Inheritance of chromatin marks during cell replication in plants

Secondary Supervisor(s): Professor Daniel Hebenstreit

University of Registration: University of Warwick

BBSRC Research Themes:

• Sustainable Agriculture and Food (Plant and Crop Science)
• Understanding the Rules of Life (Plant Science, Systems Biology)

Project Outline

The correct development of multicellular organisms entails the specifications of distinct cell types. Each of these cellular identities must be conserved during later cell divisions. At the same time, genomic integrity must be preserved in every cell and every cell cycle during the genome replication cycles that occur during the developmental differentiation. The replication of the genome requires that not only the genetic material, DNA, but also the chromatin, a macromolecular entity formed by the association of DNA with histone proteins into nucleosomes, is correctly duplicated. Therefore, both DNA and histone proteins, together with their post-translational modifications (PTMs), must be properly transferred to the daughter chromatin fibres. Since the chromatin landscape is a key component of gene regulation, the transferring process of parental histones and their association with new histone proteins is crucial to maintaining the memory of transcriptional programs of parental chromatin in the daughter chromatin after cell division. It is well established that DNA methylation is copied from the parental DNA strand to the progeny strand immediately after DNA replication and involves the cooperative interactions between DNA methylation and histone modifications.

Recent work has provided clues on how the transferring of chromatin information is mechanistically linked to the DNA replication machinery. The precise molecular mechanisms that underpin the transfer of chromatin information into the newly synthesised genome are not yet fully understood. Our knowledge in plants has been limited, in part because most studies are based on the analysis of mutants in key regulators of chromatin structure and in factors implicated in the deposition or removal of histone PTMs, which usually accumulate pleiotropic genetic and epigenetic abnormalities. Surprisingly, the precise machinery implicated in the transfer of chromatin information and how it is localised at specific genome regions during DNA replication remains currently unknown.

Objectives

The plan of work is divided into three main objectives:

(1) Define the role that histone demethylases play in balancing the inheritance of chromatin marks during replication. The student will use a new system to engineer conditional knockouts of different components of the chromatin machinery to assess their role in the inheritance of different chromatin marks.

(2) Identify the molecular components contributing to the correct transfer of chromatin information in plants. To this aim, the student will perform a targeted proteomic analysis using cell cultures to identify the factors implicated in the replication of histone marks.

(3) To use super-resolution microscopy analysis to monitor the dynamic changes in chromatin during different stages of the cell cycle and their association with components of the replisome, a macromolecular complex responsible for replicating the genomic DNA.

Collectively, this work will further our understanding of how different chromatin states are inherited in plants.

Spatiotemporal control of axillary meristem formation in wheat

Secondary Supervisor(s): Professor George Bassel

University of Registration: University of Warwick

BBSRC Research Themes:

• Sustainable Agriculture and Food (Plant and Crop Science)

• Understanding the Rules of Life (Plant Science, Systems Biology)

Project Outline

Polyploidisation and domestication have contributed to a major genetic bottleneck in most crops, which amongst other changes resulted in the reduction of axillary meristem growth and reduced secondary shoots. However, given today’s increasing global demand for food production, increased shoots could be one way to efficiently augment crop yield. Despite this, very little is known about the mechanisms underlying axillary meristem formation in most crops. Interestingly, the number of shoots varies in most wild crop relatives, but in particular among grasses. In preliminary work, we have identified a new transcriptional pathway operating in wild grass species, which controls the initiation of axillary meristems and can efficiently increase the number of shoots. In this project, we aim to further understand this pathway through computational, developmental genetic and molecular approaches, using wheat as a model species, for which we have expert knowledge and unique in-house genomic and genetic resources.

Objectives

The plan of work is divided into three main objectives:

1) To define the regulatory network that underpins AXM formation in wheat. Using single-cell RNA-seq, the student will define at a single-cell resolution the molecular network implicated in AXM initiation. The student will determine how these components function to control plant architecture, starting with the targeted mutagenesis (CRISPR) of newly identified candidates.

2) To define how the AXM pathway is regulated by changes in chromatin accessibility. Using pharmacological and genetic analyses the student will explore the role that chromatin dynamics plays in AXM initiation.

3) To understand the impact that natural variation and human selection have on AXM development. The student will use bioinformatic tools to identify cis-regulatory elements that contribute to the regulation of AXM formation.

It is anticipated that results from this work will provide fundamental knowledge of how plant architecture is controlled, providing essential know-how for engineering novel cultivars to enhance yield. 

Mechanisms controlling transgenerational stress memories in plants

Secondary Supervisor(s): Professor Guy Barker

University of Registration: University of Warwick

BBSRC Research Themes:

• Sustainable Agriculture and Food (Plant and Crop Science)
• Understanding the Rules of Life (Plant Science, Systems Biology)

Project Outline

The epigenetic mechanisms underpinning intergeneration memory are relatively well understood and are associated with the maintenance of high levels of H3K4me3 and phosphorylation of RNAP II. However, the mechanisms controlling transgenerational memory are poorly understood. Our preliminary work has discovered that environmental signals, in addition to the direct influence on plant growth, can also cause phenotypic changes that can be transmitted to the progeny, but these phenotypic changes rarely remain stable over several reproductive cycles. We found that stress directs stochastic changes in DNA methylation that are passed to offspring but actively removed by DNA demethylases during germline development. In addition, these transgenerational memories are mediated by the positive feedback of transcription factors and the histone demethylase RELATIVE OF EARLY FLOWERING 6 (REF6).

We have developed in Arabidopsis a novel strategy to elicit abiotic stress memories (extreme temperature and hyperosmotic stress) and have identified regions of the genome sensitive to epigenetic modification in response to stress. Our data indicate that these elements modulate the expression of neighbouring genes by modifying the 3D architecture of the chromatin. However, the precise mechanisms that regulate these changes remain unknown.

Objectives

The plan of work is divided into three main objectives:

(1) To define the role of stress-memory elements using targeted genome editing tools (CRISPR/Cas9). The student will generate genetic and epigenetic changes using tools developed in our laboratory and assess their effects on transcriptional memory.

(2) To assess the impact that chromatin conformation has on stress memory. The student will use targeted epigenome editing tools developed by our team to modify the chromatin landscape of stress-memory elements and determine their role in transcriptional memory.

(3) To determine the impact that genome modifications have on abiotic stress memory. The student will use an automated phenotyping image analysis platform to measure the phenotypic dynamics associated with the genomic manipulation of stress-memory elements.

Collectively, this work will further our understanding of the epigenetic mechanisms underpinning transgenerational memory in plants providing essential knowledge for engineering better adapted crop varieties. 

Targeted engineering of quantitative traits in tomato

Secondary Supervisor(s): Dr Guy Barker

University of Registration: University of Warwick

BBSRC Strategic Research Priority: Sustainable Agriculture and Food (Plant and Crop Science)

Project outline

Our ability to develop novel beneficial crop traits has significantly improved over the last century, although the ability to maintain this trajectory is limited by allelic diversity. While genetic variation at coding sequences has been heavily exploited for crop improvement, most quantitative traits reside in mutations at non-coding regulatory regions. This project aims to identify these regulatory regions in tomato using genome-wide chromatin conformation analysis and develop methodology to engineer mutations and epimutations at these regions. It is anticipated that the student will develop a platform to enhance phenotypic variation of selected agricultural traits, which will provide the basis for analyzing the link between gene-regulatory elements and quantitative traits.

Key experimental skills involved

The student will carry out a series of experiments in tomato designed to engineer mutations and epimutations, using new CRISPR variants, at the regulatory regions of genes known to be implicated in agronomic performance. These plants will be used to select allelic variants or epimutations causing enhanced agricultural performance. He/She will use computational approaches and laboratory experimentation to investigate the relationship between the temporal/spatial regulation of defined regulatory networks and trait performance.

References

Golicz et al., (2016) The pangenome of an agronomically important crop plant Brassica oleracea. Nature Communications, 7:13390

Durr et al., (2018) Highly efficient targeted genomic deletions in plants using CRISPR/Cas9 Sci Rep 8,4443

Techniques

The student will use a wide range of molecular, histological and computational techniques including: Genomic and Epigenomic analysis using Next Generation Sequencing data and customised computational analysis. Genetic analysis using classic and newly engineered mutants. Confocal microscopy analysis and cellular modelling.

Lab Techniques

CRISPR, RNA-seq and ChIP-seq. Computational-techniques: Wide range of bioinformatics tools for Next-Generation-Sequence data analysis.