Genome-wide profiling and identification of disordered transcription factors through chemical precipitation mapping [DisP-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. Overall design: DisP-seq profiles of small cell lung cancer NCI-H446 cells (wildtype, shGFP control and NFIB knockdown), Ewing sarcoma SKNMC cells (wildtype; shGFP control, EWS-FLI1 knockdown; empty expression vector, NFIB wildtype expression vector and NFIB deletion expression vector) and wild type embryonic fibroblast MRC5 cells.