Genome-wide profiling and identification of disordered transcription factors through chemical precipitation mapping [RNA-Seq]

Intrinsically disordered regions (IDRs) in DNA-associated proteins have recently been shown to play important roles in gene regulation. To better understand these domains we developed an antibody-independent assay to map disordered proteins genome-wide by combining b-isox mediated precipitation and next-generation sequencing (DisP-seq, disordered protein precipitation followed by DNA sequencing). We first analyzed Ewing sarcoma cells as a proof of principle given that the prion-like IDR of the oncogenic fusion protein EWS-FLI1 has been shown to play a critical role in this cancer. We find that DisP-seq produces thousands of strong peaks associated with diverse chromatin states in Ewing sarcoma and other cell types tested. These locations are highly enriched for DNA binding motifs matching EWS-FLI1 and other transcription factors with prominent IDRs. Moreover, depletion of EWS-FLI1 in Ewing sarcoma cells leads to a widespread reorganization of DisP-seq signals, including decreases at EWS-FLI1 binding sites and marked increases at sites associated with mesenchymal differentiation. Analysis of these new DisP-seq signals shows that they arise from redistribution of the transcription factor NFIB, which initially overlaps EWS-FLI1 and has an IDR that is required for proper localization and function. Our results thus show that antibody-independent precipitation mapping can enable genome-wide profiling and identification of disordered transcription factors to uncover their roles in complex gene regulation programs. Examination of gene expression in Ewing sarcoma SKNMC cells (empty expression vector, NFIB wildtype expression vector and NFIB deletion expression vector).