Our data reveal an unexpected link between Gcn5 and the SAGA histone modifying complex and telomere maintenance, most likely through affects on shelterin protein levels. This work also indicates for the first time that SAGA and USP22 regulate protein stability and leads to a model wherein loss of Gcn5 leads to loss of the USP22 module from SAGA in mammalian cells. This dissociation leads to lowered USP22 activity, which in turn leads to increased ubiquitination and turnover of TRF1 ().
Figure 7 A proposed model for the role of GCN5 and SAGA in shelterin protein turnover. SAGA deUb module stabilizes TRF1 protein levels by deubiquitination, thereby inhibiting degradation. Depletion of GCN5 or ATXN7L3 leads to destabilization of the deubiquitination (more ...)
In human cells, TRF1 undergoes ubiquitin mediated proteolysis after its eviction from the telomeres (Chang et al., 2003
), which is triggered by prior poly (ADP)-ribosylation mediated by tankyrase 1 (Smith et al., 1998
). We do not yet know if Gcn5 and USP22 affect TRF1 before or after its eviction from telomeres. However, our observation of similar effects of Gcn5 and Usp22 on TRF1 in mouse and human cells indicates these effects are independent of the tankyrase pathway since mouse TRF 1 lacks a tankyrase 1 binding site and over expression of tankyrase 1 in mouse cells does not induce eviction of TRF1 form telomeres (Donigian and de Lange, 2007
). Others have shown that depletion or over expression of the F-box protein FBX4, which promotes TRF1 ubiquitination, significantly impacts telomere structure by regulating the cellular levels of this shelterin component (Lee et al., 2006b
). Also, the ubiquitin ligase RLIM regulates the telomere homeostasis by affecting TRF1 steady state levels promoting its ubiquitination (Her and Chung, 2009
). Taken together these data suggest that TRF1 ubiquitination and degradation are tightly controlled and appear to provide an additional level of telomere homeostasis control. Future experiments will determine how, when, and where SAGA is directed to TRF 1, and why levels of this protein might be regulated by both ubiquitination and deubiquitination.
Our studies also revealed that Gcn5 depletion affects the steady state levels of another shelterin protein, POT 1a. Although it is not known whether POT 1a is ubiquitinated, it might also be affected in a similar way to TRF1 by Gcn5 loss. Alternatively, Pot1a may be destabilized as a secondary consequence of TRF1 depletion.
We do not formally know whether the telomere fusions we observe in Gcn5
null cells are linked to the decreased levels of TRF1 and Pot1a that we observe. However, our findings are in agreement with previous studies in mouse cells that show depletion of TRF1 leads to dysfunctional telomere induced DNA repair foci and a mild chromosome fusion phenotype (Iwano et al., 2004
; Okamoto et al., 2008
). Depletion of mTRF1 in mouse ES cells significantly diminishes the ability of another shelterin component, TIN2, to localize to telomeres. Over expression of chicken TRF1 in these cells can rescue some but not all of the TRF1 induced phenotypes and cannot restore telomeric localization of TIN2 (Okamoto et al., 2008
). To our knowledge, no one has investigated the combined effects of diminished levels of both TRF1 and POT la, but it seems reasonable that loss of a second shelterin component would enhance TRF1-related telomere dysfunction phenotypes.
Previous studies by others indicate that SAGA complex integrity and proper subdomain organization are important for its functional activity (Ingvarsdottir et al., 2005
; Lee et al., 2005
; Rodriguez-Navarro et al., 2004
; Weake et al., 2008
; Zhao et al., 2008
). In particular, the deubiquitination activity of the Ubp8 and USP22 modules towards H2B in yeast and towards H2B and H2A in mammalian cells requires other factors such as Sus1 and Sgf11 (Ingvarsdottir et al., 2005
; Kohler et al., 2006
; Lee et al., 2005
) and their mammalian orthologues, ANY2 and ATXN7L3 (Zhao et al., 2008
). Moreover, the protein structures of Ubp8/USP22 indicate that these enzymes cannot bind free ubiquitin, so the surrounding subunits may be required not only to facilitate association of the deUB enzymes with the complex but also to provide substrate binding and specificity (Bonnet et al., 2008
). Interestingly, the mammalian complexes contain multiple proteins related to yeast Sgf73, which is required for Ubp8 incorporation into SAGA (Kohler et al., 2008
). The functions of ATXN7, ATXNL2, and ATXNL3 are not known, but our data suggest that ATXNL3 is particularly important for USP22 functions, at least towards nonhistone substrates, since knock down of ATXN7 had much less effect on TRF1 levels than did knock down of ATXN7L3.
Gcn5 is closely associated with USP22 or its fly orthologue, Nonstop (Lee et al., 2005
; Weake et al., 2008
; Zhang et al., 2008
; Zhao et al., 2008
). However, the effects of Gcn5 loss on the deubiquitination activity of USP22 were not previously defined. In vitro
experiments indicate that SAGA purified from gcn5Δ
yeast is still capable of removing ubiquitin from H2B, but this activity may not be as efficient as in SAGA purified from GCN5
wild type cells (Lee et al., 2005
), consistent with our findings. Importantly, yeast H2B was recently demonstrated to be polyubiquitinated and this modification was affected by deletion of Ubp8
(Geng and Tansey, 2008
). These results highlight the ability of Ubp8, and likely USP22, to deubiquitinate polyubiquitinated substrates, as indicated by our findings with TRF1.
Overall, our results indicate that Gcn5 and SAGA affect gene expression at multiple levels, from gene transcription to protein stability. These findings will likely foreshadow discovery of additional proteins that are destabilized upon depletion of the Gcn5 or the SAGA deUB module. USP22 is part of an 11 gene signature for highly metastatic cancers with poor prognosis (Glinsky, 2006
), raising the possibility that alterations in its role in controlling protein stability may contribute to cancer progression.