The Fbw7 ubiquitin ligase targets many proteins for proteasomal degradation, which include oncogenic transcription factors (TFs) (e.g., c-Myc, c-Jun, Notch). Fbw7 is a tumor suppressor and tumors often contain mutations in FBXW7, the gene that encodes Fbw7. The complexity of its substrate network has obscured the mechanisms of Fbw7-associated tumorigenesis, yet this understanding is needed for developing therapies. We used an integrated approach employing RNA-Seq and high-resolution mapping (CUT&RUN) of histone modifications and TF occupancy (c-Jun and c-Myc) to examine the combinatorial effects of mis-regulated Fbw7 substrates in colorectal cancer cells with engineered tumor-associated FBXW7 null or missense mutations. Both Fbw7 mutations caused widespread transcriptional changes associated with active chromatin and altered TF occupancy: some were common to both Fbw7 mutant cell lines, whereas others were mutation specific. We identified loci where both Jun and Myc were co-regulated by Fbw7, suggesting that substrates may have synergistic effects. One co-regulated gene was CIITA, the master regulator of MHC Class II gene expression. Fbw7 loss increased MHC Class II expression and Fbw7 mutations were correlated with increased CIITA expression in TCGA colorectal tumors and cell lines, which may have immunotherapeutic implications for Fbw7-associated cancers. Analogous studies in neural stem cells in which FBXW7 had been acutely deleted closely mirrored the results in colorectal cancer cells. Gene set enrichment analyses revealed Fbw7-asssociated pathways that were conserved across both cell types that may reflect fundamental Fbw7 functions. These analyses provide a framework for understanding normal and neoplastic context specific Fbw7 functions.
- RNA-Seq and CUT&RUN sequencing data have been deposited in GEO under accession code GSE184041. Secure access token: cdmhkumwvtovbqn- All data generated or analyzed during the study are included in the manuscript and supporting files. Supplement figures are included as a separate PDF. Source Data files have been provided for Figures 1, 2, 3, 5 and 6. List of source data with a brief description can be found at the end of the manuscript.- Computational tools used in the study are mentioned in the Materials and Methods section. Code used are available at https://github.com/hnthirima.
Genome-wide mapping of histone modifications and transcription factor occupancy in Fbw7 mutant cellsNCBI Gene Expression Omnibus, GSE184041.
- Heshani Nayanga Thirmanne
- Bruce E Clurman
- Bruce E Clurman
- Steven Henikoff
- Patrick J Paddison
- Patrick J Paddison
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
- Wade Harper, Harvard Medical School, United States
© 2022, Thirmanne et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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