Gene expression control by protein deubiquitinases

Gene expression control by protein deubiquitinases

Available online at www.sciencedirect.com Gene expression control by protein deubiquitinases Lori Frappier1 and C Peter Verrijzer2 Protein ubiquityla...

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Gene expression control by protein deubiquitinases Lori Frappier1 and C Peter Verrijzer2 Protein ubiquitylation is involved in the regulation of virtually all aspects of eukaryotic cell biology, including gene expression. The central function of E3 ubiquitin ligases in target selection is well established. More recently, it has become appreciated that deubiquitylating enzymes (DUBs) are crucial components of ubiquitin-regulated cellular switches. Here, we discuss advances in our understanding of how DUBs regulate chromatin dynamics and gene expression. DUBs are integral components of the transcription machinery, involved in both gene activation and repression. They modulate the ubiquitylation status of histones H2A and H2B, which play pivotal roles in a cascade of molecular events that determine chromatin status. A DUB module in the SAGA coactivator complex is required for gene activation, whereas other DUBs are part of the Polycomb gene-silencing machinery. DUBs also control the level or subcellular compartmentalization of selective transcription factors, including the tumour suppressor p53. Typically, DUB specificity and activity are defined by its partner proteins, enabling remarkably versatile and sophisticated regulation. Recent findings not only underscore the pervasive and pivotal role of DUBs in gene expression control, but also raise paradoxical questions concerning the molecular mechanisms involved. Addresses 1 Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada 2 Department of Biochemistry and Centre for Biomedical Genetics, Erasmus University Medical Centre, Rotterdam, The Netherlands Corresponding authors: Frappier, Lori ([email protected]) and Verrijzer, C Peter ([email protected])

Current Opinion in Genetics & Development 2011, 21:207–213 This review comes from a themed issue on Chromosomes and expression mechanisms Edited by Peter Verrijzer and Ali Shilatifard

0959-437X/$ – see front matter # 2011 Elsevier Ltd. All rights reserved. DOI 10.1016/j.gde.2011.02.005

Introduction Ubiquitylation is a reversible post-translational modification that regulates the stability, localization or function of a target protein. Protein ubiquitylation is mediated by a cascade of three enzymatic steps, of which the last one, catalyzed by E3 ubiquitin ligases, is substrate specific and highly regulated. Initially, the processing of ubiquitin proprotein and ubiquitin recycling were considered the www.sciencedirect.com

main functions of deubiquitylating enzymes (DUBs). However, it is becoming increasingly appreciated that DUBs play a crucial regulatory role by counteracting ubiquitylation of selective targets. Inspection of the human genome suggests that it encodes for 600 E3s and nearly 100 DUBs. Of only a small minority of these enzymes do we know the physiological partners or cellular functions. There are five families of eukaryotic DUBs, of which the ubiquitin-specific protease (USP; named UBP in yeast and sometimes in other organisms) is the largest. The general properties, structure and cellular functions of DUBs have been discussed in a number of excellent recent reviews [1,2]. Here, we will concentrate on new insights into the function of DUBs in the regulation of chromatin dynamics and gene expression. Rather than providing a comprehensive overview, we will discuss selected examples that illustrate the various ways in which specialized DUBs control chromatin properties, gene activation or silencing and transcription factor levels or subcellular localization.

Chromatin control by DUBs Mono-ubiquitylation of histones H2A and H2B belongs to a plethora of post-translational histone modifications that modulate chromatin dynamics and gene transcription [3–5]. A single ubiquitin moiety is conjugated to mammalian histone H2A lysine 119 (H2Aub1) or H2B lysine 120 (H2Bub1). In other organisms, the equivalent histone residues are ubiquitylated. Histone ubiquitylation marks local chromatin status, and does not act as a degradation signal. Generalizing, H2Bub1 is associated with gene expression, whereas H2Aub1 correlates with gene silencing. The ratio of H2Aub1 versus H2Bub1 varies between different organisms, possibly reflecting the proportion of genomic euchromatin and heterochromatin. H2B ubiquitylation in Saccharomyces cerevisiae and Drosophila is mediated by the E2 ligase RAD6 (named DHR6 in flies) and the E3 ligase BRE1. Mammals have two RAD6 homologs (HR6A and HR6B), and two BRE1 homologs (RNF20 and RNF40). HR6A and HR6B appear to work redundantly, while RNF20 seems to be more critical for H2B ubiquitylation than RNF40 [3,5]. H2B ubiquitylation requires early steps in RNA pol II elongation and marks active chromatin. H2Bub1 promotes transcription through triggering additional positive histone modifications, and by stimulating nucleosome turnover by the histone chaperone FACT [3–5]. In bulk chromatin, H2B ubiquitylation stimulates methylation at H3K4 and H3K79, both active histone marks. There is also evidence, however, for a more complex gene-specific interplay between H2Bub1 and H3 methylation [4,6]. Unresolved issues include the Current Opinion in Genetics & Development 2011, 21:207–213

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Figure 1

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Chromatin control by DUBs. Simplified model illustrating the various roles of DUBs in gene activation and silencing. Note that this figure summarizes results from yeast, flies and mammals. For clarity, we did not include trans-histone pathways, down-stream effects of ubiquitylation (ubiq.) or all interacting co-regulators. Note that H2Aub1 deubiquitylation or H2Bub1 deubiquitylation (Deubiq.) can be part of gene activation as well as gene silencing, depending on the enzymes involved. See main text for details.

identification of factors binding to the H2Bub1 mark and the effect of H2Bub1 on the biophysical properties of the chromatin fiber. H2Bub1 cannot be viewed simply as a stable epigenetic chromatin mark. Optimal transcription, at least of some genes, requires a temporal cycle of H2B ubiquitylation and deubiquitylation [5,6,7]. Whereas H2B ubiquitylation acts early in the transcription cycle, subsequent steps depend on H2Bub1 deubiquitylation. This is mediated Figure 2

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Complex interplay between USP7 and the p53 pathway. Proteins in the p53 pathway whose expression levels are affected by USP7 are indicated. Solid arrows indicate effects on protein levels where black arrows are stabilization and blue arrows are destabilization. Broken arrows indicate effects on activation independent of protein levels, where black arrows are positive and blue arrows are negative effects. Current Opinion in Genetics & Development 2011, 21:207–213

by a conserved DUB within the SAGA coactivator complex, named UBP8 in S. cerevisiae, NONSTOP in flies and USP22 in mammals [6,7,8,9,10,11] (see Figure 1). H2Bub1 deubiquitylation by UBP8 facilitates serine 2 phosphorylation of the RNA polII CTD by CTK1, and subsequent activating chromatin modifications [7]. UBP8’s functioning depends critically on its integration in SAGA, as part of a DUB module together with SGF11, SUS1 and SGF73 [8,12,13]. Two insightful structural studies revealed how these attendant proteins mediate nucleosome binding, and induce allosteric changes within UBP8, required for H2Bub1 deubiquitylation [12,13]. Thus, UBP8 does not function in isolation, but its activity and specificity is controlled by associated factors. Distinct from the H2Bub1 cycle during gene activation, the maintenance of low H2Bub1 levels is a hallmark of gene silencing. S. cerevisiae UBP10 keeps telomeres and the rDNA locus depleted of H2Bub1 [14]. Consequently, the levels of H3K4 methylation and H3K79 methylation are low too. This local paucity of positive histone marks facilitates binding of Sir2, histone deacetylation and transcription repression. Another DUB, USP7, mediates gene silencing at developmental loci through H2Bub1 deubiquitylation [15]. Surprisingly, Drosophila and human USP7 associate with the biosynthetic enzyme GMP synthetase (GMPS) [15,16]. Genetic and biochemical experiments revealed that GMPS strongly simulates USP7’s ability to remove ubiquitin from H2B, but not H2A, by a mechanism that is independent of the enzymatic activity of GMPS [15,16,17]. The GMPS–USP7 complex is a gene-selective transcriptional corepressor that associates with Polycomb response elements (PREs), ecdysteroid regulated genes and the Epstein–Barr virus latent origin of replication [15,16,17]. The regulation of www.sciencedirect.com

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Figure 3

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USP7 structural organization. TRAF, catalytic and ubiquitin-like (Ubl) domains of USP7 are indicated along with amino acid numbers. USP7-binding proteins that have defined USP7-interacting regions are indicated below the regions of USP7 with which they interact.

USP7 activity by a classic biosynthetic enzyme, GMPS, might provide a relay between the metabolic state of the cell and local chromatin structure.

DUBs in Polycomb group silencing Polycomb group (PcG) proteins comprise a diverse group of transcriptional repressors that control a variety of developmental decisions in animals and plants [18–20]. They were initially identified in Drosophila as a class of genes required to maintain homeotic gene repression. Subsequent research has implicated PcG proteins in the control of a range of processes, including cellular senescence of cancer cells, stem cell pluripotency and maintenance of cell fate. PcG repression is intrinsically linked to modulation of chromatin architecture. One signature of PcG-silenced chromatin is H2Aub1, mediated by dRAF, PRC1 or related complexes (Figure 1). PRC1 and dRAF both harbour dRING/ESC (Extra sex combs), the crucial Drosophila H2A E3 ubiquitin ligase [18–20]. Of its two mammalian homologs, RING1A and RING1B, the latter appears to be the major H2A ubiquitin ligase. The downstream consequences of H2Aub1 include trans effects on other histone modifications and RNA pol II pausing [5,18]. RING1B’s role in transcription might involve more than H2A ubiquitylation, because, at least in some cells, catalytically inactive RING1B can still drive HOX gene silencing [21]. DUBs play a major role in PcG silencing. An early example was the finding that GMPS–USP7 contributes to epigenetic silencing of Drosophila homeotic genes [15]. Removal of the active H2Bub1 mark by GMPS–USP7 provides a straightforward mechanism for its cooperation with PcG silencing. However, recent research in mammalian cells revealed that USP7 also deubiquitylates selective PcG proteins [22,23]. Both USP7 and USP11 were found to deubiquitylate and stabilize mammalian PRC1-related complexes. Through modulating PcG silencing, these DUBs can modulate expression of the important human tumour suppressor p16INK4a [23]. One of USP7’s PcG targets, RING1B, is controlled by ubiquitylation in two www.sciencedirect.com

distinct ways. First, auto-ubiquitylation, generating K6, K27 and K48 mixed chains, acts as an activating modification. Second, targeting the same lysine residues, the ubiquitin E3 ligase E6-AP generates K48 poly-ubiquitin chains that target RING1B for proteasomal degradation [24]. USP7 removes both types of ubiquitin chains, conceivably recycling ‘neutral’ RING1B, which is neither degraded nor active [22]. What is the effect of H2Aub1 deubiquitylation? It seems reasonable to expect that lowering H2Aub1 levels would hamper the formation of repressive chromatin. Indeed, the mammalian DUB UBP-M/USP16 deubiquitylates H2Aub1 and counteracts PcG silencing of HOX genes [25]. However, it turned out that H2Aub1 deubiquitylation is critical for PcG repression. The Drosophila PcG gene Calypso encodes a carboxy-terminal hydrolase that deubiquitylates H2Aub1 [26,27]. CALYPSO functions in association with PcG protein ASX (additional sex combs), forming the PR-DUB complex. PR-DUB binds PREs and mediates HOX gene silencing. Thus, paradoxically, PcG repression requires H2A ubiquitylation by dRING/ESC, and H2Aub1 deubiquitylation by PRDUB. Concomitant loss of dRING/ESC and PR-DUB causes stronger homeotic effects than depletion of either one alone. Thus, reminiscent of the role of H2Bub1 in transcription activation, PcG repression might require a temporal cycle of H2A ubiquitylation and deubiquitylation. Alternatively, these antagonistic activities might be needed to fine-tune the level and localization of H2Aub1. Gross changes in H2Aub1 levels might cause the spreading and dilution of chromatin regulators, and thus affect PcG silencing indirectly. Finally, like USP7, PR-DUB might modulate the ubiquitylation status of other PcG proteins such as dRING/ESC. At this moment, the molecular events directly downstream of H2Aub1 remain enigmatic. One intriguing clue is the finding that the PcG protein RYBP can bind H2Aub1 [28]. As RYBP associates with RING1B, the enzyme responsible for H2A ubiquitylation, this might Current Opinion in Genetics & Development 2011, 21:207–213

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constitute a positive feedback loop for perpetuating or spreading the H2Aub1 mark. Collectively, the studies discussed here emphasize that ubiquitin ligases act hand in glove with DUBs during chromatin control. Rather than epigenetically stable chromatin markers, H2Aub1 and H2Bub1 appear to function as dynamic signals with a high turnover.

Transcription factor control by USPs One obvious way in which DUBs can control gene expression is by reversing transcription factor ubiquitylation, preventing degradation by the proteosome. The Drosophila DUB UBP64 controls cell fate in the developing eye by counteracting the E3 ubiquitin ligase SINA in a cell-specific manner [29]. UBP64 stabilizes the transcriptional repressor TRAMTRACK in selective precursor cells in the developing eye, driving them toward a non-neuronal identity. Thus, the balance between the activities of the E3 SINA and the DUB UBP64 constitutes a specific post-translational switch controlling cell fate. Mono-ubiquitylation can determine the subcellular localization of a transcription factor rather than its stability. The transcription factor FOXO becomes mono-ubiquitinated in response to oxidative stress, resulting in nuclear accumulation. This processs is counter-acted by USP7, which deubiquitinates FOXO and negatively regulates its transcriptional activity [30]. Another example is nuclear exclusion of the tumour suppressor PTEN, which is associated with certain cancers. PTEN subcellular localization is controlled by ubiquitylation, which in turn is modulated by a protein network involving USP7 and PML nuclear bodies [31]. USP7 also plays a central role in the control of the p53 tumour suppressor, as discussed below. Thus a single DUB, USP7, has been implicated in histone H2B ubiquitylation, hormone signaling, PcG silencing and control of three human tumour supressors, p16INK4a, PTEN and p53. As an example of the intricate roles of DUBs in transcription factor control, we discuss the regulation of p53 in detail.

USP-mediated regulation of p53 The p53 tumour suppressor can act as a transcription factor to activate the expression of specific genes involved in cell-cycle arrest or can induce apoptosis. These functional outcomes are tightly tied to p53 levels which are regulated by ubiquitylation catalyzed largely by the Mdm2 ubiquitin ligase. Conversely, accumulation of p53 in response to cellular stress involves deubiquitylation of p53 by USP7. USP7 specifically binds to and stabilizes p53 and can lead to p53-dependent cell-cycle arrest and apoptosis [32]. Such effects are interfered with by the EBV EBNA1 protein through competitive inhibition of the p53–USP7 interaction, providing a mechanism by which EBV can increase cell survival [33]. While reduced levels of USP7 result in decreased Current Opinion in Genetics & Development 2011, 21:207–213

levels of p53, ablation of the USP7 gene was unexpectedly found to stabilize p53 [34,35,36]. This effect can be attributed to the ability of USP7 to specifically bind, deubiquitylate and stabilize Mdm2. In addition, USP7 can bind, deubiquitylate and stabilize MdmX, a Mdm2 homologue that also regulates the p53 pathway [37]. Furthermore p53, Mdm2 and MdmX compete for the same binding pocket in the USP7–NTD resulting in a complicated interplay between these proteins (Figure 2). Interestingly, DNA damage that results in phosphorylation of Mdm2 and MdmX decreases the association of USP7 with Mdm2 and MdmX but not with p53, suggesting that ATM/ATR-mediated phosphorylation of Mdm2 and MdmX favours p53 stabilization [37]. The ability of USP7 to affect p53 function is not limited to deubiquitylation. USP7 binds p53 through p53 regulatory sequences known to modulate p53–DNA interactions and this interaction was recently shown to stimulate p53 binding to its recognition sites [38]. This effect is independent of the USP7 catalytic domain and involves an interaction with the USP7–CTD. In keeping with these observations, catalytically inactive USP7 can increase p53 occupancy at its target promoters and the USP7–CTD can drive p53-mediated reporter gene expression [38]. In addition, USP7 can negatively regulate PML nuclear bodies, which are important for the activation of p53. USP7 has long been known to be partially associated with PML bodies [39] and has recently been shown to induce polyubiquitylation and degradation of PML proteins independently from its catalytic activity [40]. This is in keeping with previous observations that USP7 binding is required for EBNA1 to induce PML degradation [41]. The data as a whole indicate that USP7 affects p53 function at multiple levels. Finally, although USP7 is responsible for p53 deubiquitylation in the nucleus, a recent report identified USP10 as important for deubiquitylating p53 that has been exported to the cytoplasm [42].

USP structure USPs make up more than half of all deubiquitinating enzymes and with more than 50 USPs predicted to exist in human cells [1,2]. USPs are papain-like cysteine proteases that share homology in residues surrounding the catalytic cysteine and histidine residues (Cys-box and His-box) but differ in their protein interaction domains that are largely responsible for targeting the enzyme to specific substrates. Structures of the catalytic domains of several USPs (including USP7) have been determined, revealing considerable structural homology despite limited sequence homology. The conserved USP fold consists of finger, palm and thumb subdomains resembling a right hand, with the Cys and His boxes located in the cleft separating the palm and thumb [43,44–47]. Structures determined in complex with ubiquitin or a ubiquitin derivative showed that ubiquitin is oriented with its Cwww.sciencedirect.com

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terminus at the active site cleft while its globular portion interacts with the finger domain [43,44,46]. Ubiquitin binding induces changes in conformation of the catalytic domain, in particular at the catalytic cleft [43]. The catalytic domain of USPs often accounts for a small proportion of the protein with additional domains for protein interactions and less well defined functions. In the case of USP7, the catalytic domain comprises less than a third of the protein and partial proteolysis analyses revealed a stable domain N-terminal to the catalytic domain and two protease-resistant domains C-terminal to the catalytic domain [48] (Figure 3). The N-terminal domain (NTD) has been shown to mediate USP7 interactions with Epstein–Barr nuclear antigen 1 (EBNA1), p53, Mdm2 and Mdmx [33,43,48,49,50,51]. This region was predicted to form a TRAF domain [52] and this prediction was later confirmed by several crystal structures. Saridakis et al. [33] determined the crystal structure of the USP7 NTD on its own and bound to its recognition sequence in EBNA1. While the EBNA1 peptide was positioned in a groove typically responsible for peptide interaction in other TRAF domains, the peptide adopted an unusual conformation which involved several additional contacts with b-strand 7, not previously seen in TRAF domain interactions. Comparisons of changes in the NMR resonance frequencies of USP7 upon binding EBNA1 and p53 peptides also indicated that EBNA1 made more extensive contacts with the USP7 NTD than did p53 [33] and this was later confirmed by crystal structures of the USP7–NTD bound to p53 peptides [49,50]. This difference in the USP7– NTD interaction is thought to underlie the observations that EBNA1 can interfere with p53–USP7 interactions in vitro and p53 stabilization in vivo [33,48], properties that likely contribute to the cell immortalization associated with Epstein–Barr virus latent infection. Interactions between the USP7–NTD and p53 have been shown to occur through two closely spaced peptides in the p53 regulatory region and, similarly USP7–Mdm2 interactions also involve two closely spaced peptides in Mdm2 as well as a third distant peptide [49,50,51]. Crystal structures of the p53 and Mdm2 complexes showed that the p53 and Mdm2 peptides occupied the same surface groove of the TRAF domain and hence were in competition for this site [49,50,51]. The same groove can also bind two MdmX peptides albeit with lower affinity than the p53 or Mdm2 peptides [51]. Comparisons of the peptides bound by the USP7 TRAF domain points to a consensus-binding sequence of P/AxxS where the S makes a critical contacts with USP7 [49,50,51]. The P/A residues can also be an E as found in the EBNA1 peptide bound by USP7. The simplicity of this consensus sequence does not fully account for the much higher specificity of USP7 for its target proteins and therefore it is likely that the context of the consensus sequence is www.sciencedirect.com

important in allowing the appropriate conformation of the peptide backbone needed to hydrogen bond to USP7 bstrand 7. USP7 sequences C-terminal to the catalytic domain are the least understood but they have been found to mediate interactions with at least 2 proteins, the ICP0 protein of herpes simplex virus and the cellular FOXO protein (Figure 3), the transcriptional activity of which is negatively regulated by USP7 through removal of monoubiquitin [30,31,32,33]. In addition, recent reports indicate that p53 and Mdm2 interactions can also be mediated by sequences in the USP7 C terminal region [38,53]. The C-terminal region of USP7 has also been shown to greatly stimulate ubiquitin cleavage by the catalytic domain although the mechanism is not understood [53,54]. Finally, the USP7 C-terminal region is predicted to contain a series of four ubiquitin-like domains which appear to be present at high frequency in USPs [55], and the NMR structure of one of these has been recently determined (PDB accession number 2KVR). It is not yet clear what role these ubiquitin-like domains may play in stimulating ubiquitin cleavage or mediating protein interactions.

Concluding remarks It has become clear that the reversal of histone or transcription factor ubiquitylation by DUBs plays a critical role in gene expression control. Chromatin ubiquitylation does not act as a static mark but is part of a highly dynamic regulatory circuitry. A temporal cycle of H2B ubiquitylation followed by deubiquitylation is required for optimal gene activation. Likewise, both histone H2A ubiquitylation and deubiquitylation have been implicated in PcG silencing. Gene control by DUBs involves a wide variety of distinct mechanisms. (De)ubiquitylation can control the level or subcellular localization of key transcription factors in response to signaling. Another emerging theme is that associated partner proteins control the activity and specificity of DUBs. Selective DUBs can be targeted to specific genomic loci by transcription factors, sometimes involving cooperative DNA binding. Generally, DUBs appear to be part of extensive protein-interaction networks [56]. Topics for future studies include analysis of the many DUBs whose function remains unknown, dissection of the dynamic protein networks of which they are part, and structural analysis of the conformational changes that control DUB activity. Finally, DUBs are involved in major disease pathways and are likely to provide selective targets for pharmacological intervention.

Acknowledgements This is not a comprehensive review and we apologize to authors whose work we did not discuss because of space limitations. This work was supported by a grant from NWO Chemical Sciences, ECHO 700.55.001 to CPV and by a Canadian Cancer Society research grant to LF. LF is a tier 1 Canada Research Chair in Molecular Virology. Current Opinion in Genetics & Development 2011, 21:207–213

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11. Zhao Y, Lang G, Ito S, Bonnet J, Metzger E, Sawatsubashi S,  Suzuki E, Le Guezennec X, Stunnenberg HG, Krasnov A et al.: A TFTC/STAGA module mediates histone H2A and H2B deubiquitination, coactivates nuclear receptors, and counteracts heterochromatin silencing. Mol Cell 2008, 29:92-101. See the annotation in Ref. [6].

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12. Kohler A, Zimmerman E, Schneider M, Hurt E, Zheng N: Structural  basis for assembly and activation of the heterotetrameric SAGA histone H2B deubiquitinase module. Cell 2010, 141:606617. See the annotation in Ref. [13].

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Transcriptional Control by Ubiquitin Proteases Frappier and Verrijzer 213

deubiquitylates H2Aub1. Thus, PcG repression involves both H2A ubiquitylation by dRING and deubiquitylation by PR-DUB. 28. Arrigoni R, Alam SL, Wamstad JA, Bardwell VJ, Sundquist WI, Schreiber-Agus N: The Polycomb-associated protein Rybp is a ubiquitin binding protein. FEBS Lett 2006, 580:6233-6241. 29. Bajpe PK, van der Knaap JA, Demmers JA, Bezstarosti K, Bassett A, van Beusekom HM, Travers AA, Verrijzer CP: Deubiquitylating enzyme UBP64 controls cell fate through stabilization of the transcriptional repressor tramtrack. Mol Cell Biol 2008, 28:1606-1615. 30. van der Horst A, de Vries-Smits AM, Brenkman AB, van Triest MH, van den Broek N, Colland F, Maurice MM, Burgering BM: FOXO4 transcriptional activity is regulated by monoubiquitination and USP7/HAUSP. Nat Cell Biol 2006, 8:1064-1073. 31. Song MS, Salmena L, Carracedo A, Egia A, Lo-Coco F, Teruya Feldstein J, Pandolfi PP: The deubiquitinylation and localization of PTEN are regulated by a HAUSP-PML network. Nature 2008, 455:813-817. Implicates USP7/HAUSP in the regulation of PTEN. Thus, USP7 plays a role in the control of three important human tumour suppressor pathways: p53 [32], INK4a [23] and PTEN. 32. Li M, Chen D, Shiloh A, Luo J, Nikolaev AY, Qin J, Gu W:  Deubiquitination of p53 by HAUSP is an important pathway for p53 stabilization. Nature 2002, 416:648-653. The first report that p53 associates with and is regulated by USP7/ HAUSP. Under the conditions in this study, USP7 was shown to deubiquitylate and stabilize p53. 33. Saridakis V, Sheng Y, Sarkari F, Holowaty MN, Shire K, Nguyen T,  Zhang RG, Liao J, Lee W, Edwards AM et al.: Structure of the p53 binding domain of HAUSP/USP7 bound to Epstein–Barr nuclear antigen 1 implications for EBV-mediated immortalization. Mol Cell 2005, 18:25-36. Together with [49] and [50], this paper provides the structural basis for how the N-terminal domain of USP7 binds EBNA1, p53 and Mdm2. Importantly it also reveals a mechanism by which Epstein–Barr virus can down-regulate p53 to promote cell survival. 34. Li M, Brooks CL, Kon N, Gu W: A dynamic role of HAUSP in the  p53–Mdm2 pathway. Mol Cell 2004, 13:879-886. This study, together with [35], reported the unexpected finding that complete ablation of USP7/HAUSP stabilized p53, implicating Mdm2 as another USP7 target. USP7 was shown to stabilize Mdm2 revealing a dynamic role of USP7 in the p53–Mdm2 pathway. 35. Cummins JM, Rago C, Kohli M, Kinzler KW, Lengauer C,  Vogelstein B: Tumour suppression: disruption of HAUSP gene stabilizes p53. Nature 2004, 428:486-487. See the annotation in Ref. [34]. 36. Cummins JM, Vogelstein B: HAUSP is required for p53 destabilization. Cell Cycle 2004, 3:689-692. 37. Meulmeester E, Maurice MM, Boutell C, Teunisse AF, Ovaa H,  Abraham TE, Dirks RW, Jochemsen AG: Loss of HAUSPmediated deubiquitination contributes to DNA damageinduced destabilization of Hdmx and Hdm2. Mol Cell 2005, 18:565-576. This paper identifies Mdmx as another USP7/HAUSP target and shows that DNA damage-induced phosphorylation of Mdmx and Mdm2 inhibits their interaction with USP7. This provides an interesting explanation of how USP7 favours p53 binding and stabilization in response to DNA damage. 38. Sarkari F, Sheng Y, Frappier L: USP7/HAUSP promotes the  sequence-specific DNA binding activity of p53. PLoS ONE 2010, 5:e13040. Describes a highly surprising mode of p53 regulation by USP7 that is independent of its deubiquitylation activity. This study reminds us that there can be more to DUB function than ubiquitin removal. 39. Everett R, Meredith M, Orr A, Cross A, Kathoria M, Parkinson J: A novel ubiquitin-specific protease is dyamically associated with the PML nuclear domain and binds to a herpesvirus regulatory protein. EMBO J 1997, 16:1519-1530. 40. Sarkari F, Wang X, Nguyen T, Frappier L: The herpesvirus associated ubiquitin specific protease, USP7, is a negative regulator of PML proteins and PML nuclear bodies. PLoS One 2011, 6:e16598.

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