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Challenges of epigenetic studies in plants: focus on ChIP technique

Agnieszka Zelisko-Schmidt from Diagenode elaborates on their poster presented at the 6th Plant Genomics & Gene Editing Congress: Europe, explaining how regulatory pathways in plants can be unravelled using the universal plant ChIP-seq kit.

The study of epigenetics in plants is both fascinating and challenging. Several plant development processes such as vegetative growth, reproduction, and biotic and abiotic stress response are controlled by epigenetic mechanisms. The environmental factors induce numerous changes at the level of the chromatin that lead to activating or repressing specific gene expression. Understanding epigenetic mechanisms in plants can potentially help improve agricultural practices especially in the context of climate change. However, the study of epigenetic mechanisms in plants is known to be challenging.

One of the most powerful techniques to study epigenetic variation is chromatin immunoprecipitation (ChIP), a technique which enables the study of protein-DNA interaction in vivo. ChIP performed on plant material is known to be complicated and time-consuming: the protocol consists of many critical steps which need to be optimised to ensure high-quality results. Although the number of citations with plant-specific ChIP protocols has been increasing, researchers are still less familiar with using ChIP in plant versus mammalian cells and tissues.

The most important challenges in ChIP related to the plant material

The first step of ChIP – chromatin preparation – is the most critical step in ChIP experiments. The qualitative (e.g. species, tissue, age) and quantitative variability of starting material adds a level of complexity. A multitude of model plant species, such as monocots, dicots, and trees, and many organs or tissues of interest, such as leaves, roots, buds, reproductive organs, may be available in variable quantities depending on the origin, the age and the nature of the plant material. In some cases (e.g. trees, floral organs, specific tissues, rare plants) the availability of plant tissue can be very limited in both quantity and the time needed for maturation.

Additionally, it is necessary to optimise the cross-linking and the shearing step to get enough high quality sheared chromatin for downstream applications. Furthermore, the presence of the cell wall in plant cells constitutes a significant constraint compared to mammalian cells and makes the protocol longer and more cumbersome due to the addition of extra steps such as fixation under vacuum and plant tissue grinding prior to chromatin extraction. The optimisation steps typically require large amounts of plant material, which is not ideal when quantities are limited.

The immunoprecipitation step

Once high-quality chromatin has been prepared, it can be used in the next step – immunoprecipitation (IP). The immunoprecipitation step should also be optimised, which includes:

  • The type of IP method, be it direct or indirect, etc.
  • The quality and quantity of the antibody
  • The reagents to use (agarose or magnetic beads, buffer composition, etc.)
  • The duration of incubations
  • The DNA purification
  • And elution step.

The choice of the antibody to be used is the most critical for the IP. The antibody should be specific and validated, marked as ChIP or ChIP-seq grade (depending on downstream analyses) and preferably tested on plants. It is important to use the antibody for which batch-specific validation data are available. The antibody should be tested on the material of interest to define the optimal quantity per reaction. Moreover, there are fewer antibodies available that are compatible with plant tissue compared to mammalian cells and tissue. Also, the validation of these antibodies is usually difficult, due to lack of data on the identity of the qPCR control regions. This requires antibody validation by sequencing without any qPCR validation, which is an expensive approach.

Analysing sequencing data

Another challenge of ChIP experiments is the analysis of sequencing data. The analysis parameters should be adapted depending on plant species due to the size of the genome and the presence of excessive repetitive regions. In addition, some plant genomes are polypoid and are challenging for bioinformatics analysis. Furthermore, despite the increase in the number of sequenced plant genomes, the genomes of many species are still unsequenced, causing a significant barrier to data analysis.

Diagenode strives to develop state-of-the-art solutions for epigenetics studies in plants. With more than 10 years of experience in ChIP and high profile collaborations with plant consortiums, Diagenode has the expertise to develop new protocols and overcome the difficulties and challenges related to the use of ChIP on plant material. We focus on optimising each step of the protocol unique to plant-specific challenges (see the first slide in the video below). Diagenode develops also the antibodies, solutions for DNA methylation, library preparation and instrument for chromatin and/or DNA fragmentation.

A) PCR validation data of ChIP assays performed with Diagenode solution on maize, B) rice, C, D) poplar using ChIP-seq grade anti-histone marks Diagenode antibodies. ChIP-seq data of tomato – H3K4me3 E) and poplar F) using Diagenode solution – H3K4me3.

Diagenode

 

Diagenode is a leading global provider of complete solutions for epigenetics research, biological sample preparation, and diagnostics assays based in Liege, Belgium and NJ, USA.

 

The agenda for the 7th Plant Genomics and Gene Editing Congress: Europe has launched. Returning to Rotterdam in May, the agenda is shaping up to be a great one. Download the agenda to find out more.

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