Imaging in vivo chromatin dynamics in Drosophila.

Lead Research Organisation: University of Birmingham
Department Name: Immunity and Infection

Abstract

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Description The goal of this grant was to establish a system for live imaging of chromatin protein dynamics in primary cells isolated directly from biological niches. This was achieved as the grant facilitated reciprocal transfers of experimental methods between Birmingham University and Janelia Research Campus and latterly John's Hopkins University in the USA. In particular Badenhorst (Birmingham) provided methods for isolation and immobilization of a pure population of primary cells of a single cell type - hemocytes (Drosophila macrophages) - to use as a system for analysis of chromatin protein dynamics. The Lavis laboratory (Janelia Research Campus) provided bright, photostable, cell permeant Janelia Farm dyes to be used for labeling and visualization of single molecules of histone variants, insulator proteins and chromatin remodelling proteins in hemocytes. The Wu Laboratory (Janelia Research Campus and John's Hopkins University) provided imaging systems and data analysis methods to visualize and track single molecules of these factors in hemocytes. Badenhorst (Birmingham) generated transgenic strains expressing HALO-tagged variants of insulator proteins and chromatin remodelling complexes that can be labeled with JF dyes. Wu (Janelia Research Campus and John's Hopkins University) generated transgenic strains expressing HALO-tagged histone H2A.Z variants.

The specific objectives of the grant were firstly to examine histone variant H2A.Z dynamics in hemocytes. This was achieved using hemocyte isolation and immobilization methods provided by Badenhorst, labels provided by Lavis and imaging facilities provided by Wu.

The second objective to define the contribution of SRCAP/SWR-C chromatin remodelling complex subunits to H2A.Z dynamics is on-going using HALO-tagged strains developed by the Wu laboratory. This objective has also been expanded to include the contribution of the NURF remodelling complex to H2A.Z dynamics given genetic interactions between NURF and H2A.Z mutants observed in the Badenhorst laboratory.

The third objective was to investigate in vivo dynamics of recruitment of the NURF chromatin remodelling complex and has been achieved using NURF-HALO tagged constructs and single molecule imaging on Wu laboratory microscopes. We have also expanded this objective to include imaging dynamics of related ISWI-family chromatin remodelers and have gathered data on ACF complexes. We are currently generating HALO-tagged variants of other described ISWI complexes.

The fourth objective was to investigate effect of histone post-translational modifications on NURF recruitment. This has been achieved by examining in vivo dynamics of tagged NURF isoforms that contain or lack histone binding domains, have mutations in rheostat histone modification binding pockets and also of wild-type NURF complexes in cells deficient for histone modifications (genetic mutants in histone modification writer complexes).

The final objective was to investigate effect of chromatin remodelling enzymes to genome organisation. To address this goal we have generated HALO-tagged variants of the major Drosophila insulator protein CP190 and determined binding kinetics in wild-type hemocytes. We are expanding this objective to include other insulator proteins CTCF, SuHw and BEAF and are in the process of introducing these strains into NURF mutant backgrounds to determining insulator dynamics in remodeller-deficient blood cells. We anticipate completion by the end of the grant period.

The award of this grant has made a significant contribution to our research by opening up a vital new avenue of research in chromatin biology. In particular the system for single molecule imaging/single particle tracking we have established in collaboration with Janelia Research Campus and John's Hopkins University provides a facile method for interrogating the dynamics of chromatin binding of transcription factors, histones variants and insulator proteins in live single cells. It provides a much-needed compliment to existing genomics technologies like ChIP-Sequencing that while providing high-resolution maps of chromatin landscapes, generate static, time- and population-averaged readouts. By capturing the dynamic properties of chromatin proteins both in wild type and genetic mutant backgrounds allows regulatory hierarchies to be better defined and intermediate states in biological processes to be captured.

Moreover this award has contributed more widely by serving as demonstrator of the power of single molecule imaging and single particle tracking methods to analyze biological processes. It has enabled the transfer of reagents and skills to Birmingham University and helped drive the acquisition of new single particle imaging facilities by the Medical School. Labelling and imaging approaches and analysis techniques established during this award provide a skills base that can be disseminated to a broad spectrum of UK users of this facility working on problems as diverse as chromatin organisation, membrane receptor signaling and transcriptional splicing, furthering the UK science base.

Finally, the travel facilitated by this award has provided the opportunity to make additional research contacts both Janelia Research Campus and John's Hopkins University beyond our current collaboration partners and has served to leverage additional collaborative research beyond the scope of this grant. As examples, an ongoing collaborative research project with John's Hopkins University Biophysics Department to determine the crystal structure of histone modification-reader interactions and collaborations to use HALO-tagging systems and Janelia Farm dyes for labeling of cell types for FACS sorting of Drosophila macrophage sub-populations.
Exploitation Route Chromatin remodeling enzymes are emerging drug targets for therapeutic intervention in cancer. New families of reader blocking drugs, target these type of complexes and block their association with chromatin. Single molecule imaging techniques we develop here could be used in the characterisation and classification of these drugs in assays for functional activity.

This award has enabled the transfer of reagents and skills to UK researchers and helped drive the acquisition of new single particle imaging facilities by Birmingham Uiversity. Labelling and imaging approaches and analysis techniques established during this award provide a skills base that can be disseminated to a broad spectrum of UK users of this facility working on problems as diverse as chromatin organisation, membrane receptor signaling and transcriptional splicing, furthering the UK science base.

Research conducted during this grant has established a facile system for interrogating the dynamics of chromatin binding of transcription factors, histones variants and insulator proteins in live single cells. This provides a compliment to existing high-resolution genomics mapping technologies that provide only static, time- and population-averaged readouts of chromatin processes. Capturing the dynamic properties of chromatin proteins both in wild type and genetic mutant backgrounds will allow regulatory hierarchies to be better defined and intermediate states in biological processes to be captured.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology
 
Description The award of a BBSRC USA collaboration award was instrumental in providing us access to advance single molecule labelling and imaging tools and technology. This included access to bright, photostable and cell permeant dyes - Janelia Farm (JF dyes) - that have allowed us to label and visualise single molecules of the chromatin remodelling complex NURF in live cell. In addition it provided access to custom microscopes that have enabled imaging and tracking of the dynamics of NURF in live cells to be performed (initially at Janelia Research Campus, more recently at John's Hopkins University and in the future at Birmingham University). Using this technology we have been able to discriminate how histone modifications affect dynamics of chromatin targeting of epigenetic readers like the chromatin remodelling factor NURF. Epigenetic readers are emerging drug targets for therapeutic intervention in diseases like chronic inflammation and cancer. The best characterised of these are BET inhibitors which bind Bromodomains to block acetylated histone tail recognition. NURF contains three reader domains: a Bromodomain (that is potentially targetable by BET-like inhibitors) as well as two PHD fingers. We have shown the PHD2 and PHD1 fingers recognise the primary marks histone H3K4me3 and H3K23me3 respectively. Trimethylated histone tail recognition by PHD fingers is mediated through a well-described aromatic cage of tyrosine and tryptophan residues that, while well defined-structurally, has proven to be difficult to target through small molecule inhibitors. In fact it has been argued that PHD fingers are not generally draggable targets. Our single particle tracking experiments - using wild-type NURF as well as mutated NURF variants - show that trimethylated histone tail recognition by the PHD1 and PHD2 domains is supplemented and stabilised by recognition of rheostat modifications by structurally distinct pockets on both the PHD1 and PHD2 fingers. This is a vital result as it provides an alternative, and druggable, pocket through which to influence NURF-histone tail interactions using small-molecule inhibitors. Emerging data from human studies indicate that levels of human NURF are elevated in a number of cancers, including breast and prostate cancer and melanoma. Interventions that knock-down NURF levels appear to have benefit in treatment of these cancers. Our histone modification reader studies have identified targets for drug discovery to identify small molecule inhibitors of the reader-rheostat interactions. We are attempting to source extended funding through Confidence-in-concept funding to validate these rheostat recognition pockets as targets for drug discovery. An additional outcome of this award has been a new collaboration with Umea University to adapt protein labelling methods for single particle tracking developed during this award to generated new methods for labelling of hemocyte (macrophage) subsets in Drosophila. We had already developed a system 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 offer enhanced flexibility in 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. Finally a lasting impact of this award has been its use as a demonstrator of the power of single molecule imaging and single particle tracking methods in the analysis of in vivo protein dynamics. This award provided a conduit for the transfer of reagents and skills from the USA to Birmingham University and served partly, in combination with recent appointments at Birmingham University, to drive the acquisition of new imaging facilities by the Medical School. An enhanced 4-color single particle tracking system has been purchased and has been operational since September 2018. We have used in house facilities to characterise chromatin dynamics of insulator proteins and are working through systematic analysis of Drosophila insulator proteins and their dependencies on other insulator complex components. Labelling and imaging approaches and analysis techniques acquired during the course of this award are then being disseminated to a broad spectrum of users of this facility working on problems as diverse as chromatin organisation, membrane receptor signaling and transcriptional splicing, furthering the UK science base. We have used this system to characterise the in vivo chromatin binding kinetics of 4 Drosophila insulator proteins: CP190, CTCF, BEAF and SuHW. This has allowed functional discrimination of distinct mechanisms of activity amongst the core Drosophila insulator proteins. Halo-technology used in this proposal has also been adapted to allow high-resolution genomic mapping of insulator proteins through the generation of HALO-ligandable Tn5 transposes.
First Year Of Impact 2016
Sector Chemicals,Healthcare,Pharmaceuticals and Medical Biotechnology
 
Description BBSRC IAA
Amount £29,659 (GBP)
Funding ID 21599 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 03/2020 
End 10/2020
 
Description Responsive Mode Project Grant
Amount £585,980 (GBP)
Funding ID BB/P021816/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2017 
End 09/2020
 
Description Small molecule inhibitors of the epigenetic reader BPTF as onco-therapeutics
Amount £59,685,201 (GBP)
Funding ID 1002829 
Organisation Evotec 
Sector Private
Country Germany
Start 01/2022 
End 07/2026
 
Title Fly macrophage isolation for live imaging of HALO-tagged proteins 
Description We have developed methods for isolation of primary macrophages and immobilisation on supports for live imaging. 
Type Of Material Technology assay or reagent 
Year Produced 2015 
Provided To Others? Yes  
Impact Procedure has allowed the first visualisation of chromatin remodeller single molecule dynamics in metazoan systems 
 
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 Halo-liganded Tn5 transpose 
Description Production of a Halo-liganded hyperactive Tn5 transposes that can be covalently linked to Halo-tagged proteins (Transcription factors and epigenetic regulators) to allow "cut 'n tag" based profiling of genomic distribution of Halo-tagged proteins. 
Type Of Material Technology assay or reagent 
Year Produced 2022 
Provided To Others? No  
Impact Ability to profile at high resolution that genomic distribution of drosophila insulator and chromatin remodelling complexes. Potentially generalisable to any Halo-tagged nuclear protein. 
 
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 HALT (Halo-liganded transposase tagging): a novel technique for high-resolution whole-genome profiling of dynamic epigenetic regulators 
Organisation Promega
Country United States 
Sector Private 
PI Contribution Synthesis and purification of hyperactive Tn5 transpose that is conjugated to HALO ligand. Allows transposes tagging/mapping of HALO-tagged transcription factors and chromatin remodellers.
Collaborator Contribution Synthesis of HALO-ligand derivative that can be chemically conjugated to Tn5 transposes.
Impact Project is still ongoing.
Start Year 2021
 
Description NURF histone H3K4me3 interaction 
Organisation National Institutes of Health (NIH)
Country United States 
Sector Public 
PI Contribution Showed interactions between Drosophila NURF and the histone H3K4me3 mark
Collaborator Contribution Identified interaction between NURF and H3K4me3
Impact PMID: 16728976 PMID: 19629165
Start Year 2006
 
Description NURF histone H3K4me3 interaction 
Organisation Rockefeller University
Country United States 
Sector Academic/University 
PI Contribution Showed interactions between Drosophila NURF and the histone H3K4me3 mark
Collaborator Contribution Identified interaction between NURF and H3K4me3
Impact PMID: 16728976 PMID: 19629165
Start Year 2006
 
Description NURF inhibitors 
Organisation University of Minnesota
Department Department of Applied Economics
Country United States 
Sector Academic/University 
PI Contribution Validation of NURF bromodomain-reader blockers
Collaborator Contribution Design and synthesis of chemical inhibitors of the NURF301/BPTF bromodomain histone H3K16Ac interaction
Impact Ongoing collaboration. Long term potential cancer therapeutics
Start Year 2017
 
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 Role of bromodomain inhibitor on NURF recruitment 
Organisation University of Minnesota
Country United States 
Sector Academic/University 
PI Contribution Screening of novel BPTF bromodomain inhibitors using single particle tracking assays and primary spermatocyte phenotypes.
Collaborator Contribution Provision of custom synthesised inhibitors of BPTF bromomodomains
Impact Collaboration has just commenced
Start Year 2022
 
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 Small molecule inhibitors of the epigenetic reader BPTF as onco-therapeutics 
Organisation Evotec
Country Germany 
Sector Private 
PI Contribution Design and testing of small molecule inhibitors of BPTF PHD domain recognition of histone tail modifications. Assay of effectiveness of inhibitors against a panel of melanoma and triple negative breast cancer cell lines. Functional characterisation of BPTF knockdown cell lines.
Collaborator Contribution Synthesis and toxicity screening of novel compounds. Structural characterisation of drug interactions with PHD domains.
Impact Programme has just commenced
Start Year 2022
 
Description Structure and dynamics of histone modification-reader interactions 
Organisation Johns Hopkins University
Country United States 
Sector Academic/University 
PI Contribution We have defined novel histone modifications bound by the NURF chromatin remodelling enzyme. Function of these in recruitment of remodellers to targets in chromatin is being determined by a combination of single particle tracking (SPT) methods (developed as part of the USA BBSRC Collaboration award) as well as chip-sea chromatin profiling methods. We are using SPT to examine residence times of NURF remodellers in the presence and absence of targeting histone modifications. This is achieved by tagging wild-type and mutated complexes as well as behaviours of wild-type complexes in mutant Drosophila backgrounds that lack enzymes that put histone modifications in place.
Collaborator Contribution The Bowman lab are experts in X-ray crystallography of chromatin associated proteins. They are determining structure of NURF reader domains in complex with histone modifications identified in our work. This collaboration was initiated as a result of interactions enabled by travel funded by our USA Collaboration award.
Impact Crystals of the NURF PHD2 finger in combination with triply-modified histone tail peptides have been generated. Structural determination is underway. HALO-Tagged NURF complexes wicket-type or mutant for amino acids required for histone tail recognition have been generated and transgenic lines that express these have been generated. Mutant complexes that lack tail recognition entirely have also been generated and dynamics determined by single particle tracking (using USA Collaboration grant funded travel). Data indicates clear requirement of histone tail recognition in stable chromatin binding.
Start Year 2017
 
Description Birmingham Green Heart Festival 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Stand at Birmingham Green Heart Festival in which general public was invited to the University of Birmingham to see full spectrum of research on offer. Approximately 3000 people visited during the day. Our stand demonstrated the use of Drosophila as a model for human disease.
Year(s) Of Engagement Activity 2019
 
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