BACKGROUND : Histone modifications play an integral role in plant development, but have been poorly studied in
woody plants. Investigating chromatin organization in wood-forming tissue and its role in regulating gene expression
allows us to understand the mechanisms underlying cellular differentiation during xylogenesis (wood formation) and
identify novel functional regions in plant genomes. However, woody tissue poses unique challenges for using
high-throughput chromatin immunoprecipitation (ChIP) techniques for studying genome-wide histone modifications
in vivo. We investigated the role of the modified histone H3K4me3 (trimethylated lysine 4 of histone H3) in gene
expression during the early stages of wood formation using ChIP-seq in Eucalyptus grandis, a woody biomass model.
RESULTS : Plant chromatin fixation and isolation protocols were optimized for developing xylem tissue collected from
field-grown E. grandis trees. A “nano-ChIP-seq” procedure was employed for ChIP DNA amplification. Over 9 million
H3K4me3 ChIP-seq and 18 million control paired-end reads were mapped to the E. grandis reference genome for
peak-calling using Model-based Analysis of ChIP-Seq. The 12,177 significant H3K4me3 peaks identified covered ~1.5%
of the genome and overlapped some 9,623 protein-coding genes and 38 noncoding RNAs. H3K4me3 library coverage,
peaking ~600 - 700 bp downstream of the transcription start site, was highly correlated with gene expression levels
measured with RNA-seq. Overall, H3K4me3-enriched genes tended to be less tissue-specific than unenriched genes
and were overrepresented for general cellular metabolism and development gene ontology terms. Relative expression
of H3K4me3-enriched genes in developing secondary xylem was higher than unenriched genes, however, and highly
expressed secondary cell wall-related genes were enriched for H3K4me3 as validated using ChIP-qPCR.
CONCLUSIONS : In this first genome-wide analysis of a modified histone in a woody tissue, we optimized a ChIP-seq
procedure suitable for field-collected samples. In developing E. grandis xylem, H3K4me3 enrichment is an indicator
of active transcription, consistent with its known role in sustaining pre-initiation complex formation in yeast. The
H3K4me3 ChIP-seq data from this study paves the way to understanding the chromatin landscape and epigenomic
architecture of xylogenesis in plants, and complements RNA-seq evidence of gene expression for the future
improvement of the E. grandis genome annotation.