Three-dimensional interrogation of gene regulation using next generation chromatin conformation capture and super-resolution imaging.

Lead Research Organisation: University of Oxford
Department Name: Weatherall Inst of Molecular Medicine

Abstract

To form the cells and tissues that make up our bodies requires exquisite control of the activity of our genes. The protein products of genes form the functional and structural components of our cells. Gene expression is controlled by a subgroup of proteins called transcription factors which bind specific sequences in our DNA (also called the "genome"). If this control is altered by sequence changes in the genome then the correct function of our cells is affected and can lead to disease: the particular disease or diseases depend on the types of cells affected. Scientists have become very good at finding the ~ 2% of our DNA that encodes for protein, however it has become clear that other parts of our genome (called regulatory elements) play a critical role in controlling our genes.

Regulatory elements are scattered throughout the genome and novel methods that identify such elements show that it is very difficult to understand which gene they control as they do not necessarily control nearby genes, but may affect quite distant genes. A technique called chromosome conformation capture can link regulatory elements to the genes they control by mapping their physical interactions, which are necessary for their activity, but this still does not explain why certain elements can contact and regulate some specific genes but not others. This is an important question because changes in the genome sequence can prevent regulatory elements talking to the correct genes or cause them to talk to the wrong genes with very damaging consequences. The mechanism that makes these interactions specific is unknown, but is thought to be due to the three dimensional arrangement of the genome in the nucleus. The work in this proposal adapts technologies developed by a collaboration between molecular biologists, chemists and computational scientists to visualize these interactions in 3D to understand how certain regulatory elements work with specific genes and how changes in the genome can alter this and cause disease.

Technical Summary

Next Generation Sequencing has allowed us to investigate the structure and function of the genome at unprecedented detail. In particular, chromatin accessibility assays (DNAse-seq and ATAC-seq) produce comprehensive maps of the active regions of the genome and ChIP-seq allows sub-classification by chromatin signature which infers potential function. This has revealed an unsuspected level of complexity in the regulatory landscape of the genome with estimates of ~ 2M potential regulatory elements to control ~20,000 genes. GWAS studies have mapped the majority of disease susceptibility variants to the non-coding portion of the genome suggesting that regulatory elements may be their major targets. However finding which genes these regulatory elements are controlling and how this is achieved is a major challenge, mainly due to the unpredictability of both their number and distribution around their cognate genes. It has been shown that although these elements affect multiple genes, they may "ignore" neighbouring genes in favour of more distal genes. Chromosome conformation capture-based technologies link regulatory elements and genes via their physical interaction, but do not explain why some interactions are favoured and others prevented. As the physical interactions of these elements are thought to be required for their function, this represents a fundamental mechanism for specificity and selectivity. Such interactions, in turn, are likely related to the overall 3D structure of the genome. As small changes in non-coding sequence can have major effect on gene expression, and considering the amount of observed genome variation, it is essential to understand the mechanisms by which regulatory elements work. We propose to develop new technologies to visualize specific regulatory interactions at unprecedented levels of sensitivity and resolution, so that regulatory elements can be seen interacting within the context of the surrounding chromosomal landscape.

Planned Impact

Beneficiaries of this research will be:
1. Research scientists in academia who are working to develop a better understanding of biology in healthy cells and organisms.
2. Biomedical scientists in academia and industry who are endeavouring to understand the biological processes that occur in the development and progression of human diseases.
3. In particular the project will train a highly skilled postdoctoral researcher who will develop new inter-disciplinary skills.
4. Scientists in the pharmaceutical and biotech industries who are working in discovery biology to determine new targets for diagnostics and therapy in a wide range of diseases.
5. Companies that manufacture and supply oligonucleotide probes to academia and industry for use in the above applications.
6. The UK economy through pharmaceutical and industrial development enhancing business revenue and innovative capacity.
7. The general public who could benefit from new and improved diagnostics and therapies.
8. The general public in gaining knowledge from the modelling and visualisation outcomes proposed.
9. Schools and colleges through outreach activities.

Publications

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Oudelaar AM (2017) Between form and function: the complexity of genome folding. in Human molecular genetics

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Paralkar V (2016) Unlinking an lncRNA from Its Associated cis Element in Molecular Cell

 
Title Activation of alpha-globin transcription in primary mouse erythroid progenitors 
Description Mammalian gene expression patterns are controlled by regulatory elements, which interact within Topologically Associating Domains (TADs). The relationship between activation of regulatory elements, formation of structural chromatin interactions and gene expression during development is unclear. Here, we present Tiled-C, a low-input Chromosome Conformation Capture (3C) technique. We use this approach to study chromatin architecture at high spatial and temporal resolution through in vivo mouse erythroid differentiation. In this dataset, we measure nascent transcription of the mouse alpha-globin genes by FISH using oligonucleotide probes that are specific for the alpha-globin introns. We measure the initial stages of alpha-globin upregulation in three subsets of early erythroid progenitors (S0-low, S0-medium and S1) and compare these to the levels observed in a positive control (mature, Ter119+ mouse erythroblasts) and in two negative controls (mouse primary brain, which does not express alpha-globin, and a no primary antibody control). 
Type Of Art Image 
Year Produced 2020 
URL https://idr.openmicroscopy.org/webclient/?show=project-1151
 
Title Chromatin arranges in chains of mesoscale domains with nanoscale functional topography independent of cohesin 
Description Here we use 3D super-resolution and scanning electron microscopy to analyze structural and functional nuclear organization in somatic cells. We identify linked chromatin domains (CDs) composed of irregular ~200-300-nm-wide aggregates of nucleosomes that can overlap with individual topologically associating domains and are distinct from a surrounding RNA-populated interchromatin region. High-content mapping uncovers confinement of cohesin and active histone modifications to surfaces and enrichment of repressive modifications towards the core of CDs in both hetero- and euchromatic regions. This nanoscale functional topography is temporarily relaxed in postreplicative chromatin, but remarkably persists after ablation of cohesin. Our findings establish CDs as physical and functional modules of mesoscale genome organization. 
Type Of Art Image 
Year Produced 2020 
URL https://idr.openmicroscopy.org/webclient/?show=project-1159
 
Title Chromatin arranges in chains of mesoscale domains with nanoscale functional topography independent of cohesin 
Description Here we use 3D super-resolution and scanning electron microscopy to analyze structural and functional nuclear organization in somatic cells. We identify linked chromatin domains (CDs) composed of irregular ~200-300-nm-wide aggregates of nucleosomes that can overlap with individual topologically associating domains and are distinct from a surrounding RNA-populated interchromatin region. High-content mapping uncovers confinement of cohesin and active histone modifications to surfaces and enrichment of repressive modifications towards the core of CDs in both hetero- and euchromatic regions. This nanoscale functional topography is temporarily relaxed in postreplicative chromatin, but remarkably persists after ablation of cohesin. Our findings establish CDs as physical and functional modules of mesoscale genome organization. 
Type Of Art Image 
Year Produced 2020 
URL https://idr.openmicroscopy.org/webclient/?show=project-1161
 
Title Chromatin arranges in chains of mesoscale domains with nanoscale functional topography independent of cohesin 
Description Here we use 3D super-resolution and scanning electron microscopy to analyze structural and functional nuclear organization in somatic cells. We identify linked chromatin domains (CDs) composed of irregular ~200-300-nm-wide aggregates of nucleosomes that can overlap with individual topologically associating domains and are distinct from a surrounding RNA-populated interchromatin region. High-content mapping uncovers confinement of cohesin and active histone modifications to surfaces and enrichment of repressive modifications towards the core of CDs in both hetero- and euchromatic regions. This nanoscale functional topography is temporarily relaxed in postreplicative chromatin, but remarkably persists after ablation of cohesin. Our findings establish CDs as physical and functional modules of mesoscale genome organization. 
Type Of Art Image 
Year Produced 2020 
URL https://idr.openmicroscopy.org/webclient/?show=project-1160
 
Title Chromatin arranges in chains of mesoscale domains with nanoscale functional topography independent of cohesin 
Description Here we use 3D super-resolution and scanning electron microscopy to analyze structural and functional nuclear organization in somatic cells. We identify linked chromatin domains (CDs) composed of irregular ~200-300-nm-wide aggregates of nucleosomes that can overlap with individual topologically associating domains and are distinct from a surrounding RNA-populated interchromatin region. High-content mapping uncovers confinement of cohesin and active histone modifications to surfaces and enrichment of repressive modifications towards the core of CDs in both hetero- and euchromatic regions. This nanoscale functional topography is temporarily relaxed in postreplicative chromatin, but remarkably persists after ablation of cohesin. Our findings establish CDs as physical and functional modules of mesoscale genome organization. 
Type Of Art Image 
Year Produced 2020 
URL https://idr.openmicroscopy.org/webclient/?show=project-1158
 
Title Chromatin arranges in chains of mesoscale domains with nanoscale functional topography independent of cohesin 
Description Here we use 3D super-resolution and scanning electron microscopy to analyze structural and functional nuclear organization in somatic cells. We identify linked chromatin domains (CDs) composed of irregular ~200-300-nm-wide aggregates of nucleosomes that can overlap with individual topologically associating domains and are distinct from a surrounding RNA-populated interchromatin region. High-content mapping uncovers confinement of cohesin and active histone modifications to surfaces and enrichment of repressive modifications towards the core of CDs in both hetero- and euchromatic regions. This nanoscale functional topography is temporarily relaxed in postreplicative chromatin, but remarkably persists after ablation of cohesin. Our findings establish CDs as physical and functional modules of mesoscale genome organization. 
Type Of Art Image 
Year Produced 2020 
URL https://idr.openmicroscopy.org/webclient/?show=project-1152
 
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 £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 Dynamics in the regulatory genome.
Amount £162,000 (GBP)
Funding ID 220046/Z/19/Z 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2019 
End 09/2022
 
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. VISION
Amount £247,699 (GBP)
Organisation National Institutes of Health (NIH) 
Sector Public
Country United States
Start 07/2016 
End 05/2022
 
Description Quinquennial Revew
Amount £29,000,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 04/2017 
End 03/2022
 
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 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 RASER-FISH 
Description RASER-FISH stands for resolution after single-strand exonuclease resection and maximally retains nuclear structure while performing FISH experiments. Published in Nature Communications/ 
Type Of Material Technology assay or reagent 
Year Produced 2018 
Provided To Others? Yes  
Impact We show that this region forms an erythroid-specific, decompacted, self-interacting domain, delimited by frequently apposed CTCF/cohesin binding sites early in terminal erythroid differentiation, and does not require transcriptional elongation for maintenance of the domain structure. Formation of this domain does not rely on interactions between the a-globin genes and their major enhancers, suggesting a transcription-independent mechanism for establishment of the domain. However, absence of the major enhancers does alter internal domain interactions. Formation of a loop domain therefore appears to be a mechanistic process that occurs irrespective of the specific interactions within. 
 
Title TRI-C 
Description TRI-C is a multiplex multiways 3C assay, published in Nature Genetics that maps the coincident and simultaneous interaction between related regulatory elements in the mammalian genome. 
Type Of Material Technology assay or reagent 
Year Produced 2019 
Provided To Others? Yes  
Impact TRI-C showed for the first time that regulatory elements cluster in 3D space, which has profound implications for our understanding of mammalian gene regulation. In follow up work it was used to produce a revised model for promoter competition, published in Nature Comummincations. 
 
Title Tiled-C 
Description Tiled-C is an adaptation of the Capture-C technologies that generate ultrdeep Hi-C like data and is applicable to very small cells numbers. At present under consideration in nature communications. 
Type Of Material Technology assay or reagent 
Year Produced 2020 
Provided To Others? Yes  
Impact Tiled-C has revised our current understanding of the link between the regulatory structure or the genome, epigenetic activity and gene expression. 
 
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 MRC Human Genetic Unit Edinburgh 
Organisation Medical Research Council (MRC)
Department MRC Human Genetics Unit
Country United Kingdom 
Sector Academic/University 
PI Contribution This is a scientific collaboration with Prof Nick Gilbert and Davide Marenduzzo, based around an application for a Wellcome Trust Investigator award to work on the molecular basis of 3D genome interaction that direct gene expression.
Collaborator Contribution All three group are expert in different aspect of the 3D genome and have formed a collaboration to use their expertise in collaboration and to ask for funding from the Wellcome trust to support this work.
Impact The collaboration has just been initiated.
Start Year 2021
 
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 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