Abstract
A specialised set of transcription factors called pioneer factors are able to bind their targets in previously inaccessible chromatin and upon binding create accessible regions of DNA. Their activity allows non-pioneer transcription factors to also bind their targets, regulate downstream gene expression and establish gene regulatory networks during development. In the developing mammalian cortex, one of the most illustrative examples of a stable yet versatile system, proneural transcription factors of the bHLH family represent key determinants of neural cell fate and differentiation. Among the proneural proteins, ASCL1 has been proposed to act as a pioneer transcription factor by programming the epigenome and establishing new transcriptional networks during development and cellular reprogramming in both mouse and human models. The mSWI/SNF ATP-dependent chromatin remodelling complexes play critical roles in controlling chromatin dynamics, therefore facilitating rapid transcriptional events. Proper functioning of the mSWI/SNF complexes is essential for the establishment, maintenance and functionality of neural cells during development.The overlapping activity of ASCL1 and mSWI/SNF remodellers during neurogenesis led us to investigate the hypothesis of a mutual interaction between them. Using an in vitro model of human cortical neuronal differentiation from iPSCs, I have established that ASCL1 interacts physically with multiple subunits of the mSWI/SNF complexes. To further characterise this interaction, I investigated whether ASCL1 requires the mSWI/SNF remodellers to regulate its targets. By comparing the DNA binding landscapes of ASCL1 and mSWI/SNF core subunit SMARCB1, I found that approximately 70% of ASCL1 binding sites are also genomic targets of SMARCB1. This finding suggests that ASCL1 may functionally interact with mSWI/SNF complexes in order to regulate a large subset of its targets.
I then performed reciprocal disruption of ASCL1 and mSWI/SNF assemblies at different time points during corticogenesis to investigate the mutual requirement of ASCL1 for mSWI/SNF recruitment. Correlation of DNA binding and chromatin accessibility in ASCL1 knockout, mSWI/SNF-lacking and wild-type neuronal cells re- vealed that approximately one third of ASCL1 direct targets are also direct targets of the mSWI/SNF remodellers. In addition, 55% of the ASCL1-dependent genes are also misregulated upon mSWI/SNF removal. Association of ASCL1-mSWI/SNF direct genomic targets with the transcriptional changes observed in the two mutants led to the identification of 61 ASCL1-mSWI/SNF-dependent genes with essential roles during cortical neuronal differentiation whose regulation is linked to the sites where ASCL1 and SMARCB1 bind to regulate chromatin accessibility. However, more than 80% of the ASCL1-mSWI/SNF direct targets represent distal genomic sites with enhancer-specific histone modification signatures. As a consequence, looking at the nearest annotated promoter to associate these genomic regions with their transcriptional output might only explain a subset of the ASCL1-dependent genes. More extensive bioinformatics approaches that take into consideration the 3D organisation of the genome are likely to link these distal regulatory regions with a larger proportion of the ASCL1-mSWI/SNF-dependent genes. Overall, this work advances our understanding of the mechanisms behind ASCL1 pioneer activity. The essential roles of both ASCL1 and mSWI/SNF remodellers during cortical development point towards their interaction having vast implications for human health and disease.
| Date of Award | 1 Jan 2022 |
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| Original language | English |
| Awarding Institution |
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| Supervisor | François Guillemot (Supervisor) & Cristina Dias (Supervisor) |