Epigenetic Landscape Global Nuclear Positioning Tools

The protein packaging of the genome inside our cell nuclei, known as chromatin, controls the access to genes and helps to regulate their activity. Modifications on the DNA or on the histone proteins inside chromatin determine whether it has an open or condensed structure. These marks act as a bar code for gene activity, which can be passed on in an epigenetic (not DNA encoded) way, as cells divide. The epigenetic marks are 'read' and interpreted by a collection of proteins with essential roles in chromatin regulation.

This explanation combines the insights from two perspectives of epigenetics that we aim to bridge with new methodology: the epigenome map of epigenetic marks and its influence on chromatin structural folding in the nucleus. It reflects the technological gaps that we aim to address with the novel methodology we propose to develop. For instance from the chromatin structure-function viewpoint, we have still fragmentary understanding of how histone and DNA modifications influence chromatin structure or control gene access to regulate transcription. From the epigenetic code and heritability perspective, we have only partial insight into how the bar coding is set up, read and implemented, and how it is propagated.

The study of genetics explains the organism and its cells in terms of nuclear function directed by genes on the chromosomes, which eventually has culminated in the whole genome sequencing project. This milestone achievement led to the realisation that the linear sequence knowledge had to be deepened with epigenetic information as well as gene annotations in order to be read as a functional genomic road map. Epigenetic maps increase the level of complexity of the whole genome information several fold, as theoretically there may be as many epigenomes as there are cell types, each composed of dozens of epigenetic marks. The advent of functional genomics has seen a genome-wide approach to this richly annotated genomic sequence. It often analyses collective gene function rather than single genes, within a multilevel context of the 'epigenetic landscape', an expression based on Conrad Waddington's multi-dimensional model for describing developmental transitions. Yet, it is now recognized that genomic function involves essential contacts between chromosomes and other nuclear structures, which are not easily understood from maps.

How nuclear organization relates to genomic function remains unclear. We will develop tools than can begin to interpret the epigenomic maps in terms of their three-dimensional nuclear chromatin context and function. Why is this important? To explain why tissues have normally stable cell types during life, it was thought that gene expression patterns are maintained by heritable patterns of epigenetic modifications. Recent discoveries have shown that the developmental identity of a cell is in fact less fixed than was previously assumed. Cells can now be reprogrammed to other cell types in a lab dish. This has changed our view on epigenetics. We need to know more about how epigenetic patterns define cell types, in particular the dynamic properties of the epigenetic marks and the mechanisms that link these patterns to the control of gene function. This is important in understanding the biology of how different tissues and organs are made. Furthermore cell reprogramming has important applications in regenerative medicine and biotechnology.

In eukaryotic nuclei, genomic DNA is complexed with histone proteins into the chromatin structure. DNA and histone modifiying enzymes leave epigenetic marks on the chromatin that can be passed on through cell division and help to determine the chromatin state of a gene. Genes can be active in open euchromatin or silenced in condensed heterochromatin. Epigenetic DNA and histone modifications are recognised by various binding complexes involved in this gene regulatory process.

Chromatin immunoprecipitation sequencing (ChIP-seq) projects have mapped a range of epigenetic marks along the whole genome DNA sequence. This vast information has given much insight into the epigenetic landscape of various cell types, but it remains largely unclear how these modifications are functionally and spatially organised in the nucleus, and how epigenetic patterns are set up and function in this 3D nuclear context. Global nuclear epigenetic remodelling events occur during development and reprogramming, which can be observed by immunofluorescence microscopy. Despite technological advances, this provides little information on the genomic regions involved, beyond genome wide correlations with epigenomics mapping data.

Recent discoveries of cell reprogramming and DNA methylation turnover have contributed to a more dynamic view of the epigenetic identity of a cell. To address the mechanisms involved, we need new research tools that can begin to localise mapped chromatin epigenetic regions in the 3D nucleus. We propose an original method for imaging the nuclear distributions of epigenetic marks in condensed chromatin regions, using fluorescent probes that can provide a narrower reference to ChIP-seq mapping data. In combination with novel fluorescent reporters for epigenetic epitopes and super resolution microscopy, the methodology will enable to visualise epigenetic modification changes and contribute towards a three-dimensional nuclear interpretation of epigenomic maps.

Our proposal is situated in a high impact research area and fits many Strategic Priority criteria, as well as the general BBSRC strategic research priority of 'bioscience underpinning health'.

1. Strategic Priority of 'Technology development for the biosciences'
The proposed research will develop new research methodology for epigenetics research, which has the potential for excellent impact under the Strategic Priority of 'Technology development for the biosciences'. While the methodology is primarily involved with 'bioimaging and functional analysis', it is also directly connected to 'omics technologies, including sequence'-based epigenomics. In fact, its goal is to initiate better synergy between 3D nuclear bioimaging and epigenomics experimental platforms. This will 'fulfil an unmet need in the biosciences': current epigenomic maps provide exquisite multivariate detail of mapped locations for DNA methylation and various chromatin modifications along the whole genome, but these maps cannot currently be functionally interpreted in terms of the 3D eukaryotic nucleus. Despite great advances in microscopy, bioimaging of eukaryotic nuclei still gives too little insight into the underlying DNA sequences to be very useful to epigenomics researchers.

2. Strategic Priority of 'Systems approaches to the biosciences'
The current 'technological gap' stands in the way of understanding the functional mechanisms and connections behind the complex epigenomic and 3D chromosomal patterns. How the nuclear organization relates to genomic function remains unclear, but we can start to develop new tools to begin tracing the epigenomic landscape in 3D. Our proposal shows 'new ways of working' to tackle this challenging problem and involves both 'improved molecular biology methodologies' and 'tools for metagenomics'. Although these match criteria recommended under 'Technology development for the biosciences', this proof-of-concept project will eventually necessitate complex pattern analysis of 3D nuclear distributions of histone and DNA modifications against the complexity of the epigenome. We hope this will drive a systems analysis towards an integrated understanding of nuclear function and functional genomics under the remit of the Strategic Priority of 'Systems approaches to the biosciences'

3. Strategic Priority of 'Data driven biology'
Although this proposal does not include Bioinformatics approaches, it is concerned with 'analysis and interrogation of next generation sequencing datasets', as well as 'extracting quantitative information from large or complex image sets', so it matches the scientific scope of the 'Data driven biology' Strategic Priority

4. Strategic Priority 'Living with environmental change'
The tools and resources this proposal will provide will be of 'potential application to broad communities in the biosciences'. This includes the growing epigenetics community, but it will also be of great interest to the toxicology community interested in environmental epigenetics. The co-investigator's research is in the area of non genotoxic carcinogens. As his research is of high impact in this field we have ticked the Strategic Priority 'Living with environmental change'

5. Strategic Priority 'Ageing research: lifelong health and wellbeing'
This is an area of high impact for the epigenetics field, as epigenetic factors are involved in many aspects of maintaining a lifelong healthy life, from foetal development to ageing. The development of a new research tool such as this can be the watershed to new observations, which may have significant impact on scientific advance in the field. The emerging role of epigenetics in cell type identity and cell reprogramming will be underpinned by insights from these and other new tools.