The people behind the papers – Anna Philpott and William Beckman Free

Anna Philpott (left) and William Beckman (right)

Anna, what question is your lab trying to answer?

AP: Our lab is concerned with understanding how lineage-defining transcription factors, such as ASCL1, drive cell specification and associated epigenetic transitions in certain contexts, and yet fail to do so in others. We use a range of biological systems to address this, including neuroblastoma cell lines, where ASCL1 levels remain high but the differentiation machinery is not engaged, to Xenopus (frog) embryos, where certain tissue types exhibit varying permissiveness in response to ASCL1 overexpression during early development. We are also very interested in elucidating how these lineage-defining transcription factors interact with the cell cycle machinery, and the implications of these interactions for cell fate specification across various biological contexts. For example, we have shown that ASCL1 is phosphorylated in a cell cycle-dependent manner during Xenopus embryogenesis and in mammalian cells, and that differentially modified forms of ASCL1 have an altered capacity to drive neuronal differentiation (Ali et al., 2014, 2020; Azzarelli et al., 2024).

William, how did you come to work in the lab and what drives your research today?

WB: I remember looking for postdoc positions while writing my PhD thesis in Amsterdam, Netherlands, during the COVID-19 lockdown of 2020 and finding an open position in Anna's lab at Cambridge Stem Cell Institute, which immediately grabbed my attention. At the time, Anna had just been awarded a grant to study the mechanisms behind tissue-specific responses to ASCL1 in Xenopus embryos. One of the experiments (within the grant application) involved transferring permissive or resistant nuclei to Xenopus oocytes overexpressing ASCL1 to compare the response when transcription factors are controlled. It was this initial experiment that fascinated me and made me want to join the lab. My research today is driven by an acknowledgement that there is much more to uncover regarding how lineage-defining transcription factors operate during distinct stages of the cell cycle, and how this might differ at various stages throughout development.

Tell us about the background of the field that inspired your work

AP & WB: The proneural lineage-defining transcription factor, ASCL1, has been studied extensively due to its key role in specifying neuronal subtype identity in the central and peripheral nervous systems during development, as well as due to its capacity for reprogramming various cell types to the neuronal lineage. During development, ASCL1 expression levels rise in stem cells as they exit quiescence and divide, followed by a decline (in their levels) when these amplified progenitors exit the cell cycle and differentiate. In neuroblastoma, a paediatric cancer of the peripheral nervous system, we have shown that cell proliferation is reduced when ASCL1 is knocked out, but overexpression of ASCL1 drives potent neuronal differentiation of the same neuroblasts (Parkinson et al., 2022). Clearly ASCL1 is playing a role in both proliferation and differentiation of these cells in various contexts. Our work set out to investigate how ASCL1 could be playing a role in both of these potentially opposing functions.

Can you give us the key results of the paper in a paragraph?

AP & WB: We found that ASCL1 binding patterns during the G1 phase of the cell cycle were distinct from those during the S/G2/M phases. Specifically, we saw that sites showing higher levels of ASCL1 binding during the G1 phase tended to be associated with neuronal loci, in particular at intergenic sites, while sites showing higher levels of ASCL1 binding during SG2M tended to be associated with cell cycle genes, with binding more likely to occur at gene promoters. In cycling cells, G1 binding was generally non-productive in terms of target gene expression and associated with lower levels of chromatin accessibility, whereas S/G2/M binding was generally productive and associated with higher levels of accessibility. Intriguingly, stalling these neuroblastoma cells in G1 for extended periods allowed ASCL1 to activate these neuronal genes, triggering neuronal differentiation. This revealed how ASCL1 can have distinct functions by having cell cycle stage-dependent activity, which may further imply that other transcription factors have similar cell cycle stage-dependent activities.

William, when doing the research, did you have any particular result or eureka moment that has stuck with you?

WB: We had a hypothesis that ASCL1 may have different chromatin binding patterns during the different phases of the cell cycle, partly due to what our neuroblastoma experiments were telling us, but also because ASCL1 concentrations were different during G1 versus S/G2/M. When we saw the results from the first gene ontology analysis performed on the G1 and S/G2/M enriched ASCL1 chromatin immunoprecipitation sequencing (ChIP-seq) peaks, it was extremely exciting. However, we still didn't know whether the result was a side-effect of the cell synchronisation process. Subsequently, I conducted ChIP-qPCR on cell cycle-sorted cells (a process where immune-enriched DNA fragments are identified and quantified), and when that mirrored the initial ChIP-seq results, it was even more exciting.

And what about the flipside: any moments of frustration or despair?

WB: When I first started this project, we wanted to assess ASCL1 binding in G1 versus S/G2/M cells, which meant using fairly low cell numbers after fluorescence-activated cell sorting. So, we thought CUT&RUN and CUT&Tag would be better suited. However, after many attempts and much despair, we decided that the levels of ASCL1 were too low or the binding was too transient to reliably detect ASCL1 binding events, which led us back to ChIP-seq and cell cycle synchronisation.

Why did you choose to submit this paper to Development?

AP & WB: We chose to submit this paper to Development because the journal has a reputation for publishing high quality papers and we felt that the findings of our research were most relevant to the Development community.

Anna, where will this story take your lab next?

AP: We are keen to build on from this work to look at how lineage-defining transcription factor binding and activity differ throughout the cell cycle in embryonic stem cells, and what the implications are for cell fate specification. We want to test whether our findings represent general features of these types of factors.

Finally, let's move outside the lab – what do you like to do in your spare time?

WB: When I'm not working in the lab, I am often rowing for the City of Cambridge Rowing Club. I also love film photography and watching football.

AP: I'm also a rowing fan but rarely make it out onto the river these days. Instead, I spend many hours tending to my garden on a regular basis, which is hugely relaxing and feeds my love of nature.