Dataset Information

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

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE190962 | GEO | 2023/02/20

REPOSITORIES: GEO

ACCESS DATA

Similar Datasets

Project description:Ewing sarcoma is a highly aggressive tumor characterized by a translocation between members of the FET family of RNA binding proteins and one of several ETS transcription factors, with the most common translocation being EWS-FLI1. EWS-FLI1 leads to changes in gene expression through mechanisms that are not completely understood. We performed RNA sequencing analysis on primary pediatric human mesenchymal progenitor cells (pMPCs) expressing EWS-FLI1 in order to identify novel target genes. This analysis identified lnc277 as a previously uncharacterized long non-coding RNA upregulated by EWS-FLI1 in pMPCs. Inhibiting the expression of lnc277 diminished the ability of Ewing sarcoma cell lines to proliferate and form colonies in soft agar whereas inhibiting lnc277 had no effect on other cell types tested. By analyzing gene expression after shRNA knockdown, we found that both EWS-FLI1 and lnc277 repressed many more genes that they induced and that a significant fraction of EWS-FLI1 repressed targets were also repressed by lnc277. Analysis of primary human Ewing sarcoma RNA sequencing data further supports a role for lnc277 in mediating gene repression. We identified hnRNPK as an RNA binding protein that interacts directly with lnc277. We found a significant overlap in the genes repressed by hnRNPK and those repressed by both EWS-FLI1 and lnc277, suggesting that hnRNPK participates in lnc277 mediated gene repression. Thus, lnc277 is a previously uncharacterized long non-coding RNA downstream of EWS-FLI1 that facilitates the development of Ewing sarcoma via the repression of target genes. Our studies identify a novel mechanism of oncogenesis downstream of a chromosomal translocation and underscore the importance of lncRNA-mediated gene repression as a mechanism of EWS-FLI1 transcriptional regulation. RNA from primary human bone marrow derived mesenchymal cells either control or with inducible expression of EWS-FLI1 for 13 days was used to prepare PolyA selected cDNA libraries.

Dataset Information

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure