Supervisors and Projects

Project Code: M1

Research theme: Using novel experimental and computational approaches to understand adaptive immunity across disease contexts to defined novel therapeutic targets

We are working on deciphering how the adaptive immune system can be used as diagnostics and therapeutics. We are investigating how defects in the ability to mount effective immune responses lead to infectious disease susceptibility, impaired surveillance of cancer and immunodeficiencies, compared to the mechanisms leading to a breakdown of immunological tolerance causing autoimmune diseases. This will lead us to:

o understand why certain individuals are at greater risk of developing immunological disease

o highlight key interactions of immune cells within sites of inflammation or disease

o define novel therapeutic targets or optimal therapeutic combinations

o identify blood biomarkers immune status

o stratify patients for improved clinical management

This is being achieved through the development and application of novel experimental and computational approaches, working in partnership with a global network of clinicians, immunologists and sample cohorts. We prioritise translational- and patient-focussed research to work towards bridging fundamental biology to early phase clinical trials.

How are different B cell populations developmentally linked in human health and disease?

We are investigating the generation, function and plasticity of B cell populations in human health. In particular, we are interested in how different lymphocyte subsets are developmentally linked and differences in function, and therefore providing a platform to understand how B cell fate may be different in human disease. We are defining how B cells select a particular developmental pathway, and will use this information to develop methods for modulating B cell function as potential therapeutic approaches.

How can B and T cells may be therapeutically modulated across cancers and autoimmune diseases

There is accumulating evidence for the role of both T and B cells in modulating immune responses to both solid tumours and haematological malignancies. We are investigating the contributions, function and heterogeneity of B and T cells on the immune responses to tumours and their potential role in cancer detection and treatment. We are determining the nature of B and T cell immuno-surveillance, regulation and activation across cancers and autoimmune diseases, as well as the immunological features associated with better prognosis and immunomodulation. With this, we aim to highlight novel therapeutic avenues. Our lab is affiliated with the Oxford Cancer Centre ( https://www.cancer.ox.ac.uk/research/research-themes/developments-in-immuno-oncology ) and non-cancer clinicians, with strong clinical links to a wide range of hard-to-treat diseases.

What is the effect of genetic and environmental variation on B and T cell fate?

Immunological health relies on a balance between the ability to mount an immune response against potential pathogens and tolerance to self. B and T cells are key to the immune response by producing antibodies and cytotoxic T cells. B/T cell clones selectively expand following antigen recognition by B and T cell receptors (BCR and TCR) respectively. BCRs are the membrane-form of antibodies and are generated through DNA recombination resulting in the potential to recognise a vast array of pathogens. Defects in the ability to mount effective B cell or T cell responses have been implicated in infectious susceptibility, impaired surveillance of cancer and immunodeficiencies, whereas a breakdown of immunological tolerance has been attributed to autoimmune diseases such as through autoantibody production and reduced numbers of regulatory B/T cells. Through integrating genomics, bulk and single-cell transcriptomics, and metabolomics data, serological, B /T cell repertoire and viromics datasets we will investigate the effect of both genetic variation and environmental factors on B cell fate, regulation, and the relationship to disease susceptibility.

Technology Development

We aim to develop novel experimental and computational tools to investigate the function of immune responses through advances in high-throughput and genetic technologies. These technologies can be readily applied to existing cohorts to investigate the immune system from unique perspectives.

Associate Prof Rachael Bashford-Rogers | Biochemistry (ox.ac.uk)

For informal enquiries: rachael.bashford-rogers@bioch.ox.ac.uk 

Project Code M2

Building models of infection and commensalism for electron cryotomography

This research aims to understand how bacterial responses to other cells and small molecules such as antibiotics depend on their environment. Due to their size, bacteria can be challenging to study by light microscopy. However, electron cryotomography (cryoET), which uses frozen hydrated samples to capture native structural details about cells, viruses and organelles at molecular resolution, is perfectly suited for imaging at this scale. In this project, we will adapt co-culture protocols for bacteria and mammalian cells to be suitable for cryoET. We will use a combination of mammalian growth factors and coatings with a solid medium approach for bacteria growth developed in the lab to produce spatially stabilised co-cultures suitable for cryoET and fluorescence microscopy. Initially, we will focus on models of bacterial infection and commensalism in the gut including immune and epithelial cells, but there is potential to be comparative with other microbial communities in the body, including the lungs and skin. In particular, the solid medium approach to bacterial sample preparation adapts itself well to studying the effects of the mucus layers, important in many gastrointestinal and respiratory tract diseases. Building on previous results from other projects in the lab, we will use mutations and knockout strains to understand how bacteria behave in response to their environment, especially after treatment with antimicrobial drugs.

Dr Lindsay Baker | Biochemistry (ox.ac.uk)

For informal enquiries: lindsay.baker@bioch.ox.ac.uk

Project Code M3

DNA repair, genome stability and cancer in humans

Integrity of the genome is critical to both the development and health of humans. Our chromosomes are constantly exposed to various types of DNA damage, which if unrepaired can cause diseases such as cancer. To meet these challenges, several DNA repair pathways have evolved. Our laboratory is focused on understanding how these pathways work in human cells. We have discovered several new proteins playing important roles in these pathways. By applying state-of-the-art techniques in biochemistry, molecular biology and cell biology, combined with world-class mass spectrometry and high-resolution live-cell imaging and structural biology, we are continuously elucidating the role of new components of the DNA repair pathways. Examples of methods used are recombinant protein purification, in vitro assays, CRISPR/Cas9 genome engineering, various cell-based assays including sophisticated live-cell imaging, and cryo-EM. Please contact Martin Cohn (martin.cohn@bioch.ox.ac.uk) for details on the newest and most interesting ongoing projects.

Recent papers published by DPhil students from our group:

Liang. C-C. and Cohn, M.A. (2021). Purification of DNA repair protein complexes from mammalian cells. STAR Protoc. 2(1):100348.

Socha, A., Yang, D., Bulsiewicz, A., Yaprianto, K., Kupculak, M., Liang. C-C., Hadjicharalambous, A., Wu, R., Gygi, S.P. and Cohn, M.A. (2020). WRNIP1 is recruited to DNA interstand crosslinks and promotes repair. Cell Rep. 32(1):107850.

Lopez-Martinez, D., Kupculak, M., Yang, D., Yoshikawa, Y., Liang, C-C., Wu, R., Gygi, S.P. and Cohn, M.A. (2019). Phosphorylation of FANCD2 inhibits the FANCD2/FANCI complex and suppresses the Fanconi anemia pathway in the absence of DNA damage. Cell Rep. 27(10):2990-3005.

Liang, C-C., Li, Z., Lopez-Martinez, D., Nicholson, W., Venien-Bryan, C. and Cohn, M.A. (2016). The FANCD2-FANCI complex is recruited to DNA interstrand crosslinks prior to monoubiquitination of FANCD2. Nature Commun. 7:12124.

Schwab, R., Nieminuszczy, J., Shah, F., Langton, J., Lopez Martinez, D., Liang, C-C., Cohn, M.A., Gibbons, R., Deans, A. and Niedzwiedz, W. (2015). The Fanconi anaemia pathway maintains genome stability by coordinating replication and transcription. Mol. Cell. 60(3):351-61.

Liang, C-C., Zhan, B., Yoshikawa, Y., Haas, W., Gygi, S.P. and Cohn, M.A. (2015). UHRF is a sensor for DNA interstrand crosslinks and recruits FANCD2 in the Fanconi Anemia pathway. Cell Rep. 10(12):1947-56.

For more information about the Cohn lab see: https://cohn.web.ox.ac.uk

Associate Professor Martin Cohn | Biochemistry (ox.ac.uk)

For informal enquires: martin.cohn@bioch.ox.ac.uk

Project Code M19

The role of glial post-transcriptional regulation in synaptic plasticity

The regulation of gene expression after transcription is central to many biological processes and it is particularly critical in the nervous system. Neuronal and glial cells are elongated with projections often some distance away from the nucleus. The rapid nature of synaptic growth, crucial to memory formation, requires expression of genes at these distal regions in a timely manner that would often preclude the transport of RNA or proteins from the nucleus. RNAs located at these peripheral locations in the cell, locally translated into proteins, are required for the cellular components needed to construct new synaptic connections.

While inter-neuronal synaptic connections form a key part of this system, it is becoming more apparent that glia are also centrally involved in a ‘tri-partite synapse’ system. Glia have been relatively poorly studied in comparison to neurons, yet they play a vital role in synaptic function. Previously considered to have a primarily structural role, glia are not understood to have signalling (both chemical and electrical) and other functions that are key to synaptic plasticity. Gaining a full understanding of the fundamental mechanisms of synaptic plasticity during memory and learning underpins research into neurodegenerative disorders. In both of these areas, the fruit fly, Drosophila, has proven an invaluable model, being much more experimentally accessible than its mammalian equivalent while expressing a large set of genes that are highly conserved. On-going work in the lab focuses on systematically examining the mechanisms of post transcriptional regulation in these processes, specifically, mRNA transport and localised translation, as well as mRNA stability and processing. The Davis lab is highly interdisciplinary, bringing together biologists, chemists, physicists and computer programmers to tackle fundamental biological questions.

Current research topics available for projects include:

• Screening for novel localising RNAs in the larval brain, glia and NMJ

A wide-ranging screen using single molecule FISH techniques to identify and characterise RNA that localise in the Drosophila nervous system. This work takes advantage of a library of YFP-protein trap fly-lines to systematically screen for both candidate protein and RNA localization.

• The role of translational regulation in Activity dependent synaptic plasticity

Synaptic plasticity is important as a proxy for memory and learning. This project combines electrophysiology protocols with optogenetics techniques and advanced imaging to address the role of translational regulation. In collaboration with MICRON we are developing a dedicated upright 3D-SIM super-resolution microscope to study protein-RNA interactions at the neuromuscular junction

Training opportunities : Work in the Davis lab involves routinely involves genetics (Drosophila), computational genomics, biochemistry, molecular biology, single molecule in situ hybridization techniques and advanced imaging. There is also the opportunity to be involved in developing imaging techniques and image analysis algorithms

For more information about the Davis lab see: www.ilandavis.com

Prof Ilan Davis | Biochemistry (ox.ac.uk)

For informal enquiries: ilan.davis@bioch.ox.ac.uk (please cc darragh.ennis@bioch.ox.ac.uk )

Project Code M4

Mechanistic understanding of the ubiquitin code during cell survival and programmed cell death

Use of structural biology techniques: cryo-EM, NMR and X-ray crystallography, along with biochemical assays and biophysical techniques (including SEC-MALS, ITC and MST), to understand at a molecular level how the ubiquitin code is responsible for maintaining the balance between cell survival and cell death. Our lab has recently determined the cryo-EM structure of a large ubiquitin ligase that regulates apoptosis. Current and future research projects will follow on this and other ubiquitin ligase complexes. In addition, we investigate how binding of non-degradative ubiquitin chains can activate master kinases of inflammation, and how deubiquitinating enzymes that reverse the ubiquitin code, are regulated.

For more information about the Elliott lab see https://elliottlab.web.ox.ac.uk

Dr Paul Elliott | Biochemistry (ox.ac.uk)

For informal enquiries: paul.elliott@bioch.ox.ac.uk

Project code M5

Molecular mechanisms underlying the function of ATPases involved in proofreading and disassembly of pre-mRNA splicing by the human spliceosome

Mammalian genes are transcribed into precursor messenger RNAs (pre-mRNAs), from which non-coding introns are spliced out in the nucleus before the mRNA is exported to the cytoplasm and translated into proteins. Introns expand proteomic diversity by allowing a single gene to encode multiple mRNA isoforms, coding for multiple protein isoforms with distinct activities. Introns are excised by the spliceosome – a dynamic assembly of RNA and proteins and splicing errors are implicated in up to 30% of human diseases.

The spliceosome assembles de novo on each pre-mRNA and catalyses two sequential transesterifications at a single RNA-based active site to excise a specific intron and ligate the flanking exons into mRNA ¹. During catalysis, several trans-acting ATPases modulate the transitions between different conformations spliceosome. These ATPases promote the exchange of reactants at the active site, allow exchange of protein factors that stabilize each conformation, and proofread fidelity of splice site choice². Following mRNA synthesis, the mRNA is released by the action of the ATPase Prp22, while the ATPase Prp43 disassembles the resulting intron-lariat spliceosome (ILS) to release the excised intron for degradation.

In the last six years, structures of different conformation of the yeast spliceosome have provided a molecular view of the basic mechanism of splicing in yeast, showing how the splice sites are recognised and how specific factors stabilize each catalytic conformation². The structure of the yeast post-catalytic spliceosome (P complex) suggested a mechanism by which Prp22 releases the mRNA, while the structure of the yeast ILS provided some structural insights into disassembly of the spliceosome by Prp43 and its associated co-factors³. Importantly, in yeast, proofreading of correct splice site usage by Prp22 is coupled to a discard pathway in which Prp43 disassembles spliceosomes that utilise incorrect splice sites and are rejected by Prp22^4,5 . It remains unclear where Prp43 binds in the human ILS⁶ and it is not known what specific RNA component of the spliceosome is targeted by Prp43 during discard and disassembly in mammals.

Although the active site and basic splice site recognition is conserved from yeast to humans, several additional ATPases associate with the human spliceosome and have been implicated in splicing fidelity^7,8. Indeed, in mammals fidelity of splice site choice often relies on recognition of only 1-2 nucleotides around highly variable splice sites, which must be balanced with alternative splicing. Thus, how Prp22, Prp43, and other mammalian ATPases act to safeguard splicing fidelity remains poorly understood.

We aim to establish new biochemical systems in vitro, and potentially in vivo, to study proofreading of splice site use and spliceosome disassembly in humans. Our goals are to identify the specific complexes involved in these processes and to use this system to trap intermediates during proofreading, discard, and disassembly. Electron cryomicroscopy will then be employed to obtain molecular insights into the mechanism of action of Prp22, Prp43, and associated co-factors. These structural studies will be complemented by biochemical assays and new CLIP RNA crosslinking and sequencing approaches⁹ to elucidate the mechanisms underlying proofreading of correct splice site choice.

References

1. Wilkinson, M. E., Charenton, C. & Nagai, K. RNA Splicing by the Spliceosome. Annual Review of Biochemistry 89, 1–30 (2019).

2. Fica, S. M. & Nagai, K. Cryo-electron microscopy snapshots of the spliceosome: structural insights into a dynamic ribonucleoprotein machine. Nature structural & molecular biology 24, 791–799 (2017).

3. Wan, R., Yan, C., Bai, R., Lei, J. & Shi, Y. Structure of an Intron Lariat Spliceosome from Saccharomyces cerevisiae. Cell 171, 120-132.e12 (2017).

4. Mayas, R. M., Maita, H., Semlow, D. R. & Staley, J. P. Spliceosome discards intermediates via the DEAH box ATPase Prp43p. Proceedings of the National Academy of Sciences 107, 10020–10025 (2010).

5. Mayas, R. M., Maita, H. & Staley, J. P. Exon ligation is proofread by the DExD/H-box ATPase Prp22p. Nature structural & molecular biology 13, 482–490 (2006).

6. Zhang, X. et al. Structures of the human spliceosomes before and after release of the ligated exon. Cell Research 29, 1–285 (2019).

7. Fica, S. M. Cryo-EM snapshots of the human spliceosome reveal structural adaptions for splicing regulation. Current Opinion in Structural Biology 65, 139–148 (2020).

8. Sales-Lee, J. et al. Coupling of spliceosome complexity to intron diversity. Biorxiv 2021.03.19.436190 (2021) doi:10.1101/2021.03.19.436190.

9. Strittmatter, L. M. et al. psiCLIP reveals dynamic RNA binding by DEAH-box helicases before and after exon ligation. Nat Commun 12, 1488 (2021).

For more information about the Fica lab see https://snrnpsplicingbiochemlab.web.ox.ac.uk

Dr Sebastian Fica | Biochemistry (ox.ac.uk)

For informal enquiries: sebastian.fica@bioch.ox.ac.uk

Project Code M6

Understanding and engineering microbial communities, including the human microbiome

Microbial communities contain many evolving and interacting species, which makes them difficult to understand and predict. The Foster lab combines molecular and cellular biology with ecological and evolutionary approaches to break down this complexity. Using both theory and experiment, we study how bacteria cooperate and compete in order to succeed in their communities. The lab also studies the ecological networks formed by interacting bacteria, with the goal of predicting and manipulating gut communities for better health. We have a range of projects on offer within the remit of the lab, which can be tailored to the student’s interests.

For more information about the Foster lab see: https://zoo-kfoster.zoo.ox.ac.uk/

Prof Kevin Foster | Biochemistry (ox.ac.uk)

For informal enquiries: Kevin.foster@zoo.ox.ac.uk

Project Code M7

mRNA metabolism during stress and disease progression

The research in the Furger laboratory aims to understand the molecular mechanisms that enables cells to change their gene expression programs when they are exposed to biotic and abiotic stressors and identify factors that can trigger disease associated gene expression changes. A current main focus in the laboratory is to understand the processes that are activated when cells or tissues are exposed to cold temperatures, for example during a number of medical procedures that require controlled cooling of organs or the whole patient. We want to know how they reprogram gene expression and how these changes affect the physiology of the cells. To identify and understand these processes and their physiological consequences, we use a wide range of methodologies and work closely with a number of longstanding national and international collaborators.

Available projects:

Molecular Characterisation of the Human Cold Shock Response: “Cooling the Cellular Clock”.

Despite the widespread use of controlled cooling in clinical and emergency settings, we know surprisingly little about the impact that cold temperatures have on human cells at the molecular level. We have discovered that cooling and subsequent rewarming triggers unexpected temperature specific responses in human cells, including changes to the chromosome architecture and the functioning of the circadian clock. The circadian clock is an important inbuilt time keeper that controls the alignment of cell physiology and organism behaviour with the earths day and night cycle. The DPhil project aims to unravel how cold induced structural changes, altered mRNA localisation and changes to mRNA metabolism, affect the workings and mechanics of the cellular clock.

Membrane less organelles and the spatial distribution of mRNAs in eukaryotic cells . Membrane less organelles (MLOs) are biological condensates of protein and RNA that are visible by microscopy as “droplet-like” structures in the nucleus and cytoplasm of all eukaryotic cells. Well-known eukaryotic MLOs include the nucleolus, stress granules and processing bodies. MLOs can form by assemblies of protein and RNA, creating distinct spherical liquid droplets. They play a key role in the subcellular organisation of the cellular space by creating distinct cellular compartments. Unlike membrane delimited organelles, MLOs can be dynamic and rapidly assemble or dissolve in response to specific cues. Whilst such RNA granules are common in cells, their function and how some mRNAs are selected whilst others are excluded, remains largely elusive. This project, using in vitro model condensates and in vivo approaches, aims to unravel the physical properties that determines the solvency of specific mRNAs for a particular condensate.

What do the projects offer?

The projects offer training in a wide range of state of the art methodologies including, super resolution microscopy, high through-put sequencing technologies, bioinformatics, in vitro mRNA production, tissue culture, cell biology and classic biochemistry and molecular biology techniques.

Recent key publication:

• Cold-induced chromatin compaction and nuclear retention of clock mRNAs resets the circadian rhythm. Fischl H, McManus D, Oldenkamp R, Schermelleh L, Mellor J, Jagannath A, Furger A. EMBO J. 2020 Nov 16;39(22):e105604. doi: 10.15252/embj.2020105604

Associate Prof Andre Furger | Biochemistry (ox.ac.uk)

For informal enquiries: andre.furger@bioch.ox.ac.uk

Project Code M8

Title: Control of centrosome function during the cell cycle and beyond

Background:

Centrosomes are small cytoplasmic organelles with a wide variety of cellular roles; they nucleate microtubules, serve as mitotic spindle poles, template cilia formation, while also contributing to proteostasis, signalling, trafficking and organelle positioning. They are essential for proliferation of normal cells. Indeed, congenital mutations in centrosomal genes cause growth failure syndromes (1).

Centrosomes are membrane-free organelles that concentrate up to 200 different proteins into specific structures; each centrosome contains a pair of centrioles embedded in a pericentriolar matrix. These complex multiprotein assemblies undergo dynamic changes during the cell cycle (2, 3). For instance, a single new centriole is built on each old centriole during S phase, whereas the pericentriolar matrix, the main site of microtubule nucleation, undergoes expansion and remodelling before mitosis to boost microtubule production for spindle formation.

Phosphorylation-dependent regulation of centrosome components is likely to be vital for a range of centrosome functions, including microtubule nucleation. Although several kinases including Aurora-A, Nek2, Cdk1 and Polo-like kinases 1, 2 and 4, have been implicated in centrosome regulation, a comprehensive list of their targets in the organelle is still lacking. We are currently undertaking an unbiased proteomic survey to elucidate centrosomal targets for these kinases.

Aims:

The ultimate goal of this DPhil project will be to improve our understanding of how centrosome function, and in particular centrosomal microtubule production, is controlled during the cell cycle.

First, we will functionally characterise newly identified phosphorylation sites in centrosomal proteins with an established role in microtubule nucleation. By developing tools such as specific antibodies against phosphorylation sites along with cell lines where the sites are genetically edited, we will obtain mechanistic insight into how these post-translational modifications contribute to centrosome function. Second, we will ask if these phosphorylation sites are present across multiple cell types, and if so, whether their impact on microtubule nucleation is conserved. Results from our laboratory suggest that certain cell types of the hematopoietic lineage do not expand their centrosome during mitosis (4). In these cell types, post-translational modifications of centrosome components could play an even more important part in controlling microtubule production. This hypothesis will be tested with the tools generated in the first aim, and proteomic survey of centrosomes from these unusual cell lineages will provide further insight. Finally, armed with knowledge from the first two aims, we will ask whether and how centrosomal microtubule nucleation capacity impacts on cellular processes such as trafficking, signalling and spindle formation.

Visit us on https://gergelylab.com

(1) Chavali, Putz and Gergely. 2014. The role of centrosomes in development and disease. Philos Trans R Soc Lond B Biol Sci.

(2) Tischer, Carden and Gergely. 2021. Accessorizing the centrosome: new insights into centriolar appendages and satellites. Current Opinion in Structural Biology.

(3) Vasquez-Limeta and Loncarek. 2021. Human centrosome organization and function in interphase and mitosis. Seminars in Cell & Developmental Biology.

(4) Tatrai and Gergely. 2022. Centrosome function is critical during terminal erythroid differentiation. The EMBO Journal.

For more information about the Gergely lab see https://gergelylab.com

Dr Fanni Gergely | Biochemistry (ox.ac.uk)

For informal enquires: fanni.gergely@bioch.ox.ac.uk

Project Code M9

Molecular basis of Traboulsi syndrome caused by AspH mutations

Mutations in the AspH gene are associated with Traboulsi syndrome, a rare recessive disorder with ocular clinical symptoms which are similar to those seen in Marfan syndrome patients. Since AspH encodes an EGF domain -specific hydroxylase this suggests that these symptoms may arise due to defective b-hydroxylation of EGF domains of fibrillin-1, a connective tissue protein which forms 10-12nm microfibrils in the extracellular matrix. This study aims to analyse b-hydroxylation of fibrillin-1 in relevant cell lines and to use genome-editing techniques and specific inhibitors to observe the effect of defective b-hydroxylation on fibrillin biosynthesis, secretion and assembly. In parallel, patient fibroblasts either lacking AspH or containing enzymatically deficient forms will also be examined.

Since AspH resides in the endoplasmic reticulum, and has been shown by high resolution structural studies to bind wrongly-disulphide-bonded EGF domain substrates, it is presumed that the b-hydroxylation modification is required for protein folding surveillance. However the temporal and spatial hierarchy of post-translational modifications to this domain which also include O—glycosylation of folded EGF substrates is undetermined, as is any impact of hypoxia.

Refs

1) Nisha Patel et al.,

Mutations in ASPH Cause Facial Dysmorphism, Lens Dislocation, Anterior-Segment Abnormalities, and Spontaneous Filtering Blebs, or Traboulsi Syndrome,

The American Journal of Human Genetics,Volume 94, Issue 5, 2014, Pages 755-759,

ISSN 0002-9297, https://doi.org/10.1016/j.ajhg.2014.04.002 .

2) Pfeffer, I., Brewitz, L., Krojer, T. et al. Aspartate/asparagine-β-hydroxylase crystal structures reveal an unexpected epidermal growth factor-like domain substrate disulfide pattern. Nat Commun 10, 4910 (2019). https://doi.org/10.1038/s41467-019-12711-7

3) Williamson et al., POGLUT2 and POGLUT3 O-glucosylate multiple EGF repeats in fibrillin-1, -2, LTBP-1 and promote secretion of fibrillin-1. J.Biol.Chem. 297, 101055, (2021). |https://doi.org/10.1016/j.jbc.2021.101055

For more information about the Handford lab see: http://www2.bioch.ox.ac.uk/handfordlab/

Prof Penny Handford | Biochemistry (ox.ac.uk)

For informal enquiries: penny.handford@bioch.ox.ac.uk

Project Code M10

lipid transport in membrane adaptation to temperature

Lipid bilayer membranes play a prime role in compartmentalising life and biochemistry. The self-assembly of lipids into a liquid crystalline bilayer is conceptually and biochemically simple. Robust membranes can be obtained by self-assembly of one single type of lipid molecule (for instance dioleyl-phosphatidylcholine). This simplicity contrasts with the humongous diversity of lipids found in biological membranes; thousands of different lipids can be found in the human lipidome, differing in their headgroup, backbone, fatty-moiety lengths and number and position of unsaturations. This diversity is likely useful to tune the biophysical and biochemical properties of the membrane, such as thickness and fluidity, to the function of the membrane or to changing environmental conditions. We have identified a yeast mutant that cannot adapt its membranes to changing temperature, and as a result cannot grow at low temperature. What are the molecular processes that are disrupted by maladapted lipid composition? Using yeast genetic screening for enhancers and suppressors, and in vitro reconstitution of membrane transporters, this project will address a fundamental question in biochemistry; why so many lipids, why so diverse and why so dynamic?

For more information about the Kornmann lab see: http://www.kornmann.group

Prof Benoit Kornmann | Biochemistry (ox.ac.uk)

For informal enquiries: benoit.kornmann@bioch.ox.ac.uk

Project Code M11

Detection, Signalling and Repair of DNA Damage

Background

Genomes are under continual assault from a variety of agents that cause DNA damage. Preserving genome integrity through repair of this damage is critical for human health and defects in these pathways leads to a variety of pathologies including neurodegeneration and cancer. Therefore, understanding the mechanistic basis of DNA repair will provide insights into the causes of these conditions and, importantly, strategies for their treatment. Our research aims to understand the mechanistic basis of DNA repair, with specific reference to how a family of proteins called Poly(ADP-ribose) polymerases (PARPs) regulate these processes. Our long-term vision is to exploit this knowledge to treat a variety of diseases including neurodegeneration and cancer.

Research Projects

PARPs are a cornerstone of the DNA damage response that promote repair of breaks in the DNA helix by modifying proteins at the damage site with ADP-ribose - a process known as ADP-ribosylation. These pathways are critical to maintain genome integrity and are attractive clinical targets, with PARP inhibitors being used to treat breast and ovarian tumours. However, despite the success of PARP inhibitors in the clinic, the mechanistic basis of how PARPs regulate DNA repair is unclear, representing a fundamental barrier to refine and broaden the application of these agents in the clinic. The overall goal of our research is to address these fundamentally important questions by defining the mechanistic basis of how PARPs regulate a variety of DNA repair mechanisms. DPhil projects are available that integrate cutting edge genome engineering, proteomics and cell biology to address the following:

a) How do PARPs become activated in response to DNA damage?

b) What proteins do they modify at sites of DNA damage?

c) How do these modifications regulate DNA repair?

These multidisciplinary hypothesis-driven research projects will increase our understanding of how PARPs regulate DNA repair and provide critical information to develop novel strategies that target PARPs to treat a variety of pathologies.

Prof Nick Lakin | Biochemistry (ox.ac.uk)

For informal enquiries: nicholas.lakin@bioch.ox.ac.uk

Project Code: M21

 Cytoskeletal organisation of the malaria parasite

The malaria parasite, Plasmodium falciparum, infects various cell types within its insect and human hosts as part of a complex life cycle (1). In the human bloodstream, the parasite invades red blood cells to replicate inside, hidden away from the host immune system. To invade these cells, the parasite uses specialised invasive organelles, the rhoptries and micronemes (2).

The parasite cell needs to be precisely organised to successfully invade the host. The rhoptries and micronemes are held at the apical end of the cell, whereas its nucleus, mitochondria and other organelles are held toward the basal end (3). It is not known how the cell sets up and maintains this striking polarisation. One class of proteins proposed to contribute are the various cytoskeletal filaments that form fascinating structures throughout the parasite. This is exemplified by the microtubules, which form a beautiful array along the parasite membrane (4). However how these filaments link to other structures in the cell to carry out their function remains mysterious. Our lab is interested in how these filament systems position and shape organelles throughout the parasite cell.

The aim of this DPhil project is to further our understanding into how organelles are linked to the microtubule cytoskeleton in the malaria parasite. We will identify and characterise novel microtubule-binding proteins to see how they link organelles to microtubules in vitro and in vivo. We will examine how they recruit organelles to the cytoskeleton using electron microscopy, then design assays to test their functional dynamics using biophysical techniques including TIRF microscopy. To dissect the protein function in cells we will use CRISPR/Cas9-based strategies to modify the protein inside the parasite. We will explore their localisation in cells and examine the consequences of knocking out the protein using light microscopy. Please contact clinton.lau@bioch.ox.ac.uk for more details.

References

1. Venugopal, K. et al. Nat. Rev. Microbiol. 2020 183 18, 177–189 (2020). https://doi.org/10.1038/s41579-019-0306-2

2. Schrevel, J. et al. Parasitology 135, 1–12 (2008). https://doi.org/10.1017/S0031182007003629

3. Fowler, R. E. et al. Parasitology 117, 425–433 (1998). https://doi.org/10.1017/S003118209800328X

4. Ferreira, J. et al. bioRxiv (2022). https://doi.org/10.1101/2022.04.13.488170

Dr Clinton Lau | Biochemistry (ox.ac.uk)

For informal enquiries: Clinton.lau@bioch.ox.ac.uk

Project Code M12

Drosophila as a model to study risk genes for human neurodegenerative diseases

Identifying DNA sequence variation in immune genes has allowed us to better predict the chances of an individual developing Late Onset Alzheimer's Disease (LOAD). These risk genes are connected to inflammation in the immune cells of the brain, called microglia. Nevertheless, separating cause and consequence is still an intractable problem since neurodegeneration itself increases inflammation while how the immune system responds to such changes may determine whether neurons live or die.

For rapid progress in assessing genetic risk, we will use the fruit fly Drosophila, which has a 2-month lifespan. We will test loss or alteration of function in fly homologues of LOAD risk genes in glia, which has the same immune and neurological properties as mammalian microglia. We will do this in 1) healthy-aged flies and 2) flies genetically predisposed to higher levels of inflammation. We will ask how risk genes influence increasing levels of age-dependent neurodegeneration going from condition 1 to 2 and verify results in mouse organotypic slices. Given the conservation in immunity and basic brain development between flies and mammals, we hope to uncover mechanisms underscoring LOAD and provide space for testing drugs at the whole animal level.

Prof Petros Ligoxygakis | Biochemistry

For informal enquiries: petros.ligoxygakis@bioch.ox.ac.uk

Project Code M13

Epigenetics and Evolution

Epigenetic gene regulation refers to changes in the expression of genes, which can be maintained through cell division. The ability of epigenetic changes to be passed on when cells divide makes them indispensable for development. Thus the fundamental processes that mediate epigenetic inheritance are conserved across millions of years of eukaryotic evolution. Despite their ancient origin, however, epigenetic inheritance mechanisms evolve very rapidly in individual lineages. For example, many epigenetic mechanisms that are present across the eukaryotes have been lost multiple times independently in different species. We are fascinated by this diversity and are attempting to understand the reasons for it. Previously we have used evolutionary approaches to identify genes that co-evolve with the epigenetic modification 5-methyl-cytosine, which enabled us to identify that the DNA methyltransferases that introduce 5-methyl-cytosine can also introduce the highly toxic 3-methyl-cytosine into DNA (see, for example, Rosic et al., Nature Genetics 2018: https://doi.org/10.1038/s41588-018-0061-8 ). In this project, we will use a computational co-evolution approach to identify genes that co-evolve with other epigenetic pathways that show rapid evolution. Our approach will be bolstered by searching for co-expression signatures across different human cancers, as cancers evolve rapidly. Together, these approaches will enable us to identify genes that co-evolve with epigenetic pathways, and thus may be required for these epigenetic pathways. Having identified possible candidates, we will then turn to experiments in cultured mammalian cells to test our hypotheses by using CRISPR/Cas9 to delete genes that we predict are required for certain epigenetic mechanisms, and investigating whether this results in negative consequences for cells that express these epigenetic pathways. The project will thus utilise both computational and experimental techniques. Our results will help us to understand some of the diversity in epigenetic gene regulation across mammals. Moreover, by identifying co-expression signatures across cancer, we may be able to understand why epigenetic regulation diversifies so rapidly in the development of cancer, with possible implications for treatment.

For more information about the Sarkies lab see Home | Epievo (psarkies.wixsite.com)

Associate Prof Peter Sarkies | Biochemistry (ox.ac.uk)

For informal enquiries Peter.sarkies@bioch.ox.ac.uk

Project Code 14

Interrogating nuclear structure-function relationships in mammalian cells by advanced super-resolution imaging

Three-dimensional (3D) chromatin organisation plays a crucial role in regulating mammalian genome functions such as RNA transcription, replication and DNA repair. Population-based sequencing approaches (e.g. Hi-C) have highlighted the compartmentalisation of chromatin into 0.5-1 MB sized topologically associating domains (TADs). However, many of the physical features at the single-cell level are still underexplored. Our primary research objective is to identify principles and underlying mechanisms of functional chromatin organisation in mammalian cells. Specifically, we aim to understand the interplay between biophysical forces, epigenetic memory, and cohesin complex activity to modulate cell-type-specific transcriptional programmes by directly visualising dynamic nuclear organisation and gene activity in living or 3D-preserved cells. To this end, we employ a combination of genetic editing with innovative in vivo/in situ fluorescence labelling and super- resolution imaging approaches. Our activities are closely linked to the Micron Oxford Advanced Bioimaging Unit and supported by our well-established ties to leading chromatin and epigenetic research groups within the Department and across Oxford.

For a MSc/PhD project, we seek (an) enthusiastic, proactive and adventurous student(s) eager to immerse in the latest imaging technologies to study topographical and biophysical aspects of gene regulation in an interdisciplinary environment. The topic of the project can be along the lines of either (1) studying the effect of directed phase separation on mesoscale domain organisation and transcriptional modulation, (2) examining mechanisms of gene reactivation during pathological de- differentiation processes (e.g. liver fibrosis, EMT), or (3) examining enhancer-promoter interactions e.g. in the alpha-globin locus, using multiplexed RNA-DNA-Immuno-FISH and correlative 3D super- resolution light end electron microscopy. The details of any project will be subject to personal preferences and be worked out closer to start date.

Main techniques: Mammalian tissue culture, molecular cloning, transfection, immunofluorescence labelling, fluorescence in situ hybridisation (DNA/RNA FISH), super-resolution structured illumination microscopy, single-molecule imaging, focussed ion beam scanning electron microscopy (FIB-SEM), computational image analysis.

Relevant papers:
 

Miron E, ..., Schermelleh L. 2020. Chromatin arranges in chains of mesoscale domains with nanoscale functional topography independent of cohesin. Science Advances 6, eaba8811.

Brown JM, … Schermelleh L, Buckle VJ. 2022. RASER-FISH, a non-denaturing fluorescence in situ hybridization for preservation of three-dimensional interphase chromatin structure. Nat Protoc 17, 1306-1331.

Rodermund L,..., Schermelleh L#, Brockdorff N#. 2021. Time-resolved structured illumination microscopy reveals key principles of Xist RNA spreading. Science. 372: eabe7500
 

Ochs F, ..., Schermelleh L#, Lukas J#, Lukas C. 2019. Stabilization of chromatin topology safeguards genome integrity. Nature, 594: 571-574.
 

Schermelleh L et al. 2019. Super-resolution microscopy demystified. Nat Cell Biol 21: 72-84.

Associate Prof Lothar Schermelleh | Biochemistry (ox.ac.uk)

For informal enquiries: lothar.schermelleh@bioch.ox.ac.uk

Project Code M15

Protein structure and interactions in health and disease .

Our laboratory seeks to understand how protein functions arise from their molecular structure and interactions, and how these processes are involved in human health and disease. Projects in at least two areas are possible. (1) We have a long-standing interest in the molecular mechanisms by which Influenza virus proteins function. We are studying the protein-protein and lipid interactions of ‘flu’ proteins to better understand their role in the virus life cycle and to identify potential therapeutic targets. (2) More recently, we have begun to study the structure and function of chaperone proteins, especially J-domain proteins, to understand how they interact with misfolded/unfolded clients and with the HSP70 machinery.

A central technique of our laboratory is solution nuclear magnetic resonance (NMR) spectroscopy, which allows atomic-level studies of protein structures and their interactions. NMR can be uniquely informative in situations where the molecular conformations or interactions are dynamic or heterogeneous. However, we also use a wide variety of other biochemical and biophysical tools as needed for these investigations. We have collaborations with various research groups including virologists, cell biologists, and computational biologists.

Associate Prof Jason Schnell | Biochemistry (ox.ac.uk)

For informal enquiries: Jason.Schnell@bioch.ox.ac.uk

Project Code M18

Molecular mechanisms in the brain and vascular system. Structural biology meets cell biology.

Project Code M16

Deciphering molecular mechanism that control transcription of non-coding RNAs

Recent technological advances have revealed a plethora of diverse long non-coding (nc) RNA molecules produced from eukaryotic genomes. Mutations in non-coding regions of the genome and altered expression of ncRNAs underpins a number of pathologies including cancer. Yet, very little is known about mechanisms involved in production of ncRNAs preventing us from understanding their role in health and disease. Our previous work lead to discovery that in contrast to mRNAs, nc transcripts rely on distinct and poorly understood mechanisms that control their RNA polymerase II (Pol II) transcription. As a result, ncRNAs are non-polyadenylated and targeted by the cellular RNA degradation machinery, RNA exosome.

The PhD project aims to fill the key gaps in our understanding of the transcriptional mechanisms involved in regulation of ncRNA. This will be achieved through the Aims 1-3. A PhD student will identify and characterise transcription complexes linked to production of ncRNA biochemically (Aim 1) and investigate how these complexes are recruited to Pol II during transcription and how they control biogenesis of ncRNA in human cells using state-of-the-art genomic approaches (Aim 2 and 3).

For more information about the Vasilieva lab see: www.vasilievalab.com

Associate Prof Lidia Vasilieva | Biochemistry (ox.ac.uk)

For informal enquiries: lidia.vasilieva@bioch.ox.ac.uk

Project Code M17

Replication barriers and genome stability

A hallmark of ageing is the accumulation of genomic mutations and rearrangements through mistakes made during the normal processes of DNA replication, repair and chromosome segregation. It is thought that this gradual corruption of the genome results in gene regulatory changes, which cause cellular degeneration and functional decline that ultimately drives ageing and its associated diseases. Accordingly, the pace of genomic deterioration is likely to be a key determinant of healthy lifespan, which is strongly influenced by both environmental and genetic factors. Through a complete understanding of how mutations and genome rearrangements arise, as well as the factors that mitigate their occurrence, we will be better placed to develop new approaches to improve the healthy ageing of humankind.

Conflicts between replication forks and single-strand DNA breaks (SSBs) and protein-DNA complexes (PDCs) are a major threat to genome stability through their potential to cause fork collapse and failure of complete genome duplication. By exploiting state-of-the-art fission yeast genetics, advanced microscopy, protein biochemistry, advanced proteomics and genomic approaches, we aim to elucidate the different pathways that limit genome instability arising from replication fork- SSB/PDC conflicts, and how pathway choice is influenced by the nature and context of the SSB/PDC. This work will make a seminal contribution to our understanding of how genome deterioration, and consequent ageing and age related disorders, is driven by problems that arise during S phase.

For more information about the Whitby lab see: https://whitbylab.com

Prof Matthew Whitby | Biochemistry (ox.ac.uk)

For informal enquiries: matthew.whitby@bioch.ox.ac.uk

Project Code: M20

How does UDP-glucose get into the ER?

It is not known how UDP-glucose enters the endoplasmic reticulum (ER), where it is needed e.g. as substrate for the glycoprotein misfold sensor UGGT (UDP-glucose glucosyl transferase) to help in the folding and quality control of N-glycosylated proteins in the ER. This ER-quality control (ERQC) pathway is not only needed by our own cells but is also exploited by viruses during infection. We would like to understand the basic biology of the UDP-glucose transport process into the ER, and investigate its role also in viral infection. The UDP-glucose transporter is not known, and the main aim of this project is to identify the protein responsible, to describe its role in health and infection, and assess the possibility of interfering with it.

UDP-glucose is also one of the UDP-sugars involved in extracellular signalling mediated by P₂Y₁₄ G-protein coupled cell surface receptors. P₂Y₁₄ receptors that are activated by UDP-glucose are primarily expressed in cells involved in immune and inflammatory responses.

The presence of a novel neuronal vesicular transporter, SLC35D3, that concentrates UDP-glucose in synaptic vesicles indicates its role in signalling, including in brain. Although SLC35D3 is not a classical ER/Golgi membrane protein, it has been found in synaptic vesicles in mouse brain and we would like to investigate whether it may perhaps also be found in the ER/Golgi.

If SLC35D3 is not the transporter responsible for UDP-glucose entry into the ER, we intend to identify and study the elusive UDP-glucose transporter through a variety of biochemical, biophysical, structural and functional studies.

For more information about the Zitzmann lab see: https://zitzmannlab.web.ox.ac.uk

Prof Nicole Zitzmann | Biochemistry (ox.ac.uk)

For informal enquiries: Nicole.Zitzmann@bioch.ox.ac.uk

Project Code: D1

Research theme: Using novel experimental and computational approaches to understand adaptive immunity across disease contexts to defined novel therapeutic targets

We are working on deciphering how the adaptive immune system can be used as diagnostics and therapeutics. We are investigating how defects in the ability to mount effective immune responses lead to infectious disease susceptibility, impaired surveillance of cancer and immunodeficiencies, compared to the mechanisms leading to a breakdown of immunological tolerance causing autoimmune diseases. This will lead us to:

o understand why certain individuals are at greater risk of developing immunological disease

o highlight key interactions of immune cells within sites of inflammation or disease

o define novel therapeutic targets or optimal therapeutic combinations

o identify blood biomarkers immune status

o stratify patients for improved clinical management

This is being achieved through the development and application of novel experimental and computational approaches, working in partnership with a global network of clinicians, immunologists and sample cohorts. We prioritise translational- and patient-focussed research to work towards bridging fundamental biology to early phase clinical trials.

How are different B cell populations developmentally linked in human health and disease?

We are investigating the generation, function and plasticity of B cell populations in human health. In particular, we are interested in how different lymphocyte subsets are developmentally linked and differences in function, and therefore providing a platform to understand how B cell fate may be different in human disease. We are defining how B cells select a particular developmental pathway, and will use this information to develop methods for modulating B cell function as potential therapeutic approaches.

How can B and T cells may be therapeutically modulated across cancers and autoimmune diseases

There is accumulating evidence for the role of both T and B cells in modulating immune responses to both solid tumours and haematological malignancies. We are investigating the contributions, function and heterogeneity of B and T cells on the immune responses to tumours and their potential role in cancer detection and treatment. We are determining the nature of B and T cell immuno-surveillance, regulation and activation across cancers and autoimmune diseases, as well as the immunological features associated with better prognosis and immunomodulation. With this, we aim to highlight novel therapeutic avenues. Our lab is affiliated with the Oxford Cancer Centre ( https://www.cancer.ox.ac.uk/research/research-themes/developments-in-immuno-oncology ) and non-cancer clinicians, with strong clinical links to a wide range of hard-to-treat diseases.

What is the effect of genetic and environmental variation on B and T cell fate?

Immunological health relies on a balance between the ability to mount an immune response against potential pathogens and tolerance to self. B and T cells are key to the immune response by producing antibodies and cytotoxic T cells. B/T cell clones selectively expand following antigen recognition by B and T cell receptors (BCR and TCR) respectively. BCRs are the membrane-form of antibodies and are generated through DNA recombination resulting in the potential to recognise a vast array of pathogens. Defects in the ability to mount effective B cell or T cell responses have been implicated in infectious susceptibility, impaired surveillance of cancer and immunodeficiencies, whereas a breakdown of immunological tolerance has been attributed to autoimmune diseases such as through autoantibody production and reduced numbers of regulatory B/T cells. Through integrating genomics, bulk and single-cell transcriptomics, and metabolomics data, serological, B /T cell repertoire and viromics datasets we will investigate the effect of both genetic variation and environmental factors on B cell fate, regulation, and the relationship to disease susceptibility.

Technology Development

We aim to develop novel experimental and computational tools to investigate the function of immune responses through advances in high-throughput and genetic technologies. These technologies can be readily applied to existing cohorts to investigate the immune system from unique perspectives.

Associate Prof Rachael Bashford-Rogers | Biochemistry (ox.ac.uk)

For informal enquiries: rachael.bashford-rogers@bioch.ox.ac.uk

Project Code D2

Nanomachines in the bacterial cell envelope

The cell envelope of bacteria comprises the cell wall and either one or two membranes, and provides a formidable barrier to the movement of macromolecules between the bacterial cytoplasm and the external environment. Our group aims to understand the molecular mechanisms by which proteins, nucleic acids, and mechanical force are transferred across and along these barriers. As part of this work we characterise the dedicated nanomachines that carry out these processes.

We use a wide range of methodologies in our work, in some cases via collaboration. These approaches include protein purification and characterisation, bacterial cell biology, bacterial genetics, live cell single molecule fluorescence imaging and other biophysical analysis, bioinformatics, and structural biology.

Specific research areas in which projects could be offered include:

• Protein transport. We study the transport of folded proteins across the bacterial inner membrane by the Tat transport system and protein export across the outer membrane by the recently discovered Type 9 Secretion System. Both systems are important for bacterial pathogenesis. We are also interested in the export of lipoproteins to the surface of Gram-negative bacteria.
• DNA transport. We study the mechanisms by which genes move between bacteria, thereby contributing to antibiotic resistance and other adaptive traits. We are interested in the processes of Conjugation, in which DNA is transferred between bacteria either by direct contact or via a retractile pilus, and Transformation (natural competence) in which the bacterium takes up naked DNA molecules directly from their environment.
• Gliding motility in which bacteria move rapidly across solid surfaces using surface adhesins running on mobile tracks located in the cell envelope.
• Physical properties of the cell envelope. In particular, we are interested in the functional properties of the periplasm, which is the compartment lying between the inner and outer membrane of Gram-negative bacteria and which contains the cell wall.

More details about my group can be found at: https://benberksgroup.web.ox.ac.uk

Example references showcasing some of our technical approaches:

• Hennell James et al. (2021) Structure and mechanism of the proton-driven motor that powers type 9 secretion and gliding motility. Nat Microbiol 6:
• Lauber et al. (2018) Type 9 secretion system structures reveal a new protein transport mechanism. Nature 564:
• Alcock et al. (2016) Assembling the Tat protein translocase. Elife 5:
• Alcock et al. (2013) Live cell imaging shows reversible assembly of the TatA component of the twin-arginine protein transport system. PNAS 110:
• Silale et al. (2021) The DNA transporter ComEC has metal-dependent nuclease activity that is important for natural transformation. Mol Microbiol 116:

Prof Ben Berks | Biochemistry (ox.ac.uk)

For informal enquiries: ben.berks@bioch.ox.ac.uk

Project Code D3

Selectivity and modulation of ion channels

We are interested in various ion channels, but in particular those that are allosterically modulated in complex ways and can even change their selectivity according to which ligand is bound. Projects will use molecular dynamics simulations and related computational methods to investigate modulation and selectivity. Projects are likely to be formulated around the following channels Twin Pore Channel 2, TRPA1, cysloop receptor channels and ionotropic glutamate receptors.

For more information about the Biggin lab see: https://bigginlab.web.ox.ac.uk

Prof Phil Biggin | Biochemistry (ox.ac.uk)

For informal enquiries: Philip.biggin@bioch.ox.ac.uk

Project Code D4

DNA repair, genome stability and cancer in humans

Integrity of the genome is critical to both the development and health of humans. Our chromosomes are constantly exposed to various types of DNA damage, which if unrepaired can cause diseases such as cancer. To meet these challenges, several DNA repair pathways have evolved. Our laboratory is focused on understanding how these pathways work in human cells. We have discovered several new proteins playing important roles in these pathways. By applying state-of-the-art techniques in biochemistry, molecular biology and cell biology, combined with world-class mass spectrometry and high-resolution live-cell imaging and structural biology, we are continuously elucidating the role of new components of the DNA repair pathways. Examples of methods used are recombinant protein purification, in vitro assays, CRISPR/Cas9 genome engineering, various cell-based assays including sophisticated live-cell imaging, and cryo-EM. Please contact Martin Cohn (martin.cohn@bioch.ox.ac.uk) for details on the newest and most interesting ongoing projects.

Recent papers published by DPhil students from our group:

Liang. C-C. and Cohn, M.A. (2021). Purification of DNA repair protein complexes from mammalian cells. STAR Protoc. 2(1):100348.

Socha, A., Yang, D., Bulsiewicz, A., Yaprianto, K., Kupculak, M., Liang. C-C., Hadjicharalambous, A., Wu, R., Gygi, S.P. and Cohn, M.A. (2020). WRNIP1 is recruited to DNA interstand crosslinks and promotes repair. Cell Rep. 32(1):107850.

Lopez-Martinez, D., Kupculak, M., Yang, D., Yoshikawa, Y., Liang, C-C., Wu, R., Gygi, S.P. and Cohn, M.A. (2019). Phosphorylation of FANCD2 inhibits the FANCD2/FANCI complex and suppresses the Fanconi anemia pathway in the absence of DNA damage. Cell Rep. 27(10):2990-3005.

Liang, C-C., Li, Z., Lopez-Martinez, D., Nicholson, W., Venien-Bryan, C. and Cohn, M.A. (2016). The FANCD2-FANCI complex is recruited to DNA interstrand crosslinks prior to monoubiquitination of FANCD2. Nature Commun. 7:12124.

Schwab, R., Nieminuszczy, J., Shah, F., Langton, J., Lopez Martinez, D., Liang, C-C., Cohn, M.A., Gibbons, R., Deans, A. and Niedzwiedz, W. (2015). The Fanconi anaemia pathway maintains genome stability by coordinating replication and transcription. Mol. Cell. 60(3):351-61.

Liang, C-C., Zhan, B., Yoshikawa, Y., Haas, W., Gygi, S.P. and Cohn, M.A. (2015). UHRF is a sensor for DNA interstrand crosslinks and recruits FANCD2 in the Fanconi Anemia pathway. Cell Rep. 10(12):1947-56.

For more information about the Cohn lab see: https://cohn.web.ox.ac.uk

Associate Professor Martin Cohn | Biochemistry (ox.ac.uk)

For informal enquires: martin.cohn@bioch.ox.ac.uk

Project Code D29

The role of glial post-transcriptional regulation in synaptic plasticity

The regulation of gene expression after transcription is central to many biological processes and it is particularly critical in the nervous system. Neuronal and glial cells are elongated with projections often some distance away from the nucleus. The rapid nature of synaptic growth, crucial to memory formation, requires expression of genes at these distal regions in a timely manner that would often preclude the transport of RNA or proteins from the nucleus. RNAs located at these peripheral locations in the cell, locally translated into proteins, are required for the cellular components needed to construct new synaptic connections.

While inter-neuronal synaptic connections form a key part of this system, it is becoming more apparent that glia are also centrally involved in a ‘tri-partite synapse’ system. Glia have been relatively poorly studied in comparison to neurons, yet they play a vital role in synaptic function. Previously considered to have a primarily structural role, glia are not understood to have signalling (both chemical and electrical) and other functions that are key to synaptic plasticity. Gaining a full understanding of the fundamental mechanisms of synaptic plasticity during memory and learning underpins research into neurodegenerative disorders. In both of these areas, the fruit fly, Drosophila, has proven an invaluable model, being much more experimentally accessible than its mammalian equivalent while expressing a large set of genes that are highly conserved. On-going work in the lab focuses on systematically examining the mechanisms of post transcriptional regulation in these processes, specifically, mRNA transport and localised translation, as well as mRNA stability and processing. The Davis lab is highly interdisciplinary, bringing together biologists, chemists, physicists and computer programmers to tackle fundamental biological questions.

Current research topics available for projects include:

• Screening for novel localising RNAs in the larval brain, glia and NMJ

A wide-ranging screen using single molecule FISH techniques to identify and characterise RNA that localise in the Drosophila nervous system. This work takes advantage of a library of YFP-protein trap fly-lines to systematically screen for both candidate protein and RNA localization.

• The role of translational regulation in Activity dependent synaptic plasticity

Synaptic plasticity is important as a proxy for memory and learning. This project combines electrophysiology protocols with optogenetics techniques and advanced imaging to address the role of translational regulation. In collaboration with MICRON we are developing a dedicated upright 3D-SIM super-resolution microscope to study protein-RNA interactions at the neuromuscular junction

Training opportunities : Work in the Davis lab involves routinely involves genetics (Drosophila), computational genomics, biochemistry, molecular biology, single molecule in situ hybridization techniques and advanced imaging. There is also the opportunity to be involved in developing imaging techniques and image analysis algorithms

For more information about the Davis lab see: www.ilandavis.com

Prof Ilan Davis | Biochemistry (ox.ac.uk)

For informal enquiries: ilan.davis@bioch.ox.ac.uk (please cc darragh.ennis@bioch.ox.ac.uk )

Project Code D5

Analysis of the human antibody repertoire induced in vaccinated volunteers by novel vaccines targeting the essential RH5-CyRPA-RIPR invasion complex used by P. falciparum malaria parasites to invade red blood cells.

Description

Traditionally the analysis of human antibody responses following vaccination has involved global approaches, with assays measuring the total polyclonal serum antibody response in terms of titre, concentration, subtype response or avidity. Although such measures remain useful readouts, the development of highly effective vaccines against difficult and complex pathogens, such as the Plasmodium parasite that causes human malaria, requires a much greater understanding of the fine specificity of the vaccine-induced antibody response. In recent years, significant advances have been made in terms of ability to analyse the human antibody repertoire generated in response to vaccination, and to assess the identification of key neutralising epitopes.

The aim of this project will be to analyse the human antibody response induced by novel candidate vaccines targeting the RH5-CyRPA-RIPR blood-stage malaria invasion complex in Phase I clinical trials undertaken in Oxford and Tanzania. This will build upon on-going research in the Draper Group to develop vaccines against this critical target complex within the parasite and to better understand its biology. A variety of techniques will be used to isolate antigen-specific B cell subsets from volunteers immunised with the RH5-CyRPA-RIPR antigen using different vaccine delivery regimens in humans (adults and infants). The isolated B cells will be used to analyse the human antibody response via sequencing and cloning of the B cell receptor gene repertoire. Key analyses will include variable region gene usage, mutation, and clonality of the response over time post-immunisation. Identified sequences will be used to generate human monoclonal antibodies (hu-mAbs) which will be assessed for functional anti-parasitic activity, affinity and epitopes mapped on the antigen using structural and immunological approaches. The outputs of this work should identify key epitopes around this protein complex and determinants within it that are recognised following human vaccination with these novel vaccines. Next steps could include rational structure-guided design of improved next-generation vaccine immunogens for onward clinical development. The project will be benefit from the group’s extensive experience of clinical immunology, B cell immuno-monitoring, protein engineering and parasitology, as well as from strong collaborations with other leading labs globally.

For more information on the Draper Lab, please see: https://draperlab.web.ox.ac.uk/

Prof Simon Draper | Biochemistry (ox.ac.uk)

For informal enquiries: simon.draper@bioch.ox.ac.uk

Project Code D6

Mechanistic understanding of the ubiquitin code during cell survival and programmed cell death

Use of structural biology techniques: cryo-EM, NMR and X-ray crystallography, along with biochemical assays and biophysical techniques (including SEC-MALS, ITC and MST), to understand at a molecular level how the ubiquitin code is responsible for maintaining the balance between cell survival and cell death. Our lab has recently determined the cryo-EM structure of a large ubiquitin ligase that regulates apoptosis. Current and future research projects will follow on this and other ubiquitin ligase complexes. In addition, we investigate how binding of non-degradative ubiquitin chains can activate master kinases of inflammation, and how deubiquitinating enzymes that reverse the ubiquitin code, are regulated.

For more information about the Elliott lab see https://elliottlab.web.ox.ac.uk

Dr Paul Elliott | Biochemistry (ox.ac.uk)

For informal enquiries: paul.elliott@bioch.ox.ac.uk

Project Code D7

Molecular mechanisms underlying the function of ATPases involved in proofreading and disassembly of pre-mRNA splicing by the human spliceosome

Mammalian genes are transcribed into precursor messenger RNAs (pre-mRNAs), from which non-coding introns are spliced out in the nucleus before the mRNA is exported to the cytoplasm and translated into proteins. Introns expand proteomic diversity by allowing a single gene to encode multiple mRNA isoforms, coding for multiple protein isoforms with distinct activities. Introns are excised by the spliceosome – a dynamic assembly of RNA and proteins and splicing errors are implicated in up to 30% of human diseases.

The spliceosome assembles de novo on each pre-mRNA and catalyses two sequential transesterifications at a single RNA-based active site to excise a specific intron and ligate the flanking exons into mRNA ¹. During catalysis, several trans-acting ATPases modulate the transitions between different conformations spliceosome. These ATPases promote the exchange of reactants at the active site, allow exchange of protein factors that stabilize each conformation, and proofread fidelity of splice site choice². Following mRNA synthesis, the mRNA is released by the action of the ATPase Prp22, while the ATPase Prp43 disassembles the resulting intron-lariat spliceosome (ILS) to release the excised intron for degradation.

In the last six years, structures of different conformation of the yeast spliceosome have provided a molecular view of the basic mechanism of splicing in yeast, showing how the splice sites are recognised and how specific factors stabilize each catalytic conformation². The structure of the yeast post-catalytic spliceosome (P complex) suggested a mechanism by which Prp22 releases the mRNA, while the structure of the yeast ILS provided some structural insights into disassembly of the spliceosome by Prp43 and its associated co-factors³. Importantly, in yeast, proofreading of correct splice site usage by Prp22 is coupled to a discard pathway in which Prp43 disassembles spliceosomes that utilise incorrect splice sites and are rejected by Prp22^4,5 . It remains unclear where Prp43 binds in the human ILS⁶ and it is not known what specific RNA component of the spliceosome is targeted by Prp43 during discard and disassembly in mammals.

Although the active site and basic splice site recognition is conserved from yeast to humans, several additional ATPases associate with the human spliceosome and have been implicated in splicing fidelity^7,8. Indeed, in mammals fidelity of splice site choice often relies on recognition of only 1-2 nucleotides around highly variable splice sites, which must be balanced with alternative splicing. Thus, how Prp22, Prp43, and other mammalian ATPases act to safeguard splicing fidelity remains poorly understood.

We aim to establish new biochemical systems in vitro, and potentially in vivo, to study proofreading of splice site use and spliceosome disassembly in humans. Our goals are to identify the specific complexes involved in these processes and to use this system to trap intermediates during proofreading, discard, and disassembly. Electron cryomicroscopy will then be employed to obtain molecular insights into the mechanism of action of Prp22, Prp43, and associated co-factors. These structural studies will be complemented by biochemical assays and new CLIP RNA crosslinking and sequencing approaches⁹ to elucidate the mechanisms underlying proofreading of correct splice site choice.

References

1. Wilkinson, M. E., Charenton, C. & Nagai, K. RNA Splicing by the Spliceosome. Annual Review of Biochemistry 89, 1–30 (2019).

2. Fica, S. M. & Nagai, K. Cryo-electron microscopy snapshots of the spliceosome: structural insights into a dynamic ribonucleoprotein machine. Nature structural & molecular biology 24, 791–799 (2017).

3. Wan, R., Yan, C., Bai, R., Lei, J. & Shi, Y. Structure of an Intron Lariat Spliceosome from Saccharomyces cerevisiae. Cell 171, 120-132.e12 (2017).

4. Mayas, R. M., Maita, H., Semlow, D. R. & Staley, J. P. Spliceosome discards intermediates via the DEAH box ATPase Prp43p. Proceedings of the National Academy of Sciences 107, 10020–10025 (2010).

5. Mayas, R. M., Maita, H. & Staley, J. P. Exon ligation is proofread by the DExD/H-box ATPase Prp22p. Nature structural & molecular biology 13, 482–490 (2006).

6. Zhang, X. et al. Structures of the human spliceosomes before and after release of the ligated exon. Cell Research 29, 1–285 (2019).

7. Fica, S. M. Cryo-EM snapshots of the human spliceosome reveal structural adaptions for splicing regulation. Current Opinion in Structural Biology 65, 139–148 (2020).

8. Sales-Lee, J. et al. Coupling of spliceosome complexity to intron diversity. Biorxiv 2021.03.19.436190 (2021) doi:10.1101/2021.03.19.436190.

9. Strittmatter, L. M. et al. psiCLIP reveals dynamic RNA binding by DEAH-box helicases before and after exon ligation. Nat Commun 12, 1488 (2021).

For more information about the Fica lab see https://snrnpsplicingbiochemlab.web.ox.ac.uk

Dr Sebastian Fica | Biochemistry (ox.ac.uk)

For informal enquiries: sebastian.fica@bioch.ox.ac.uk

Project Code D8

Understanding and engineering microbial communities, including the human microbiome

Microbial communities contain many evolving and interacting species, which makes them difficult to understand and predict. The Foster lab combines molecular and cellular biology with ecological and evolutionary approaches to break down this complexity. Using both theory and experiment, we study how bacteria cooperate and compete in order to succeed in their communities. The lab also studies the ecological networks formed by interacting bacteria, with the goal of predicting and manipulating gut communities for better health. We have a range of projects on offer within the remit of the lab, which can be tailored to the student’s interests.

For more information about the Foster lab see: https://zoo-kfoster.zoo.ox.ac.uk/

Prof Kevin Foster | Biochemistry (ox.ac.uk)

For informal enquiries: Kevin.foster@zoo.ox.ac.uk

Project Code D9

mRNA metabolism during stress and disease progression

The research in the Furger laboratory aims to understand the molecular mechanisms that enables cells to change their gene expression programs when they are exposed to biotic and abiotic stressors and identify factors that can trigger disease associated gene expression changes. A current main focus in the laboratory is to understand the processes that are activated when cells or tissues are exposed to cold temperatures, for example during a number of medical procedures that require controlled cooling of organs or the whole patient. We want to know how they reprogram gene expression and how these changes affect the physiology of the cells. To identify and understand these processes and their physiological consequences, we use a wide range of methodologies and work closely with a number of longstanding national and international collaborators.

Available projects:

Molecular Characterisation of the Human Cold Shock Response: “Cooling the Cellular Clock”.

Despite the widespread use of controlled cooling in clinical and emergency settings, we know surprisingly little about the impact that cold temperatures have on human cells at the molecular level. We have discovered that cooling and subsequent rewarming triggers unexpected temperature specific responses in human cells, including changes to the chromosome architecture and the functioning of the circadian clock. The circadian clock is an important inbuilt time keeper that controls the alignment of cell physiology and organism behaviour with the earths day and night cycle. The DPhil project aims to unravel how cold induced structural changes, altered mRNA localisation and changes to mRNA metabolism, affect the workings and mechanics of the cellular clock.

Membrane less organelles and the spatial distribution of mRNAs in eukaryotic cells . Membrane less organelles (MLOs) are biological condensates of protein and RNA that are visible by microscopy as “droplet-like” structures in the nucleus and cytoplasm of all eukaryotic cells. Well-known eukaryotic MLOs include the nucleolus, stress granules and processing bodies. MLOs can form by assemblies of protein and RNA, creating distinct spherical liquid droplets. They play a key role in the subcellular organisation of the cellular space by creating distinct cellular compartments. Unlike membrane delimited organelles, MLOs can be dynamic and rapidly assemble or dissolve in response to specific cues. Whilst such RNA granules are common in cells, their function and how some mRNAs are selected whilst others are excluded, remains largely elusive. This project, using in vitro model condensates and in vivo approaches, aims to unravel the physical properties that determines the solvency of specific mRNAs for a particular condensate.

What do the projects offer?

The projects offer training in a wide range of state of the art methodologies including, super resolution microscopy, high through-put sequencing technologies, bioinformatics, in vitro mRNA production, tissue culture, cell biology and classic biochemistry and molecular biology techniques.

Recent key publication:

• Cold-induced chromatin compaction and nuclear retention of clock mRNAs resets the circadian rhythm. Fischl H, McManus D, Oldenkamp R, Schermelleh L, Mellor J, Jagannath A, Furger A. EMBO J. 2020 Nov 16;39(22):e105604. doi: 10.15252/embj.2020105604

Associate Prof Andre Furger | Biochemistry (ox.ac.uk)

For informal enquiries: andre.furger@bioch.ox.ac.uk

Project Code D10

Title: Control of centrosome function during the cell cycle and beyond

Background:

Centrosomes are small cytoplasmic organelles with a wide variety of cellular roles; they nucleate microtubules, serve as mitotic spindle poles, template cilia formation, while also contributing to proteostasis, signalling, trafficking and organelle positioning. They are essential for proliferation of normal cells. Indeed, congenital mutations in centrosomal genes cause growth failure syndromes (1).

Centrosomes are membrane-free organelles that concentrate up to 200 different proteins into specific structures; each centrosome contains a pair of centrioles embedded in a pericentriolar matrix. These complex multiprotein assemblies undergo dynamic changes during the cell cycle (2, 3). For instance, a single new centriole is built on each old centriole during S phase, whereas the pericentriolar matrix, the main site of microtubule nucleation, undergoes expansion and remodelling before mitosis to boost microtubule production for spindle formation.

Phosphorylation-dependent regulation of centrosome components is likely to be vital for a range of centrosome functions, including microtubule nucleation. Although several kinases including Aurora-A, Nek2, Cdk1 and Polo-like kinases 1, 2 and 4, have been implicated in centrosome regulation, a comprehensive list of their targets in the organelle is still lacking. We are currently undertaking an unbiased proteomic survey to elucidate centrosomal targets for these kinases.

Aims:

The ultimate goal of this DPhil project will be to improve our understanding of how centrosome function, and in particular centrosomal microtubule production, is controlled during the cell cycle.

First, we will functionally characterise newly identified phosphorylation sites in centrosomal proteins with an established role in microtubule nucleation. By developing tools such as specific antibodies against phosphorylation sites along with cell lines where the sites are genetically edited, we will obtain mechanistic insight into how these post-translational modifications contribute to centrosome function. Second, we will ask if these phosphorylation sites are present across multiple cell types, and if so, whether their impact on microtubule nucleation is conserved. Results from our laboratory suggest that certain cell types of the hematopoietic lineage do not expand their centrosome during mitosis (4). In these cell types, post-translational modifications of centrosome components could play an even more important part in controlling microtubule production. This hypothesis will be tested with the tools generated in the first aim, and proteomic survey of centrosomes from these unusual cell lineages will provide further insight. Finally, armed with knowledge from the first two aims, we will ask whether and how centrosomal microtubule nucleation capacity impacts on cellular processes such as trafficking, signalling and spindle formation.

Visit us on https://gergelylab.com

(1) Chavali, Putz and Gergely. 2014. The role of centrosomes in development and disease. Philos Trans R Soc Lond B Biol Sci.

(2) Tischer, Carden and Gergely. 2021. Accessorizing the centrosome: new insights into centriolar appendages and satellites. Current Opinion in Structural Biology.

(3) Vasquez-Limeta and Loncarek. 2021. Human centrosome organization and function in interphase and mitosis. Seminars in Cell & Developmental Biology.

(4) Tatrai and Gergely. 2022. Centrosome function is critical during terminal erythroid differentiation. The EMBO Journal.

For more information about the Gergely lab see https://gergelylab.com

Dr Fanni Gergely | Biochemistry (ox.ac.uk)

For informal enquires: fanni.gergely@bioch.ox.ac.uk

Project Code D11

Mechanisms of ubiquitin signalling in DNA repair

Background:

Research in the lab is focussed on understanding the mechanisms of DNA repair, which is a cellular process that functions to maintain the integrity of DNA and, in doing so, prevents the transformation of a normal cell into a cancer cell. Importantly, we know that failure to properly regulate DNA repair leads to various cancers, so understanding this regulation is essential. One class of enzymes that plays a major role in DNA repair regulation is deubiquitinating enzymes or DUBs. The lab has recently identified a novel DUB class that functions in DNA repair. However, there are still key gaps in our mechanistic understanding of this DUB that we aim to address using an integrated approach of biochemistry and cell biology. Promisingly, targeting DUBs with small molecule inhibitors is an emerging and attractive therapeutic avenue in cancer research.

There are currently opportunities for students to join our cell biology efforts to either work on the novel DUB that we have recently discovered or explore new cancer-relevant avenues in ubiquitin-dependent signalling.

A project may involve one of three areas of on-going research in the lab.

(1) Use advanced single-cell high-content microscopy approaches in combination with engineered cell lines to understand the steps in a cellular ubiquitin-dependent signalling process regulated by the recently discovered DUB, as well as use a range of cellular assays available in the lab.

(2) Follow-up on hits from genome-wide CRISPR-Cas9 screen that we have performed to explore the mechanistic basis for synthetic lethal interactions using advanced cell biology, genetics and proteomics tools available in the lab.

(3) Confirm a predicted paralog synthetic lethality in the ubiquitin system using tools and cell lines generated in the lab and then explore this further using genome-wide CRISPR-Cas9 screens and/or base editing approaches to understand which cellular pathways are particularly susceptible to this synthetic lethal interaction, followed by mechanistic dissection of these observations.

For more information see: Dr Ian Gibbs-Seymour | Biochemistry (ox.ac.uk)

For informal enquiries: Ian.gibbs-seymour@bioch.ox.ac.uk

Project Code D12

Molecular basis of Traboulsi syndrome caused by AspH mutations

Mutations in the AspH gene are associated with Traboulsi syndrome, a rare recessive disorder with ocular clinical symptoms which are similar to those seen in Marfan syndrome patients. Since AspH encodes an EGF domain -specific hydroxylase this suggests that these symptoms may arise due to defective b-hydroxylation of EGF domains of fibrillin-1, a connective tissue protein which forms 10-12nm microfibrils in the extracellular matrix. This study aims to analyse b-hydroxylation of fibrillin-1 in relevant cell lines and to use genome-editing techniques and specific inhibitors to observe the effect of defective b-hydroxylation on fibrillin biosynthesis, secretion and assembly. In parallel, patient fibroblasts either lacking AspH or containing enzymatically deficient forms will also be examined.

Since AspH resides in the endoplasmic reticulum, and has been shown by high resolution structural studies to bind wrongly-disulphide-bonded EGF domain substrates, it is presumed that the b-hydroxylation modification is required for protein folding surveillance. However the temporal and spatial hierarchy of post-translational modifications to this domain which also include O—glycosylation of folded EGF substrates is undetermined, as is any impact of hypoxia.

Refs

1) Nisha Patel et al.,

Mutations in ASPH Cause Facial Dysmorphism, Lens Dislocation, Anterior-Segment Abnormalities, and Spontaneous Filtering Blebs, or Traboulsi Syndrome,

The American Journal of Human Genetics,Volume 94, Issue 5, 2014, Pages 755-759,

ISSN 0002-9297, https://doi.org/10.1016/j.ajhg.2014.04.002 .

2) Pfeffer, I., Brewitz, L., Krojer, T. et al. Aspartate/asparagine-β-hydroxylase crystal structures reveal an unexpected epidermal growth factor-like domain substrate disulfide pattern. Nat Commun 10, 4910 (2019). https://doi.org/10.1038/s41467-019-12711-7

3) Williamson et al., POGLUT2 and POGLUT3 O-glucosylate multiple EGF repeats in fibrillin-1, -2, LTBP-1 and promote secretion of fibrillin-1. J.Biol.Chem. 297, 101055, (2021). |https://doi.org/10.1016/j.jbc.2021.101055

For more information about the Handford lab see: http://www2.bioch.ox.ac.uk/handfordlab/

Prof Penny Handford | Biochemistry (ox.ac.uk)

For informal enquiries: penny.handford@bioch.ox.ac.uk

Project Code D13

Molecular Parasitology

Description

Understanding the fundamental processes which underlie host-parasite interactions and/or structure-guided vaccine design

For more information about the Higgins lab see: https://higginslab.web.ox.ac.uk

Prof Matt Higgins | Biochemistry (ox.ac.uk)

For informal enquiries matthew.higgins@bioch.ox.ac.uk

Project Code D14

Understanding mechanisms underlying chromatin-based inheritance and cellular memory

DNA sequences that constitute the genome are replicated in a self-templating manner and transmitted through cell division resulting in fateful inheritance of genetic information. In addition to the primary DNA-sequence, chromosome structure and patterns of gene expression are also maintained through mitotic and sometimes even meiotic divisions.

How components other that primary DNA sequence, such as proteins, that are part of chromatin and govern gene activities and chromosome structure are maintained and replicated through cell division is not understood. We are interested in resolving this.

Research in our lab revolves around two main research themes:

1. The Human Centromere

Centromeres are chromosomal loci that form the attachment site for spindle microtubules, driving chromosome segregation in mitosis. Centromeres are defined by a unique chromatin structure featuring the histone H3 variant CENP-A. Nucleosomes containing CENP-A are at the center of an epigenetic feedback loop where chromatin-bound CENP-A is sufficient to initiate self-templated inheritance of centromeres, even on ectopic genomic loci, called neocentromeres. A DPhil project can be developed on determining how CENP-A chromatin domains form de novo, how CENP-A chromatin can be transmitted through cell division, how its assembly is cell cycle controlled.

2. Interferon-induced Transcriptional Memory

Cytokine signalling leads to gene induction. Strikingly, exposure to cytokines such as interferons can lead to long-term potentiation of transcription, called transcriptional memory. In such a scenario, re-exposure of cells to the inducing signal leads to an enhanced response in primed cells, even after multiple cell division cycles.

We aim to understand the mechanistic basis of transcriptional memory and define the role of chromatin, transcription factor networks and cytokine signalling. We recently identified a unique chromatin structure that is maintained at primed genes for multiple cell divisions in the absence of ongoing target gene transcript. Potential DPhil projects will revolved around understanding how such chromatin is fatefully replicated contributing to “memory" of prior interferon exposure, which may form the basis for short-term innate immune memory.

Key methodologies include mammalian cell culture, microscopy-based fluorescent pulse labelling technologies to visualize both ancestral as well as nascent pools of CENP-A histones, chromatin immunoprecipitation (ChIP), CUT&RUN, NGS experiments, as well as CRISPR-based genome editing to establish cell biological tools.

We run a dynamic team of postdocs and students that offer expertise and support. See www.jansenlab.org for more information. http://www.jansenlab.org/people.html for team members and http://www.jansenlab.org/papers.html for recent publications on these topics. Do get in touch if you have any queries.

Prof Lars Jansen | Biochemistry (ox.ac.uk)

For informal enquiries lars.jansen@bioch.ox.ac.uk

Project Code D15

Deciphering the molecular choreography within bacterial cell envelopes

The cell envelope that surround Gram-negative bacteria is a complex tripartite architecture composed of two membranes and the periplasm. The cell envelope provides protection in various forms, against dangers from the external environment. In order to achieve these protective functions, molecular organisation and communication within each of the compartments as well as across all three compartments must be coordinated and regulated. While we know many of the structures of the proteins, lipids and glycans that are involved in these processes, less is known about how they work together.

The Khalid group use computational methods (mostly molecular dynamics simulations) in collaboration with experimental groups (e.g. working in structural biology and biophysics) both within Oxford and outside to study the molecular interactions within the cell envelope of Gram-negative bacteria. We typically construct computational models of the cell envelope by taking structures/homology models of proteins, embedding them in a model of their native environment (membrane or aqueous) and adding key molecules such as the cell wall, osmolytes, water and ions. We then use molecular dynamics to animate the model. The underlying biochemistry is combined with classical physics by the computer program to predict how the molecules should move. The resulting movie is then analysed to explore key molecular interactions. Molecular graphics and images play a big role in our work.

A typical DPhil project would involve working on one or more proteins (often in collaboration with the team that determined the structure of the protein(s)) to understand how the conformational dynamics of the individual proteins and also how they interact with other proteins. The ability to write code or previous experience of molecular dynamics simulations is not a requirement; it is however important that you are enthusiastic about the structures and dynamics of proteins and happy to work with computers.

For more information about the Khalid lab see: Syma Khalid Research Group – Computational Microbiology (wordpress.com)

Prof Syma Khalid | Biochemistry (ox.ac.uk)

For informal enquiries: syma.khalid@bioch.ox.ac.uk

Project Code D16

Functional ramifications of outer membrane organisation in Gram-negative bacteria

The outer membrane (OM) of Gram-negative bacteria adapts to a changing environment, supports cellular integrity, adheres to host cells during pathogenesis and is a major factor in antibiotic resistance.  The textbook description of the OM is as a well-mixed asymmetric bilayer in which lipids and proteins are freely interspersed.  Recent work from our laboratory on the model organism Escherichia coli has shown that this is far from reality.  Using bacteriocins as specific labels, we have shown that outer membrane proteins (OMPs) are instead organised both temporally and spatially into supramolecular islands (Rassam et al (2015) Nature; Rassam et al (2018) Nat Commun).  OMP islands move as tectonic plates towards the old poles of growing cells where OMP biogenesis is absent.  As a result, the bacterium replenishes the protein composition of its OM simply by division.  What is less well understood however is how such organisation impacts other aspects of the organism’s biology.

 The aims of this project are to use experimental and computational methods to probe the functional consequences of OMP organisation in bacteria.  The Kleanthous & Khalid labs have collaborated extensively using similar integrated approaches (e.g. Szczepaniak et al (2020) Nat Commun).  Specifically, the project will investigate how OMP island formation impacts antibiotic expulsion through TolC-efflux channels and cell envelope stabilisation via the peptidoglycan-binding protein, OmpA.  Experimental approaches will use a combination of photoactivated crosslinking coupled with LC/MS-MS, and super-resolution fluorescence microscopy in live bacteria (e.g. White et al (2017) PNAS).  Computational approaches will exploit recently developed coarse grain and atomistic molecular dynamics simulations of the bacterial cell envelope (e.g. Khalid et al (2019) Acc Chem Res).

For more information about the Kleanthous lab see: https://kleanthouslab.web.ox.ac.uk

Prof Colin Kleanthous | Biochemistry (ox.ac.uk)

For informal enquiries: colin.kleanthous@bioch.ox.ac.uk

Project Code D17

Discovering how epigenetics regulate gene expression in stem cells

Controlling how our genes are expressed is fundamental to cell function and development. While the molecular biology revolution of the mid-twentieth century defined the central dogma which states that DNA instructs the production of RNA and then protein, the mechanisms that unpin how the earliest steps of this cascade are controlled, namely how DNA is read and translated into the RNA remains very poorly understood. In eukaryotes, the ability to read DNA sequence is profoundly influenced by histones that package DNA into chromatin, and chromatin constitutes a central epigenetic regulator of RNA production and gene expression. However, our understanding of the mechanisms that enable epigenetic systems to control gene expression remain rudimentary at best. To address this fundamental problem, we use embryonic stems cells as a model to study how chromatin and epigenetic systems regulate gene expression to ensure stem cells remain pluripotent and can also support cellular differentiation and organismal development.

In the context of this overarching focus of the laboratory, a project is available to examine how proteins that function at CpG island elements (epigenetically defined elements associated with gene promoters in mammals) contribute to regulation of gene expression. The student will use and develop (via CRISPR-based genome engineering) embryonic stem cell lines containing degrons to rapidly deplete epigenetic regulators. They will then use genomic approaches coupled with next generation sequencing (ChIP-seq, RNA-seq, etc.) and/or live-cell imaging to determine how these systems affect chromatin modifications and gene expression. In particular, this will be applied to components of either the Polycomb repressive or Trithorax activator systems, which are paradigms of epigenetic gene regulation in mammals. As the project progresses there will also be an opportunity to dissect how these systems are used to regulate gene expression during cellular differentiation. The student will benefit from extensive support and training in the approaches necessary to tackle these fascinating problems, and will be at the forefront of uncovering how the epigenome shapes gene regulation and genome function.

For more details, see Kloselab.co.uk or our recently published papers:

(1) BAP1 constrains pervasive H2AK119ub1 to control the transcriptional potential of the genome. Fursova NA, Turberfield AH, Blackledge NP, Findlater EL, Lastuvkova A, Huseyin MK, Dobrinić P, Klose RJ. Genes and Development, 2021.

(2) Live-cell single particle tracking of PRC1 reveals a highly dynamic system with low target site occupancy. Huseyin MK and Klose RJ. Nature Communications, 2021.

(3) Cohesin Disrupts Polycomb-Dependent Chromosome Interactions in Embryonic Stem Cells.

Rhodes JDP, Feldmann A, Hernández-Rodríguez B, Díaz N, Brown JM, Fursova NA, Blackledge NP, Prathapan P, Dobrinic P, Huseyin MK, Szczurek A, Kruse K, Nasmyth KA, Buckle VJ, Vaquerizas JM, Klose RJ. Cell Reports, 2020.

(4) PRC1 Catalytic Activity Is Central to Polycomb System Function. Blackledge NP, Fursova NA, Kelley JR, Huseyin MK, Feldmann A, Klose RJ. Molecular Cell, 2020.

(5) Synergy between Variant PRC1 Complexes Defines Polycomb-Mediated Gene Repression. Fursova NA, Blackledge NP, Nakayama M, Ito S, Koseki Y, Farcas AM, King HW, Koseki H, Klose RJ. Molecular Cell, 2019.

For more information about the Klose lab see: Klose Lab – Learning how chromatin impinges on gene expression

Prof Rob Klose | Biochemistry (ox.ac.uk)

for informal enquiries rob.klose@bioch.ox.ac.uk

Project Code D18

lipid transport in membrane adaptation to temperature

Lipid bilayer membranes play a prime role in compartmentalising life and biochemistry. The self-assembly of lipids into a liquid crystalline bilayer is conceptually and biochemically simple. Robust membranes can be obtained by self-assembly of one single type of lipid molecule (for instance dioleyl-phosphatidylcholine). This simplicity contrasts with the humongous diversity of lipids found in biological membranes; thousands of different lipids can be found in the human lipidome, differing in their headgroup, backbone, fatty-moiety lengths and number and position of unsaturations. This diversity is likely useful to tune the biophysical and biochemical properties of the membrane, such as thickness and fluidity, to the function of the membrane or to changing environmental conditions. We have identified a yeast mutant that cannot adapt its membranes to changing temperature, and as a result cannot grow at low temperature. What are the molecular processes that are disrupted by maladapted lipid composition? Using yeast genetic screening for enhancers and suppressors, and in vitro reconstitution of membrane transporters, this project will address a fundamental question in biochemistry; why so many lipids, why so diverse and why so dynamic?

For more information about the Kornmann lab see: http://www.kornmann.group

Prof Benoit Kornmann | Biochemistry (ox.ac.uk)

For informal enquiries: benoit.kornmann@bioch.ox.ac.uk

Project Code D19

Detection, Signalling and Repair of DNA Damage

Background

Genomes are under continual assault from a variety of agents that cause DNA damage. Preserving genome integrity through repair of this damage is critical for human health and defects in these pathways leads to a variety of pathologies including neurodegeneration and cancer. Therefore, understanding the mechanistic basis of DNA repair will provide insights into the causes of these conditions and, importantly, strategies for their treatment. Our research aims to understand the mechanistic basis of DNA repair, with specific reference to how a family of proteins called Poly(ADP-ribose) polymerases (PARPs) regulate these processes. Our long-term vision is to exploit this knowledge to treat a variety of diseases including neurodegeneration and cancer.

Research Projects

PARPs are a cornerstone of the DNA damage response that promote repair of breaks in the DNA helix by modifying proteins at the damage site with ADP-ribose - a process known as ADP-ribosylation. These pathways are critical to maintain genome integrity and are attractive clinical targets, with PARP inhibitors being used to treat breast and ovarian tumours. However, despite the success of PARP inhibitors in the clinic, the mechanistic basis of how PARPs regulate DNA repair is unclear, representing a fundamental barrier to refine and broaden the application of these agents in the clinic. The overall goal of our research is to address these fundamentally important questions by defining the mechanistic basis of how PARPs regulate a variety of DNA repair mechanisms. DPhil projects are available that integrate cutting edge genome engineering, proteomics and cell biology to address the following:

a) How do PARPs become activated in response to DNA damage?

b) What proteins do they modify at sites of DNA damage?

c) How do these modifications regulate DNA repair?

These multidisciplinary hypothesis-driven research projects will increase our understanding of how PARPs regulate DNA repair and provide critical information to develop novel strategies that target PARPs to treat a variety of pathologies.

Prof Nick Lakin | Biochemistry (ox.ac.uk)

For informal enquiries: nicholas.lakin@bioch.ox.ac.uk

Project Code: D31

Cytoskeletal organisation of the malaria parasite

The malaria parasite, Plasmodium falciparum, infects various cell types within its insect and human hosts as part of a complex life cycle (1). In the human bloodstream, the parasite invades red blood cells to replicate inside, hidden away from the host immune system. To invade these cells, the parasite uses specialised invasive organelles, the rhoptries and micronemes (2).

The parasite cell needs to be precisely organised to successfully invade the host. The rhoptries and micronemes are held at the apical end of the cell, whereas its nucleus, mitochondria and other organelles are held toward the basal end (3). It is not known how the cell sets up and maintains this striking polarisation. One class of proteins proposed to contribute are the various cytoskeletal filaments that form fascinating structures throughout the parasite. This is exemplified by the microtubules, which form a beautiful array along the parasite membrane (4). However how these filaments link to other structures in the cell to carry out their function remains mysterious. Our lab is interested in how these filament systems position and shape organelles throughout the parasite cell.

The aim of this DPhil project is to further our understanding into how organelles are linked to the microtubule cytoskeleton in the malaria parasite. We will identify and characterise novel microtubule-binding proteins to see how they link organelles to microtubules in vitro and in vivo. We will examine how they recruit organelles to the cytoskeleton using electron microscopy, then design assays to test their functional dynamics using biophysical techniques including TIRF microscopy. To dissect the protein function in cells we will use CRISPR/Cas9-based strategies to modify the protein inside the parasite. We will explore their localisation in cells and examine the consequences of knocking out the protein using light microscopy. Please contact clinton.lau@bioch.ox.ac.uk for more details.

References

1. Venugopal, K. et al. Nat. Rev. Microbiol. 2020 183 18, 177–189 (2020). https://doi.org/10.1038/s41579-019-0306-2

2. Schrevel, J. et al. Parasitology 135, 1–12 (2008). https://doi.org/10.1017/S0031182007003629

3. Fowler, R. E. et al. Parasitology 117, 425–433 (1998). https://doi.org/10.1017/S003118209800328X

4. Ferreira, J. et al. bioRxiv (2022). https://doi.org/10.1101/2022.04.13.488170

Dr Clinton Lau | Biochemistry (ox.ac.uk)

For informal enquiries: Clinton.lau@bioch.ox.ac.uk

Project Code D20

Drosophila as a model to study risk genes for human neurodegenerative diseases

Identifying DNA sequence variation in immune genes has allowed us to better predict the chances of an individual developing Late Onset Alzheimer's Disease (LOAD). These risk genes are connected to inflammation in the immune cells of the brain, called microglia. Nevertheless, separating cause and consequence is still an intractable problem since neurodegeneration itself increases inflammation while how the immune system responds to such changes may determine whether neurons live or die.

For rapid progress in assessing genetic risk, we will use the fruit fly Drosophila, which has a 2-month lifespan. We will test loss or alteration of function in fly homologues of LOAD risk genes in glia, which has the same immune and neurological properties as mammalian microglia. We will do this in 1) healthy-aged flies and 2) flies genetically predisposed to higher levels of inflammation. We will ask how risk genes influence increasing levels of age-dependent neurodegeneration going from condition 1 to 2 and verify results in mouse organotypic slices. Given the conservation in immunity and basic brain development between flies and mammals, we hope to uncover mechanisms underscoring LOAD and provide space for testing drugs at the whole animal level.

Prof Petros Ligoxygakis | Biochemistry

For informal enquiries: petros.ligoxygakis@bioch.ox.ac.uk

Project Code D21

Solute Carrier transporters as targets for cancer and inflammation therapies

Amino acids are the building blocks of proteins and precursors for a number of essential metabolites, such as nucleotides, glutathione, polyamines, hexosamines, creatine, and myriad additional metabolites. Amino acids are classified into essential and nonessential, with this definition based on the demand of organisms to grow and thrive, a concept which can also be applied to cancer cells. Cancer cells undergo significant adaptations during their shift to uncontrolled cell growth, termed metabolic reprogramming. Amino acid and vitamin transporters play an essential role in cancer cell metabolism. Although metabolic reprogramming enables cancer cells to decouple from normal homeostatic control, they also open up unique opportunities for disease therapy. This project will build on ongoing work in the Newstead group (https://newsteadgroup.org/) to understand the role of amino acid and vitamin transporters on cancer cell survival and inflammation. Specifically, the student will undertake structural (cryo-EM), biochemical and cell-based studies.

For more information about the Newstead lab see www.newsteadgroup.org

Prof Simon Newstead | Biochemistry (ox.ac.uk)

For informal enquiries from prospective students: simon.newstead@bioch.ox.ac.uk

Project Code D30

Mechanisms of Wnt pathway activation

Wnt signalling is one of the most important cell signalling pathways in metazoans. It regulates key processes from embryonic development through to adult tissue homeostasis. As a result, alterations in this pathway are often linked to severe developmental defects, cancer, and other disorders. Most of our understanding as to how the pathway functions is derived from the Wnt/beta-catenin signalling cascade. To remain in an inactive state, when it is not needed, cells continuously degrade the transcriptional co-activator beta-catenin through the beta-catenin destruction complex (DC) in the cytoplasm. Thus, preventing its nuclear translocation and activation of gene expression. Conversely, pathway activation is initiated by the simultaneous binding of secreted Wnt proteins to FZD and LRP5/6 membrane receptors. The subsequent signal transduction recruits and tethers the beta-catenin destruction complex to the Wnt-bound receptors at the cell membrane ultimately resulting in its inactivation. Beta-catenin can now escape degradation, translocate to the nucleus, and drive gene expression.

Despite our broad understanding of how the pathway operates there is still a limited understanding of the molecular mechanisms driving Wnt/beta-catenin signalling. Our recent work (Ranes et al. 2021) on the biochemical and biophysical characterisation of the beta-catenin destruction complex, delivered unique insights into this process and how it is deregulated in colorectal cancers. Building on this achievement, the PhD project will focus on deciphering the molecular mechanisms which underpin inactivation of the beta-catenin destruction complex upon pathway activation. The student will functionally reconstitute Wnt receptor complexes to explore and dissect molecular mechanisms driving this DC inactivation. In this exciting endeavour the student will benefit from close support to gain experience in core techniques such as biochemical assay development, structure-based (cryo-EM, cross-link mass spectrometry) and biophysical approaches (mass photometry, SEC-MALS, BLI) to tackle this research question.

Reference:

Ranes M, Zaleska M, Sakalas S, Knight R & Guettler S. Reconstitution of the destruction complex defines roles of AXIN polymers and APC in β-catenin capture, phosphorylation, and ubiquitylation. Mol Cell, 2021.

For informal enquiries: michael.ranes@icr.ac.uk

Project Code D22

Epigenetics and Evolution

Epigenetic gene regulation refers to changes in the expression of genes, which can be maintained through cell division. The ability of epigenetic changes to be passed on when cells divide makes them indispensable for development. Thus the fundamental processes that mediate epigenetic inheritance are conserved across millions of years of eukaryotic evolution. Despite their ancient origin, however, epigenetic inheritance mechanisms evolve very rapidly in individual lineages. For example, many epigenetic mechanisms that are present across the eukaryotes have been lost multiple times independently in different species. We are fascinated by this diversity and are attempting to understand the reasons for it. Previously we have used evolutionary approaches to identify genes that co-evolve with the epigenetic modification 5-methyl-cytosine, which enabled us to identify that the DNA methyltransferases that introduce 5-methyl-cytosine can also introduce the highly toxic 3-methyl-cytosine into DNA (see, for example, Rosic et al., Nature Genetics 2018: https://doi.org/10.1038/s41588-018-0061-8 ). In this project, we will use a computational co-evolution approach to identify genes that co-evolve with other epigenetic pathways that show rapid evolution. Our approach will be bolstered by searching for co-expression signatures across different human cancers, as cancers evolve rapidly. Together, these approaches will enable us to identify genes that co-evolve with epigenetic pathways, and thus may be required for these epigenetic pathways. Having identified possible candidates, we will then turn to experiments in cultured mammalian cells to test our hypotheses by using CRISPR/Cas9 to delete genes that we predict are required for certain epigenetic mechanisms, and investigating whether this results in negative consequences for cells that express these epigenetic pathways. The project will thus utilise both computational and experimental techniques. Our results will help us to understand some of the diversity in epigenetic gene regulation across mammals. Moreover, by identifying co-expression signatures across cancer, we may be able to understand why epigenetic regulation diversifies so rapidly in the development of cancer, with possible implications for treatment.

For more information about the Sarkies lab see Home | Epievo (psarkies.wixsite.com)

Associate Prof Peter Sarkies | Biochemistry (ox.ac.uk)

For informal enquiries Peter.sarkies@bioch.ox.ac.uk

Project Code D23

Interrogating nuclear structure-function relationships in mammalian cells by advanced super-resolution imaging

Three-dimensional (3D) chromatin organisation plays a crucial role in regulating mammalian genome functions such as RNA transcription, replication and DNA repair. Population-based sequencing approaches (e.g. Hi-C) have highlighted the compartmentalisation of chromatin into 0.5-1 MB sized topologically associating domains (TADs). However, many of the physical features at the single-cell level are still underexplored. Our primary research objective is to identify principles and underlying mechanisms of functional chromatin organisation in mammalian cells. Specifically, we aim to understand the interplay between biophysical forces, epigenetic memory, and cohesin complex activity to modulate cell-type-specific transcriptional programmes by directly visualising dynamic nuclear organisation and gene activity in living or 3D-preserved cells. To this end, we employ a combination of genetic editing with innovative in vivo/in situ fluorescence labelling and super- resolution imaging approaches. Our activities are closely linked to the Micron Oxford Advanced Bioimaging Unit and supported by our well-established ties to leading chromatin and epigenetic research groups within the Department and across Oxford.

For a MSc/PhD project, we seek (an) enthusiastic, proactive and adventurous student(s) eager to immerse in the latest imaging technologies to study topographical and biophysical aspects of gene regulation in an interdisciplinary environment. The topic of the project can be along the lines of either (1) studying the effect of directed phase separation on mesoscale domain organisation and transcriptional modulation, (2) examining mechanisms of gene reactivation during pathological de- differentiation processes (e.g. liver fibrosis, EMT), or (3) examining enhancer-promoter interactions e.g. in the alpha-globin locus, using multiplexed RNA-DNA-Immuno-FISH and correlative 3D super- resolution light end electron microscopy. The details of any project will be subject to personal preferences and be worked out closer to start date.

Main techniques: Mammalian tissue culture, molecular cloning, transfection, immunofluorescence labelling, fluorescence in situ hybridisation (DNA/RNA FISH), super-resolution structured illumination microscopy, single-molecule imaging, focussed ion beam scanning electron microscopy (FIB-SEM), computational image analysis.

Relevant papers:
 

Miron E, ..., Schermelleh L. 2020. Chromatin arranges in chains of mesoscale domains with nanoscale functional topography independent of cohesin. Science Advances 6, eaba8811.

Brown JM, … Schermelleh L, Buckle VJ. 2022. RASER-FISH, a non-denaturing fluorescence in situ hybridization for preservation of three-dimensional interphase chromatin structure. Nat Protoc 17, 1306-1331.

Rodermund L,..., Schermelleh L#, Brockdorff N#. 2021. Time-resolved structured illumination microscopy reveals key principles of Xist RNA spreading. Science. 372: eabe7500
 

Ochs F, ..., Schermelleh L#, Lukas J#, Lukas C. 2019. Stabilization of chromatin topology safeguards genome integrity. Nature, 594: 571-574.
 

Schermelleh L et al. 2019. Super-resolution microscopy demystified. Nat Cell Biol 21: 72-84.

Associate Prof Lothar Schermelleh | Biochemistry (ox.ac.uk)

For informal enquiries: lothar.schermelleh@bioch.ox.ac.uk

Project Code D24

Protein structure and interactions in health and disease .

Our laboratory seeks to understand how protein functions arise from their molecular structure and interactions, and how these processes are involved in human health and disease. Projects in at least two areas are possible. (1) We have a long-standing interest in the molecular mechanisms by which Influenza virus proteins function. We are studying the protein-protein and lipid interactions of ‘flu’ proteins to better understand their role in the virus life cycle and to identify potential therapeutic targets. (2) More recently, we have begun to study the structure and function of chaperone proteins, especially J-domain proteins, to understand how they interact with misfolded/unfolded clients and with the HSP70 machinery.

A central technique of our laboratory is solution nuclear magnetic resonance (NMR) spectroscopy, which allows atomic-level studies of protein structures and their interactions. NMR can be uniquely informative in situations where the molecular conformations or interactions are dynamic or heterogeneous. However, we also use a wide variety of other biochemical and biophysical tools as needed for these investigations. We have collaborations with various research groups including virologists, cell biologists, and computational biologists.

Associate Prof Jason Schnell | Biochemistry (ox.ac.uk)

For informal enquiries: Jason.Schnell@bioch.ox.ac.uk

Project Code D28

Molecular mechanisms in the brain and vascular system. Structural biology meets cell biology

Specialised receptor and ligand proteins found on the surfaces of cells give rise to distinct molecular assemblies that instruct cell behaviour. Understanding the rules governing the assembly of these receptor-ligand complexes requires detailed knowledge of their structures and signalling properties. The lab uses cryo-electron microscopy, X-ray crystallography and a range of cell biology techniques to reveal fundamental structural and functional mechanisms by which receptors control biological processes, with a particular focus on the neural and vascular systems. Our lab is very collaborative and there is opportunity to liaise with other teams for access to specialised techniques such as super-resolution microscopy and in-vivo methods.

For more information about the Seiradake lab see: http://seiradake.web.ox.ac.uk

Prof Elena Seiradake | Biochemistry (ox.ac.uk)

For informal enquires: elena.seiradake@bioch.ox.ac.uk

Project Code D25

DNA repair and stress responses in bacteria

Fast and accurate repair of DNA damage is essential for the survival and genome stability of all organisms. Indeed, DNA damaging agents are highly effective as antibiotic treatments against bacterial pathogens and as cancer therapies. However, an unwanted side effect of these treatments is the formation of mutations that promote disease progression and eventually lead to drug resistance. Research in the Uphoff lab aims at understanding this process from the molecular scale to the cellular level, ultimately describing the mechanisms that drive evolution of cell populations.

A key aspect of our work is the development of advanced fluorescence microscopy techniques to visualise molecular processes inside bacteria. For example, we use single-molecule imaging to track the movement of DNA repair enzymes and transcription factors that regulate stress responses. We use fluorescent markers to “see” mutations in living cells. We also apply microfluidic technologies to trap individual cells and watch their behaviour over tens to hundreds of generations of growth.

Our research focuses on bacteria. Beyond their usefulness as tractable model organisms, bacteria play crucial roles in human health and the environment. Bacterial infections and rising antibiotic resistance are major burdens to society, impacting millions of lives and causing significant economic loss worldwide. Our recent work revealed unexpected mechanisms of the SOS response which plays a key role in antibiotic resistance. Projects on adaptation of bacteria to reactive oxygen species (ROS) and alkylating agents showed that bacteria modulate their mutation rates in response to stress. These and other projects in our group have opened fascinating questions about the mechanisms of bacterial genome evolution, which we will address in the coming years.

The Uphoff group is a multi-disciplinary and international team with expertise in biochemistry, molecular biology, genetics, and physics. DPhil students in the group gain experience in a range of modern research techniques, such as super-resolution microscopy, microfluidics, bacterial genetic engineering, and computer coding. The exact project focus will be discussed and matched to the interests and experience of the student.

For more information, see www2.bioch.ox.ac.uk/uphofflab/ or have a look at some of our publications:

• Single-molecule imaging of LexA degradation in Escherichia coli elucidates regulatory mechanisms and heterogeneity of the SOS response. Jones EC, Uphoff S. Nature Microbiology 6, 981–990 (2021)
• Transient non-specific DNA binding dominates the target search of bacterial DNA-binding proteins. Stracy M, Schweizer J, Sherratt DJ, Kapanidis A, Uphoff S, Lesterlin C. Molecular Cell 81, 1-16 (2021)
• Pulses and delays, anticipation and memory: seeing bacterial stress responses from a single-cell perspective. Lagage V, Uphoff S. FEMS Microbiology Reviews fuaa022 (2020)
• Real-time dynamics of mutagenesis reveal the chronology of DNA repair and damage tolerance responses in single cells. Uphoff S. Proc Natl Acad Sci U S A 115, E6516-E6525 (2018)
• Stochastic activation of a DNA damage response causes cell-to-cell mutation rate variation. Uphoff S, Lord ND, Potvin-Trottier L, Okumus B, Sherratt DJ, Paulsson J. Science, 27290, 1094-1097 (2016)

For more information about the Uphoff lab see https://www.bioch.ox.ac.uk/uphofflab

Associate Prof Stephan Uphoff | Biochemistry (ox.ac.uk)

For informal enquiries: stephan.uphoff@bioch.ox.ac.uk

Project Code D26

Deciphering molecular mechanism that control transcription of non-coding RNAs

Recent technological advances have revealed a plethora of diverse long non-coding (nc) RNA molecules produced from eukaryotic genomes. Mutations in non-coding regions of the genome and altered expression of ncRNAs underpins a number of pathologies including cancer. Yet, very little is known about mechanisms involved in production of ncRNAs preventing us from understanding their role in health and disease. Our previous work lead to discovery that in contrast to mRNAs, nc transcripts rely on distinct and poorly understood mechanisms that control their RNA polymerase II (Pol II) transcription. As a result, ncRNAs are non-polyadenylated and targeted by the cellular RNA degradation machinery, RNA exosome.

The PhD project aims to fill the key gaps in our understanding of the transcriptional mechanisms involved in regulation of ncRNA. This will be achieved through the Aims 1-3. A PhD student will identify and characterise transcription complexes linked to production of ncRNA biochemically (Aim 1) and investigate how these complexes are recruited to Pol II during transcription and how they control biogenesis of ncRNA in human cells using state-of-the-art genomic approaches (Aim 2 and 3).

For more information about the Vasilieva lab see: www.vasilievalab.com

Associate Prof Lidia Vasilieva | Biochemistry (ox.ac.uk)

For informal enquiries: lidia.vasilieva@bioch.ox.ac.uk

Project Code D27

Replication barriers and genome stability

A hallmark of ageing is the accumulation of genomic mutations and rearrangements through mistakes made during the normal processes of DNA replication, repair and chromosome segregation. It is thought that this gradual corruption of the genome results in gene regulatory changes, which cause cellular degeneration and functional decline that ultimately drives ageing and its associated diseases. Accordingly, the pace of genomic deterioration is likely to be a key determinant of healthy lifespan, which is strongly influenced by both environmental and genetic factors. Through a complete understanding of how mutations and genome rearrangements arise, as well as the factors that mitigate their occurrence, we will be better placed to develop new approaches to improve the healthy ageing of humankind.

Conflicts between replication forks and single-strand DNA breaks (SSBs) and protein-DNA complexes (PDCs) are a major threat to genome stability through their potential to cause fork collapse and failure of complete genome duplication. By exploiting state-of-the-art fission yeast genetics, advanced microscopy, protein biochemistry, advanced proteomics and genomic approaches, we aim to elucidate the different pathways that limit genome instability arising from replication fork- SSB/PDC conflicts, and how pathway choice is influenced by the nature and context of the SSB/PDC. This work will make a seminal contribution to our understanding of how genome deterioration, and consequent ageing and age related disorders, is driven by problems that arise during S phase.

For more information about the Whitby lab see: https://whitbylab.com

Prof Matthew Whitby | Biochemistry (ox.ac.uk)

For informal enquiries: matthew.whitby@bioch.ox.ac.uk