Control of insulator function and higher order genome organisation by the chromatin remodeling enzyme NURF

The development of all cells in the body is determined by a set of instructions encoded in genes, found in DNA. All cells contain the same information. The immense variety of cell types in the human body, each with distinct functions is achieved by changing the way this information is read, or "expressed". In eukaryotes, the large amount of DNA required for correct development is compacted into manageable units by wrapping like thread around a protein spool to form structures called nucleosomes. In addition to compacting DNA, nucleosomes provide additional levels of so-called epigenetic information. By varying nucleosome position, access to gene control elements can be regulated and genes turned off or on, changing cell identity and function. Moreover, nucleosomes are the fundamental units in higher orders of genome organisation through which functional interactions between often-distant gene regulatory elements are stabilized, allowing correct patterns of gene expression. As such changes in nucleosome organisation can profoundly affect genome organisation and gene regulation.

In our laboratory we study how nucleosome organisation is altered by chromatin remodeling enzymes. These large protein complexes use ATP, the cells energy transfer molecule, to change nucleosome position, allowing the wholesale reorganization ("remodeling") of genome architecture. We seek to understand how these complexes work as many human diseases are triggered by altered or disordered gene expression and global genome organization. By understanding the epigenetic mechanisms underlying genome organisation and gene regulation, new therapies to cure disease can be developed.

In our research, we use the "model organism" Drosophila melanogaster (fruit flies). Although not immediately be apparent, Drosophila and humans have evolved from a common ancestor and thus share many design principles. A useful analogy is to compare a high-performance racing car and a child's go-kart. While one is more sophisticated, the basic elements of control - steering and brakes - are the same. Similarly, Drosophila uses many of the same mechanisms to control gene expression as humans. As such, we can use Drosophila as a stand-in for humans, a so-called "model organism". This is useful as it allows us to do experiments that are impossible or unethical in humans, for example deliberately deleting or altering genes to determine their role in development. In our work we use fly strains in which we can tag, alter or delete ("knock-out") the protein complexes that establish and regulate genome organisation to determine their function in gene regulation.

In this study we will determine functions of a key chromatin remodeling enzyme called NURF in genome organisation. Drosophila provides an especially powerful system to characterize NURF, as NURF is only present in multicellular eukaryotes precluding studies in simpler model organisms like yeast. In our research we will determine how NURF regulates genome interactions critical for higher order chromatin organisation. To do this we will use variants of a technique called chromosome conformation capture (called ChIA-PET) to fish-out NURF complexes from nuclei. We can then co-purify regions of the genome brought into close physically proximity by NURF and identify these by determining their DNA sequence. Through genetic ablation of NURF and co-factor proteins at these sites we will determine the functional importance of NURF in establishing these regulatory genome interactions. This method will provide a high-resolution view of higher order regulatory genome interactions but requires many millions of cells to perform thus generating a snapshot of interactions averaged over many cells. To capture the dynamics of remodeler genome interactions in single cells over short time scales genome interactions we will also deploy advanced microscopy techniques that allow imaging of single remodeler and insulator molecules in live cells.

Development of higher eukaryotes is driven by complex gene regulatory programmes requiring functional interactions between control elements often separated over millions of base pairs. Insulators are instrumental to this, mediating spatial genome organisation that brings control elements into physical proximity. By altering chromatin structure and dynamics at insulators, wholesale remodeling of genome architecture and thus gene expression can be achieved. Here we will investigate how insulator function is regulated by the ATP-dependent chromatin remodeling factor NURF. We have developed biochemical and genetic tools and single molecule imaging methods allowing high-resolution functional analysis of higher order genome interactions in primary cells as well as analysis of remodeler and insulator dynamics in single cells over short time scales. The three core objectives of this proposal are to:

i. Identify higher-order genome interactions mediated by NURF and insulator proteins using targeted chromatin interaction analysis (ChIA-PET), and determine functional consequences of genetic ablation of NURF and insulator components on higher-order genome interactions.

ii. Investigate dynamics of NURF-insulator interactions and functional organisation of insulator bodies using single molecule and super-resolution imaging.

iii. Identify RNA species that target remodeler activity at insulators using RNA immunoprecipitation sequencing. RNA function in remodeler recruitment and genome organisation will be characterised by CRISPR-mediated ablation and subsequent chromatin interaction analysis, imaging and in vivo enhancer blocking assays.

Understanding the complex interplay between regulated changes in nucleosome positioning, spatial genome organisation and gene expression will reveal fundamental elements of higher eukaryotic gene regulation that may illuminate potential strategies for wholesale genome reprogramming and therapeutic intervention in disease.

Our research aims to define the principles controlling three-dimensional nuclear organization and how it affects gene regulation to control normal development and disease. This data enhances the knowledge economy by providing new insights into the lexicon of the epigenetic regulatory code. Data generated here will have benefit to a substantial global body of researchers in the field of chromatin and epigenetics. The size of this community (Pubmed searches on "epigenetic" identifies 49,816 publications), will ensure obvious impact of data generated here.

By revealing the basic mechanisms controlling genome spatial organisation our research benefits workers in disease fields such as Medical Genetics, Stem Cell, Developmental and Cancer Biology. Analysis of cancer mutations shows that up to 40% map to regulatory regions. Regulatory mutations have been defined human developmental disorders including campomelic dysplasia, holoprosencephaly and aniridia but wide-spread use of sequencing technology is likely to reveal the true impact of regulatory mutation in human disease. In the long term, our research may help offer new avenues for therapeutic intervention. Chromatin regulators including remodeling and modifying enzymes are emerging disease targets. A survey by Grand View Research on the Epigenetic Market (July 2016) showed the global market in epigenetic medicine in 2014, including diagnostics and therapeutics, to be valued at $3.98 billion, with compound annual growth rate of 19.3%.

Our research to understand the role of chromatin regulators in three-dimensional nuclear organization can help define new epigenetic medicine targets. Remodeling enzymes are an under-explored therapeutic target but can potentially be targeted, either by disrupting subunit interaction surfaces, blocking ATPase catalytic sites, or by disrupting HPTM - reader domain interactions. Understanding their role in spatial genome organisation and regulation will broad general interest and could have considerable impact in this lucrative market. Moreover our research to identify RNA regulators of chromatin remodeling enzymes has the potential to reveal new fundamental genome regulatory mechanisms, in turn defining new therapeutic targets.

A clear route to exploit advances made during our research is through our existing contacts with industry. We have previous links with the company Cellzome (now GSK) and through previous CRUK-funded research with Cancer Research Technology. CRT has entered into a collaboration with AstraZeneca to pursue drug development in the field of cancer metabolism, drawing on expertise in fields including epigenetics. In addition, Birmingham has been selected as one of the centres of the "Translational Research Partnerships". These provide mechanisms by which industry can engage in early phase development of novel compounds. Both CRT and the capability clusters provide contact pathways of impact to companies like AstraZeneca, which have interests in developing epigenetics targeting medicines. Through these mechanisms our research will help to enhance the research capacity and knowledge of industry, potentially contributing to wealth creation and prosperity.

Support for our research will synergize with investment in next-generation technology by Birmingham University. Technology and expertise mastered by this facility will have long-term benefits for future UK academic partners. To support future technological expansion in the field of epigenetic medicine, it is paramount that UK knowledge base in these technologies be maintained. Our work as part of the Birmingham Centre for Genome Biology (BCGB) epigenetics consortium will help establish a set of skilled researchers to provide a strong science base. This will make a significant contribution to the UK knowledge economy by delivering and training highly skilled researchers in key technologies critical to the development of the field of epigenetic therapeutics.