Accessible chromatin changes dynamically during development and harbours functional regulatory regions which are poorly understood in the context of wood development. We explored the importance of accessible chromatin in Eucalyptus grandis in immature xylem generally, and MYB transcription factor‐mediated transcriptional programmes specifically.
We identified biologically reproducible DNase I Hypersensitive Sites (DHSs) and assessed their functional significance in immature xylem through their associations with gene expression, epigenomic data and DNA sequence conservation. We identified in vitro DNA binding sites for six secondary cell wall‐associated Eucalyptus MYB (EgrMYB) transcription factors using DAP‐seq, reconstructed protein‐DNA networks of predicted targets based on binding sites within or outside DHSs and assessed biological enrichment of these networks with published datasets.
25 319 identified immature xylem DHSs were associated with increased transcription and significantly enriched for various epigenetic signatures (H3K4me3, H3K27me3, RNA pol II), conserved noncoding sequences and depleted single nucleotide variants. Predicted networks built from EgrMYB binding sites located in accessible chromatin were significantly enriched for systems biology datasets relevant to wood formation, whereas those occurring in inaccessible chromatin were not.
Our study demonstrates that DHSs in E. grandis immature xylem, most of which are intergenic, are of functional significance to gene regulation in this tissue.
Supporting Information: Fig. S1. Agarose gel electrophoresis images of immature xylem DNase‐seq library quality‐control and preparation.
Fig. S2. Performance of various peak‐calling algorithms in identifying DHSs.
Fig. S3. Irreproducible discovery rate plots for immature xylem DNase‐seq data.
Fig. S4. Absolute expression levels of genes overlapping immature xylem DHSSs in seven Eucalyptus tissues and organs.
Fig. S5. Proximal enrichment of small‐fragment, immature xylem (pooled‐fragment) and large‐fragment DHSs to H3K4me3, H3K27me3 and transcriptional start sites.
Fig. S6. Degree distributions of nodes in transcription factor‐target gene networks involving EgrMYB transcription factors.
Methods S1. DNase I treatment, DNA isolation and sequencing.
Notes S1. Irreproducible discovery rate analysis.
Table S1. Parameters for sequence read mapping and variant detection for E. grandis TAG0014 reference genome imputation.
Table S2. Immature xylem DNase‐seq mapping rates.
Table S3. Summary of peak‐calling algorithms tested.
Table S4. Biological reproducibility of immature xylem DHSs.