Regulation of the transcription cycle by co-ordinate interaction of ATP-dependent chromatin remodelling and histone post-translational modifications.
Lead Research Organisation:
University of Birmingham
Department Name: Immunity and Infection
Abstract
The development and identity of all cells in the body is determined by a set of instructions encoded in genes, found in DNA in the cell nucleus. 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, such as humans, DNA is folded and compacted into manageable units by wrapping like thread around a spool composed of proteins called histones, to form chromatin. These manageable units, called nucleosomes, offer an additional level of information to the cell as the histone proteins at their core can be selectively modified by the incorporation of acetate, phosphate, methyl or other chemical groups. These histone post-translational modifications (HPTMs) can act as so-called epigenetic "marks" to encode additional information in the genome. These marks can function to tether or recruit enzyme complexes that either silence or allow gene expression. By varying the distribution of histone post-translational modifications, and their ability to be read or decoded, genes can be selectively turned off or on in cells, controlling the development and function of cells. We seek to understand how this occurs as many human diseases, such as cancers and lymphomas, are triggered by altered or disordered gene expression. By understanding these epigenetic mechanisms of gene regulation, new therapies to cure disease can be developed.
In our research, we use both human cell lines and the "model organism" Drosophila melanogaster (fruit flies). Although at first glance it may not seem so, Drosophila and humans have evolved from a common ancestor and thus share many design principals. A useful analogy is to compare a high-performance racing car and a child's go-kart. Although one is more sophisticated, the basic elements of control - steering and brakes - are the same. In the same way, 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 interpret epigenetic marks to determine their function in gene regulation.
In this study we will determine how the distribution of a key epigenetic regulator called NURF is affected by histone post-translational modifications. We will determine how NURF then alters the positions of nucleosomes. By changing the position of nucleosomes NURF can affect the interaction of RNA polymerase (the enzyme that "expresses" genes) with DNA. To do this we will use a technique called chromatin immunoprecipitation (ChIP) to fish-out regions of the Drosophila genome to which NURF is targeted and identify these by determining their DNA sequence using a DNA sequencer that can sequence millions of DNA fragments in one go. We will examine whether NURF recruitment correlates with the presence of histone-modifications. Subsequently we will determine whether NURF recruitment to these regions is able to affect the activities of RNA polymerase by using the same technique of ChIP-Seq to map the distribution of RNA polymerase on genes in cells that contain or lack the NURF regulator and the histone modifications to which NURF can bind.
In our research, we use both human cell lines and the "model organism" Drosophila melanogaster (fruit flies). Although at first glance it may not seem so, Drosophila and humans have evolved from a common ancestor and thus share many design principals. A useful analogy is to compare a high-performance racing car and a child's go-kart. Although one is more sophisticated, the basic elements of control - steering and brakes - are the same. In the same way, 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 interpret epigenetic marks to determine their function in gene regulation.
In this study we will determine how the distribution of a key epigenetic regulator called NURF is affected by histone post-translational modifications. We will determine how NURF then alters the positions of nucleosomes. By changing the position of nucleosomes NURF can affect the interaction of RNA polymerase (the enzyme that "expresses" genes) with DNA. To do this we will use a technique called chromatin immunoprecipitation (ChIP) to fish-out regions of the Drosophila genome to which NURF is targeted and identify these by determining their DNA sequence using a DNA sequencer that can sequence millions of DNA fragments in one go. We will examine whether NURF recruitment correlates with the presence of histone-modifications. Subsequently we will determine whether NURF recruitment to these regions is able to affect the activities of RNA polymerase by using the same technique of ChIP-Seq to map the distribution of RNA polymerase on genes in cells that contain or lack the NURF regulator and the histone modifications to which NURF can bind.
Technical Summary
Regulated changes in chromatin structure and dynamics have a key role in controlling transcription by restricting RNA polymerase II (Pol II) recruitment to normal transcription start sites (TSSs) at the 5' end genes, and controlling the processive elongation of Pol II into the gene body. Chromatin dynamics are controlled by the concerted actions of ATP-dependent chromatin remodelling enzymes and histone post-translational modifications (HPTMs). In this proposal I will investigate how HPTMs act as molecular rheostats to control binding of an ATP-dependent chromatin remodeler (NURF), and how NURF regulates both transcription elongation and the suppression of cryptic initiation. I have developed experimental methods to knock-out chromatin regulators specifically in Drosophila macrophages, and then purify sufficient quantities of these cells for high-resolution ChIP-Sequencing and transcriptome analysis. I will exploit this system to:
i. Examine how a combination of the H3K4me3, H3K9Ac, H3S10p and H4K16Ac HPTMs control recruitment of NURF to the +1 nucleosome on active genes.
ii. Determine function of H3K9Ac, H3S10p and H4K16Ac on NURF recruitment by knocking-out the H3K9 and H4K16 histone acetyltransferases and H3S10 kinase.
iii. Map Pol II distributions in control and NURF knock-out macrophages to determine NURF functions in elongation and cryptic initiation.
iv. Use total RNA-Seq and CAGE-Seq to show changes in transcription elongation and cryptic initiation in NURF knock-out macrophages.
v. Confirm that loss of NURF alters recruitment of components of the Set2/Rpd3 pathway that suppresses cryptic initiation in the wake of elongating Pol II.
This research will show how combinations of HPTMs act as rheostats to dynamically tune chromatin remodelling by NURF and control transcription. Understanding the interplay between HPTMs, chromatin remodelling and Pol II activity will help in the development of new strategies for therapeutic intervention in disease.
i. Examine how a combination of the H3K4me3, H3K9Ac, H3S10p and H4K16Ac HPTMs control recruitment of NURF to the +1 nucleosome on active genes.
ii. Determine function of H3K9Ac, H3S10p and H4K16Ac on NURF recruitment by knocking-out the H3K9 and H4K16 histone acetyltransferases and H3S10 kinase.
iii. Map Pol II distributions in control and NURF knock-out macrophages to determine NURF functions in elongation and cryptic initiation.
iv. Use total RNA-Seq and CAGE-Seq to show changes in transcription elongation and cryptic initiation in NURF knock-out macrophages.
v. Confirm that loss of NURF alters recruitment of components of the Set2/Rpd3 pathway that suppresses cryptic initiation in the wake of elongating Pol II.
This research will show how combinations of HPTMs act as rheostats to dynamically tune chromatin remodelling by NURF and control transcription. Understanding the interplay between HPTMs, chromatin remodelling and Pol II activity will help in the development of new strategies for therapeutic intervention in disease.
Planned Impact
Our research aims to provide evidence for existence of combinations of histone post-translational modifications (HPTMs) that can act as rheostats to regulate recruitment of nuclear factors to chromatin. This data enhances the knowledge economy by providing new insights into the lexicon of the epigenetic code. Data generated here will have obvious benefit to researchers in the field of chromatin and epigenetics, including not only our existing collaborators, partners in the EpiCentre epigenetics consortium in Birmingham, but a substantial global body of researchers. The size of this community is indicated by the growing number of research publications that have been generated in this field, 30,164 to date.
This research benefits workers in disease fields such as Cancer Biology and Inflammation. HPTMs and chromatin remodelling and modifying enzymes that interpret them are emerging disease targets. A survey by Business Insights, "Innovations in Epigenetics: Advances in Technologies, Diagnostics & Therapeutics," deduced that the current market in so-called epigenetic medicine to be valued $560 million, derived from the sale of the anticancer products Dacogen, Vidaza, and Zolinza, with 30 epigenetic drugs under development. Our research offers an alternative way to target HPTMs and disease. Rather than removing marks it may be feasible to mask HPTMs by modification of flanking residues or by competing interactions with histone tail mimics - multiply modified tails with enhanced affinity for domains. These approaches offer attractive new paradigms for epigenetic medicine that have will broad general interest. By opening up new potential avenues to target chromatin pathways, our research will have considerable impact in this lucrative market.
A clear route to exploit advances made during our research is through our existing contacts with industry. We have links (including joint MRC CASE PhD fellowship applications) with the company Cellzome, which utilizes Episphere bead technology to study nuclear factors. Through our current CRUK-funded research we have links to Cancer Research Technology (CRUK's technology transfer arm). 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 centers of the "Translational Research Partnerships" previously known as "Therapeutic Capability Clusters". 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 by supporting UK-based centers of excellence. A key remit of our sequencing core is to use high-throughput sequencing to understand the control and function of the epigenome. This, in concert with the Birmingham EpiCentre 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 that will be critical to the development of the field of epigenetic therapeutics.
This research benefits workers in disease fields such as Cancer Biology and Inflammation. HPTMs and chromatin remodelling and modifying enzymes that interpret them are emerging disease targets. A survey by Business Insights, "Innovations in Epigenetics: Advances in Technologies, Diagnostics & Therapeutics," deduced that the current market in so-called epigenetic medicine to be valued $560 million, derived from the sale of the anticancer products Dacogen, Vidaza, and Zolinza, with 30 epigenetic drugs under development. Our research offers an alternative way to target HPTMs and disease. Rather than removing marks it may be feasible to mask HPTMs by modification of flanking residues or by competing interactions with histone tail mimics - multiply modified tails with enhanced affinity for domains. These approaches offer attractive new paradigms for epigenetic medicine that have will broad general interest. By opening up new potential avenues to target chromatin pathways, our research will have considerable impact in this lucrative market.
A clear route to exploit advances made during our research is through our existing contacts with industry. We have links (including joint MRC CASE PhD fellowship applications) with the company Cellzome, which utilizes Episphere bead technology to study nuclear factors. Through our current CRUK-funded research we have links to Cancer Research Technology (CRUK's technology transfer arm). 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 centers of the "Translational Research Partnerships" previously known as "Therapeutic Capability Clusters". 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 by supporting UK-based centers of excellence. A key remit of our sequencing core is to use high-throughput sequencing to understand the control and function of the epigenome. This, in concert with the Birmingham EpiCentre 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 that will be critical to the development of the field of epigenetic therapeutics.
Organisations
- University of Birmingham (Lead Research Organisation)
- EVOTEC (Collaboration)
- Howard Hughes Medical Institute (Collaboration)
- UNIVERSITY OF LEICESTER (Collaboration)
- Promega (Collaboration)
- Johns Hopkins University (Collaboration)
- UNIVERSITY OF BIRMINGHAM (Collaboration)
- University of Minnesota (Collaboration)
- Umea University (Collaboration)
Publications
Bruns Tony
(2015)
CMV infection of human sinusoidal endothelium regulates hepatic T cell recruitment and activation
in JOURNAL OF HEPATOLOGY
Zimmermann HW
(2016)
Bidirectional transendothelial migration of monocytes across hepatic sinusoidal endothelium shapes monocyte differentiation and regulates the balance between immunity and tolerance in liver.
in Hepatology (Baltimore, Md.)
| Description | The overall objective of the grant was to investigate how histone post-translational modifications influence recruitment of the NURF chromatin remodelling enzyme to promoter associated nucleosomes on active genes and how this impacts transcription of genes. Our data defined key role of ten histone post-translational modifications that together uniquely decorate that +1 nucleosome of active genes. Combinations of these histone marks are "read" or bound through three protein domains on the NURF chromatin remodelling complex. We have define structurally how one combination of marks - histone H3K4me3, H3K9Ac and H3S10p - are recognised by the PHD2 domain through two separate binding pockets. Using a combination of high resolution genome mapping methods as well as single molecule imaging technology we developed during an associated BBSRC USA Collaboration award we show that these marks are required for recruitment of NURF to the +1 nucleosome and remodelling of the +1 nucleosome that assists in the passage of RNA polymerase during transcription elongation. The first objective of the grant was to examine how a combination of the H3K4me3, H3K9Ac, H3S10p and H4K16Ac HPTMs control recruitment of NURF to the +1 nucleosome on active genes. To this end we have used high-throughput sequencing/chromatin profiling methods to profile the distribution of histone post-translational modifications relative to binding of the NURF remodeler on active genes. We also used structural characterization by NMR to define binding pockets on the NURF chromatin remodeling enzyme that bind histone modifications to define structural cognates for histone tail recognition by chromatin remodelling enyzmes. Key findings and associated datasets generated as part of this objective included: i) Base-pair resolution mapping of NURF remodeler binding sites ii) Base-pair resolution mapping of nucleosomes decorated by the histone post-translational modifications H3K4me3, H3K9Ac, H3S10p, H3K9AcS10p and H4K16Ac. iii) Comparison of datasets to show the +1 nucleosome is decorated by combinations of these histone modifications that cumulatively mark the +1 nucleosome for NURF binding. iv) Identification of binding pockets on the NURF PHD2 finger for the rheostat histone modifications H3K9Ac and H3S10p. The second objective of the grant was to determine function of H3K9Ac, H3S10p and H4K16Ac on NURF recruitment by knocking-out the H3K9 and H4K16 histone acetyltransferases and H3S10 kinase. To explore this we have performed base-pair resolution mapping of NURF remodeler binding sites. This was facilitated by developing a novel mass culture and isolation method that allows recovery of large numbers of primary macrophages (100-150 million per preparation) from Drosophila larvae. We and also exploited single particle tracking/single molecule imaging methods developed in associated USA BBSRC Collaboration grants to examine how the dynamics of chromatin remodelers are influenced by histone post-translational modifications. Key findings and datasets generated as part of this objective included: i) Base-pair resolution mapping of NURF remodeler binding sites in wild-type and mutant hemocytes lacking H3K9Ac, H4K16Ac and H3S10p. ii) Determined binding kinetics in live cells of single wild-type NURF complexes as well as complexes that lack the ability to bind histone post-translation modifications. Mutant complexes included domain deletes as well as point mutations critical for H3K4me3 and rheostat recognition. iii) Corroborated SPT investigations using independent imaging methods - FRAP (fluorescence recovery after photobleaching) to determine chromatin biding characteristics of YFP-tagged NURF complexes iv) Examined NURF binding to polytene chromosomes to determine how loss of H3K9Ac, H4K16Ac and H3S10p affect NURF recruitment to specific gene loci. The third objective of the grant was to determine how lack of NURF affects RNA polymerase recruitment. We used our novel larval mass culture and macrophage isolation method to profile polII distribution in wild-type and NURF deficient primary macrophages. Key findings and datasets generated as part of this objective included: i) Base-pair resolution mapping of polII binding sites in wild-type and NURF mutant hemocytes. ii) Profiling changes in polII distribution in the absence of NURF iii) Determination that lack of NURF affects transcription primarily at the level of elongation. No effects on cryptic initiation were observed The fourth objective of the grant was to verify using CAGE-Seq whether changes in transcription elongation and cryptic initiation occur in NURF knock-out macrophages. We used our novel larval mass culture and macrophage isolation method to isolate sufficient macrophages of each genotype for CAGE profiling. Key findings and datasets generated as part of this objective included: i) CAGE-Seq datasets of hemocytes from wild-type and NURF mutant backgrounds. ii) Bioiformatic analysis showing no evidence of cryptic initiation in NURF deficient macrophages. iii) Changes in CAGE tag number indicating enhanced elongation rate in NURF deficient hemocytes were observed. The final objective of the grant was to determine whether loss of NURF altered recruitment of components of the Set2/Rpd3 pathway that suppresses cryptic initiation in the wake of elongating Pol II. Key findings and datasets generated as part of this objective included: i) Generation of custom polyclonal antibodies against the MRG15 subunit of the Rpd3(S) complex. ii) ChiP profiling of distribution of the Rpd3(S) subunit in wild-type and NURF-deficient macrophages |
| Exploitation Route | Particular outcomes of the award related to histone post-translational binding by NURF is currently being extended to the development of reader blocker drugs that can be used to down-regulate NURF function. Have engaged with industry partners mediated via the University of Birmingham drug discovery pipeline to validate NURF reader domains as drug targets, in turn motivating for the design of specific inhibitors of these binding pockets. Addition outcomes relate to fly macrophage CAGE datasets that are currently being used by the Drosophila innate immune community as a resource to define macrophage-specific transcripts to target gene expression and modification technologies specifically to immune-effector cells. |
| Sectors | Chemicals,Healthcare,Pharmaceuticals and Medical Biotechnology |
| Description | Research funded during this grant has helped identify a combination of histone modifications that are bound by the chromatin remodelling factor NURF and which are required for tight binding to chromatin. These include three core modifications that are bound separately by three reader domains as well as a suite of ten flanking modifications that act as rheostats that enforce tight chromatin binding. 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. We have however shown 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. We have structurally defined these interactions by NMR. We have used whole genome profiling by ChIP-Seq, chromatin pull-down and analysis of in vivo chromatin dynamics by FRAP and single particle tracking methods to show that recognition of these rheostat modifications is critical to recruitment and tight binding of the NURF remodeller in vivo. 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. |
| First Year Of Impact | 2017 |
| Sector | Chemicals,Healthcare,Pharmaceuticals and Medical Biotechnology |
| Impact Types | Economic |
| 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 | Anti- NURF301 antibody |
| Description | Rabbit polyclonal antibody that recognises the large specificity subunit of the NURF chromatin remodelling complex |
| Type Of Material | Antibody |
| Year Produced | 2016 |
| Provided To Others? | Yes |
| Impact | Reagent has allowed mapping of NURF chromatin distribution by ChIP-Seq and discrimination of in vivo targets of this remodelling enzyme |
| URL | http://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1005969#sec012 |
| Title | Anti-MRG15 antibodies |
| Description | Antibodies against the MRG15 subunit of the Rpd3(S) cryptic repression complex |
| Type Of Material | Antibody |
| Year Produced | 2016 |
| Provided To Others? | No |
| Impact | Has allowed ChIP-Seq mapping of the distribution of the Rpd3(S) cryptic repression machinery. |
| Title | Bulk isolation of hemocytes for ChIP and RNA-Seq analysis |
| Description | We have developed Drosophila strains that allow biotin tagging of nucleosomes in hemocytes and also membrane tagging of hemocytes. In combination with mass culture methods and rapid processing techniques these allow us to isolate 100-150 million cells or nucleosomes from these within 4 hrs. |
| Type Of Material | Technology assay or reagent |
| Provided To Others? | No |
| Impact | This has allowed profiling of chromatin marks and transcriptomes of primary hemocytes. We have also adapted this method to the isolation of chromatin and RNA from hemocyte subtypes |
| Title | CTAP-tagged NURF301 strains |
| Description | Transgenic Drosophila strains that express CTAP-tagged variants of the NURF chromatin remodelling complex for immunoprecipitation and immune-localisation |
| Type Of Material | Model of mechanisms or symptoms - non-mammalian in vivo |
| Year Produced | 2015 |
| Provided To Others? | No |
| Impact | Localisation of NURF complexes on polytene chromosomes. Discrimination of differential expression of NURF isoforms. |
| Title | Fluorescently tagged NURF301 Drosophila strains |
| Description | Transgenic Drosophila strains expressing CFP, YFP and GFP tagged variants of the NURF chromatin remodelling enzyme |
| Type Of Material | Model of mechanisms or symptoms - non-mammalian in vivo |
| Year Produced | 2014 |
| Provided To Others? | No |
| Impact | We have used this tool to map NURF binding sites on chromatin, explore requirements of histone modifications for NURF recruitment and used these strains as adjuncts to single particle tracking experiments to confirm in vivo dynamics of NURF remodellers by FRAP assay |
| 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 MNase mapping |
| Description | Method to fix and isolate hemocytes and prepare soluble chromatin for nucleosome extraction |
| Type Of Material | Technology assay or reagent |
| Provided To Others? | No |
| Impact | Used to map nucleosome targets of chromatin remodelling enzymes |
| Title | Hemocyte CAGE datasets |
| Description | CAGE transcriptome datasets from wild-type and NURF deficient hemocytes. |
| Type Of Material | Database/Collection of data |
| Year Produced | 2017 |
| Provided To Others? | No |
| Impact | Precise definition of transcription start-sites in wild-type and NURF deficient hemocytes have been determined using this data. This has allowed requirements of NURF for transcription initiation to be determined and effects on cryptic repression of transcription to be assayed. In addition the CAGE datasets provide a useful resource for the Drosophila immune community by allowing identification of fly immune-specific transcripts. We are using this resource to generate reagents for tagging and identification of blood cell sub-populations in flies |
| Title | Hemocyte chromatin profiling |
| Description | High-resolution profiles of histone H3K4me3 and histone variant H2A.Z distribution in Drosophila primary hemocytes |
| Type Of Material | Database/Collection of data |
| Year Produced | 2016 |
| Provided To Others? | Yes |
| Impact | Datasets allowed characterisation of role of histone modifications in recruitment of the NURF chromatin remodeller to be determined |
| URL | https://www.ebi.ac.uk/ena/data/view/PRJEB12941 |
| Title | Hemocyte nucleosome maps |
| Description | High-resolution, base-pair nucleosome maps of wild-type and NURF deficient hemocytes. Mononucleosomes were isolated from primary wild-type and NURF-mutant hemocytes and underlying DNA sequence determined to map precise nucleosome positions. |
| Type Of Material | Database/Collection of data |
| Year Produced | 2016 |
| Provided To Others? | Yes |
| Impact | Nucleosome maps allowed nucleosome targets of NURF chromatin remodeller to be determined. Wild-type nucleosome maps were first high-resolution map of nucleosome position in a metazoan species. These have been used to investigate nucleosome positioning around insulator and transcription factor binding sites to determine effect of chromatin proteins on nucleosome organisation. We are currently exploiting these maps to determine requirements of nucleosome position for transcription splice-site selection (collaboration with Soller lab). |
| URL | https://www.ebi.ac.uk/ena/data/view/PRJEB12941 |
| Title | Mapping of histone modifications in synchronised cells |
| Description | Mapping of H3K4me3 and H3T3pK4me3 marks in G2 synchronised Drosophila cells |
| Type Of Material | Database/Collection of data |
| Year Produced | 2016 |
| Provided To Others? | No |
| Impact | The H3T3p modification is a negative rheostat that blocks in vitro recognition of H3K4me3 by the NURF PHD2 domain. This ChIP dataset allows inhibitory role of H3T3p mark in vivo to be verified. |
| Title | Modified histone profiling of Drosophila cells |
| Description | Distribution of the histone modifications H3K4me3, H3K9Ac, H3K9AcS10p and H4K16Ac in S2 cells |
| Type Of Material | Database/Collection of data |
| Year Produced | 2016 |
| Provided To Others? | No |
| Impact | Mapping of histone modifications and comparison with NURF ChIP-Seq datasets allowed verification of role of histone modifications in NURF recruitment |
| Title | NURF301 ChIP-Seq datasets |
| Description | ChIP-Seq whole genome mapping of NURF301 distribution |
| Type Of Material | Database/Collection of data |
| Year Produced | 2016 |
| Provided To Others? | Yes |
| Impact | Data allowed integration of NURF distribution with activity (determined by nucleosome mapping). |
| URL | https://www.ebi.ac.uk/ena/data/view/ERS1163815 |
| 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 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 | 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 | 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 | 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 | Unravelling the enigma of why humans require seven distinct histone deacetylase complexes - implications for therapeutic intervention |
| Organisation | University of Leicester |
| Country | United Kingdom |
| Sector | Academic/University |
| PI Contribution | Genomics analysis of HALO-tagged HDAC lines |
| Collaborator Contribution | Structural analysis by X-ray crystallography and cryo-EM of HDAC complexes |
| Impact | Project initiates in Sept 2020 |
| Start Year | 2020 |
| Description | CRUK Race for life lab tours |
| Form Of Engagement Activity | Participation in an open day or visit at my research institution |
| Part Of Official Scheme? | No |
| Geographic Reach | National |
| Primary Audience | Supporters |
| Results and Impact | Lab tours for CRUK Race for Life volunteers and donors. Helped show funded research and usefulness of model organisms in medical research. |
| Year(s) Of Engagement Activity | 2013 |
| 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 |