Global and context-specific transcriptional consequences of oncogenic Fbw7 mutations
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Evaluation Summary:
FBXW7 is a CRL controlling the abundance of numerous transcription factors through targeted degradation. The authors use isogenic cancer and normal cell lines that differ only in the functional status of FBXW7 to examine the genome-wide effects of FBXW7. The authors demonstrate that null and missense mutations have similar large-scale effects on gene expression and chromatin modification status of many loci at intergenic and intronic regions, but that there are substantial differences in affected loci between the mutant backgrounds. This study represents the first such systematic evaluation of the impact of FBXW7 functional variation on global gene expression.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Abstract
The Fbw7 ubiquitin ligase targets many proteins for proteasomal degradation, which include oncogenic transcription factors (TFs) (e.g., c-Myc, c-Jun, and 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 (cleavage under target and release using nuclease) of histone modifications and TF occupancy (c-Jun and c-Myc) to examine the combinatorial effects of misregulated Fbw7 substrates in colorectal cancer (CRC) 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 coregulated by Fbw7, suggesting that substrates may have synergistic effects. One coregulated 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 CRC cells. Gene set enrichment analyses revealed Fbw7-associated 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.
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Evaluation Summary:
FBXW7 is a CRL controlling the abundance of numerous transcription factors through targeted degradation. The authors use isogenic cancer and normal cell lines that differ only in the functional status of FBXW7 to examine the genome-wide effects of FBXW7. The authors demonstrate that null and missense mutations have similar large-scale effects on gene expression and chromatin modification status of many loci at intergenic and intronic regions, but that there are substantial differences in affected loci between the mutant backgrounds. This study represents the first such systematic evaluation of the impact of FBXW7 functional variation on global gene expression.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested …
Evaluation Summary:
FBXW7 is a CRL controlling the abundance of numerous transcription factors through targeted degradation. The authors use isogenic cancer and normal cell lines that differ only in the functional status of FBXW7 to examine the genome-wide effects of FBXW7. The authors demonstrate that null and missense mutations have similar large-scale effects on gene expression and chromatin modification status of many loci at intergenic and intronic regions, but that there are substantial differences in affected loci between the mutant backgrounds. This study represents the first such systematic evaluation of the impact of FBXW7 functional variation on global gene expression.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
Fbw7 functions to control the abundance of more than 2 dozen transcriptional regulators, but how this affects transcription at the global level is largely unknown. The authors employ RNA-Seq, CUT&RUN on H3K27ac/H3K27me3, and a detailed analysis of the loci affected to provide a global analysis of the effect of Fbw7 mutation on transcription in HCT116 cells as well as neural stem cells. The results reveal complex, but intriguing, results suggesting that Fbw7 mutation affects primarily Jun and Myc functions in distal regulatory regions rather than promoters. Although HCT116 cells employed (WT, Fbw7-/-, and Fbw7R/+) are clonal, there is significant overlap in the two mutant lines, which suggests that a substantial fraction of the effects reflect loss of Fbw7 activity. Analogous patterns related to Jun and Myc …
Reviewer #1 (Public Review):
Fbw7 functions to control the abundance of more than 2 dozen transcriptional regulators, but how this affects transcription at the global level is largely unknown. The authors employ RNA-Seq, CUT&RUN on H3K27ac/H3K27me3, and a detailed analysis of the loci affected to provide a global analysis of the effect of Fbw7 mutation on transcription in HCT116 cells as well as neural stem cells. The results reveal complex, but intriguing, results suggesting that Fbw7 mutation affects primarily Jun and Myc functions in distal regulatory regions rather than promoters. Although HCT116 cells employed (WT, Fbw7-/-, and Fbw7R/+) are clonal, there is significant overlap in the two mutant lines, which suggests that a substantial fraction of the effects reflect loss of Fbw7 activity. Analogous patterns related to Jun and Myc levels at distal regulatory regions are seen in the neural stem cells, where there is a pool of depleted cells rather than clonal cells derived from targeted mutagenesis.
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Reviewer #2 (Public Review):
This manuscript addresses two critical questions: (i) the general issue of how specific missense mutations in FBXW7 (thought to be dominant negative) differ in global gene expression phenotypes compared to null mutations and (ii) the integrated effects of FBXW7 loss-of-function due to perturbation of multiple central transcription factor substrates of FBXW7. The authors use RNAseq, histone modification (cut and run) profiles and TF occupancy profiles of three otherwise isogenic cell lines (+/+, R505C/+ and -/-) derived from the HCT116 colon cancer cell line to answer the above questions. The main conclusions of the study are (i) missense mutations cause different global effects compared complete elimination of FBWX7 and that these changes can be correlated with chromatin and TF occupancy profiles, primarily …
Reviewer #2 (Public Review):
This manuscript addresses two critical questions: (i) the general issue of how specific missense mutations in FBXW7 (thought to be dominant negative) differ in global gene expression phenotypes compared to null mutations and (ii) the integrated effects of FBXW7 loss-of-function due to perturbation of multiple central transcription factor substrates of FBXW7. The authors use RNAseq, histone modification (cut and run) profiles and TF occupancy profiles of three otherwise isogenic cell lines (+/+, R505C/+ and -/-) derived from the HCT116 colon cancer cell line to answer the above questions. The main conclusions of the study are (i) missense mutations cause different global effects compared complete elimination of FBWX7 and that these changes can be correlated with chromatin and TF occupancy profiles, primarily at intergenic and intronic loci; (ii) two of the main downstream TFs, MYC and JUN appear to co-regulate numerous genes; (iii) one of the co-regulated genes, CIITA, drives increased MHC class II gene expression in FBWX7 mutant contexts. The main trends were found to hold in non-transformed neuronal stem cell populations in which FBWX7 was deleted, including binding to the CIITA locus and increased expression of MHC II genes. This study elaborates the complex role of FBWX7 as one of the most commonly mutated tumor suppressors in human cancer and provides specific new insights into the potential effect of MHC class II gene deregulation in cancer. The design, results and analysis are of high quality and support the conclusions drawn. Although results are consistent with dosage effects of FBXW7 heterozygous mutations on gene expression profiles (called the "just enough" model), this conclusion is confounded by the absence of a wild type heterozygote (-/+) control for the dominant negative (R505C/+) heterozygote, so discussion around this particular conclusion should be tempered somewhat. The datasets in the study will be very useful resources for interrogating FBXW7 as a regulatory super-hub on cancer and investigating potential downstream therapeutic targets.
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Reviewer #3 (Public Review):
In this study, the authors aim to compare the effects of Fbw7 deletion (-/-) and Fbw7 mutation (R/+) on the transcriptional landscape. Since Fbw7 targets a number of transcription factors (TFs), including c-Myc and c-Jun, which are frequently deregulated in cancer, many downstream genes are expected to be affected in both cell lines. The authors thus examined transcriptional profiles as well as histone modifications and TF occupancy in WT, Fbw7(-/-), and Fbw7(R/+) cells. While the purpose of this study is interesting and important, the interpretation of the results is somewhat puzzling and complex. Furthermore, the details of the experimental design in some parts are not clearly described, which hinders the understanding and evaluation of this study. Overall, the biological impact of deletion or mutation of …
Reviewer #3 (Public Review):
In this study, the authors aim to compare the effects of Fbw7 deletion (-/-) and Fbw7 mutation (R/+) on the transcriptional landscape. Since Fbw7 targets a number of transcription factors (TFs), including c-Myc and c-Jun, which are frequently deregulated in cancer, many downstream genes are expected to be affected in both cell lines. The authors thus examined transcriptional profiles as well as histone modifications and TF occupancy in WT, Fbw7(-/-), and Fbw7(R/+) cells. While the purpose of this study is interesting and important, the interpretation of the results is somewhat puzzling and complex. Furthermore, the details of the experimental design in some parts are not clearly described, which hinders the understanding and evaluation of this study. Overall, the biological impact of deletion or mutation of Fbw7 on cancer still remains unclear.
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