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

Lead Research Organisation: University of Birmingham
Department Name: Institute of Cancer and Genomic Sciences

Abstract

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.

Technical Summary

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.

Planned Impact

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.

Publications

10 25 50
 
Description The project was recently initiated. However, to data key findings include:

Generation of tagged strains allowing single molecule imaging of chromatin remodelling enzymes and insulator proteins. We have generated HALO-tagged variants of the NURF chromatin remodelling complex and insulator proteins CP190, CTCF and SuHw. We have also used these to examine dynamics of chromatin association of remodellers and insulators in live Drosophila hemocytes. This has allowed enhanced comparison of binding kinetics of these distinct classes of chromatin proteins.

We have performed RNA immunoprecipitation sequencing (RIP-Seq) using as baits antibodies against NURF and the insulator protein CP190. These are currently being sequenced and will allow identification of RNA components of remodeler/insulator complexes. We have identified RNAs that are bound by NURF complexes and are characterising function of these RNAs

We are currently crossing HALO-tagged CP190, CTCF and SuHw constructs into NURF mutant backgrounds to be able to discriminate functional requirements of NURF for insulator complex assembly. We have used single particle analysis to define the ground-state dynamics of insulator proteins and their requirements for NURF for recruitment.

We have established high-resolution mapping methodologies (ChIA-PET) to discriminate insulator mediated DNA loops and are exploring the role of NURF remodellers in allowing these loops to occur.
Exploitation Route Project is ongoing and this is too early to conclude.
Sectors Pharmaceuticals and Medical Biotechnology

 
Title H3K23me3 Antibody 
Description In the course of an analysis of factors that enable NURF recruitment to chromatin we identified a novel histone post-translational modification Histone H3 Lysise 23 trim ethyl as a potential recruiter of NURF complexes. Analysis of this modification has been limited by availability of high-quality specific antibodies against this mark. We have generated polyclonal antibodies against this mark 
Type Of Material Antibody 
Year Produced 2018 
Provided To Others? No  
Impact Identification of H3K23me3 distribution profile by immunostaining and ChIP-Seq 
 
Title HALO-tagged NURF301 strains 
Description Transgenic Drosophila strains expressing HALO-tagged variants of the NURF chromatin remodelling enzyme. Strains corresponding to all naturally occurring isoforms where generated. 
Type Of Material Model of mechanisms or symptoms - non-mammalian in vivo 
Year Produced 2017 
Provided To Others? No  
Impact NURF-HALO strains have been using in combination with hemocyte isolation methods and single particle tracking methods developed during the course of BBSRC funded USA collaboration award to determine residence times of NURF chromatin remodelling enzyme. Use of mutated and naturally occurring isoforms has allowed role of histone post-translational modifications in NURF recruitment to be determined. 
 
Title HALO-tagged insulator strains 
Description CP190-HALO strains have been using in combination with hemocyte isolation methods and single particle tracking methods developed during the course of BBSRC funded USA collaboration award to determine residence times of insulator components. Use of these in wild-type and NURF mutant backgrounds allows role of chromatin remodellers in insulator function to be determined. 
Type Of Material Model of mechanisms or symptoms - non-mammalian in vivo 
Year Produced 2017 
Provided To Others? No  
Impact Determination of chromatin residence times of Insulator components and requirements of chromatin remodelling enzymes. 
 
Title Hemocyte system for single particle tracking of chromatin protein dynamics 
Description We have developed methods for isolation and immobilisation of Drosophila primary hemocytes for single particle tracking microscopy to determine binding kinetics and nuclear dynamics of chromatin associated proteins. 
Type Of Material Technology assay or reagent 
Year Produced 2016 
Provided To Others? No  
Impact Methodology has allowed in vivo dynamics of ISWI family of chromatin remodelling enzymes, including NURF and ACF, to be determined. We have used this system to demonstrate histone post-translational modifications are required for stable NURF binding to chromatin in vivo. We have used this system to characterise dynamics of Drosophila insulator components and are using this system to explore dependencies on chromatin remodelling enzymes. 
 
Description Cell-specific hemocyte labelling methods 
Organisation Umea University
Country Sweden 
Sector Academic/University 
PI Contribution We have adapted protein labelling methods for single particle tracking (SPT) developed as part of BBSRC USA Collaboration award to generated new methods for labelling of hemocyte (macrophage) subsets in Drosophila. We have expertise using mouse CD8-GFP fusions to label the cell surface of hemocytes allowing both fluorescent visualisation by microscopy and FACS as well as bulk phase cell isolation using anti-CD8 beads. However use of GFP has restricted versatility for FACS where labels that are spectrally distinct would have great benefit. We have adapted our SPT labelling methods to generate mCD8-HALO and mCD8-Snap tags that can be expressed on hemocytes and hemocyte cell subsets. Like the parental mCD8-GFP fusion these can be used both for fluorescent visualisation as well as bulk phase cell isolation, but have the great benefit of offering flexible labelling that can be achieved by simply altering the choice of fluorophore used as HALO- and Snap-ligand. We have mined CAGE datasets generated during previous analysis of wild-type and NURF mutant hemocytes to identify new hemocyte specific genes, promoters of which are used to drive mCD8-HALO and mCD8-Snap expression.
Collaborator Contribution The Hultmark laboratory (principally Ines Anderl) has developed a model infection assay that uses GFP and mCherry reporters to discriminate cell fate change during a model immune response. This assay relies on FACS analysis of fluorophore expression. Use of mCD8-HALO and mCD8-Snap tags expressed under the control of cell-specific promoters allows greater resolution of cell state intermediates during this model immune differentiation.
Impact Hemocyte specific mCD8-HALO and mCD8-Snap labels for fluorescent visualisation by microscopy and FACS as well as bulk phase cell isolation of hemocytes and hemocyte subsets.
Start Year 2016
 
Description NURF live imaging 
Organisation Howard Hughes Medical Institute
Country United States 
Sector Charity/Non Profit 
PI Contribution Generation of tagged strains allowing single molecule imaging of chromatin remodelling enzymes and insulator proteins. We have generated HALO-tagged variants of the ISWI type chromatin remodelling complexes NURF, ACF and insulator proteins CP190, CTCF and SuHw. We have also used these to examine dynamics of chromatin association of remodellers and insulators in live Drosophila cells. We have developed a facile method for extracting and immobilising primary cells of a single cell type from Drosophila larvae. This allows enhanced comparison of binding kinetics of chromatin proteins by providing a consistent and reproducible system for analysis. We have also generated mutated variants of the NURF chromatin remodelling complex to discriminate the function of histone binding in stable recruitment of remodellers. We are using this system to explore the dynamics of NURF-histone variant H2A.Z interaction. We are analysing NURF dynamics in H2A.Z mutants and vice versa as well as dynamics of interaction between NURF and H2A.Z to provide molecular basis of genetic and transcriptome interactions between NURF and H2A.Z that we have shown in other work in the laboratory.
Collaborator Contribution Access to microscope facilities and dye labels allowing single molecule imaging. In particular the provision of JF549 and JF646 HALO ligands. Collaborators have generated HALO-tagged variants of the the histone variants H2A.Z and the core Drosophila transcription factor GAGA. They are using our cell isolation and processing methods to examine binding kinetics of these components.
Impact Visualisation of chromatin remodelling enzyme and insulator protein localisation and dynamics in live cells. Single particle tracking experiments using collaborators microscopes reveal clear differences in lifetime between stable association of insulators and more transient interaction of remodellers. Within remodeling factor families SPT has allowed distinct categories of chromatin interaction to be discriminated. Mutational analysis of NURF complexes reveal key requirement of histone-tail recognition in targeting of remodellers to chromatin. This research is currently being submitted together with data on the elucidation of the full-spectrum of histone modifications bound by NURF. A manuscript surveying ISWI family binding kinetics as demonstrated by SPT is in preparation.
Start Year 2015
 
Description NURF sructural analysis and cryo-EM 
Organisation University of Leicester
Country United Kingdom 
Sector Academic/University 
PI Contribution Expression and purification chromatin reader domains on NURF complex. Isolation of tagged NURF complexes from embryos for gyro-EM
Collaborator Contribution Generation of crystals for X-ray diffraction for isolated NURF reader domains. Cryo-EM analysis of particles of NURF complexes to resolve structural information of NURF complexes ion isolation or engaged with nucleosomes
Impact Initial stages of collaboration
Start Year 2019
 
Description Nucleosome position and transcriptional splice site selection 
Organisation University of Birmingham
Department School of Biosciences
Country United Kingdom 
Sector Academic/University 
PI Contribution Nucleosome maps of hemocytes have been used to investigate effect of chromatin organisation on transcriptional splice site selection. We are generating nucleosome maps for S2 and Kc cell lines to compliment transcription splicing analysis that has been performed on these cell lines in parallel by the Soller laboratory
Collaborator Contribution Splice site selection analysis in S2, Kc cells and in primary hemocytes
Impact Collaboration is recently initiated and ongoing.
Start Year 2018
 
Description Single particle tracking 
Organisation Johns Hopkins University
Department Department of Biology
Country United States 
Sector Academic/University 
PI Contribution Collaborators in the Wu lab have developed custom microscopes for single particle tracking (SPT) of nuclear proteins. We have used these to monitor dynamics of chromatin remodelling insulator proteins in Drosophila hemocytes. Travel to JHU Department of Biology to use these facilities has been supported by BBSRC USA Collaboration travel funding.
Collaborator Contribution Collaborators in the Wu lab have developed custom microscopes for single particle tracking (SPT) of nuclear proteins. They have also generated HALO-tagged Drosophila strains for the H2A.Z histone variant.
Impact Determination of dynamics of remodelling complex members and insulator proteins in vivo.
Start Year 2016
 
Description Lab tours/tasters 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Undergraduate students
Results and Impact Undergraduate research taster activities include guided lab tours for BSc Medical Science and MbChB students. Provide students with experience of working laboratory environment as well as broad overview of research area and future directions of research in the field
Year(s) Of Engagement Activity 2016,2017,2018