EWS/FLI mediated reprogramming of 3D chromatin promotes an altered transcriptional state in Ewing sarcoma

Abstract

Ewing sarcoma is a prototypical fusion transcription factor-associated pediatric cancer that expresses EWS/FLI or a highly related FET/ETS chimera. EWS/FLI dysregulates transcription to induce and maintain sarcomagenesis, but the mechanisms utilized are not fully understood. We therefore sought to define the global effects of EWS/FLI on chromatin conformation and transcription in Ewing sarcoma cells using a well-validated ‘knock-down/rescue’ model of EWS/FLI function in combination with next generation sequencing assays to evaluate how the chromatin landscape changes with loss, and recovery, of EWS/FLI expression. We found that EWS/FLI (and EWS/ERG) genomic localization is largely conserved across multiple patient-derived Ewing sarcoma cell lines. This EWS/FLI binding signature is associated with establishment of topologically-associated domain (TAD) boundaries, compartment activation, enhancer-promoter looping that involve both intra- and inter-TAD interactions, and gene activation. In addition, EWS/FLI co-localizes with the loop-extrusion factor cohesin to promote chromatin loops and TAD boundaries. Importantly, local chromatin features provide the basis for transcriptional heterogeneity in regulation of direct EWS/FLI target genes across different Ewing sarcoma cell lines. These data demonstrate a key role of EWS/FLI in mediating genome-wide changes in chromatin configuration and support the notion that fusion transcription factors serve as master regulators of three-dimensional reprogramming of chromatin.

The DBD-α4 helix of EWSR1::FLI1 is required for GGAA microsatellite binding that underlies genome regulation in Ewing sarcoma

“…CUT&Tag experiments were carried out for 2 biological replicates of CTCF and H3K27ac and 3 biological replicates for FLAG tagged DBD and DBD+ constructs. An in-house pipeline was used to analyze CUT&Tag data (Boone et al, 2021;Showpnil et al, 2022). Quality control on raw sequencing reads were performed with FastQC (v0.11.4) (Andrews, 2010).…”

Modulation of the high-order chromatin structure by Polycomb complexes

“…Phase separation such as liquid-liquid phase separation (LLPS) was proposed to be crucial for transcriptional control and chromatin structure organization ( Hnisz et al, 2017 ). Ahn et al (2021) have demonstrated that NUP98-HOXA9, an oncogenic transcription factor (TF) with potent LLPS capability, mediates CTCF-independent DNA loops among cognate NUP98-HOXA9-bound sites, forming long-range E:P contacts, and that such a looping mechanism requires the presence of NUP98-HOXA9’s intrinsically disordered regions (IDRs), which is essential for LLPS; similar looping was also reported for another cancer-related TF, EWS-FLI ( Showpnil et al, 2022 ). Moreover, a recent work indicated that LLPS plays a role in weakening the insulation properties of a subset (∼20%) of TAD boundaries, which exhibit the feature of “house-keeping” genes, high transcription and a lack of CTCF ( Gamliel et al, 2022 ).…”

Through the lens of phase separation: intrinsically unstructured protein and chromatin looping

“…the BAF complex) and induction of an oncogenic gene-expression program [ 124 ]; also, fusing a minimal LLPS-inducing IDR sequence to FLI was sufficient to recapitulate the EWS-FLI-related activities [ 124 ]. A more recent study further reported that EWS-FLI induces looping [ 125 ], reminiscent of what was observed with NUP98-HOXA9 [ 82 ]. Specifically, new DNA loops are formed with their anchors associated with binding of EWS/FLI and not CTCF [ 125 ]; in addition, EWS/FLI binds to the GGAA-rich motifs in the genome of Ewing’s sarcoma, leading to new enhancer formation, A-type compartmentalization, and target gene upregulation [ 125 ].…”