title: Genome-wide mapping of histone H3 lysine 4 trimethylation in Eucalyptus grandis developing xylem.
abstract: 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.
authors.affiliation: Department of Genetics, Forestry and Agricultural Biotechnology Institute (FABI), Genomics Research Institute (GRI), University of Pretoria, Private Bag X20, Pretoria, 0028, South Africa. steven.hussey@fabi.up.ac.za.
authors.forename: Steven G
authors.initials: SG
authors.lastname: Hussey
authors.affiliation: Department of Genetics, Forestry and Agricultural Biotechnology Institute (FABI), Genomics Research Institute (GRI), University of Pretoria, Private Bag X20, Pretoria, 0028, South Africa. eshchar.mizrachi@fabi.up.ac.za.
authors.forename: Eshchar
authors.initials: E
authors.lastname: Mizrachi
authors.affiliation: US Forest Service, Pacific Southwest Research Station, Davis, CA, USA. agroover@fs.fed.us.|Department of Plant Biology, University of California, Davis, USA. agroover@fs.fed.us.
authors.forename: Andrew
authors.initials: A
authors.lastname: Groover
authors.affiliation: Department of Plant Science, Forestry and Agricultural Biotechnology Institute (FABI), Genomics Research Institute (GRI), University of Pretoria, Private Bag X20, Pretoria, 0028, South Africa. dave.berger@up.ac.za.
authors.forename: Dave K
authors.initials: DK
authors.lastname: Berger
authors.affiliation: Department of Genetics, Forestry and Agricultural Biotechnology Institute (FABI), Genomics Research Institute (GRI), University of Pretoria, Private Bag X20, Pretoria, 0028, South Africa. zander.myburg@fabi.up.ac.za.
authors.forename: Alexander A
authors.initials: AA
authors.lastname: Myburg
mesh_terms: D002473:Cell Wall / Q000235:genetics; D047369:Chromatin Immunoprecipitation; D002874:Chromosome Mapping / Q000379:methods*; D005052:Eucalyptus / Q000235:genetics*; D018506:Gene Expression Regulation, Plant; D017343:Genes, Plant; D056726:Genetic Association Studies; D018745:Genome, Plant; D006657:Histones / Q000378:metabolism*; D008239:Lysine / Q000378:metabolism*; D008745:Methylation; D011401:Promoter Regions, Genetic / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA; D014934:Wood / Q000235:genetics; D052584:Xylem / Q000235:genetics*
chemical_list: D006657:Histones; D008239:Lysine