Epigenetic Reprogramming of the Plant Paternal Genome

Histone proteins are a core component of chromatin that complex with DNA to facilitate nuclear packaging and gene regulation. Histone sequence variants are common in eukaryotic lineages and they generate diversity in chromatin structure to influence cellular specification and epigenetic inheritance. During spermatogenesis, animals and plants undergo a dramatic structural organization of the genome that serves to tightly package and protect paternal chromatin in preparation for its delivery to the female gametes. Studies in mice have shown that testis-specific histone variants mark specific loci to facilitate genome reorganization and poise genes for expression during embryogenesis. In plants, we have known of sperm specific-histone variants for nearly 20 years yet we still know little about their role in reprogramming the paternal genome. Arabidopsis sperm cells are characterized by the expression of two specific histone variants, HTB8 and HTR10, which encode histones H2B.8 and H3.10, respectively. Recent detailed marker analysis has shown that H2B.8 and H3.10 also accumulate in condensed chromatin of embryonic nuclei during late embryogenesis, suggesting a potential role in chromatin compaction. As such H2B.8 and H3.10 represent a fitting case study to explore the epigenetic role of histone variants in plant gametogenesis and late embryogenesis, providing insight into how chromatin architecture relates to differentiation. This proposal describes a 24-month project designed to characterize H2B.8 and H3.10 function by comparing both pollen and seed chromatin in Arabidopsis thaliana. In addition, pollen ontology will be used as a cellular system to profile spatial and temporal changes in chromatin organisation during development. To achieve the objectives of this proposal, the research methodology is designed to carry out five main objectives (1) to generate genome- wide maps of H2B.8 and H3.10 deposition by ChIP-seq analysis using both native and GFP antibodies (2) to use advance microscopy and profile gene expression in knockout mutants to determine the impact of H2B.8 and H3.10 on gene expression and chromatin condensation (3) to map the post-translational modifications H2B.8 and H3.10 accumulate in planta using mass-spectrometry (4) to compare the histone complement and global modification state of progenitor germ cell, sperm cell and vegetative cell chromatin using quantitative mass spectrometry (5) to identify H2B.8 and H3.10 orthologues in different angiosperm families and to demonstrate conserved male gamete-specificity using in situ hybridisation. The project is proposed to take place in Dr Frederic Bergers lab at the Gregor Mendel Institute in Vienna.

No additional funding sources recorded.