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Super-enhancers require a combination of classical enhancers and novel facilitator elements to drive high levels of gene expression

View ORCID ProfileJoseph Blayney, Helena Francis, Brendan Camellato, Leslie Mitchell, Rosa Stolper, View ORCID ProfileJef Boeke, View ORCID ProfileDouglas Higgs, Mira Kassouf
doi: https://doi.org/10.1101/2022.06.20.496856
Joseph Blayney
1Weatherall Institute of Molecular Medicine, University of Oxford, The John Radcliffe Hospital, Headington, Oxford, OX3 9DS, UK
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  • ORCID record for Joseph Blayney
Helena Francis
1Weatherall Institute of Molecular Medicine, University of Oxford, The John Radcliffe Hospital, Headington, Oxford, OX3 9DS, UK
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Brendan Camellato
2Institute for Systems Genetics and Department of Biochemistry and Molecular Pharmacology, NYU Langone Health, New York, NY 10016
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Leslie Mitchell
2Institute for Systems Genetics and Department of Biochemistry and Molecular Pharmacology, NYU Langone Health, New York, NY 10016
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Rosa Stolper
1Weatherall Institute of Molecular Medicine, University of Oxford, The John Radcliffe Hospital, Headington, Oxford, OX3 9DS, UK
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Jef Boeke
2Institute for Systems Genetics and Department of Biochemistry and Molecular Pharmacology, NYU Langone Health, New York, NY 10016
3Department of Biomedical Engineering, NYU Tandon School of Engineering, Brooklyn NY 11201
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Douglas Higgs
1Weatherall Institute of Molecular Medicine, University of Oxford, The John Radcliffe Hospital, Headington, Oxford, OX3 9DS, UK
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Mira Kassouf
1Weatherall Institute of Molecular Medicine, University of Oxford, The John Radcliffe Hospital, Headington, Oxford, OX3 9DS, UK
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  • For correspondence: mira.kassouf@imm.ox.ac.uk
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Abstract

Super-enhancers (SEs) are a class of compound regulatory elements which control expression of key cell-identity genes. It remains unclear whether they are simply clusters of independent classical enhancer elements or whether SEs manifest emergent properties and should therefore be considered as a distinct class of element. Here, using synthetic biology and genome editing, we engineered the well characterised erythroid α-globin SE at the endogenous α-globin locus, removing all SE constituents in a mouse embryonic stem cell-line, to create a "blank canvass". This has allowed us to re-build the SE through individual and combinatorial reinsertion of its five elements (R1, R2, R3, Rm, R4), to test the importance of each constituent's sequence and position within the locus. Each re-inserted element independently creates a region of open chromatin and binds its normal repertoire of transcription factors; however, we found a high degree of functional interdependence between the five constituents. Surprisingly, the two strongest α-globin enhancers (R1 and R2) act sub-optimally both on their own and in combination, and although the other three elements (R3, Rm and R4) exhibit no discernible enhancer activity, they each exert a major positive effect in facilitating the activity of the classical enhancers (R1 and R2). This effect depends not simply on the sequence of each elements but on their positions within the cluster. We propose that these "facilitators" are a novel form of regulatory element, important for ensuring the full activity of SEs but are distinct from conventional enhancer elements.

Competing Interest Statement

Jef Boeke is a Founder and Director of CDI Labs, Inc., a Founder of and consultant to Neochromosome, Inc, a Founder, SAB member of and consultant to ReOpen Diagnostics, LLC and serves or served on the Scientific Advisory Board of the following: Sangamo, Inc., Modern Meadow, Inc., Rome Therapeutics, Inc., Sample6, Inc., Tessera Therapeutics, Inc. and the Wyss Institute.

Footnotes

  • Typos have been revised and references added

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-ND 4.0 International license.
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Posted June 22, 2022.
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Super-enhancers require a combination of classical enhancers and novel facilitator elements to drive high levels of gene expression
Joseph Blayney, Helena Francis, Brendan Camellato, Leslie Mitchell, Rosa Stolper, Jef Boeke, Douglas Higgs, Mira Kassouf
bioRxiv 2022.06.20.496856; doi: https://doi.org/10.1101/2022.06.20.496856
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Super-enhancers require a combination of classical enhancers and novel facilitator elements to drive high levels of gene expression
Joseph Blayney, Helena Francis, Brendan Camellato, Leslie Mitchell, Rosa Stolper, Jef Boeke, Douglas Higgs, Mira Kassouf
bioRxiv 2022.06.20.496856; doi: https://doi.org/10.1101/2022.06.20.496856

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