Genome-Wide Analysis of Lysine 4-Trimethylated Histone H3 Modification during Xylogenesis in Eucalyptus

The study of transcription factor and modified histone targets in non-model woody species is challenged by a lack of high-throughput platforms. Chromatin immunoprecipitation coupled to next-generation DNA sequencing (ChIP-seq) allows for genome-wide mapping of in vivo protein-DNA interactions, but its application in non-model species and new tissues requires extensive optimization. We aimed to develop a ChIP-seq procedure for field-collected immature xylem tissue from Eucalyptus trees. We optimized several parameters for nuclei isolation, chromatin isolation, sonication, ChIP and DNA amplification. Our ChIP-seq approach was validated using an antibody specific to the trimethylated lysine 4 residue of histone H3 (H3K4me3), a histone modification associated with transcriptional activation in eukaryotes. Twenty million paired-end reads for H3K4me3-enriched and control libraries of immature xylem from two E. grandis individuals were mapped to the E. grandis reference genome, and peak-calling was performed using MACS. The vast majority of significant H3K4me3 peaks overlapped with annotated genes, showing a typical distribution approx. 500-600 bp downstream of the transcription start site. H3K4me3 enrichment was associated with high-abundance transcripts in developing xylem, but low tissue-specificity across seven tissues, supporting a role in general transcriptional activation. This first comprehensive analysis of a modified histone in a tree species is consistent with results reported in Arabidopsis, rice and mammalian systems. H3K4me3 data can aid in the identification of transcriptional start sites and improve low-confidence gene annotation of the E. grandis reference genome sequence.