Available PhD projects

Learn more about Professor Shaun Cowley

Understanding gene regulation by HDAC1 complexes in development and cancer

Packaging DNA into nucleosomes helps protect the long fragile genomes of eukaryotic species. However, in doing so it becomes an ever-present physical barrier to the machinery required for its replication, repair and transcription. Wrapped up tightly in its histone overcoat, how do cells gain access to the underlying DNA? Universally, in species as diverse as brewers’ yeast, fruit flies, worms and man, the answer is to post-translationally modify (PTM) the histones to either help open it up, or compress the chromatin still further. Lysine-acetylation (Lys-Ac) is one of the most common histone PTMs and occurs on the unstructured and Lys-rich N-terminal tails of the core histones (H2A, H2B, H3 and H4). Given the density of Lys residues within histone tails, neutralization of their positive charge by acetylation reduces the overall affinity of histones for the negatively charged DNA backbone, opening chromatin and making it more transcriptionally permissive. Histone acetylation is highly dynamic with its level regulated by the opposing action of histone acetyltransferases (HATs) and histone deacetylases (HDACs). There are 18 HDAC enzymes in mammalian cells that can be sub-divided according to the presence of Zn2+-dependent (Class I, II and IV) or NAD+-dependent (Class III/Sirtuins) catalytic domains. As master regulators of chromatin accessibility, HDACs have been implicated in almost all nuclear functions, including DNA repair, DNA replication, chromosome segregation and gene expression. There is a compelling motivation to understand the fundamental biology of HDAC enzymes and to exploit this knowledge to improve their use as drug targets.

Because of their role in cell cycle progression, HDAC inhibitors (HDACi), such as SAHA, have been utilised as anticancer agents. HDACi are also established treatments for epilepsy and bi-polar disorder, and have shown promise as therapeutics for neurodegenerative disorders, such as Huntingdon’s disease. However, the use of pan-HDACi in patients is associated with multiple debilitating side-effects. Even specific HDACi, such as Entinostat (MS-275) which target predominantly HDAC1/2, results in many of the same problems, presumably because it still targets all four HDAC1/2 containing complexes non-specifically. Given the positive therapeutic value of HDAC inhibition in numerous disease states, and the detrimental side-effects of generic HDAC inhibition, there is a strong imperative to design novel HDAC inhibitors (HDACi) with improved specificity and alternative modes of action. Or to phrase the question in another way, can we drug individual HDAC1/2 complexes? And if so, what are the consequences?

Projects in the Cowley lab

• The role of HDAC1/2 complexes in development
  • HDAC1/2 complexes are essential for embryonic development. We have therefore used gene editing methods, such as CRISPR, to generate embryonic stem (ES) cell lines in which we can specifically ‘switch off’ HDAC1 and HDAC2, or components of specific complexes e.g. LSD1 and Sin3A, to examine their critical roles in development using a number of ES cell differentiation systems. Currently, we are performing transcriptomic experiments in ‘gastruloids’, an organoid system formed from aggregating ES cells that closely mimics the gastrulating embryo.
• Targeting HDAC1/2 for degradation in cancer cells
  • In collaboration with Dr James Hodgkinson (Dept. of Chemistry) at the University of Leicester, have been developing novel PROTACs directed towards HDAC1/2. Proteolysis Targeting Chimaeras (PROTAC) are hetero-bifunctional molecules which incorporate a known binding moiety to the protein of interest (POI, e.g. an inhibitor), coupled to a ligand for an E3 ubiquitin ligase complex. Direct recruitment of the E3 ligase to the POI via the PROTAC, targets it for ubiquitination and ultimately degradation. We recently published the first PROTACs to target HDAC1/2 specifically in cancer cells and are currently optimising additional molecules that target specific complexes.

Learn more about studying for a PhD in Molecular and Cell Biology

References

• Comprehensive Transcriptomic Analysis of Novel Class I HDAC Proteolysis Targeting Chimeras, India M. Baker, Joshua P. Smalley, Khadija A. Sabat, James T. Hodgkinson*, and Shaun M. Cowley. Biochemistry 2023, 62, 3, 645–656. doi.org/10.1021/acs.biochem.2c00288
• PROTAC-mediated degradation of class I histone deacetylase enzymes in corepressor complexes, Smalley JP, Adams GE, Millard CJ, Song Y, Norris JKS, Schwabe JWR, Cowley SM, Hodgkinson JT. Chem Commun (Camb). 2020 Apr 21;56(32):4476-4479. doi: 10.1039/d0cc01485k. PMID: 32201871
• The MiDAC histone deacetylase complex is essential for embryonic development and has a unique multivalent structure. Turnbull RE, Fairall L, Saleh A, Kelsall E, Morris KL, Ragan TJ, Savva CG, Chandru A, Millard CJ, Makarova OV, Smith CJ, Roseman AM, Fry AM, Cowley SM, Schwabe JWR. Nat Commun. 2020 Jun 26;11(1):3252. doi: 10.1038/s41467-020-17078-8. PMID: 32591534
• Histone deacetylase (HDAC) 1 and 2 are essential for accurate cell division and the pluripotency of embryonic stem cells. Jamaladdin S, Kelly RD, O'Regan L, Dovey OM, Hodson GE, Millard CJ, Portolano N, Fry AM, Schwabe JW, Cowley SM. Proc Natl Acad Sci U S A. 2014 Jul 8;111(27):9840-5. doi: 10.1073/pnas.1321330111. PMID: 24958871

Learn more about Professor Cyril Dominguez Learn more about Professor Ian Eperon Learn more about Professor Andrew Hudson

Structural investigation of oncogenic splicing factors

More than 90% of human genes can and do express multiple proteins. This is achieved by a process called alternative RNA splicing, which is an essential step in gene expression in mammals. In human, more than 100,000 proteins are produced from only 20,000 genes. For example, neurexin 3 alone is believed to express 1,728 different proteins with different synaptic functions from one pre-mRNA sequence.

Alternative splicing dictates which protein to express; this varies between tissues, development stages or in response to extracellular environment and the choices made affect processes ranging from memory and differentiation to death and disease.

Alternative splicing is regulated by many RNA-binding proteins, called splicing factors, in the nucleus. Through binding to the pre-mRNA, these proteins will compete or cooperate to induce the inclusion or exclusion of certain exons. However, the molecular mechanisms governing these regulatory events are still largely unknown. Mutations of overexpression of these splicing factors are associated with cancer progression.

Our laboratory is interested in the molecular basis of splicing regulation by studying the specificity of protein-RNA interactions and the role of post-translational modifications on splicing factor functions. To that aim we use a multidisciplinary approach combining biochemistry (protein expression and purification, in vitro translation and purification of RNAs, ..), structural biology (NMR, X-ray crystallography, and cryo-EM), biophysics (single-molecule microscopy, ITC, SPR, …), and cell biology (fluorescence and confocal imaging, cell-based splicing assays, …).

The project will focus on deciphering the function and regulation of the splicing factor Sam68 that display oncogenic properties and is overexpressed in numerous cancers.

Learn more about studying for a PhD in Molecular and Cell Biology

References

• I. Malki, I. Liepina, N. Kogelnik, H. Watmuff, S. Robinson, A. Lightfoot, O. Gonchar, A. Bottrill, A.M. Fry, C. Dominguez (2022) Cdk1-mediated threonine phosphorylation of Sam68 modulates its RNA binding, alternative splicing activity, and cellular functions. Nucleic Acids Res., 50, 13045-62.
• M. Feracci, J. Foot, S.N. Grellscheid, M. Danilenko, R. Stehle, O. Gonchar, H.S. Kang, C. Dalgliesh, N.H. Meyer, Y. Liu, A. Lahat, M. Sattler, I.C. Eperon, D.J. Elliott, and C. Dominguez (2016). Structural basis of RNA recognition and dimerization by the STAR proteins T-STAR and Sam68. Nat. Commun. 7, 10355.
• C. Weldon, I. Behm-Asmant, G. Burley, L. Hurley, C. Branlant, I. Eperon*, and C. Dominguez* (2017). Specific G-quadruplex ligands regulate the alternative splicing of Bcl-x. Nat. Chem. Biol.,13, 18-20.
• C Weldon, J.G. Dacanay, V. Gokhale, P.V.L. Boddupally, I. Behm-Ansmant, G.A. Burley, C. Branlant, L.H. Hurley, C. Dominguez*, and I.C. Eperon* (2018). Specific G-quadruplex ligands modulate the alternative splicing of Bcl-x. Nucleic Acids Res., 46, 886-96

How do RNA-binding proteins control splice site selection? A multi-disciplinary approach

Almost every protein-coding gene in vertebrates can and does express multiple proteins. This is achieved primarily by RNA splicing, which is an essential step in gene expression in mammals and generates incredible diversity. There is a good correlation between the levels of alternative splicing and an organism’s complexity. Indeed, it is widely held that the development of alternative splicing has been a key enabler of the evolution of complex organisms. The numbers of isoforms peak in neuronal tissue; neurexin 3, for example, expresses over 1,700 protein variants from splicing of one pre-mRNA sequence. Switches in splicing play major roles in almost all biological processes in complex organisms, including differentiation and the development of tissues and organs, apoptosis, senescence and ageing, long-lasting memory responses and direct responses to signals, such as caffeine or thermal control of diurnal rhythms in Drosophila and mice. Splicing switches are therefore deeply embedded determinants in biological processes, with the corollary that they are therefore also often involved in mediating or causing disease, including all forms of cancer.

Splicing events are controlled by numerous RNA-binding proteins that bind to the pre-mRNA. These proteins often bind promiscuously and weakly. Some act as repressors, others as activators, and many can be either of these depending on their sites of binding. A further complication arises because many have intrinsically disordered regions that can mediate interactions or phase effects. The mechanisms by which they affect splicing are largely still unclear. We are using single molecule microscopy, single molecule mass specrometry, structural biology and chemical and biological probes of flexibility to work out the mechanisms of control. The work is a collaboration between biochemists, structural biologists and physical chemists in Leicester, all in the Leicester Institute for Structural and Chemical Biology, and colleagues in synthetic chemistry in Strathclyde (Prof. G. A. Burley) and nano-engineering in Glasgow (Dr A. Clark), and it involves using state-of-the-art methods and developing the new methods required for investigating complex systems.

This project will involve working with the multi-disciplinary team. It is concerned with developing methods for following events in real time, both in vitro and in cells. It will lead to following single molecules of labelled protein by fluorescence microscopy, and analysing the dynamics of movement and interactions. Apart from microscopy, it will involve cloning and other standard molecular biology methods, in vitro assays of splicing and complex formation, and protein purification and characterization. It will lead to a PhD in Biochemistry.

Successful applicants will have a relevant first degree at a level equivalent to an upper second class, or higher, and some knowledge of chemistry, maths and computer programming.

Learn more about studying for a PhD in Molecular and Cell Biology

References

• Cherny, D., Gooding, C., Eperon, G.E., Coelho, M.B., Bagshaw, C.R., Smith, C.W.J., & Eperon, I.C.* (2010). Stoichiometry of a regulatory splicing complex revealed by single molecule analyses. EMBO J. 29, 2161-2172.
• Hodson, M.J., Hudson, A.J., Cherny, D., & Eperon, I.C.* (2012). The transition in spliceosome assembly from complex E to complex A purges surplus U1 snRNPs from alternative splice sites. Nucleic Acids Res. 40, 6850-6862.
• Chen, L., Weinmeister, R., Kralovicova, J., Eperon, L.P., Vorechovsky, I., Hudson, A.J., and Eperon, I.C.* (2017). Stoichiometries of U2AF35, U2AF65 and U2 snRNP reveal new early spliceosome assembly pathways. Nucleic Acids Research 45, 2051-2067.
• Jobbins, A.M., Reichenbach, L.F., Lucas, C.M., Hudson, A.J., Burley, G.A., & Eperon, I.C.* (2018). The mechanisms of a mammalian splicing enhancer. Nucleic Acids Research 46, 2145-2158.
• Jobbins, A.M., Campagne, S, Weinmeister, R., Lucas, C.M., Gosliga, A.R., Clery, A., Chen, L., Eperon, L.P., Hodson, M.J., Hudson, A.J.H., Allain, F.H.T. and Eperon, I.C. (2021) Exon-independent recruitment of SRSF1 is mediated by U1 snRNP stem-loop 3. EMBO J., in press.

Learn more about Dr Joanna Fox

Structural and functional analysis of the protein complexes which regulate cell fate decisions to identify novel cancer therapies

Whether a cell dies or not has profound consequences on health and disease. In healthy tissue, cells that acquire high levels of genetic damage are safely removed via a process of programmed cell death. This highly controlled process, called apoptosis; is critical in long-lived mammals as it maintains homeostasis during healthy ageing. If it is not executed correctly, it can result in the development and progression of widespread diseases. These include neurodegenerative diseases, where too many cells die when they shouldn't, or cancer, where damaged cells don't die when required.

The commitment to apoptotic cell death therefore must be highly regulated. This regulation is achieved by the formation of multi-protein complexes and specific protein-protein interactions, which either inhibit or initiate the apoptosis pathways. Our ability to manipulate these protein-protein interactions for the treatment of disease has been hindered by a lack of a defined model of the upstream regulatory protein complexes.

This project will build on published work from the lab, which revealed that an inhibitory phosphorylation determines if BAK activation can occur initiating apoptotic cell death. This project will study the mechanism by which the upstream regulatory protein complexes interact with and phosphorylate BAK to control this commitment to apoptosis. To address this question complementary cell biology and structural biology techniques such as NMR, x-ray crystallography and Cryo-electron microscopy will be used to visualise for the first time the key multi-protein complex involved in BAK regulation. The detailed structural insight generated will be used to identify and test, via mutagenesis of critical residues in each protein, key interactions which if disrupted alter the cell fate outcome. These studies will pave the way to develop novel therapeutic strategies to modulate BAK activity to increase levels of apoptosis in cancer models.

Learn more about studying for a PhD in Molecular and Cell Biology

References

• Vinesh Dhokia, John A. Y. Moss, Salvador Macip and Joanna L. Fox. At the Crossroads of Life and Death: The Proteins That Influence Cell Fate Decisions. Cancers. 2022 Jun; 14(11): 2745.
• Joanna L Fox, Marion MacFarlane. Targetting cell death signalling in cancer: minimising ‘Collateral damage’. Br J Cancer. 2016 May 3. doi: 10.1038/bjc.2016.111.
• Joanna L Fox. Cancer chemoresistance and BAK. Oncoscience. 2015 Dec 8;2(12):932-933.
• Joanna L Fox and Alan Storey. BMX Negatively Regulates BAK Function, Thereby Increasing Apoptotic Resistance to Chemotherapeutic Drugs. Cancer Res 2015 75(7): 1345-55

Learn more about Dr Gareth Hall Learn more about Dr Fred Muskett

Structural and functional characterisation of MALT1 ubiquitination to aid in the development of novel anti-cancer therapies

Nuclear factor-kappa B (NF-kB) is a family of transcription factors that play a crucial role in regulating various cellular processes; primarily related to immune responses, inflammation, and cell survival. Abnormal and prolonged NF-kB activation is often associated with various types of cancer. The CARMA1-BCL10-MALT1 (CBM) signalosome is a protein complex that plays a critical role in the NF-kB canonical activation pathway. Formation of the CBM protein complex is triggered by antigen receptors on lymphocytes, such as T-cells and B-cells, and results in lymphocyte activation and differentiation. The protease MALT1 contributes to the activity of the CBM signalosome complex, not only through its proteolytic function, but also by acting as a scaffold protein by recruiting the E3 ligase TRAF6. The interaction of TRAF6 with MALT1 results in the non-degrading ubiquitination of the MALT1 protein and subsequent activation of downstream kinases that leads to NF-kB activation. Inhibitors targeting the proteolytic function of MALT1 have been used treat a number of different cancers, and resulted in a reduced proliferation of some tumour cell types. However, MALT1 protease inhibitors have had no effect on some lung carcinoma types, indicating that the scaffold function of MALT1 is primarily involved in the tumorigenic propagation of this cancer.

The main aim of this project is to understand the mechanisms by which TRAF6 interacts with MALT1, resulting in the subsequent ubiquitination of the protease. The secondary goal is to use this information to develop inhibitors that could modulate TRAF6 recruitment to MALT1 and impact on cellular progression. This project will advance the research recently published by the group, which demonstrated that MALT1 protease activation initially results from the ubiquitination of a primary ubiquitination site, and that leads to the dimerization of the MALT1 protein. More recently, the research group demonstrated that this dimerization of the MALT1 protein results in the formation of a secondary ubiquitination site that is hypothesised to recruit and promote activation of downstream kinases, leading to NF-kB activation.

Objectives

• Characterise the potential secondary ubiquitin binding site on the MALT1 dimer, using structural biology and biophysical techniques.
• Mutagenesis of critical residues in protein complexes to determine which are the key interactions that govern complex formation.
• Potentially utilise single-chain antibodies to probe the surface of MALT1, with the aim of developing novel inhibitors of the MALT1 ubiquitination sites.

This project will use structural biology techniques such as NMR, X-ray crystallography and Cryo-electron microscopy, along with biophysical techniques, such as circular dichroism and biolayer interferometry, to analyse protein-protein interactions.

Learn more about studying for a PhD in Molecular and Cell Biology

References

• Schairer, R., et al. (2019) Allosteric activation of MALT1 by its ubiquitin-binding Ig3 domain, PNAS, 10:1073.
• Solsana, B.G., et al. (2022) The Paracaspase MALT1 in Cancer, Biomedicines, 10(2): 344.
• Düwel, M. et al. (2010) A20 negatively regulates T cell receptor signalling to NF-kB by cleaving MALT1 ubiquitin chains, J. Immunology, 182(12):7718-7728.
• Jaworski, M. and Thome, M. (2016) The Paracaspase MALT1: biological function and potential for therapeutic inhibition, Cell Mol. Life Sci., 73:459-473.

Learn more about Professor Salvador Macip

Targeting senescence to ameliorate ageing and treat cancer and other age-related diseases

Ageing is a biological process that affects all humans. Despite the scientific advances of the past decades, the mechanisms that lead to ageing are not fully understood. Evidence suggests that accumulation of old (senescent) cells in tissues plays a critical role in the appearance of the symptoms associated with age, as well as age related diseases such as cancer, Alzheimer’s, diabetes or fibrosis. Indeed, recent experiments in mice have showed that when senescent cells are eliminated from tissues, healthspan and lifespan increases substantially. Clinical trials have already started to test drugs that kill senescent cells (called senolytics), although they have many off-target effects.

Our work on senescence is aimed at (i) better understanding why organisms age and (ii) providing the basis for new treatments that could be applied to slow down and improve ageing and age-related diseases. We have been the first to identified novel membrane markers of senescence (the “senescent surfacome”) that can be used to detect and selectively kill senescent cells^1,2. We were one of the firsts to use nanoparticles to achieve this³ and next we designed an antibody-drug conjugate against senescent cells that is the first in a new class of targeted senolytics⁴. Moreover, we have used drugs that inhibit the formation of senescent cells to extend the lifespan and healthspan of mice and improve their cognitive functions in old age⁵. We are characterizing these and other novel targeted therapies and we are testing them in vitro and in vivo, in order to define novel anti-senescent strategies that could be applied clinically in the near future and thus improve ageing and age-related diseases in humans, with specific emphasis on cancer.

Learn more about studying for a PhD in Molecular and Cell Biology

References

1. Althubiti M, Lezina L, Carrera S, Jukes-Jones R, Giblett SM, Antonov A, Barlev N, Saldanha GS, Pritchard C, Cain K and Macip S. Characterization of novel markers of senescence and their prognostic potential in cancer. Cell Death Dis. 2014 Nov 20;5:e1528.
2. Althubiti M, Macip S. Detection of Senescent Cells by Extracellular Markers Using a Flow Cytometry-Based Approach. Methods Mol Biol. 2017;1534:147-153.
3. AE Ekpenyong-Akiba, F Canfarotta, B Abd H, M Poblocka, M Casulleras, ... S Macip. Detecting and targeting senescent cells using molecularly imprinted nanoparticles. Nanoscale Horizons 4 (3), 757-768
4. Poblocka M, Bassey AL, Smith VM, Falcicchio M, Manso AS, Althubiti M, Sheng X, Kyle A, Barber R, Frigerio M, Macip S. Targeted clearance of senescent cells using an antibody-drug conjugate against a specific membrane marker. Sci Rep. 2021 Oct 13;11(1):20358. doi: 10.1038/s41598-021-99852-2.
5. Ekpenyong-Akiba AE, Poblocka M, Althubiti M, Rada M, Jurk D, Germano S, Kocsis-Fodor G, Shi Y, Canales JJ, Macip S. Amelioration of age-related brain function decline by Bruton's tyrosine kinase inhibition. Aging Cell. 2020 Jan;19(1):e13079. doi: 10.1111/acel.13079. Epub 2019 Nov 17.

Learn more about Dr Robert Mahen

Understanding centrosome assembly in healthy and cancerous tissue with imaging

This is an opportunity for a scientist to join the lab of Dr Robert Mahen in the Department of Molecular and Cell Biology at the University of Leicester.

Cell division is one of the most fundamental features of life. Centrosomes are cellular organelles that allow accurate cell division, through formation of the mitotic spindle. They also form hair-like appendages called cilia that have important functions in human tissues. Defects in centrosomes result in a range of human developmental disorders and are also implicated in cancer. In our lab we seek to understand the assembly and function of centrosomes – with a long-term goal of understanding their critical roles in human physiology.

Despite their importance, the fundamental structural and biophysical properties of centrosomes are still poorly understood. Work in the Mahen lab has proposed new models for how centrosomes are constructed from component parts during mitosis (Mahen, PNAS, PMID 21576470), and for the control of centrosome position and number during interphase (Mahen, PLoS BIO PMID 29649211, PMID 36282799).

Building on our previous work, this project will use combined advances in genome editing and super resolution imaging to directly image centrosome activity in living cells during cell division. We will use state of the art imaging technologies including expansion microscopy and volume electron microscopy to understand the structure and assembly of centrosomes at unprecedented resolution, both in healthy and cancerous tissue.

Training opportunities

You will receive multidisciplinary training in techniques including super resolution imaging, expansion microscopy, quantitative live-cell imaging, molecular cloning, genome editing and flow cytometry. In parallel, there will be opportunity to collaborate with colleagues across the University of Leicester and in other institutions to use diverse cell culture systems and genomics analyses. The research will be conducted in a friendly and supportive atmosphere with access to advanced facilities at the University of Leicester.

Learn more about studying for a PhD in Molecular and Cell Biology

References

• Mahen, R. cNap1 bridges centriole contact sites to maintain centrosome cohesion. PLOS Biology. 2022. 20(10).
• Mahen R. Stable centrosomal roots disentangle to allow interphase centriole independence. PLOS Biology. 2018. 16(4).
• Mahen R, Koch B, Wachsmuth M, Politi AZ, Perez-Gonzalez A, Mergenthaler J, Cai Y, Ellenberg J. Comparative assessment of fluorescent transgene methods for quantitative imaging in human cells. Molecular Biology of the Cell. 2014 25(22).
• Mahen R, and Venkitaraman, AR. Pattern formation in centrosome assembly. Current Opinion in Cell Biology. 2012 24(1).
• Mahen R, Jeyasekharan AD, Barry NP, Venkitaraman AR. Continuous polo-like kinase 1 activity regulates diffusion to maintain centrosome self-organization during mitosis. Proceedings of the National Academy of Sciences, USA. 2011 108(22).

Learn more about Dr Yolanda Markaki Learn more about Professor John Schwabe Learn more about Dr James Hodgkinson

Epigenetics of early human development: From molecular mechanisms to regenerative medicine

The 46th chromosome in XX female mammals, the second X, has to be switched off. That way females equalize gene dosage of X-linked genes with XY males. The process of X-inactivation is a fascinating mechanism of whole chromosome silencing which initiates during early embryonic development and is maintained for life in somatic tissues thereafter. Female embryos do not survive without inactivating the X, while the importance of robust regulation of X-inactivation is evident by hundreds of X-linked diseases, including cancer, ageing and female-specific vulnerabilities to diseases when the process goes awry. Importantly, maintenance of X-inactivation is necessary for the epigenetic stability of human pluripotent stem cells, which often reactivate the inactive X in culture and thus their applicability to cell-based regenerative therapies becomes compromised.

The non-coding RNA, XIST, is expressed on the X chromosome and is the critical factor required to establish X-inactivation. We previously identified that XIST triggers the silencing process through the recruitment of chromatin regulators and the formation of molecular nanomachines termed XIST-SMACs (XIST-Supramolecular Complexes). SMACs have never been studied during human development and we have no knowledge of their molecular organization, which will be critical in order to devise chemical strategies to fight dysregulation of X-inactivation.

Studying X-inactivation in human embryonic stem cell lines (hESCs) or induced pluripotent stem cells (hiPSCs) reprogrammed from somatic cells is essential to understand fundamental gene-regulatory events of early human development. Naïve and primed hPSCs correspond to pre- and post-implantation, a time of human development we know very little about and when X-inactivation occurs. In this project, we will conduct for the first time super-resolution microscopy investigations of the epigenetic changes occurring on the inactivating X using chemical reprogramming of hPSCs as a model system. We will reconstitute RNA-protein supercomplexes and study them through cryo-electron microscopy to elucidate their organization at the molecular level. Finally, we will test highly specific chemical tools to reset the process of X-inactivation and to generate a platform for the generation of epigenetically improved hPSCs to fight X-linked diseases.

These studies will offer an unprecedented view into gene regulation during this critical timepoint in humans, which is when many pregnancies terminate. Ultimately, understanding the molecular mechanisms underpinning formation of XIST-supercomplexes will allow the development of therapeutic applications to tackle dysregulation of XCI in disease or the production of epigenetically stable human pluripotent stem cells.

Environment

The candidate will be embedded in the Department of Cell and Molecular Biology and become a member of the Leicester Institute of Structural and Chemical Biology (LISCB), a research institute of excellence offering access to world class facilities. They will join the LISCB doctoral training programme, which will offer training on both technical and transferable career-development skills. The project will be supervised by our expert team in developmental epigenetics, imaging, structural and chemical biology that will equip the candidate with unique skills on stem cell biology, genome editing, super-resolution microscopy and structural biology methods. As part of this multidisciplinary project the candidate will make use of the multi-million pound state-of-the-art cryo-electron and super-resolution microscopy suites and our human pluripotent stem cell facility.

Pre-requisites

The candidate should be:

• familiarized with molecular and cellular biology and have experience with cell culture
• highly motivated and organized
• fluent in English
• holding (by the start date) a Master's degree (300 ECTS credits)

Application procedure

Applications must include:

• a motivation letter addressed to Dr Yolanda Markaki
• a complete CV including contact details
• contact details of two referees

Learn more about studying for a PhD in Molecular and Cell Biology

References

• 1. Markaki Y*, Chong JG, Wang Y, Jacobson EC, Luong C, Tan SYX, Jachowicz JW, Strehle M, Maestrini D, Dror I, Mistry BA, Schöneberg J, Banerjee A, Guttman M, Chou T*, Plath K*. Xist nucleates local protein gradients to propagate silencing across the X chromosome. Cell. 2021.
• Pandya-Jones A, Markaki Y, Serizay J, Chitiashvili T, Mancia Leon WR, Damianov A, Chronis C, Papp B, Chen CK, McKee R, Wang XJ, Chau A, Sabri S, Leonhardt H, Zheng S, Guttman M, Black DL, Plath K. A protein assembly mediates Xist localization and gene silencing. Nature. 2020;587(7832):145-51.doi:10.1038/s41586-020-2703-0.
• Kraus F, Miron E, Demmerle J, Chitiashvili T, Budco A, Alle Q, Matsuda A, Leonhardt H, Schermelleh L, Markaki Y. Quantitative 3D structured illumination microscopy of nuclear structures. Nat Protoc. 2017;12(5):1011-28.
• Patel U, Smalley JP, Hodgkinson JT. PROTAC chemical probes for histone deacetylase enzymes. RSC Chem Biol. 2023; 4(9):623-634.

Learn more about Professor Daniel Panne

Structural insights into genome control by cohesion

Our basic goal is to understand how chromatin structure influences gene regulation. Chromatin is generally repressive in nature but its structure is manipulated by cells in a regulated way to determine which genes are potentially transcriptionally active and which genes remain repressed in a given cell type. This regulation depends on interactions between DNA sequence-specific transcription factors, chromatin enzymes and chromatin. The structural subunit of chromatin is the nucleosome core, which contains 147 bp of DNA wrapped 1.7 times around a central histone octamer composed of two molecules each of the four core histones (H2A, H2B, H3 and H4). Generally, nucleosomes are regularly spaced along the DNA, like beads on a string. Gene activation involves the recruitment of a set of factors to a promoter in response to appropriate signals, ultimately resulting in the formation of an initiation complex by RNA polymerase II (Pol II) and transcription. These events occur in the presence of nucleosomes, which are compact structures capable of blocking transcription at every step. To circumvent and regulate this chromatin block, eukaryotic cells possess dedicated enzymes, including ATP-dependent chromatin remodeling machines, histone modifying complexes and histone chaperones. The remodeling machines use ATP to move nucleosomes along or off DNA, or to exchange histone variants between nucleosomes. The histone modifying complexes contain enzymes which modify the histones post-translationally to alter their DNA-binding properties and to mark them for recognition by other complexes, which have activating or repressive roles. Histone-modifying enzymes include histone acetylases (HATs), deacetylases (HDACs), methylases and kinases. Histone chaperones mediate histone transfer reactions that occur during transcription and DNA replication (e.g. Asf1 and the CAF-1 complex). In addition, the genome is spatially organised to allow for DNA-based processes. Key to this organisation is the cohesin complex which organises the genome by reeling chromatin into loops which grow in size until stopped by CTCF. The enzymes that regulate chromatin modification and structure are central to epigenetics. Many human diseases have been linked to chromatin remodeling enzymes and epigenetic modifications. For example, aberrant regulation of the HAT p300 leads to aggressive forms of squamous carcinoma. A full understanding of the structure and mechanism of functions of chromatin structure, enzymes and modifications is therefore vital and enables new strategies in pharmacological targeting. Our aim is to dissect the machinery of such chromatin regulators and elucidate their contributions to gene regulation.

Our current efforts are focused on elucidating the structure and function of cohesin and its regulators, chromatin modifiers, ATP-dependent chromatin remodeling complexes and histone chaperones. We have made significant progress towards understanding a number of these key chromatin regulators. The major aim of this PhD project is to build on these key insights. We will use structural biology approaches including cryoEM and biochemical studies of key chromatin regulatory complexes including Cohesin and its regulators.

Learn more about studying for a PhD in Molecular and Cell Biology

References

• García-Nieto A., Patel A., Li Y., Oldenkamp R., Feletto L., Graham J.J., Willems L., Muir K.W., Panne D.* & Rowland B.D.* Structural basis of centromeric cohesion protection. Nature Struct Mol Biol (2023). * co-correspondence; Journal Cover.
• Ibrahim Z. Wang T., Destaing O., et al. Panne D. Structural insights into p300 regulation and acetylation-dependent genome organisation Nature Commun13, 7759 (2022).
• Li Y., Haarhuis J., Cacciatore A.S., Willems L., TeunissenH., Muir K.W., de Wit E., Rowland, B.D. & Panne D. (2020) The structural basis for cohesin-CTCF anchored loops. Nature 578, 472–476.
• Muir K.W., Li Y., Weiss F., Panne D. (2020) The structure of the cohesin ATPase elucidates the mechanism of SMC-kleisin ring opening. Nature Struct Mol Biol 27: 233-239.
• Ortega E., Rengachari S., Ibrahim Z., Hoghoughi N., Gaucher J., Holehouse A.S., Khochbin S., Panne D. (2018) Transcription factor dimerization activates the p300 acetyltransferase. Nature 562: 538–544.
• Sauer P., Timm J., Sitbon D., Ochsenbein F., Almouzni. G, Panne D. (2017) Insights into the molecular architecture and histone H3-H4 deposition mechanism of the Chromatin assembly factor 1., eLife, doi:10.7554/eLife.23474.

Learn more about Professor Thomas Schalch

Join our quest to unravel the secrets of eukaryotic genomes

We're on the lookout for passionate PhD students who are eager to delve deep into the mysteries of our DNA. Our quest? To understand the subtle changes that determine how our genes function, both in sickness and in health. Just as a music score dictates the notes, the structure and signals on our chromosomes play a crucial role in the symphony of life. But many nuances of this complex system remain unknown. That's where you come in!

Why join us?

• Cutting-edge research: We're trying to understand how chromosomal proteins (also known as chromatin) affect our DNA's behaviour. Think of it as understanding how different musicians play their part in an orchestra, impacting the final melody.
• Skill development: You'll gain hands-on experience in areas like biochemistry, structural biology, and genome analysis. Imagine being an investigator, scientist, and analyst all rolled into one.
• Dynamic environment: We operate in a fast-paced realm of research. It's not just another PhD; it's a thrilling adventure in a competitive arena.
• State-of-the-art tools: We've got some of the coolest gadgets in the world of biology! From high-tech microscopes to powerful spectrometers, we're equipped with the best.
• Global collaboration: Our work spans borders! You'll be part of a global team, amplifying your exposure and networking opportunities.

Our mentorship and lab culture

We believe in nurturing talent. Our mentorship is hands-on and supportive, ensuring you're never lost in the vast world of research. Our lab is not just well-equipped but also a well-organized hive of innovation. And we don't just focus on the hard science! With our weekly group meetings, you'll develop transferable skills, refining your ability to present findings and work harmoniously within a team.

About us

We're proud members of the Leicester Institute for Structural and Chemical Biology. Our experts in biophysics, biochemistry, chemical and structural biology will instruct you in our doctoral training program, transforming you into an expert in the realm of molecular biology research.

Are you ready to contribute to ground breaking research and potentially shape the future of medicine and therapeutic approaches? Join us in this exciting journey into the very core of life – our DNA.

Learn more about studying for a PhD in Molecular and Cell Biology

References

• Stirpe A., Guidotti N., Northall S., Kilic, S., Hainard, A., Vadas, O., Fierz, B., Schalch, T. (preprint). SUV39 SET domains mediate crosstalk of heterochromatic histone marks. eLife 10, e62682, Open Access Article, bioRxiv Preprint
• Bailey, L.T., Northall, S.J., and Schalch, T. (2021). Breakers and amplifiers in chromatin circuitry: acetylation and ubiquitination control the heterochromatin machinery. Current Opinion in Structural Biology 71, 156–163. Open Access Article
• Leopold K., Stirpe A. and Schalch, T. (2019). Transcriptional gene silencing requires dedicated interaction between HP1 protein Chp2 and chromatin remodeler Mit1. Genes & Development 33, 565-577 Open Access Article
• Moraru, M and Schalch, T. (2019). Chromatin fiber structural motifs as regulatory hubs of genome function? Essays In Biochemistry, EBC20180065. Open Access Article

Learn more about Dr Ralf Schmid

Molecular Modelling of P2X receptor function

P2X receptors (P2XR) are a family of ligand-gated ion channels. P2XRs are activated upon binding of extracellular ATP and allow the influx of small cations after channel opening. The human genome encodes seven P2XR paralogs (P2XR1-P2XR7) that have distinct roles and show tissue specific expression raising their therapeutic potential. Structurally, P2XRs are characterised by a large extracellular ligand binding domain, two transmembrane helices, and intracellular N- and C-termini. Key questions for receptor function are how ATP triggers the opening of the channel, how the receptor transits back from the open to the closed state via a desensitized state, and how small molecule agonists and antagonists affect receptor function. We will use computational techniques such as homology and Alphafold modelling, molecular dynamics simulations, ligand docking and evolutionary analysis to understand the mechanistic details of the transitions between these states and to inform drug design. Depending on the interests of the applicant there may be project opportunities that explore alternative protein targets using the lab’s technologies.

Learn more about studying for a PhD in Molecular and Cell Biology

References

• M Tian et al. Discovery and structure relationships of salicylanilide derivatives as potent, non-acidic P2X1 receptor antagonists (2020) J. Med. Chem. 63, 6164-6178.
• A Stavrou, RJ Evans, R Schmid. Identification of a distinct desensitisation gate in the ATP-gated P2X2 receptor (2020) Biochem. Biophys. Res. Comm. 523, 190-195.
• R Schmid, RJ Evans. ATP-gated P2X receptor channels: molecular insights into functional roles (2019) Ann. Rev. Phsiol. 81, 43-62.

Learn more about Dr Christopher Switzer

The role of Reactive Sulfur Species in Ageing and Disease

Oxidative stress (OS) is a major driver of cellular dysfunction in ageing and disease. While Reactive Oxygen Species (ROS) have been solely linked to oxidative stress in the past, this project will investigate the roles of Reactive Sulfur Species (RSS), such as persulfides and polysulfides, as major contributors to cellular oxidative stress. This revolutionary idea will spark new discoveries into ageing and chronic disease aetiology and reveal novel molecular targets for advanced therapeutics.

Classic models of elevated OS will be utilised to study the role of RSS in OS-associated cellular effects:

• Ageing
Ageing and OS are intimately intertwined; OS increases cellular ageing, while aged cells have higher levels of OS. SOD1 knockout mice are a murine model of ageing, as these mice exhibit elevated OS and other hallmarks of ageing. While this has been assumed to be due to elevated ROS, my recent findings indicate that RSS are also elevated in this model of OS. Therefore, to model the ageing process, SOD1 KO will be compared to WT littermates. As we are interested in the ageing process, we are particularly interested in studying middle aged/pre-elderly subjects, to see if RSS play a role during ageing.

Circulating blood cells will be analysed via the following Aim methodologies at intervals to capture the entire ageing process. Additional tissues from middle-aged/pre-elderly mice (12-18 months) will be analysed. To examine if elevated RSS production alters the ageing process, an inducible CARS2 overexpressing WT and SOD1 KO mice will be generated and analysed as above, and RSS-donor compounds will be administered to WT and SOD1 KO mice.

• Motor Neuron Disease
Motor neurone diseases (MND) are conditions of elevated oxidative stress that result in motor neurone cell death. Amyotrophic Lateral Sclerosis (ALS) is the most common form of MND and SOD1 mutations are known drivers of inherited ALS, however, the biochemical or cellular mechanisms of this are not clear. My preliminary data shows that ALS-associated SOD1 mutants have decreased H2S metabolic capacity and elevated RSS, which points to H2S and/or RSS as potential culprits in ALS pathology.

• Tumour Biology
Tumours are associated with high levels of oxidative stress and hypoxia. Using cellular models and patient tumour explants, the role of RSS in driving hypoxic tumour progression will be studied at the chemical, molecular and cellular level.

Learn more about studying a PhD in Molecular and Cell Biology

Learn more about Dr Kayoko Tanaka

Obtaining an integrated understanding of oncogenic RAS signalling

The RAS family of small GTPases act as signalling hubs regulating cell proliferation and differentiation. The physiological importance of RAS signalling is evident as about 25% of all human cancers harbour mutations in ras genes, where kras is most frequently mutated (about 18%) (COSMIC, v94). However, there is no anti-Ras inhibitor available except the one that targets G12C oncogenic mutation through the thiol group of the cysteine 12. As G12C mutation contributes to just about 10% of kras oncogenic mutations, it is vital to develop effective inhibitors against other oncogenic Ras variants. Towards this goal, we need to understand the mode of action of oncogenic RAS molecules.

The way Ras activates downstream effectors is through direct protein-protein interactions. 56 human proteins are found to have a domain termed either Ras Binding Domain (RBD) or Ras Association domain (RA), that features a ubiquitin-like ββαββαβ fold. Some of them are experimentally proven to act as Ras effectors. Representative examples include RAF kinases that prime ERK pathway activation, PI3 Kinase that leads to Akt activation and RalGEFs that act as a GDP-GTP exchange factor (GEF) for small GTPases, RalA and RalB.

Although the essentiality of RAS-effector interactions in the oncogenic RAS signalling is well-recognised, the dynamic nature of these interactions has been elusive.

• Does one molecule of RAS simultaneously interact with multiple effectors?
• Does the RAS molecule jump between different effectors?
• Does interaction with one of the effectors influence the next interaction?
• Do all effectors contribute equally to cause the oncogenic-Ras phenotype? Or, some of them play a more important role than others?

We will address these questions in the PhD project by combining various techniques, including single-molecule analysis using optical microscopy, structural biology (X-ray crystallography, NMR and cryo-electron microscopy), biochemistry (protein purification, biolayer interferometry and surface plasmon resonance) and live-cell imaging of human culture cells where genes encoding relevant signalling molecules are to be edited by CRISPR-Cas9 technology. Successful delivery of the project will bring a novel concept of RAS signalling and help design inhibitors targeting RAS signalling.

Learn more about studying for a PhD in Molecular and Cell Biology

References

• Hindul NL, Abbott LR, Adan SMD, Straatman KR, Fry AM, Hirota K and Tanaka K (2023) Endogenous oncogenic KRAS expression increases cell proliferation and motility in near-diploid hTERT RPE-1 cells. bioRxiv, doi.org/10.1101/2023.09.08.556827
• Tariq M, Ikeya T, Togashi N, Fairall L, Bueno-Alejo C, Kamei S, Romartinez-Alonso B, Muro Campillo MA, Hudson AJ, Ito Y, Schwabe JWR, Dominguez C, Tanaka K (2022) Structrual and biochemical insights into heterotetramer formation between human oncogenic K-Ras4BG12V and Rgl2, a RalA/B activator. bioRxiv, dio.org/10.1101/2022.10.10.511529.
• Hindul NL, Jhita A, Oprea DG, Hussain TA, Gonchar O, Campillo MAM, O’Regan L, Kanemaki MT, Fry AM, Hirota K, Tanaka K (2022) Construction of a human hTERT RPE-1 cell line with inducible Cre for editing of endogenous genes. Biol Open, 11: bio059056
• Kelsall EJ, Vértesy Á, Straatman K, Tariq M, Gadea R, Parmar C, Schreiber G, Randhawa S, Dominguez C, Klipp E, Tanaka K (2019) Constitutively active RAS in S.pombe causes persistent Cdc42 signalling but only transient MAPK activation. bioRxiv, doi.org/10.1101/380220.

Transcriptional kinases in cancer and developmental disorders

Cyclin-dependent kinases (CDKs) are protein kinases performing essential functions in regulation of cell cycle and transcription. Each CDK requires an interaction with a specific cyclin partner for switching on their kinase activity. Mutations in different CDKs or associated cyclins have been linked to developmental defects. For example, mutations in CDK10 and in cyclin Q are both causing developmental disorders, named Al Kaissi syndrome and STAR syndrome, respectively, that are only partially overlapping. In addition, CDK10 is misregulated and mutated in several types of cancer, including breast and gastro-intestinal cancer. For example, CDK10 has been linked to tamoxifen resistance in breast cancer through its regulation of the ETS2 protein; a transcription factor which can promote cellular proliferation, invasion, and metastasis in cancers. However, the functions of CDK10 and its associated cyclin Q (previously cyclin M) are poorly understood, mostly due to the absence of specific inhibitors.

To determine the functions of CDK10, we used CRISPR/Cas9 genome engineering to generate a human cancer cell line in which the endogenous CDK10 can be selectively and rapidly inhibited using a bulky ATP analogue. Preliminary data has shown that CDK10 inhibition affects gene expression in a potentially ETS2-independent manner, indicating a potential direct role of CDK10 on transcription and/or RNA processing. The CDK10 analogue-sensitive cell line will be therefore used to define by phospho-proteomics the proteins phosphorylated by CDK10 and the effects of CDK10 inhibition on different cellular processes, including cell cycle and gene expression.

CRISPR/Cas9 will also be used to generate novel cell lines with the Al Kaissi and STAR pathogenic mutations found in CDK10 and cyclin Q to understand the cellular consequences of these mutations. This project will provide important insights into the mechanisms behind these two developmental disorder syndromes and will determine if the cellular consequences of CDK10 and cyclin Q mutations are due only to a loss of CDK10 activity or also from additional reasons.

Investigating the roles of CDK10 in ETS2 factor stability and phosphorylation of other targets provides the opportunity to exploit our expertise in basic research to identify druggable pathways regulated by CDK10 for potential clinical benefit. This project will also determine whether inhibiting CDK10 could be clinically relevant for the treatment of specific cancers (gastro-intestinal, cutaneous melanomas, and breast) and pave the way for the development of CDK10 specific inhibitors.

Techniques for this project

Human cell culture, CRISPR/Cas9, phospho-proteomics, RNA-seq, nascent transcription techniques (POINT-seq, PRO-seq), flow cytometry, immunofluorescence, bioinformatics, and standard molecular biology techniques (western blot, co-immunoprecipitation, cloning, PCR, qPCR, …).

Learn more about studying for a PhD in Molecular and Cell Biology

References

• Iorns E, Turner NC, Elliott R, Syed N, Garrone O, Gasco M, et al. Identification of CDK10 as an important determinant of resistance to endocrine therapy for breast cancer. Cancer Cell. 2008;13(2):91-104.
• Guen VJ, Gamble C, Flajolet M, Unger S, Thollet A, Ferandin Y, et al. CDK10/cyclin M is a protein kinase that controls ETS2 degradation and is deficient in STAR syndrome. Proc Natl Acad Sci U S A. 2013;110(48):19525-30.
• Windpassinger C, Piard J, Bonnard C, Alfadhel M, Lim S, Bisteau X, et al. CDK10 Mutations in Humans and Mice Cause Severe Growth Retardation, Spine Malformations, and Developmental Delays. Am J Hum Genet. 2017;101(3):391-403.

Learn more about Dr Jonathon Willets

Investigating the roles that G protein coupled receptors play in hypertension

G protein-coupled receptors (GPCRs) constitute a very large family of heptahelical, integral membrane proteins that mediate a wide variety of physiological processes ranging from the transmission of light and odorant signals to the mediation of neurotransmission and hormonal actions. GPCR signalling plays a vital role in the regulation of smooth muscle excitability to control a wide range of physiological processes including blood pressure and uterine quiescence during pregnancy. Dis-regulation of these GPCR signalling pathways leads to pathophysiological changes associated with conditions such as hypertension and pre-term labour. Our work is focused on identifying and comparing the molecular mechanisms underlying GPCR regulation of smooth muscle excitability in arterial and uterine smooth muscle both in normal and diseased tissue. Present emphasis centres around G protein-coupled receptor kinases and arrestin proteins which were originally identified as mediators of GPCR desensitization, but now are increasingly identified as complex signalling molecules that regulate diverse process such as cell proliferation, migration, metastasis, transcription regulation and apoptosis.

PhDs are available in the following areas of research:

• GPCR regulation of hypertension/ vascular smooth muscle growth and migration
• GPCR regulation in pre-term labour
• The role of endocannabinoid receptor signalling in reproduction

Work is primarily concerned with the roles that G-protein coupled receptor kinases (GRKs) and arrestin proteins play in the regulation of endogenously expressed GPCRs. GPCRs are a large family of cell surface proteins that decode a plethora of external signals to enable cellular communication. Research focuses on regulation of endogenous GPCR signalling in smooth muscle excitability, with relevance to vascular disease. Here, we are interested in two families of proteins, GRK and non-visual arrestins, which are known not only to negatively regulate GPCR signalling but control signalling pathways involved in increasingly diverse cellular processes e.g. migration, growth, metastasis and hypertension.

We combine fluorescent bioprobes/confocal imaging to examine GPCR regulation in ‘real-time’ in primary cell cultures, within days of isolation. We routinely utilise molecular manipulations of protein levels or function to determine their involvement in multiple GPCR signalling cascades and physiological outputs. Combining these powerful techniques enables unique identification of specific interactions of individual endogenous GRKs/arrestins (or other proteins) and endogenously expressed receptors, often only days after isolation. Recently research has examined the role that GRK and arrestins play in the regulation of MAPK signalling pathways in vascular diseases.

Learn more about studying for a PhD in Molecular and Cell Biology

References

• Alonazi, A. S. A., and Willets, J. M. (2021). G protein-coupled receptor kinase 2 is essential to enable vasoconstrictor-mediated arterial smooth muscle proliferation. Cell Signal, 88, 110152. doi:10.1016/j.cellsig.2021.110152
• Nash, C. A., Nelson, C. P., Mistry, R., Moeller-Olsen, C., Christofidou, E., Challiss, R. A. J., and Willets, J. M. (2018). Differential regulation of beta2-adrenoceptor and adenosine A2B receptor signalling by GRK and arrestin proteins in arterial smooth muscle. Cell Signal, 51, 86-98. doi:10.1016/j.cellsig.2018.07.013
• Rainbow, R. D., Brennan, S., Jackson, R., Beech, A. J., Bengreed, A., Waldschmidt, H. V., … Willets, J. M. (2018). Small-Molecule G Protein-Coupled Receptor Kinase Inhibitors Attenuate G Protein-Coupled Receptor Kinase 2-Mediated Desensitization of Vasoconstrictor-Induced Arterial Contractions. Mol Pharmacol, 94(3), 1079-1091. doi:10.1124/mol.118.112524