Nuclear organisation and erythropoiesis

Lead Research Organisation: University of Oxford
Department Name: UNLISTED

Abstract

Basic DNA sequence is only a starting block for understanding the complex interplay of factors that regulate the ability of a gene to make the correct protein at the correct time and in the correct cell type. We have detailed maps of a region of chromosome 16 around the alpha globin genes with regulatory features within the sequence and additional (epigenetic) factors which can influence when and where a gene becomes active. DNA coils down within a cell nucleus with several proteins to form chromatin. We now want to look inside individual cells at the way chromatin is organised around the alpha globin genes when they are switched on and off. We would like to understand how this chromatin is arranged within a nucleus and what changes in conformation and condensation may be necessary to allow genes to become active. We will also look at how DNA replicates itself in this region and whether that alters when the alpha globin genes are very active. Understanding these very basic concepts of how genes are regulated is an important foundation to developing new ways to modify gene activity for patients with inherited anaemias.

Technical Summary

The organisation of chromatin within the nucleus has a key role to play in the processes of transcription and replication but the mechanisms governing how chromatin domains are established and altered during the course of development and differentiation remain poorly understood. There is a broad consensus that chromatin is organised in dynamic loops but many questions remain about what may drive their formation, how long they are stable for and how they may alter with the cell cycle. The ? globin gene region is one of the best-characterised segments of the genome and it is an ideal model with which to investigate such issues. Analysis of single cells will be an essential complement to the genome-wide studies of chromatin configurations described in Professors Higgs & Gibbons’ programme. Our ultimate aim is to gain better insight into transcriptional regulation. The questions we plan to address are – What are the kinetics of transcription from the ? globin and nearby genes during erythroid differentiation? What are the concurrent spatial relationships across the ? globin regulatory region? What mechanisms underlie the striking decompaction of chromatin around these heavily transcribed genes? What contribution may replication make to gene regulation and also to misregulation in disease?

People

ORCID iD

Publications

10 25 50

Related Projects

Project Reference Relationship Related To Start End Award Value
MC_UU_00016/1 01/04/2017 31/03/2022 £3,035,000
MC_UU_00016/2 Transfer MC_UU_00016/1 01/04/2017 31/03/2022 £3,411,000
MC_UU_00016/3 Transfer MC_UU_00016/2 01/04/2017 31/03/2022 £1,366,000
MC_UU_00016/4 Transfer MC_UU_00016/3 01/04/2017 31/03/2020 £3,017,000
MC_UU_00016/5 Transfer MC_UU_00016/4 01/04/2017 31/03/2020 £497,000
MC_UU_00016/6 Transfer MC_UU_00016/5 01/04/2017 31/03/2022 £2,530,000
MC_UU_00016/7 Transfer MC_UU_00016/6 01/04/2017 31/03/2022 £2,018,000
MC_UU_00016/8 Transfer MC_UU_00016/7 01/04/2017 31/03/2018 £1,131,000
MC_UU_00016/9 Transfer MC_UU_00016/8 01/04/2017 31/03/2022 £2,500,000
MC_UU_00016/10 Transfer MC_UU_00016/9 01/04/2017 31/03/2018 £1,171,000
MC_UU_00016/11 Transfer MC_UU_00016/10 01/04/2017 31/03/2022 £1,387,000
MC_UU_00016/12 Transfer MC_UU_00016/11 01/04/2017 31/03/2022 £446,000
 
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-1152
 
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 Educational dance 'Cell' 
Description The performance CELL explores human cells through intricate choreography. Using giant inflatable nucleus and cells, it is a playful exploration of cell biology and immunology, inspired by life in the lab, the actions of cells and the folding of DNA. The production features a soundtrack by Grammy award-winner Tchad Blake and an inflatable set designed by renowned environmental artist Steve Messam. The installation-like cells come alive with movement to make audiences marvel at the body's power, quirks and hidden landscapes and to allow young people to discover the life-giving world inside their bodies; a universe smaller than a grain of sand. 
Type Of Art Performance (Music, Dance, Drama, etc) 
Year Produced 2019 
Impact As supporting material, the dance company have produced a comic that is being given to all attendees as well as explanatory videos to help teachers and Q&A sessions, all featuring an avatar of Prof Buckle. 
URL https://www.imm.ox.ac.uk/about/news/arts-meets-science-in-dance-exploration-of-cell-biology
 
Description Independent charity reg no: 1108692
Amount £82,000 (GBP)
Organisation Reuben and Friends 
Sector Charity/Non Profit
Country United Kingdom
Start 09/2017 
End 02/2019
 
Description Understanding the basis of Rare Anaemias
Amount £38,342 (GBP)
Funding ID RCF18/033 
Organisation University of Oxford 
Sector Academic/University
Country United Kingdom
Start 03/2019 
End 03/2020
 
Title Hudep cell lines as a model for Congenital Dyserythropoietic Anaemia Type I (CDA-I) 
Description The Hudep cell lines are erythroid progenitor lines that can be induce to differentiate into erythroblasts. In separate lines we have tagged the two genes known to underlie CDA-I, CDAN1 and C15ORF41. We have also introduced one of the common CDAN1 mutations into a tagged line and now have two mutant lines for CDAN1 that faithfully replicate the diagnostic nuclear morphology of patient erythroblasts. We have now also introduced a mutation into the tagged C15ORF41 line. These cell lines means we avoid the need for mouse models. 
Type Of Material Model of mechanisms or symptoms - human 
Year Produced 2017 
Provided To Others? Yes  
Impact One paper on the Hudep cell lines has been published (Olijnik et al PMID 32518175). Another paper (on the ultrastructural analysis of abnormal erythroblast nuclei found in CDA-I, using the mutated Hudep cell lines) is in preparation. 
 
Title RASER-FISH 
Description RASER-FISH is a fluorescence in situ hybridisation method that avoids the need for denaturing DNA before a DNA probe can hybridise. In this way, chromatin structure can be optimally maintained. 
Type Of Material Technology assay or reagent 
Year Produced 2016 
Provided To Others? Yes  
Impact Papers published: Brackley et al 2016 PMID: 27036497 Brown et al 2018 PMID: 30242161 Oudelaar et al 2018 PMID: 30374068 Ochs et al 2019 PMID: 31645724 Rhodes et al 2020 PMID: 31968256 Miron et al 2020PMID: 32967822 Chiariello et al 2020 PMID: 32075757 A detailed methods paper has now been accepted by Nature Protocols: Brown JM, Parisi E, De Ornellas S, Schermelleh L, Buckle VJ. (2022) RASER-FISH: non-denaturing fluorescence in situ hybridization for preservation of three-dimensional interphase chromatin structure. Nat Protocols in press. 
 
Title Use of oligonucleotide probe pools to elucidate chromatin organisation 
Description Massive scale synthesis and novel synthetic labelling of oligonucleotide pools covering up to 3Mb of the human and mouse genomes, for use in both chromosome conformation capture and super-resolution imaging. 
Type Of Material Technology assay or reagent 
Year Produced 2018 
Provided To Others? Yes  
Impact Papers on: Dynamics of the 4D genome during lineage specification, differentiation and maturation in vivo (doi.org/10.1101/763763) Chromatin arranges in chains of mesoscale domains with nanoscale functional topography independent of cohesin (doi.org/10.1101/566638) Targeted high-resolution chromosome conformation capture at genome-wide scale (doi.org/10.1101/2020.03.02.953745) A tissue-specific self-interacting chromatin domain forms independently of enhancer-promoter interactions (doi.org/10.1101/234427) 
 
Description Cohesin and polycomb-dependent chromatin interactions 
Organisation University of Oxford
Country United Kingdom 
Sector Academic/University 
PI Contribution We used single cell RASER-FISH analysis to investigate the effects of cohesin and PCR1 removal on chromatin interactions at specific loci.
Collaborator Contribution Our collaborators Dr James Rhodes and Prof Rob Klose (Biochemistry) established the degron ES cell lines for removal of SCC1 and RING1B, identified enhanced polycomb interactions, investigated the effect on gene expression.
Impact Rhodes JDP, Feldmann A, Hernández-Rodríguez B, Díaz N, Brown JM, Fursova NA, Blackledge NP, Prathapan P, Dobrinic P, Huseyin M, Szczurek A, Kruse K, Nasmyth KA, Buckle VJ, Vaquerizas JM, Klose RJ. (2020) Cohesin disrupts polycomb-dependent chromosome interactions. Cell Reports 30 (3): 820-835.
Start Year 2018
 
Description New fluorescent probes for labelling nucleic acids 
Organisation University of Oxford
Country United Kingdom 
Sector Academic/University 
PI Contribution The main objective of this proposal is to develop new tools for the fluorescent imaging of specific DNA and RNA sequences in cells. The aim is to make a step change in sensitivity and resolution of fluorescent imaging relative to existing nucleic acid probe technologies. The new technology will be evaluated in an important and demanding biological imaging application, the objective of which is to understand regulation of the alpha globin gene. To achieve this we will determine the relative spatial organisation of alpha globin enhancers and promoters as the gene becomes transcriptionally active during erythroid differentiation.
Collaborator Contribution The approach involves the large scale synthesis of rigid crosslinked double stranded DNA constructs containing multiple fluorescent labels, to be undertaken at the Dept of Chemistry. These constructs are designed to be highly fluorescent by avoidance of dye-dye quenching interactions. They are chemically linked in a single tube reaction to pools of unlabelled probes made on a high-throughput DNA synthesiser to produce densely labelled probes. This modular strategy is designed to be highly cost-effective and labour saving. It is also generic and can be applied to a wide variety of imaging projects.
Impact Brown JM, Roberts NA, Graham B, Waithe D, Lagerholm C, Telenius JM, De Ornellas S, Oudelaar AM, Scott C, Szczerbal I, Babbs C, Kassouf MT, Hughes JR, Higgs DR, Buckle VJ. (2018) A tissue-specific self-interacting chromatin domain forms independently of enhancer-promoter interactions. Nat Commun 9 (1):3849. Oudelaar AM, Beagrie RA, Gosden M, De Ornellas S, Georgiades E, Kerry J, Hidalgo D, Carrelha J, Shivalingam A, El-Sagheer AH, Telenius JM, Brown T, Buckle VJ, Socolovsky M, Higgs DR, Hughes JR. (2020) Dynamics of the 4D genome during in vivo lineage specification and differentiation. Nat Commun 11 (1): 2722. Miron E, Oldenkamp R, Pinto DMS, Brown JM, Faria AR, Shaban HA, Rhodes JDP, Innocent C, de Ornellas S, Buckle V, Schermelleh L. (2020) Chromatin arranges in chains of nanodomains with 3D functional zonation. Sci Adv 6: eaba8811. Brown JM, Parisi E, De Ornellas S, Schermelleh L, Buckle VJ. (2022) RASER-FISH: non-denaturing fluorescence in situ hybridization for preservation of three-dimensional interphase chromatin structure. Nat Protocols in press.
Start Year 2014
 
Description RASER-FISH and Super-resolution Imaging 
Organisation University of Oxford
Country United Kingdom 
Sector Academic/University 
PI Contribution This project is to assess the applicability of RASER-FISH (Resolution After Single-stranded Exonuclease Resection) for characterising the organisation of chromatin within the nucleus at a single cell level, particularly in relation to gene activity. RASER-FISH avoids the use of denaturing and maintains optimal chromatin structure. My lab performs the hybridisations by various methods and analysis by STED. We assess the degree of single strandedness achieved and other factors defining different approaches to fluorescence in-situ hybridisation.
Collaborator Contribution Dr Lothar Schermelleh of Micron and the Dept of Biochemistry undertakes analysis of our hybridised material using Structured Illumination Microscopy and is quantifying the chromatin structure after various FISH approaches.
Impact We present this method to the community as a greatly improved way to assess chromatin structure at high resolution. Brown JM, Parisi E, De Ornellas S, Schermelleh L, Buckle VJ. (2022) RASER-FISH: non-denaturing fluorescence in situ hybridization for preservation of three-dimensional interphase chromatin structure. Nat Protocols in press. Ochs F, Karemore G, Miron E, Brown J, Sedlackova H, Rask M-B, Lampe M, Buckle V, Schermelleh L, Lukas J, Lukas C. (2019) Stabilization of chromatin topology by 53BP1 and RIF1 safeguards genome integrity. Nature 574: 571-574. Rhodes JDP, Feldmann A, Hernández-Rodríguez B, Díaz N, Brown JM, Fursova NA, Blackledge NP, Prathapan P, Dobrinic P, Huseyin M, Szczurek A, Kruse K, Nasmyth KA, Buckle VJ, Vaquerizas JM, Klose RJ. (2020) Cohesin disrupts polycomb-dependent chromosome interactions. Cell Reports 30 (3): 820-835.
Start Year 2015
 
Description The use of single copy INT sequences for fluorescently tagging DNA in live cells 
Organisation University of Toulouse
Department Department of Molecular Biology
Country France 
Sector Academic/University 
PI Contribution We are testing the use of INT sequences as an alternative to using large banks of repeats such as Lac or Tet as a DNA tag. The INT sequences are being integrated into the alpha globin locus in mouse ES cells by site-directed targetting. Our aim is the analysis of chromatin organisation at the alpha globin genes in live cells during erythroid differentiation.
Collaborator Contribution Our partners have provided us with the INT sequences. They are currently patenting this system.
Impact The system is currently being tested.
Start Year 2012
 
Description Three-dimensional interrogation of gene regulation using next generation chromatin conformation capture and super-resolution imaging 
Organisation University of Oxford
Department Department of Chemistry
Country United Kingdom 
Sector Academic/University 
PI Contribution We plan to look at the chromatin conformation of an entire 1.5Mb region around the alpha globin locus in both human and mouse. We propose to use massive scale DNA synthesis and novel synthetic labeling to develop the complementary approaches of chromatin capture and fluorescence in situ hybridisation as tools to understand how perturbations of 3D nuclear structure contribute to gene expression and human disease. This MRC Project Grant employs a chemist who will be responsible for oligo synthesis in the Dept Chemistry with Prof Tom Brown and subsequent super-resolution imaging in my lab using the tiled oligo pools.
Collaborator Contribution Prof Tom Brown will supervise the production of directly labelled pools of oligonucleotides. Prof Jim Hughes (primary grant holder) will supervise the chromatin capture experiments and subsequent analysis. Dr Dominic Waithe will support the image analysis.
Impact Multi-disciplinary: 1. Chemistry for massively parallel oligonucleotide synthesis 2. Genomic analysis for chromatin interaction frequencies 3. Super-resolution imaging for analysis of chromatin organisation in single cells 4. Image analysis Brown JM, Roberts NA, Graham B, Waithe D, Lagerholm C, Telenius JM, De Ornellas S, Oudelaar AM, Scott C, Szczerbal I, Babbs C, Kassouf MT, Hughes JR, Higgs DR, Buckle VJ. (2018) A tissue-specific self-interacting chromatin domain forms independently of enhancer-promoter interactions. Nat Commun 9 (1):3849. Oudelaar AM, Beagrie RA, Gosden M, De Ornellas S, Georgiades E, Kerry J, Hidalgo D, Carrelha J, Shivalingam A, El-Sagheer AH, Telenius JM, Brown T, Buckle VJ, Socolovsky M, Higgs DR, Hughes JR. (2020) Dynamics of the 4D genome during in vivo lineage specification and differentiation. Nat Commun 11 (1): 2722. Miron E, Oldenkamp R, Pinto DMS, Brown JM, Faria AR, Shaban HA, Rhodes JDP, Innocent C, de Ornellas S, Buckle V, Schermelleh L. (2020) Chromatin arranges in chains of nanodomains with 3D functional zonation. Sci Adv 6: eaba8811. Chiariello AM, Bianco S, Oudelaar AM, Esposito A, Annunziatella C, Fiorillo L, Conte M, Corrado A, Prisco A, Larke MSC, Telenius JM, Sciarretta R, Musella F, Buckle VJ, Higgs DR, Hughes JR, Nicodemi M. (2020) A dynamic folded hairpin conformation is associated with a-globin activation in erythroid cells. Cell Rep 30 (7): 2125-2135. Waithe D, Brown JM, Reglinski K, Diez-Sevilla I, Roberts D, Eggeling C. (2020) Object detection networks and augmented reality for cellular detection in fluorescence microscopy. J Cell Biol. 219 (10): e201903166. Downes DJ, Beagrie RA, Gosden ME, Telenius J, Carpenter SJ, Nussbaum L, De Ornellas S, Sergeant M, Eijsbouts CQ, Schwessinger R, Kerry J, Roberts N, Shivalingam A, El-Sagheer A, Oudelaar AM, Brown T, Buckle VJ, Davies JOJ, Hughes JR. (2021) High-resolution targeted 3C interrogation of cis-regulatory element organization at genome-wide scale. Nat Commun 12(1):531. Jeziorska DM, Tunnacliffe EAJ, Brown JM, Ayyub H, Sloane-Stanley J, Sharpe JA, Lagerholm C, Babbs C, Smith AJH, Buckle VJ, Higgs DR. (2022) On-microscope staging of live cells reveals changes in the dynamics of transcriptional bursting during differentiation. Nat Commun in revision. Brown JM, Parisi E, De Ornellas S, Schermelleh L, Buckle VJ. (2022) RASER-FISH: non-denaturing fluorescence in situ hybridization for preservation of three-dimensional interphase chromatin structure. Nat Protocols in press.
Start Year 2015
 
Description Advising Powys Dance on developing an educational ballet on The Cell 
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 Powys Dance ( mainly funded by the Arts Council for Wales) are developing an educational ballet to tour schools and science festivals. Last year their ballet was physics-based. This year they are focussing on gene regulation and cellular development and differentiation, all based on blood cells. They held a workshop over three days with dancers, set designers, musicians and scientific advisors where I spoke about my research and helped plan how that could be turned into dance!
Year(s) Of Engagement Activity 2018,2019
URL http://dawnspowysdance.org/powys-dance-news/2018/12/21/professors-on-tour-the-lowdown
 
Description Impelo performances of The Cell 
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 After advising on choreography for the educational ballet Cell by Impelo (https://www.impelo.org.uk/cell) I attended several performances of the ballet in London and Oxford to take Q&A sessions after the performance. In addition, Impelo have produced supporting material in the form of a comic and explanatory videos featuring an avatar of Prof Buckle (https://www.imm.ox.ac.uk/about/news/mrc-wimm-research-shines-at-this-years2019-oxford-science-and-ideas-festival).
Year(s) Of Engagement Activity 2019
URL https://www.imm.ox.ac.uk/about/news/arts-meets-science-in-dance-exploration-of-cell-biology
 
Description Royal Society Summer Science 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 The Royal Society Summer Exhibition is a prestigious event, showcasing science from only 22 groups, so we were very pleased to be selected. It has an extremely wide audience and consequent impact, including a media day where we recorded several interviews.
Year(s) Of Engagement Activity 2017
URL https://www.medsci.ox.ac.uk/folding-genome
 
Description ed 41st JP Morgan Healthcare conference 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact ed 41st JP Morgan Healthcare conference. Interaction with leading Pharma industry partners
Year(s) Of Engagement Activity 2023
URL https://www.jpmorgan.com/solutions/cib/insights/health-care-conference