Abstract B024: The EWSR1 low complexity domain drives cooperative DNA binding of EWSR1::FLI1 to multiple GGAA repeats

Abstract

Ewing Sarcoma is defined by its hallmark mutation, a t(11;22)q24;q12) translocation which results in fusion of the EWSR1 and FLI1 genes. The fusion product, EWSR1::FLI1, contains the N-terminal low complexity domain (LCD) of EWSR1 and the DNA-binding Domain (DBD) of FLI1. EWSR1::FLI1 binds at microsatellites (µsats), defined as (GGAA)n sequences, found throughout the genome. The fused EWSR1 LCD promotes a network of homo- and heterotypic protein-protein interactions, which renders EWSR1::FLI1 a potent transcription factor at loci containing these µsats. Current literature suggests that EWSR1::FLI1 activity is dependent on the number of µsats present, with in vitro data supporting an unclear threshold of consecutive GGAA repeats that are needed for transcriptional activity mediated by EWSR1::FLI1. Further, many mechanistic studies into EWSR1::FLI1 binding omit the LCD, which does not fully recapitulate the true DNA binding mechanism of EWSR1::FLI1. In this work, we hypothesize that the DNA binding mechanisms and sequence specificity of EWSR1::FLI1 utilized on two constructs: the full-length EWSR1::FLI1, and an LCD deletion mutant (Del22 EWSR1::FLI1). Both constructs were subject to fluorescence polarization assays and electrophoretic mobility shift assays (EMSAs) to reveal mechanistic contributions of the LCD to DNA oligomers containing increasing numbers of µsats. We also investigated the stoichiometry of binding for both constructs and determined the influence of ionic strength and macromolecular crowders on DNA binding interactions. Preliminary data suggests that both EWSR1::FLI1 constructs bind to increasing numbers of µsats with greater affinity. The full-length EWSR1::FLI1 also exhibits cooperativity that is dependent on number of µsats whereas the Del22 mutant does not. Additionally, we show here that the LCD does not significantly govern the stoichiometry of binding. Further experiments reveal that the observed cooperativity is dependent on the number of nucleotides between adjacent µsats, as cooperativity is abolished when µsats are more than eight nucleotides away from each other. This supports a mechanistic definition of a functional µsat based on LCD-derived cooperativity. Cooperative DNA binding is also greatly enhanced with the introduction of macromolecular crowders, and both constructs exhibit different dependencies on monovalent and divalent cations. We observe that maximal DNA binding occurs at physiological ionic strength and crowding, which corresponds as well with increased cooperativity. Taken together, this project suggests that the EWSR1 LCD is involved mechanistically in the binding of consecutive µsats through LCD-LCD interactions, highlighting a novel mechanistic contribution of this domain to EWSR1::FLI1 function. Our observations also note that the cooperativity contributed by the LCD correlates with biologically significant findings pertaining to EWSR1::FLI1-mediated transcriptional regulation. Further mechanistic insight is needed, however, to prove the biological relevance of these findings.