The TRRAP transcription cofactor represses interferon-stimulated genes in colorectal cancer cells
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**Evaluation Summary:
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This manuscript will be of interest to those studying gene transcription and its regulation by co-activator complexes in metazoans. The identification of the large TRAPP subunit shared between two conserved histone modification complexes as a negative regulator of the interferon type I pathway in colorectal cancer cell line provides novel insights into the functions of this TRAPP subunit, which was previously known only for this positive effects on gene transcription.(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. Reviewer #1 agreed to share their name with the authors.)
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Abstract
Transcription is essential for cells to respond to signaling cues and involves factors with multiple distinct activities. One such factor, TRRAP, functions as part of two large complexes, SAGA and TIP60, which have crucial roles during transcription activation. Structurally, TRRAP belongs to the phosphoinositide 3 kinase-related kinases (PIKK) family but is the only member classified as a pseudokinase. Recent studies established that a dedicated HSP90 co-chaperone, the triple T (TTT) complex, is essential for PIKK stabilization and activity. Here, using endogenous auxin-inducible degron alleles, we show that the TTT subunit TELO2 promotes TRRAP assembly into SAGA and TIP60 in human colorectal cancer cells (CRCs). Transcriptomic analysis revealed that TELO2 contributes to TRRAP regulatory roles in CRC cells, most notably of MYC target genes. Surprisingly, TELO2 and TRRAP depletion also induced the expression of type I interferon genes. Using a combination of nascent RNA, antibody-targeted chromatin profiling (CUT&RUN), ChIP, and kinetic analyses, we propose a model by which TRRAP directly represses the transcription of IRF9 , which encodes a master regulator of interferon-stimulated genes. We have therefore uncovered an unexpected transcriptional repressor role for TRRAP, which we propose contributes to its tumorigenic activity.
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**Evaluation Summary:
**
This manuscript will be of interest to those studying gene transcription and its regulation by co-activator complexes in metazoans. The identification of the large TRAPP subunit shared between two conserved histone modification complexes as a negative regulator of the interferon type I pathway in colorectal cancer cell line provides novel insights into the functions of this TRAPP subunit, which was previously known only for this positive effects on gene transcription.(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. Reviewer #1 agreed to share their name with the authors.)
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Reviewer #1 Public Review:
Using auxin-inducible degron alleles the manuscript shows that the TTT chaperone component TELO2 is required to stabilize TRRAP and facilitate its interactions with SAGA and TIP60 complex components. These data are convincing. Through genomic analysis they found that TRRAP plays a role in the repression of a number of interferon inducible genes through direct interaction with the IRF9 master regulator gene. They then try to demonstrate that both SAGA and TIP60 are involved in repression of IRF9, however, this aspect is incomplete and not convincing.
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Reviewer #2 Public Review:
The manuscript by Detilleux and coworkers continues studies by the Helmlinger group on the conserved TRAPP subunit of the SAGA and TIP60 (NuA4 in yeast) complexes. In this manuscript the authors create auxin-inducible degron allelles of TRAPP and of its TTT chaperone TELO2 in the HCT116 colorectal cancer cell line. In previous work the group showed that the TTT complex stabilizes TRAPP, and indeed degradation of TELO2 reduces nuclear accumulation of TRAPP. As expected direct auxin-induced degradation of TRAPP is more rapid, and inhibits HTC116 cell growth already after one day instead of two days for TELO2. Loss of TRAPP from the SAGA and TIP60 is significant, but not complete. RNAseq analysis showed that reduced TELO2 and TRAPP mostly leads to a reduced expression of genes, including MYC and E2F target genes, …
Reviewer #2 Public Review:
The manuscript by Detilleux and coworkers continues studies by the Helmlinger group on the conserved TRAPP subunit of the SAGA and TIP60 (NuA4 in yeast) complexes. In this manuscript the authors create auxin-inducible degron allelles of TRAPP and of its TTT chaperone TELO2 in the HCT116 colorectal cancer cell line. In previous work the group showed that the TTT complex stabilizes TRAPP, and indeed degradation of TELO2 reduces nuclear accumulation of TRAPP. As expected direct auxin-induced degradation of TRAPP is more rapid, and inhibits HTC116 cell growth already after one day instead of two days for TELO2. Loss of TRAPP from the SAGA and TIP60 is significant, but not complete. RNAseq analysis showed that reduced TELO2 and TRAPP mostly leads to a reduced expression of genes, including MYC and E2F target genes, which is expected given the documented role of TRAPP as a co-activator for MYC ad E2F.
Unexpected is the increase expression of the interferon type 1 group of genes (ISGs). The authors investigated regulation of the ISG pathway to find that TRAPP depletion mostly affects IRF9 expression at the mRNA and protein levels and to a lesser extent IRF7 expression. IRF9 and IRF7 are critical transcription factors for the ISG pathway and these observations offer an explanation for the induction of interferon type 1 genes after TRAPP depletion. The authors continue to show by 4SU labeling that IRF9 and IRF7 are transcriptionally induced and by CUT&RUN-PCR that TRAPP binds to the promoter regions of these genes. Re-expression of TRAPP reverses these effects. In order to dissect which of the TRAPP containing complexes several SAGA and TIP60 complexes are targeted by siRNA knock-down, but this does not provide clear distinction between SAGA and TIP60. In general, the exact mechanistic details of TRAPP-mediated repression of gene transcription have not been worked out, but the current work provides a strong basis for future studies addressing this.
In conclusion, this study clearly demonstrates that TRAPP directly inhibits expression of the IRF9 and IRF7 transcription factors and thereby inhibits the ISG pathway in colorectal tumor cells. This is an interesting observation by itself as until now TRAPP was only known for its positive effects on gene transcription.Major open issues:
- it is unclear why the authors did not choose to sequence the DNA from the TRAP CUT&RUN experiment, but rather performed (a more cumbersome) PCR analysis. A genome-wide CUT&RUN dataset for TRAPP would have allowed a direct comparison with their TELO2 and TRAPP depletion RNAseq datasets.
- it would be interesting to know the mechanistic basis for TRAPP as an inhibitor of IRF9 and IRF7 gene expression. I.e. how is TRAPP recruited to the promoters of these genes, is there a correlation with altered chromatin stats and/or histone modifications or variants at these promoters?
- while the data clearly show that TRAPP acts a repressor of the IRF9 and IRF7 genes, it is not entirely clear whether this relies on the SAGA and/or TIP60 complexes. To delineate the contribution of (other subunits of) these complexes one would need to create auxin-inducible degradation alleles of core subunits like, SUPT20H, SUPT7L, TAF5L of SAGA and EP400, TIP60, MRG15 etc of TIP60.
In particular, the issues 2 and 3 should be guiding future work.
Minor issues:
- The siRNA knockdown data are inconclusive and can be removed without a loss of impact of this work.
- The number of subunits shared between yeast and human SAGA depends on the organism. While in yeast five subunits (Taf5, Taf6, Taf9, Taf10 and Taf12) are shared, human SAGA shares only four (TAF9, TAF9b, TAF10 and TAF12) with human TFIID. This should be corrected on page 3, line 14.
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