are unknown [1], in part due to a lack of optimised experimental methods to be carried out prior to genome-wide data analyses.By contrast, a great deal of research is currently focused on epigenetic events in plants. This topic is particularly interesting because in plants, unlike animals, acquired epigenetic changes can be transmitted to progeny since germ cells differentiate from somatic tissues present in an adult individual. In addition, stable patterns of gene expression necessary for differentiation and long-term adaptation can be mitotically and meiotically inherited in the form of active or silent epigenetic gene variants via mechanisms associated with chromatin structure [2]. In this respect the role of histone modifications is becoming increasingly appreciated.Chromatin immunoprecipitation (ChIP) is a widely used procedure both to identify novel TF-target genes and to examine histone modifications. It is currently used in Arabidopsis [3] but is not yet developed for use with tomato (Solanum lycopersicum), which is considered a model plant species for a group of economically important crops such as potato, pepper and

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