NIHRDataInsights/HRCSData huggingface dataset:
Title: Mechanisms of selection against cells with mitochondrial dysfunction during mammalian development
Funder: Medical Research Council

Abstract: Mitochondria are cellular organelles that contain their own genome (mtDNA) that encodes for vital components of the bioenergetic machinery and that are found in all eukaryotic organisms. Mitochondrial dysfunction plays a role in a wide range of diseases, from mitochondrial related pathologies to cancer, and are also thought to contribute to ageing. Given the impact of dysfunctional mitochondria, stringent quality controls exist to ensure optimal mitochondrial performance. How these mechanisms act is largely unknown. We have recently demonstrated that in the mouse embryo, just prior to gastrulation, there is an active selection against cells with mitochondrial dysfunction and mtDNA mutations. In this proposal we aim to establish the molecular mechanism underlying this selection and its importance. For this we will do three things. First, by using a combination of candidate and unbiased approaches we will determine which stress responses become activated by cells with mitochondrial defects in the embryo and how manipulating these stresses affects the fate of cells with mitochondrial dysfunction.Second, as we have found that repression of the mTOR pathway is important for the elimination of defective cells during development, we will focus on understanding the importance of mTOR for the elimination of cells with mitochondrial dysfunction and mtDNA mutations. Finally, we will determine the importance that the elimination of cells with mitochondrial dysfunction has for development. For this we will analyse what happens to the patterning and metabolic performance of the different tissues of the early embryo if these cells are not eliminated.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: GENERIC HEALTH RELEVANCE
Title: New embryological perspectives on imprinting disease
Funder: Medical Research Council

Abstract: We have combined micro-whole genome bisulphite sequencing of uniparental mouse embryos with RNA-seq of reciprocal crosses to identify 71 novel blastocyst-imprinted expressed (nBiX) genes with transient allelic expression asymmetry. Asymmetry is lost after implantation and mostly H3K27me3-dependent. The human orthologues of 85% of nBiX genes are associated with disease. However, the relationship between preimplantation imprinted gene expression and disease is poorly understood. We hypothesise that nBiX expression levels in preimplantation embryos are critical. This will be tested by reversibly altering nBiX transcript levels exclusively during preimplantation development, thereby isolating consequences due to early disruption from subsequent effects. nBiX transcript levels will be increased by coinjecting oocytes with sperm plus different concentrations of nBiX cRNA, and depleted by coinjecting oocytes with sperm plus either siRNA (RNAi) or morpholinos (blocking translation) that target nBiX transcripts. Outcomes will be assessed in a battery of tests to characterise molecular, cellular and developmental perturbations that occur during pre- and post-implantation development, and combining them with disease metrics (eg weight gain) post-term. This will relate specific transient preimplantation nBiX RNA perturbation with downstream phenotypic outcomes. We will determine parental expression bias in human blastocysts by ultra-deep RNA-seq with associated whole-exome sequencing. This will generate a unique human dataset, delineate imprinted human gene expression to reveal the degree of conservation to mouse imprinted genes, and inform the selection of nBiX targets. It will also reveal the relationship between early embryonic imprinted (nBiX) gene dysregulation and disease, illuminating regenerative processes (eg pluripotency induction), mechanisms of reprogramming in nuclear transfer cloning and epigenetic inheritance.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING;2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: GENERIC HEALTH RELEVANCE
Title: Regulation of DNA repair pathways by monoubiquitin signals
Funder: Medical Research Council

Abstract: Monoubiquitin signals regulate many essential cellular activities, in particular DNA replication and repair processes. Multiple DNA repair pathways are controlled by highly specific single ubiquitin signals. These include the Fanconi Anemia (FA) pathway which repairs DNA interstrand crosslinks, and the Translesion Synthesis (TLS) pathway which permits replication past the site of damage. Although each pathway has distinct dedicated enzymes for assembling the modification, they share a common regulator which removes the signal. The deubiquitinase (DUB) Ubiquitin specific protease-1 (USP1) is dysregulated in several cancers. In conjunction with its activating co-factor, UAF1, it deubiquitinates FANCD2 and FANCI in the FA pathway, and the proliferating cell nuclear antigen (PCNA) in the TLS pathway. We recently identified mutations in USP1 that disrupt its activity towards FANCD2, but not FANCI and PCNA. This gives us a unique opportunity to uncouple the repair pathways and investigate the influence of each pathway on the other, and how perturbations might modulate the pathway responses to replication stress and DNA damaging agents. This is exciting because of the role of the DNA damage response as a target for anticancer therapy, but also as a mechanism for development of drug resistance. Understanding the molecular mechanisms of interplay between DNA repair pathways may provide new possibilities for targeted therapies. We aim to address this with the following objectives: 1) Determine the molecular mechanism of deubiquitination of specific substrates by USP1-UAF1. 2) Define the molecular basis of the regulatory posttranslational modifications and DNA on substrate targetting. 3) Understand the functional consequences of signalling in the FA pathway on TLS and vice versa. These studies will show what is important for substrate recognition, signal removal, and how this might be regulated in individual targets.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE
Title: The Roadmap of Human Pluripotency
Funder: Medical Research Council

Abstract: We will deconstruct the cellular and molecular progression of human pluripotency using the experimentally tractable system of pluripotent stem cells in defined culture conditions. We will take advantage of naïve stem cell lines representing the earliest stage of pluripotency, developed and characterised with current MRC funding. As a reference for the human embryo, we will exploit our naïve stem cell blastoid model to generate cultures containing embryonic disc-like structures that capture features of peri- and early post-implantation embryogenesis. We will also use human embryos donated for research. We will apply our feeder-free culture system to delimit signal input and use flow sorting to purify cells at similar stages in transition. We will generate knock-in fluorescent reporters for live imaging and quantitation of lineage induction by flow cytometry. To measure signalling pathway activities we will use immunoblotting, immunostaining and gene expression assay by qRT-PCR. Additionally, we will deploy fluorescent reporters for live cell detection of signalling dynamics. We will undertake clonal analysis of lineage potential via DNA barcoding. We will generate scRNA-seq time series data to reveal transcriptome landscapes and will integrate lineage analysis using expressed barcodes. Bioinformatics and computational tools will be applied to deconvolve cell trajectories. Histone modification and transcription factor binding profiles will be generated by Cut & Run and integrated with chomatin profiling by ATAC-seq. We will use bioinformatics and experimental approaches to characterise interactions between SMADs downstream of NODAL and BMPs, ETV factors downstream of ERK, and core pluripotency factors. We will use ChIP-PCR for quantitative measurement of binding dynamics at specific regulatory sites. For functional interrogation, genetic perturbations generated via CRISPR/Cas9 will include rapid and reversible protein elimination using degron technology.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE
Title: Mechanical robustness during tissue development and repair
Funder: Medical Research Council

Abstract: Tissues must develop and maintain their correct morphology to function effectively throughout life. Mechanical forces play an instructive role in tissue morphogenesis, yet tissues are constantly exposed to additional fluctuating external forces, ranging from the continuous 'noise' of daily motion to the extreme perturbations associated with wounding. Despite this, most tissues develop to their target morphology that they maintain and repair throughout life. Tissues, however, are less able to cope with extreme mechanical perturbations, such as a wound, especially in the presence of additional mechanical fluctuations, which can lead to scars. How do tissues respond to different external mechanical fluctuations to achieve tissue robustness during development and repair? We aim to determine: 1. How tissues buffer mechanical 'noise' during development. 2. How the physical properties of tissues enable effective repair after a wound. 3. The role of mechanical noise during tissue repair. We will use a combination of genetics, imaging, biophysics and mathematics to address these aims. Our novel tools to measure, manipulate and model the mechanical forces impacting on tissues put us in a unique position to carry out this programme. This work will determine the impact of mechanical stress on tissue robustness, increase our understanding of the diversity of biological form, improve the treatment of developmental defects affecting tissue morphology, and aid the design of new therapies for more efficient and seamless wound repair.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING;2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: GENERIC HEALTH RELEVANCE
Title: MRC Transition Support Award: Cell biological mechanisms underlying stem cell competition
Funder: Medical Research Council

Abstract: Clonal analyses have shown that, to remain at the niche, a stem cell must continuously compete with neighbours for space. While in control conditions this competition is neutral, such that each stem cell has an equal chance of replacing its neighbour, mutant stem cells carrying an oncogenic mutation leading to constitutive signalling activity can bias replacement in their favour and displace all wild type cells. Building on work showing that competition bias depended on increased proliferative ability, I explored the links between cell cycle-dependent transcription and cell fate during my Fellowship. I showed that the cell cycle-dependent E2f1/Dp transcription factor prevents differentiation by controlling metabolic activity. Additionally, activity of this transcription factor in stem cells is required non-autonomously to maintain quiescence of the niche, revealing an unexpected signalling from stem cells to their niche. An outstanding aim of the proposal is to determine how signals regulating stem cell competitiveness affect proliferation. To this end, we will analyse the transcriptional targets of these signals and determine a gene expression signature of competitiveness. Secondly, we studied the inputs into differentiation, and showed that the Tor pathway induces a permissive state for differentiation, but that commitment requires input from another lineage in the tissue, allowing coordination of differentiation across the lineages. Additionally, we have shown that differential mechanisms of translation initiation operate in differentiation and self-renewal. An outstanding aim is to determine which transcripts are differentially translated in stem cells to gain insight into post-transcriptional mechanisms allowing stem cells to react rapidly to environmental signals and undergo differentiation. Altogether, this work will provide a complete picture of the mechanisms regulating stem cell competitiveness, from the upstream signals to the downstream cell biology.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE
Title: Epigenetic control of Microhomology Mediated End Joining (MMEJ) in heterochromatin of Lamina Associated Domains.
Funder: Medical Research Council

Abstract: Cells are subjected to thousands of DNA lesions a day, which require high-fidelity repair to avoid genetic mutations and genomic rearrangements. These deleterious effects of DNA damage can contribute to tumour formation, and they are frequently exploited in anticancer therapies. A key feature of the mammalian cell nucleus is the non-random arrangement of the genome within the nuclear space. It is becoming increasingly clear that the nuclear organization of genomic DNA affects how cells cope with DNA damage by determining the balance of error free and error prone DNA repair. DSBs at heterochromatin and late replicate domains such as Lamina Associated domains (LADs), are prone to highly erroneous DNA repair which is in line with the high mutation rates detected in heterochromatin of cancer genomes. What shifts the balance towards mutagenic DNA repair in heterochromatin of LADs in expense of an error free DNA repair pathway and what are the consequences of this for the integrity of LADs and nuclear lamina are largely unexplored. We have developed novel experimental tools that enable us to induce specific DSBs at LADs in a tightly controlled manner, to princely measure the kinetics of different DNA repair pathways in real time and to identify the proteome of LADs in the absence and presence of DNA damage. These tools will allow us to carefully dissect the epigenetic mechanisms that promote the activation mutagenic DNA repair at LADs and how these impact on maintenance of heterochromatin and nuclear lamina. Furthermore, they will allow us to understand how we can modify chromatin to shift DNA repair pathways choice and exploit it for cancer treatment. We will employ cutting-edge approaches such as Dam-ID, Bio-ID, TIDE/NGS and HT-microscopy to provide the first systematic account of DSB repair dynamics and fidelity in LADs, answering long-standing questions on the causative or passenger role of the underlying chromatin structure in genome stability.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE
Title: The regulation of transcriptional bursting by superenhancers
Funder: Medical Research Council

Abstract: Progress in biomedicine requires us to obtain a complete understanding of how individual genes are switched on and off during differentiation and development. This involves understanding how the fundamental cis-regulatory elements of the genome (enhancers, promoters and insulators) interact with each other within the 3-dimensional genome, over time. Although there may be an almost infinite number of ways in which such elements may interact at different loci, the principles by which they do so are likely to be conserved throughout evolution. Here we will analyse the well characterised regulatory elements controlling the alpha globin locus whose expression is regulated throughout well-defined cellular stages of development and differentiation. We have previously identified all cis regulatory elements in the locus and shown that five enhancers constituting a "superenhancer", control expression of both embryonic and adult genes. As these genes are activated, the superenhancer comes into close physical contact and plays a key role in recruiting the pre-initiation complex and Polymerase II. Of interest, we have recently shown that alpha-gene expression occurs in transcriptional bursts; a common, well recognised but poorly understood mechanism of gene expression. To address how the superenhancer regulates transcriptional bursting at its natural locus, we have made models in which each enhancer is removed, individually and in combination. More recently, we have rebuilt the superenhancer, element by element, in a series of models and shown that individual components underpin different roles. Here we propose to use single molecule imaging, live imaging and super-resolution imaging to analyse these models to determine each element's contribution to the process of transcriptional bursting. Finally, we will test our findings in their clinical context by examining how natural mutations of the human enhancers are correlated with changes in the red cells of affected individuals.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE
Title: MLTC-M Community of Practice in ECR training on best practice patient and public involvement with diverse populations
Funder: Medical Research Council

Abstract: The work plan will be defined based on individual needs but provisionally will include a combination of face-to-face and online meetings, focus groups and one-to-one interviews. It is anticipated that two face-to-face meetings will be conducted; one workshop at the start of the project and a second at the end to discuss dissemination and sustainability strategies. PPI meetings, discussions with public representatives, expert-led seminars and strategy sharing workshops will be conducted online. A Working Group will be established to: oversee the delivery of the project and reflect, report on, and disseminate learnings; plan for how the CoP will be sustained beyond the lifetime of the Collaboratives; and identify funding streams and strategies for ongoing future engagement of the CoP. A 'sandpit' workshop will take place to understand the needs, expectations and challenges of ECRs around PPI in MLTC research. Sandpits are "workshops that bring together researchers from different institutions and disciplines to discuss a specific topic or problem" to create "intensive discussion forums where free thinking is encouraged" and where "lateral thinking through role play, small group work and brainstorming activities" are arranged. Following this, focus groups or interviews will be conducted to understand the perspectives of patients, and skills-building workshops will be delivered to enable ECRs to be better informed and equipped to enhance their own PPI practices. Collaboratives will share strategies on ways to embed diverse voices within PPI and identify areas for future improvement; and a strategy will be developed for the dissemination of guidelines and tools for evaluating the impact of PPI, based on shared learning across Collaboratives. Our preliminary discussions have already highlighted some challenges that participating Collaboratives are currently facing and the need for shared learning on strategies to embed diverse populations into PPI in MLTC research.


Research activity codes: 1.5 RESOURCES AND INFRASTRUCTURE (UNDERPINNING)
Health categories: GENERIC HEALTH RELEVANCE
Title: MLTC-M Community of Practice in Statistical Methods for Multimorbidity Research
Funder: Medical Research Council

Abstract: The CoP will define best practices and coordination of effort for the following themes: Defining multimorbidity: Each Research Collaborative will identify patients with multiple long term conditions. Defining these robustly from electronic medical records and cohort data is critical. Resources exist with published code lists, but consistency is lacking. Sharing best practice will add considerably to the robustness and efficiency in documenting and publishing these. Statistical Genetics: Genome-wide association studies (GWAS) aim to determine association between genetic variants and a specific outcome. Extending GWAS to multimorbidity is an active area for the research collaboratives. "Genetic correlation" determines the overall shared genetics between traits, while "Mendelian randomisation" uses genetic instrumental variables to test for causality. The CoP will identify collaborative opportunities and share knowledge to apply these methods. Translation to treatment: Each consortium aims to improve the treatment of individuals with multimorbidity. Genetic discoveries can identify targetable biological mechanisms and pharmacogenetics investigates the effect of genetic variants on treatment response, potentially aiding the development of personalised treatments. The Research Collaboratives will use diverse forms of clinical data, such as imaging, which are difficult to analyse using traditional methods. However, techniques such as machine learning (ML) can be used. ML aims to develop computer algorithms that improve with experience. Examples include processing and normalising raw data, integrating genomic information, exploring data structure, predictive modelling, generative modelling, and prioritising experiments. Data resource curation: The CoP will actively work to share best practices, new data and data analysis code between the research consortiums. Output will be circulated to the wider scientific community through reports and educational resources.


Research activity codes: 2.6 RESOURCES AND INFRASTRUCTURE (AETIOLOGY)
Health categories: GENERIC HEALTH RELEVANCE
Title: MLTC-M Community of Practice in Clinical Context and Pathways (Clinical Community of Practice)
Funder: Medical Research Council

Abstract: This clinical Community of Practice (CoP) will enable meaningful networking and collaboration between clinician researchers working with multiple long-term conditions/multimorbidity (MLTC-M) and aid dissemination and pathways to impact. The clinical CoP is flexible to address the clinical challenges of the MLTC-M Research Collaboratives. We have initiated dialogue with the NIHR Clinical Research Network (CRN) project on MLTC-M, and plan to develop an online platform with NIHR Learn to support online networking and communication for clinicians in MLTC-M. A monthly newsletter will include professional development opportunities and events, and a specific hashtag will facilitate social media connections. The clinical CoP will host two face-to-face events: a launch event, to co-develop initial priorities with members and a clinical showcase for cross-Collaborative presentations. Between these events we will host 3 symposiums of cross-Collaborative research tailored to specific conferences, eg. the British Geriatrics Society and the Society of Academic Primary Care, from whom we have support in principle. We will conduct a survey to understand clinicians' key priorities for MLTC-M research, which we anticipate will be published and further developed to form a Delphi exercise through writing groups for external funding. We will organise 6 online workshops, focussed on priorities established by CoP members, working synergistically and responsively with other CoPs. These might include complex phenotypes or disease clusters and pathways to impact. We will support the formation of topic-specific working groups, which may overlap in content and timing with these workshops, and writing groups to co-develop external grant applications, for example on patient-focussed pathways to impact. The platform and community will outlast the funded CoP through stakeholder partnership, engagement with the wider MLTC-M clinical research community and collaborative funding bids.


Research activity codes: 7.4 RESOURCES AND INFRASTRUCTURE (DISEASE MANAGEMENT)
Health categories: GENERIC HEALTH RELEVANCE