ChIP-seq and RNA-seq for complex and low-abundance tree buds reveal chromatin and expression co-dynamics during sweet cherry bud dormancy

Noémie Vimont,François Roudier,David Guillaume Schöepfer,Sandra Cortijo,Philip A Wigge,Bénédicte Wenden,Fu Xiang Quah,Elisabeth Dirlewanger

doi:10.1007/s11295-019-1395-9

Abstract

Chromatin immunoprecipitation-sequencing (ChIP-seq) is a robust technique to study interactions between proteins, such as histones or transcription factors and DNA. This technique in combination with RNA-sequencing (RNA-seq) is a powerful tool to better understand biological processes in eukaryotes. We developed a combined ChIP-seq and RNA-seq protocol for tree buds (Prunus avium L., Prunus persica L Batch, Malus x domestica Borkh.) that has also been successfully tested on Arabidopsis thaliana and Saccharomyces cerevisiae. Tree buds contain phenolic compounds that negatively interfere with ChIP and RNA extraction. In addition to solving this problem, our protocol is optimised to work on small amounts of material. Furthermore, one of the advantages of this protocol is that samples for ChIP-seq are cross-linked after flash freezing, making it possible to work on trees growing in the field and to perform ChIP-seq and RNA-seq on the same starting material. Focusing on dormant buds in sweet cherry, we explored the link between expression level and H3K4me3 enrichment for all genes, including a strong correlation between H3K4me3 enrichment at the DORMANCY-ASSOCIATED MADS-BOX 5 (PavDAM5) loci and its expression pattern. This protocol will allow analysis of chromatin and transcriptomic dynamics in tree buds, notably during its development and response to the environment.

Highlights

The term ‘epigenetics’ has traditionally been used to refer to heritable changes in gene expression that take place without altering DNA sequence (Wolffe and Matzke 1999), but it is used, in a broader sense, to refer to modifications of the chromatin environment (Miozzo et al 2015)
We find that dormancy-associated PavDAM6 and PavDAM5 genes are more expressed in dormant buds than in non-dormant cherry buds and that H3K4me3 occupancy is associated with PavDAM5 expression level
We demonstrated the correlation between chromatin status and gene expression for AGAMOUS (AG) and ELONGATION FACTOR 1 (EF1) that are known to be under control of H3K27me3 and H3K4me3, respectively (Saito et al 2015)

Summary

Introduction

The term ‘epigenetics’ has traditionally been used to refer to heritable changes in gene expression that take place without altering DNA sequence (Wolffe and Matzke 1999), but it is used, in a broader sense, to refer to modifications of the chromatin environment (Miozzo et al 2015). The numerous ChIP protocols published in plants and mammals [(Cortijo et al 2018; Kaufmann et al 2010; Li et al 2014; Nelson et al 2006; Ricardi et al 2010; Saleh et al 2008; Wal and Pugh 2012; Xie and Presting 2016; Yamaguchi et al 2014) to cite just some] cannot be directly used for plant materials with high phenolic content (alkaloids or lignified cell walls) like tree buds (Bílková et al 1999) Such chemicals need to be chelated during the chromatin extraction to prevent inhibition of downstream processes. While several studies include ChIPseq performed in trees, some with improvements described in this protocol such as using a chelator of interfering

Methods

Results

Conclusion