Deciphering the structure and dynamics of the polyA/polyQ tract in neuronal pioneer factor Ascl1

Abstract

During the process of development, stem cells give rise to cells that go forth on a path of specification and determination. As each cell shares the same genetic material, they resort to various methods for concealing genes from the transcriptional machinery, thereby controlling each cell's identity. This is facilitated by the formation of nucleosome core particles that collectively assemble into condensed chromatin, making genes accessible only to those transcription factors (TFs) with pioneering activity. The sequence-specific binding of these pioneer transcription factors (pTFs) to the tightly packed chromatin is the spark that ignites the subsequent recruitment of other factors needed for transcription initiation. The discovery of this unique pTF activity has enabled the reprogramming of fibroblasts to induced pluripotent stem cells (iPSCs), but it has also led to discoveries of other pTFs that can directly induce specific cell fates beyond undifferentiated states. One such pTF is achaete-scute homolog 1 (Ascl1), capable of reprogramming mouse- and human embryonic fibroblasts to induced neuronal cells. However, the current status of such reprogramming can be achieved with limited efficiency. Ascl1 is essential for neurogenesis during development where it is expressed by neural crest cells for their differentiation into neurons. Little is known about the structural properties and mechanisms of Ascl1 as the protein is disordered in nature and prone to aggregation, making conventional structural biology methods inapplicable. In this project, an N-terminal only construct of Ascl1 (residues 1-117) was purified and fluorescently labelled close to the termini. The fluorescent labelling of the protein allowed for the application of single-molecule Förster resonance energy transfer (smFRET) for probing structural features of the construct, including comparison to the full-length protein. The resulting transfer efficiency histogram based on the signal from thousands of single molecules yielded a well-defined population with lower mean FRET than full-length Ascl1, providing strong evidence for intramolecular interactions between the N-terminal disordered region and the DNA binding domain. This work lays the foundation for deciphering the molecular properties of Ascl1, which could aid designs of more efficient cell reprogramming mechanisms.