Gene regulation by distal regulatory elements in Erythropoiesis and the effect of natural sequence variants.

Lead Research Organisation: University of Oxford

Abstract

Biology has undergone a revolution over the last decade as we can now sequence all of an organism's DNA and reconstruct its complete blueprint. A fundamental question in biology is how are different parts of this DNA blueprint used in different situations (a nerve cell compared to a blood cell) when the both contain the same DNA sequence. The most basic expression of a genome's activity is which genes are turned on to produce the proteins of a cell and so determine which type of cell it is. Technical advances in sequencing technologies now allow us to investigate how and in what situations particular parts of that blueprint are used. This shows a very complex control mechanism where small sections of DNA are scattered around the DNA of the genes they control. This distribution makes it difficult to know which elements control which gene and so decode the instructions in the blueprint. We have developed an approach that allows us to see which control regions interact with which genes. We intend to use this to work out how these regions control genes, but also what happens when these region are damaged and leave us vulnerable to certain diseases.

Technical Summary

It has become clear over the last 10 years that control of gene expression is very often not determined by the promoter of the gene, but rather by variable numbers of regulatory elements, which are unpredictably distributed in and around the genes themselves. These so called distal regulatory elements are situate away from the genes the control, often over large genomic distances and are separated by intervening unrelated genes or even embedded in the introns of other genes. The inability to link any given gene with its regulatory elements is a fundamental problem which has greatly hampered our understanding of gene regulation. Although of great biological importance, the role of these elements in health and disease is becoming increasing clear as the effect of mutations in these regions begins to come to light. In particular the vast majority of predicted variants in the normal population, which from GWAS studies, are associated with disease susceptibility are not found in coding regions and are thought to be acting through changes in distal regulatory elements. To address this problem have developed a high resolution and high throughput method to robustly map the physical interactions between genes and their regulatory elements. Using this Capture-C method we have mapped en masse the regulatory interactions of over 450 genes in the mouse erythroid system. The Capture-C method massively increases the number of genes with linked regulatory elements and we intend to use this ability to study how the molecular events at regulatory elements coordinate expression from linked gene promoters. Our experimental model is the extremely well characterised murine erythroid differentiation system. This model provides large numbers of cells from well defined stages of differentiation which will allow us to study the transcriptional regulation of these genes in a dynamic fashion and includes two extremely well studied paradigms of gene regulation, the ? and ? globin loci. To study changes in protein binding we use high-resolution versions of the DNAse-seq and ChIP-seq assays and to determine the expression status of the genes under study we have developed highly quantitative RNA assays using metabolic labelling of nascent RNA. Additionally we will use our human erythroid model to investigate the effect of previously characterised GWAS SNPs responsible for normal variation of erythroid parameters to determine the underlying principals of how regulatory variants can affect human health and disease susceptibility.

Publications

10 25 50
 
Title • CELL- Dance project with Powys Dance 
Description The MRC WIMM was approached in March 2018 by Theatre Powys Dance Company (now known as Impelo), regarding the possibility of providing scientific input to a new dance collaboration. The aim of the project was to deliver key science curriculum objectives in the area of biology via a novel and engaging medium. During 2017/18 MRC HIU collaborated with Abingdon Science Partnership to establish a course for teachers. Abingdon Science Partnership were also involved in the Impelo project, and suggested that our PEC Officer might be able to identify researchers specialising in the area of DNA and genomics. Two members of MRC MHU working in this area were involved in the project: Prof Veronica Buckle and PhD student Caroline Harrold (Higgs/Hughes group). 
Type Of Art Performance (Music, Dance, Drama, etc) 
Year Produced 2019 
Impact The resulting shows is fun and interactive, including inflatable cells that come alive with dance, and make children and adults marvel at the power, quirk and hidden world inside our bodies. The show is also accompanied by a comic book that audiences can take away with them, which expands upon and reinforce the visual information in the show. Prof Veronica Buckle is a character in this book. There will also be a set of digital resources. The project has now reached its final stages, and the show CELL had its first performances on the 1st and 2nd of July 2019 at Imperial College. The show will then go on tour around the UK to schools, venues and festivals from Autumn 2019. We hope to be able to bring CELL to our local community as part of the 2019 Oxford Science and Ideas Festival. 
 
Description 3D genome MRC Project Grant
Amount £382,298 (GBP)
Funding ID MR/N00969X/1 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 02/2016 
End 02/2019
 
Description DPhil Studentship
Amount £82,863 (GBP)
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 10/2014 
End 09/2018
 
Description DPhil Studentship
Amount £161,673 (GBP)
Funding ID 109110/Z/15/Z 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2016 
End 09/2020
 
Description MRC Centenary Award
Amount £30,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 09/2012 
 
Description MRC Project Grant
Amount £382,298 (GBP)
Funding ID MR/N00969X/1 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 02/2016 
End 01/2019
 
Description National Institute of Health USA
Amount £917,526 (GBP)
Organisation National Institutes of Health (NIH) 
Sector Public
Country United States
Start 05/2015 
End 04/2020
 
Description National Institute of Health USA. VISION
Amount £247,699 (GBP)
Organisation National Institutes of Health (NIH) 
Sector Public
Country United States
Start 07/2016 
End 05/2022
 
Description PhD Studentship
Amount £161,117 (GBP)
Funding ID 105281/Z/14/Z 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2014 
End 09/2018
 
Description Quinquennial Revew
Amount £29,000,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 04/2017 
End 03/2022
 
Description Wellcome Trust Research Training Fellowship
Amount £265,927 (GBP)
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2015 
End 09/2018
 
Description Wellcome Trust Strategic Award
Amount £2,664,612 (GBP)
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 03/2015 
End 02/2019
 
Description wellcome Trust Institutional Strategic Support Fund
Amount £50,679 (GBP)
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2014 
End 09/2019
 
Title CSynth 
Description Pilot project for the dynamic visualization of 3D nuclear structure 
Type Of Material Technology assay or reagent 
Year Produced 2017 
Provided To Others? Yes  
Impact Developed in collaboration with Stephen Taylor and Goldsmiths university this tool allows for the interaction and interrogation of 3-Dimensional Chromatin structure. The aim being to provide and an intuitive way of humans interacting with complex 3D structure in the nucleus to further our understanding of gene regulation. 
URL http://www.csynth.org/
 
Title Capsequm Design Tool 
Description The Capsequm tool is a publicly available web tools used to design capture probe for the Capture-C method 
Type Of Material Technology assay or reagent 
Year Produced 2015 
Provided To Others? Yes  
Impact People and now using this tool worldwide to design and perform Capture-C experiments 
URL http://apps.molbiol.ox.ac.uk/CaptureC/cgi-bin/CapSequm.cgi
 
Title Capture-C 
Description Capture-C is a method to link regulatory elements in the mammalian genome to the genes the regulate. The method is a massively multiplexed version of the Chromatin Conformation Capture Assay 
Type Of Material Technology assay or reagent 
Year Produced 2014 
Provided To Others? Yes  
Impact The method now form the basis of a new initiative to understand how variants identified in in Genome-Wide association studies mediate their effect. 
URL http://slave03.molbiol.ox.ac.uk/CaptureC/cgi-bin/CapSequm.cgi
 
Title Next Generation Capture-C 
Description A vast more sensitive version of the original Capture-C assay. 
Type Of Material Technology assay or reagent 
Year Produced 2016 
Provided To Others? Yes  
Impact This approach is now being used world-wide to interrogate question of gene regulation and the effect of sequence changes associated with common disease. 
 
Title Sasquatch 
Description An online version of our computational tools for high throughput prediction of the effect of SNPs on transcription factor binding in non-coding regulatory regions using DNase footprint meta-analysis 
Type Of Material Technology assay or reagent 
Year Produced 2017 
Provided To Others? Yes  
Impact This tool allows for the prioritization of genomic sequence variants based of the impact on the formation of DNA foorprints 
URL http://apps.molbiol.ox.ac.uk/sasquatch/cgi-bin/foot.cgi
 
Title scaRNA-seq 
Description A method to detect and quantify the amount of promoter-proximal pausing found at the level of individual genes. 
Type Of Material Technology assay or reagent 
Year Produced 2021 
Provided To Others? Yes  
Impact Gene transcription occurs via a cycle of linked events, including initiation, promoter-proximal pausing, and elongation of RNA polymerase II (Pol II). A key question is how transcriptional enhancers influence these events to control gene expression. Here, we present an approach that evaluates the level and change in promoter-proximal transcription (initiation and pausing) in the context of differential gene expression, genome-wide. This combinatorial approach shows that in primary cells, control of gene expression during differentiation is achieved predominantly via changes in transcription initiation rather than via release of Pol II pausing. Using genetically engineered mouse models, deleted for functionally validated enhancers of the a- and ß-globin loci, we confirm that these elements regulate Pol II recruitment and/or initiation to modulate gene expression. Together, our data show that gene expression during differentiation is regulated predominantly at the level of initiation and that enhancers are key effectors of this process. 
URL https://www.sciencedirect.com/science/article/pii/S1097276521000022?via%3Dihub
 
Title Sasquatch 
Description Based on an approached developed by my group the Sasquatch tool and database uses DNase Footprinting to assess the damaging potential of a SNP in a tissue-specific manner. 
Type Of Material Data analysis technique 
Year Produced 2016 
Provided To Others? Yes  
Impact The tool is now being used by many external groups via our public web interface 
URL http://apps.molbiol.ox.ac.uk/sasquatch/cgi-bin/foot.cgi
 
Description Chemistry and imaging 
Organisation University of Oxford
Department Department of Chemistry
Country United Kingdom 
Sector Academic/University 
PI Contribution This collaboration brought together a multidisciplinary team or molecular biologists, microscopy experts and chemistry to generate synthetic probes for DNA capture and imaging
Collaborator Contribution As PI on the grant I coordinate the collaboration and bring our bioinformatic and molecular skills
Impact We submitted and were awarded an MRC project Grant to further this collaboration
Start Year 2016
 
Description Dark Matter Project 
Organisation New York University
Country United States 
Sector Academic/University 
PI Contribution I have been made a member of the Dark Matter project based on our work to predict 3D genome structure using deep neural network approaches and build genome regulatory domains from scratch to understand the principles of gene regulation in the mammalian genome.
Collaborator Contribution The Dark Matter Project are expert in the use of large-scale synthetic biology approaches and have agreed to build from scratch 10 complete regulatory domains guided by our machine learning approaches. These will be integrated into the mouse genome to test basic principles of mammalian gene regulation
Impact The partnership has only just been initiated.
Start Year 2021
 
Title NG Capture C 
Description The IP covers the Next Generation Capture-C method for massively multiplexed 3C analysis of genome interactions. 
IP Reference WO2017068379 
Protection Patent application published
Year Protection Granted 2016
Licensed No
Impact None so far
 
Description Epigenesys meeting 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Undergraduate students
Results and Impact xx
Year(s) Of Engagement Activity 2014
 
Description Methods & Techniques Course 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Postgraduate students
Results and Impact This is a yearly lecture given to University of Oxford DPhil students to teach them the principals and concepts of genome analysis.
Year(s) Of Engagement Activity 2016,2017,2018
 
Description New Scientist Live 2017 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact We organised and presented an interactive exhibit on the how the 3-Dimension Genome is linked to common human disease. At the center of this was a specific implementation of our CSynth tool for structural visualisation of genome structure http://www.csynth.org/.
Year(s) Of Engagement Activity 2016
URL https://live.newscientist.com/exhibitors/weatherall-institute-of-molecular-medicine/
 
Description Royal Society Summer Exhibition 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact XXX
Year(s) Of Engagement Activity 2017
 
Description Student Internship 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Undergraduate students
Results and Impact I organised a short unpaid internship for a student who wished to extend their practical experience of scientist. This student Miriam O'Hanlon came an worked in my lab for a period of 6 weeks, performing supervised experiments and joining in the group presentations, lectures and discussions.
Year(s) Of Engagement Activity 2015
 
Description Student work experience 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact School children aged 16-18 have the opportunity to work in the lab for a period of 1 week

Some have gone on to study medicine of biomedical sciences at University
Year(s) Of Engagement Activity 2014
 
Description Thought leader Ship article covering our research 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact We were the subject of and interviewed for an article for Research Features covering thought leadership.
Year(s) Of Engagement Activity 2017
URL http://cdn.researchfeatures.com/3d_issues/issue104/index.html
 
Description Visits to Schools 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Based on the demonstration and science we developed for the Royal society and New Scientist live events, we have restructured this into a more mobile experience which a rolling series of volunteers have been taking to various schools in and around the Oxford area.
Year(s) Of Engagement Activity 2017,2018