Scalable in vitro production of defined mouse erythroblasts

Francis, Helena; Harold, Caroline L; Beagrie, Robert A.; King, Andrew; Gosden, M; Blayney, Joseph; Jeziorska, Danuta M.; Babbs, Christian; Higgs, Douglas R.; Kassouf, Mira

doi:10.1371/journal.pone.0261950

Cited by 9 publications

(38 citation statements)

References 52 publications

(81 reference statements)

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“…This provides a "blank canvas" into which any combination of elements, in any position, can be re-introduced and the effects on α-globin expression assessed. Contrary to expectation, in in vitro differentiated erythroid cells (Francis et al, 2022), reinsertion of the strongest constituent of the α-SE, R2 (R2-only), resulted in five-fold lower levels of transcription than predicted in the absence of R1, R3, Rm and R4. To investigate this further, we generated an R2-only mouse model.…”

Section: Introductioncontrasting

confidence: 84%

“…An in vitro EB-based differentiation system was used to generate erythroid cells (Francis et al, 2022). Briefly, 24 hours pre-differentiation, mouse ES cells were passaged into adaptation medium.…”

Section: Methodsmentioning

confidence: 99%

“…An in vitro EB-based differentiation system was used to generate erythroid cells (Francis et al, 2022).…”

Section: Mouse Model Generationmentioning

confidence: 99%

“…A third genetic model was made by deleting the remaining R2 element from R2-only ES cells using a CRISPR-Cas9 approach, creating a model in which all elements of the α-globin SE had been removed from the locus (Δα−SE) ( fig 1, C ). We then used an embryoid body (EB)-based in vitro differentiation and erythroid purification system recently developed in our lab (Francis et al, 2022) to produce hemizygous WT, Δα−SE and R2-only erythroid cells in which the single remaining α-globin locus is derived from a synthetic construct.…”

Section: Introductionmentioning

confidence: 99%

“…We next set out to rebuild the α-SE in various configurations to determine the role of each of the other SE elements. Because rebuilding the SE entailed the generation of numerous genetic models – too many to reasonably study in mice – we moved back to the orthogonal in vitro embryoid body (EB)-based erythroid differentiation system (Francis et al, 2022). This allowed rapid engineering of hemizygous WT and R2-only ES cells, followed by production of genetically engineered mouse erythroid cells, in which we could study the activity of the α-SE.…”

Section: Introductionmentioning

confidence: 99%

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

Blayney

Kassouf

Francis

et al. 2022

Preprint

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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.

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Section: Introductioncontrasting

confidence: 84%

Section: Methodsmentioning

confidence: 99%

“…An in vitro EB-based differentiation system was used to generate erythroid cells (Francis et al, 2022).…”

Section: Mouse Model Generationmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Blayney

Kassouf

Francis

et al. 2022

Preprint

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Super-enhancers include classical enhancers and facilitators to fully activate gene expression

Blayney,

Francis,

Rampasekova

et al. 2023

Cell

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On-microscope staging of live cells reveals changes in the dynamics of transcriptional bursting during differentiation

et al. 2022

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Determining the mechanisms by which genes are switched on and off during development is a key aim of current biomedical research. Gene transcription has been widely observed to occur in a discontinuous fashion, with short bursts of activity interspersed with periods of inactivity. It is currently not known if or how this dynamic behaviour changes as mammalian cells differentiate. To investigate this, using an on-microscope analysis, we monitored mouse α-globin transcription in live cells throughout erythropoiesis. We find that changes in the overall levels of α-globin transcription are most closely associated with changes in the fraction of time a gene spends in the active transcriptional state. We identify differences in the patterns of transcriptional bursting throughout differentiation, with maximal transcriptional activity occurring in the mid-phase of differentiation. Early in differentiation, we observe increased fluctuation in transcriptional activity whereas at the peak of gene expression, in early erythroblasts, transcription is relatively stable. Later during differentiation as α-globin expression declines, we again observe more variability in transcription within individual cells. We propose that the observed changes in transcriptional behaviour may reflect changes in the stability of active transcriptional compartments as gene expression is regulated during differentiation.

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Scalable in vitro production of defined mouse erythroblasts

Cited by 9 publications

References 52 publications

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

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

Super-enhancers include classical enhancers and facilitators to fully activate gene expression

On-microscope staging of live cells reveals changes in the dynamics of transcriptional bursting during differentiation

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