Stefan Schoenfelder

Dr Schoenfelder holds a Babraham Institute Career Progression Fellowship which provides two years of support for his research.

Research Summary

Functional organisation of the genome in 3D
98% of the DNA in our body is non-coding, i.e. does not carry the information needed to build proteins. Non-coding has sometimes been equated with ‘non-functional’, or called ‘junk’ in the past; today we know that this is far from the truth. Scattered throughout non-coding DNA is a plethora of so-called regulatory elements, including enhancers, silencers and insulators. These regulatory elements function like molecular switches to control which genes are active (and thus produce proteins) in which cells. This process of gene expression control is vital to allow cells – which all contain the same genes – to specialise to carry out different tasks, and to help them respond to changes.

Enhancers are a type of regulatory element that control gene expression over long distances. They contact their target genes via chromosomal interactions, often bridging large distances in the genome, with the intervening DNA ‘looping out’. To understand how enhancers work, we study them in the context of the three-dimensional organisation of the genome.
 
Our aim is to find regulatory elements and to understand which genes they control. We also aim to uncover the molecular mechanisms by which regulatory elements find their target genes in the three-dimensional space of the cell nucleus, and to understand how altering the function of regulatory elements can lead to developmental malformations and disease.
 
We study these questions in pluripotent stem cells – cells that have the potential to create all cell types in the adult body. We use a combination of molecular, genetic, biochemical and imaging approaches to study pluripotent stem cells in their ‘ground state’, and when they start to form new cell types – a process called cell lineage specification.
 
Techniques and Methods

Through high-resolution mapping and experimental perturbation of the spatial genome architecture, we aim to reveal gene regulatory principles that underpin cell states and cell fate transitions. This may ultimately pave the way for us to experimentally engineer 3D genome folding to achieve predictable outcomes on gene expression and cell fate choice, with potential implications for gene therapy and regenerative medicine.
 

Latest Publications

High-resolution three-dimensional chromatin profiling of the Chinese hamster ovary cell genome.
Bevan S, Schoenfelder S, Young RJ, Zhang L, Andrews S, Fraser P, O'Callaghan PM

Chinese hamster ovary (CHO) cell lines are the pillars of a multi-billion dollar biopharmaceutical industry producing recombinant therapeutic proteins. The effects of local chromatin organisation and epigenetic repression within these cell lines result in unpredictable and unstable transgene expression following random integration. Limited knowledge of the CHO genome and its higher-order chromatin organisation has thus far impeded functional genomics approaches required to tackle these issues. Here, we present an integrative three-dimensional (3D) map of genome organisation within the CHOK1SV® 10E9 cell line in conjunction with an improved, less fragmented CHOK1SV® 10E9 genome assembly. Using our high-resolution chromatin conformation datasets, we have assigned ≈ 90% of sequence to a chromosome-scale genome assembly. Our genome-wide 3D map identifies higher-order chromatin structures such as topologically associated domains, incorporates our chromatin accessibility data to enhance the identification of active cis-regulatory elements and importantly links these cis-regulatory elements to target promoters in a 3D promoter interactome. We demonstrate the power of our improved functional annotation by evaluating the 3D landscape of a transgene integration site and two phenotypically different cell lines. Our work opens up further novel genome engineering targets, has the potential to inform vital improvements for industrial biotherapeutic production, and represents a significant advancement for CHO cell line development. This article is protected by copyright. All rights reserved.

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Biotechnology and bioengineering, 1, 1, 23 Oct 2020

PMID: 33095445

Cohesin-Dependent and -Independent Mechanisms Mediate Chromosomal Contacts between Promoters and Enhancers.
Thiecke MJ, Wutz G, Muhar M, Tang W, Bevan S, Malysheva V, Stocsits R, Neumann T, Zuber J, Fraser P, Schoenfelder S, Peters JM, Spivakov M

It is currently assumed that 3D chromosomal organization plays a central role in transcriptional control. However, depletion of cohesin and CTCF affects the steady-state levels of only a minority of transcripts. Here, we use high-resolution Capture Hi-C to interrogate the dynamics of chromosomal contacts of all annotated human gene promoters upon degradation of cohesin and CTCF. We show that a majority of promoter-anchored contacts are lost in these conditions, but many contacts with distinct properties are maintained, and some new ones are gained. The rewiring of contacts between promoters and active enhancers upon cohesin degradation associates with rapid changes in target gene transcription as detected by SLAM sequencing (SLAM-seq). These results provide a mechanistic explanation for the limited, but consistent, effects of cohesin and CTCF depletion on steady-state transcription and suggest the existence of both cohesin-dependent and -independent mechanisms of enhancer-promoter pairing.

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Cell reports, 32, 3, 21 Jul 2020

PMID: 32698000

ESCO1 and CTCF enable formation of long chromatin loops by protecting cohesin from WAPL.
Wutz G, Ladurner R, St Hilaire BG, Stocsits RR, Nagasaka K, Pignard B, Sanborn A, Tang W, Várnai C, Ivanov MP, Schoenfelder S, van der Lelij P, Huang X, Dürnberger G, Roitinger E, Mechtler K, Davidson IF, Fraser PJ, Lieberman-Aiden E, Peters JM

Eukaryotic genomes are folded into loops. It is thought that these are formed by cohesin complexes extrusion, either until loop expansion is arrested by CTCF or until cohesin is removed from DNA by WAPL. Although WAPL limits cohesin's chromatin residence time to minutes, it has been reported that some loops exist for hours. How these loops can persist is unknown. We show that during G1-phase, mammalian cells contain acetylated cohesin which binds chromatin for hours, whereas cohesin binds chromatin for minutes. Our results indicate that CTCF and the acetyltransferase ESCO1 protect a subset of cohesin complexes from WAPL, thereby enable formation of long and presumably long-lived loops, and that ESCO1, like CTCF, contributes to boundary formation in chromatin looping. Our data are consistent with a model of nested loop extrusion, in which acetylated cohesin forms stable loops between CTCF sites, demarcating the boundaries of more transient cohesin extrusion activity.

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eLife, 9, 1, 17 Feb 2020

DOI: 10.7554/eLife.52091

PMID: 32065581