Loci-specific phase separation of FET fusion oncoproteins promotes gene transcription

Abstract

Abnormally formed FUS/EWS/TAF15 (FET) fusion oncoproteins are essential oncogenic drivers in many human cancers. Interestingly, at the molecular level, they also form biomolecular condensates at specific loci. However, how these condensates lead to gene transcription and how features encoded in the DNA element regulate condensate formation remain unclear. Here, we develop an in vitro single-molecule assay to visualize phase separation on DNA. Using this technique, we observe that FET fusion proteins undergo phase separation at target binding loci and the phase separated condensates recruit RNA polymerase II and enhance gene transcription. Furthermore, we determine a threshold number of fusion-binding DNA elements that can enhance the formation of FET fusion protein condensates. These findings suggest that FET fusion oncoprotein promotes aberrant gene transcription through loci-specific phase separation, which may contribute to their oncogenic transformation ability in relevant cancers, such as sarcomas and leukemia.

FUS oncofusion protein condensates recruit mSWI/SNF chromatin remodeler via heterotypic interactions between prion‐like domains

“…A major consequence of FET oncofusions is the transfer of the prion‐like domain from an RNA‐binding protein to a DNA‐binding protein. Our results described here along with two recent reports suggest that FET‐fusion oncoproteins can form ectopic nuclear condensates 18,19 . These reports also suggest that FET‐fusion protein condensates can activate transcription at DNA enhancer sites by engaging with essential transcriptional co‐activators such as BRD4 and by recruiting RNA polymerase II 18,19 .…”

“…Our results discussed above suggest that the PLD of FUS enables phase separation of the oncogenic FUS‐DDIT3 transcription factor. Transcription factors can interact with the surface of DNA and such interactions can contribute to their phase separation at specific genomic loci 19,57,58 . To test if FUS‐DDIT3 can form condensates on DNA, we tethered a single double‐stranded (ds) λ‐phage genomic DNA between two optically‐trapped polystyrene beads using laminar‐flow in a microfluidic glass chamber (see Materials & Methods for further details and Figure 4a, Figure S8).…”

“…The FUS‐DDIT3 clusters formed at multiple loci on a single DNA chain. This is likely because FUS‐DDIT3 interacts promiscuously with the λ‐phage genomic DNA 19 . Next, to determine if these FUS‐DDIT3 condensates can recruit BRG1, the single‐molecule DNA tether with FUS‐DDIT3 condensates was transferred to a microfluidic channel containing 10 nM of RED‐tris‐NTA‐labeled BRG1 (Figure S8).…”

“…Transcription factors typically utilize their DNA‐binding domain (DBD) to bind to specific gene loci and use their activation domains to recruit additional regulatory coactivators and RNA Polymerase II 14–17 . For FET oncofusion transcription factors, PLDs can act as the activation domain and provide additional functionalities to the fusion transcription factors including the capacity to form phase‐separated transcriptional hubs 18,19 . Moreover, these fused PLDs may establish new interactions or modulate interactions with existing partners of the parent transcription factor.…”

Regulation of RNA Polymerase II Transcription Initiation and Elongation by Transcription Factor TFII-I

“…FET proteins contain IDRs that drive phase separation ( Wang et al, 2018 ). Loci-specific phase separation mediated by Fet proteins recruits RNA Pol II to promoters and activates transcription ( Wei et al, 2020 ; Zuo et al, 2021 ). Thus, TFII-I may regulate transcription negatively through NELF-driven phase separation, or positively through TAF15-driven phase separation.…”