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.

Complex multicellular organisms are made up different cell types that become organised into tissues and organs. A fundamental question in biology is how different cell types can arise when they have the same genomic DNA sequence. Gene regulation plays an important role in this process and controls the activation of different genes at any given time point in particular cell types. A major way that gene expression is regulated is through a dynamic structure called chromatin, which is a complex of DNA wrapped around specialised proteins called histones. These histones are the target of various types of chemical modifications that imply whether the chromatin state of genes is active or silent. Different types of histone proteins are common in complex organisms and they generate diversity in chromatin structure to influence both gene expression and DNA packaging. Flowering plants are the most dominant form of plant life on earth and produce the seeds, grains and vegetables that are staples of human consumption. Key to the evolutionary success of flowering plants is the innovation of the pollen grain, which is made up of two sperm cells encased within a large cell called the vegetative cell. Because of such a simple three-celled structure, pollen is an attractive system to understand the molecular basis for differential gene regulation and how this ultimately specifies different cell types. Chromatin undergoes dramatic structural reorganization in pollen, with the vegetative and sperm cells having very different chromatin states visible by eye under a microscope. It has been known that pollen chromatin contains a specific class of histones for nearly 20 years yet we know nothing about their influence on chromatin organisation. This project has focussed on addressing the molecular basis for the different chromatin states between pollen cell types and the function of this special class of pollen-specific histones. This FWF Lise Meitner fellowship project was awarded to Dr Michael Borg and Dr Frederic Berger at the Gregor Mendel Institute in Vienna. They have shown that a sperm-specific histone widely-conserved in flowering plants plays an important role in reorganising sperm cell chromatin by changing modification states that allows the activation of sperm-specific genes. They have also used a special technique called ATAC-seq to study what parts of the chromatin are open to indicate active parts of the genome. Their work shows how the different open chromatin states of each pollen cell type activates a unique set of genes important for pollen function and thus plant fertility. This has implications not only socially for improving agricultural sustainability of crops but also more fundamentally since it enlightens our understanding of how chromatin states exert influence on gene expression and ultimately specify cell types in complex multicellular organisms.