Supporting data for Dynamic Pipsqueak Amyloid Regulates Polycomb Complex Activity in Drosophila Embryos
Prion-like proteins challenge conventional protein structure-function paradigms by assuming multiple stable conformations capable of recruiting and converting other proteins to alternative states through self-perpetuating mechanisms. This remarkable property, governed by prion domains (PrDs), furnishes cells with a mechanism to store information long-term despite protein turnover, serving crucial roles in processes ranging from stress adaptation to memory persistence.
To broaden our understanding of functional prion-like proteins, we conducted a comprehensive screen of the Drosophila melanogaster proteome for PrDs, identifying several promising candidates including Pipsqueak (psq), a transcription regulator with diverse developmental functions. Psq recruits Polycomb group (Pc-G) complexes to specific DNA elements termed Polycomb Response Elements, thereby maintaining transcriptional patterns that preserve cellular identity.
The psq locus encodes various isoforms categorised into long (psqL) and short (psqS) variants. Both share a DNA binding domain and two predicted PrDs, whilst psqL contains an additional PrD and a BTB domain for protein-protein interactions. Utilising a yeast-based assay, we demonstrated that two of psq's PrDs drive formation of self-sustaining states.
In early embryonic development, psqL exists in both monomeric and SDS-resistant aggregated forms. CRISPR/Cas9-mediated fluorescent tagging revealed that psq forms stable nuclear foci that rapidly assemble following nuclear division and disassemble during cell division. Fluorescence recovery analysis indicates these are likely amyloid assemblies.
We propose that psqL amyloid-like aggregates serve as an epigenetic mechanism perpetuating the repressive transcriptional state of Pc-G target loci. The dynamic nature of these assemblies suggests regulated processes controlling their formation and disassembly during development. Characterising the structure and physiological relevance of psq aggregation could reveal novel gene regulation mechanisms and distinguish features of functional versus pathological amyloids.
