Profiling of histone acetylation and methylation marks associated with embryo and aleurone tissue-specific epigenetic regulation of seed dormancy in wheat

Chromatin remodeling via histone posttranslational modification (PTM) plays a fundamental role in genome silencing in quiescent seeds. Several recently recognized dormancy controlling epigenetic mechanisms result in either enhancement or suppression effects. Moreover, evidence suggests that epigenetic regulation could be a robust mechanism for altering levels of phytohormones associated with dormancy ethylene, ABA, and GA. The complex panorama of histone PTMs requires further studies to define the combinatorial preferences of histone variants for dynamic gene regulation. We used lines of spring wheat (Triticum aestivum L.) doubled haploid population with marginal levels of seed dormancy to investigate global changes in histone PTM landscape in embryo and aleurone tissues during early hours of imbibition upon induction or suppression of germination. Histone-enriched fractions were prepared using stepwise nuclei enrichment, chromatin isolation, salt- and acid-labile extractions, and 1D Tricine-SDS-PAGE fractionation, followed by a large scale GeLC-MS/MS analysis for in-depth characterization of histone core proteins and their variants. A total of 197 protein bands were subject to in-vitro propionylation prior to trypsin digestion, nanoLC-MS/MS analysis on the Q-Exactive, followed by iterative MASCOT searches for characteristic histone-specific PTMs.
We utilized customized annotated wheat EST database representing all possible expressed sequences of wheat transcriptome to profile histone variants, and Scaffold Q+ for validation of protein IDs, clustering analysis and PTMs filtering. Bioinformatics perl scripts were developed to display the PTM site locations and types among histone variant clusters in each of the two genotypes.
The histone purification procedure resulted in significant enrichment with embryo- and aleurone-specific histone variants and PTM forms. In each tissue, embryo and aleurone, 30 identified unique protein clusters represented core histones and their variants with characteristic dormant and non-dormant genotype-specific changes in dynamic PTMs acetylation, methylation, dimethylation, and thrimethylation. Altogether, 731 and 771 unique EST contigs (protein forms) were found for aleurone and embryo tissues, respectively. Sets of unique protein forms were identified for each histone cluster. Thus, in aleurone 4 different variant clusters were found to have a range of 45 to 60 unique protein forms, and cluster H2B.2 had 149 identified unique protein forms. Dramatic differences were found in PTMs between dormant and non-dormant aleurone with over 4-fold higher number of sites in dormant aleurone for all tested PTMs in the majority of histone variants. One of the most abundant in aleurone protein forms of core histone H2A was found to have 9 acetylation, 6 methylation, 2 dimethylation, and 1 trimethylation PTM sites unique to dormant tissue, whereas 2 acetylation, 4 methylation, and 1 dimethylation modification sites were found to be unique to non-dormant aleurone. An abundant variant of another core histone H2B had 4 acetylation, 1 methylation, and 1 dimethylation sites unique to dormant, whereas 1 acetylation and 1 methylation sites unique to non-dormant aleurone. In both tissues the highest PTM level was found for H2B.1 to 3 variants. Embryo PTM landscape was in general comparable between dormant and non-dormant tissues. In embryo, 9 variant clusters were identified with a range of 1 to 5, four clusters with 40 to 60, and one cluster H2B.2 with 157 unique protein forms. Significantly higher number of Me1 (6-fold) and Me2 (4-fold) combinatorial sites was found in H2B.3 variant from non-dormant as compared to dormant embryo.