ASCL1 is a pioneer factor that can reprogram somatic cells to produce neurons. Preventing ASCL1 from being phosphorylated appears to enhance its reprogramming abilities, but the reason for this is unclear. A new paper in Development explores how ASCL1 activity is affected by different cellular contexts and reveals that the basis of the reprogramming efficiency of ASCL1 is more complicated than it first appears. To learn more about the story behind the paper, we caught up with first author Roberta Azzarelli, who is now a Lecturer in Pharmacology at University College London, UK, and corresponding author Anna Philpott, Professor of Cancer and Developmental Biology at the University of Cambridge, UK.
Anna Philpott (left) and Roberta Azzarelli (right)
Anna, what questions are your lab trying to answer?
AP: We are interested in how cells choose their fate in development and how a stable fate decision is maintained in the face of challenge. We are also fascinated by mechanisms that coordinate the cell cycle with the decision of developmental progenitors to divide or differentiate.
Roberta, how did you come to work in the lab and what drives your research today?
RA: During my PhD I worked on proneural basic helix-loop-helix (bHLH) transcription factors in brain development, and soon realized that these factors are highly regulated to ensure accurate balance of proliferation and differentiation. At the time, Anna's lab had just started to explore phosphorylation as a regulatory mechanism and was coupling this work with cancer research. I decided to join her lab, because I found all this highly fascinating and I could see the potential of taking this research in many in different directions, since it was applicable to other bHLH transcription factors, other tissues, and in diseases. My research today is very much driven by a continuation of these studies, as there are still so many open questions. I am particularly interested in the acquisition and maintenance of cellular identities in development, and in the mechanisms underlying cell fate plasticity in cancer.
Tell us about the background of the field that inspired your work
RA & AP: We have been working on bHLH phospho-regulation and activity for quite a long time and many other labs have now adopted the phosphomutant form of these transcription factors (including Ascl1, Neurog2 and Neurog3) to enhance differentiation. The general assumption is that these phosphomutant proteins enhance differentiation because they are more stable when unphosphorylated; however, we could not find data supporting this for ASCL1. So, we decided to actually test this in the lab, resulting in this paper, which challenges some of these initial assumptions.
The general assumption is that these phosphomutant proteins enhance differentiation because they are more stable when unphosphorylated; however, we could not find data supporting this for ASCL1
Can you give us the key results of the paper in a paragraph?
RA & AP: In this paper, we address why the phosphomutant form of ASCL1 is more active at driving neurogenesis than wild-type ASCL1. We find that while unphosphorylated ASCL1 protein is more stable, the mere accumulation of ASCL1 protein is not the main cause of the enhanced neurogenic activity. Instead, unphosphorylated ASCL1 is better able to remodel and open the chromatin at neuronal loci than the phosphorylated protein.
When doing the research, did you have any particular result or eureka moment that has stuck with you?
RA: We were quite struck when the results from the ATACseq came back (analysis performed by our colleague and co-author Dr Sarah Gillen). These data showed that the ASCL1 phosphomutant exhibits an increased ability to remodel and open chromatin in comparison to wild-type ASCL1, even when matched for protein levels, while different protein levels of ASCL1 phosphomutant showed absolutely no differences in chromatin opening. We did not expect such a clear result from the data, which reinforced all the other findings we generated before and after that experiment.
The ASCL1 phosphomutant exhibits an increased ability to remodel and open chromatin in comparison to wild-type ASCL1, even when matched for protein levels
Phosphomutant ASCL1-mediated neurogenesis. Embryonic stem cell-derived neuroectodermal cells exposed to a phosphomutant version of ASCL1 turn into neurons. Nuclei are shown in blue and the neuronal marker Tubb3 is shown in magenta. Image courtesy of Frances Connor.
And what about the flipside: any moments of frustration or despair?
RA: When we performed the experiment with the phospho-mimetic form of ASCL1, we thought that this ASCL1 form would be less active than the wild type, as it cannot undergo the dephosphorylation necessary for differentiation. However, the data did not support our hypothesis and showed a puzzling picture, where the phospho-mimetic somehow had an intermediate activity between the wild type and the more neurogenic phosphomutant. This was quite frustrating, as it challenged our assumptions from many years of previous research. However, we took on board the new result and concluded that adding constitutive negative charges on ASCL1 did not abolish its neurogenic activity. These data instead supported other dynamic regulatory mechanisms, likely relying on steric hindrance and other electrostatic properties, that would influence the interaction of ASCL1 with other ASCL1 molecules, co-factors or the DNA itself.
Why did you choose to submit this paper to Development?
RA & AP: We decided that Development was the right journal for our work long before we had even finished all the experiments. Development is the perfect fit for this study, is highly regarded in the field and is the right platform to reach our broad audience of developmental and stem cell biologists, as well as scientists interested in fate reprogramming and differentiation for regenerative medicine and cancer. Development is always a top pick for us as it is run by scientists for scientists.
What is next for you after this paper?
RA: Early in 2024, I moved to a lectureship at University College London to open my lab, so I finished working on this project while in that transition. My future research stems from this work, and I will continue to collaborate with Anna to address some of the key open questions in the field: what is phosphorylation actually doing mechanistically? How would cells interpret multiple cell fate decisions driven by diverse bHLH transcription factors? How would the mutational landscape of cancer cells influence the response to bHLHs? And how does this impact the fate plasticity of cancer cells? These are some of the key questions I will address in my lab in the future, and I am sure they will keep me busy for years to come.
Where will this story take your lab next?
AP: In the Philpott lab, we are now moving to three-dimensional human stem cell-derived models to explore in more depth the epigenetic and co-factor interactions that determine differential competency to reprogramming in different tissues and at different times during early development. We also continue to work on these same problems in my beloved Xenopus frog system.
Finally, let's move outside the lab – what do you like to do in your spare time?
AP: I love gardening and find growing flowers from seed to be hugely rewarding and relaxing.
RA: Most of my spare time is occupied by a little one in the family. However, when I manage, I love to go to art or photography exhibitions; that is where my brain regenerates.
R.A. & A.P.: Cambridge Stem Cell Institute, Jeffrey Cheah Biomedical Centre, Cambridge Biomedical Campus, Cambridge CB2 0AW, UK.
R.A.: Department of Pharmacology, UCL School of Pharmacy, University College London, 29-39 Brunswick Square, London WC1N 1AX, UK.
A.P.: Department of Oncology, University of Cambridge, Cambridge CB2 0XZ, UK.
E-mail: [email protected]; [email protected]
