The Role of Deubiquitinating Enzymes in Chromatin Regulation

Abstract

Post-translational modifications of the histones are centrally involved in the regulation of all DNA-templated processes, including gene transcription, DNA replication, recombination, and repair. These modifications are often dynamic, and their removal is just as important as their addition in proper regulation of cellular functions. Although histone acetylation/deacetylation and histone methylation/demethylation are highly studied, the functions and regulation of histone ubiquitination and deubiquitination are less well understood. This review highlights our current understanding of how histone ubiquitination impacts gene transcription, DNA repair, and cell cycle progression, and stresses the importance of deubiquitinases to normal cellular functions as well as to disease states such as cancer.

Introduction

The conjugation of ubiquitin molecules to intracellular proteins has emerged as a critical regulatory process in virtually all aspects of cell biology. Ubiquitin is attached to target proteins as a means of regulating the half-life, localization and activity of many polypeptides. Through a cascade including a ubiquitin activating enzyme (E1), dozens of ubiquitin conjugating enzymes (E2s), and hundreds of ubiquitin ligases (E3s), ubiquitin is specifically attached to targeted proteins in a precisely timed and accurate manner. Ubiquitin addition may take the form of single molecule attachment to one or multiple lysines (mono-ubiquitination) or may occur as Ub chains (poly-ubiquitination) with each subsequent Ub attached to a lysine of the prior. These Ub chains may exist in several formats depending on the lysine that is used for these inter-Ub linkages. Several lysines are utilized for chain formation including Lysine 6, (K6), K11, K29, K48 and K63. Proteasome targeting is accomplished primarily through the formation of K48-linked chains. K63 linkages perform more diverse functions by altering target protein structure, localization or activity.

Deubiquitination is the removal of ubiquitin molecules from targeted substrates and is mediated by a group of enzymes that belong to the superfamily of proteases, known as deubiquitinating enzymes (DUBs). These DUBs have been grouped into five subfamilies: 1) the ubiquitin C-terminal hydrolases (UCHs), 2) the ubiquitin-specific proteases/ubiquitin-specific processing proteases (USPs/UBPs), 3) the ovarian tumor proteases (OTUs), 4) the Josephin or Machado-Joseph disease protein domain proteases (MJDs) and 5) the Jab1/MPN domain-associated metalloisopeptidase (JAMM) domain proteins. The first four subfamilies are cysteine peptidases, while the last one is zinc metalloisopeptidase [1]. The mechanistic details of deubiquitination are still much less understood than the mechanism of the ubiquitination pathway. An important role of the deubiquitinating enzymes is emerging from a) the identification of a growing number of substrates and b) from the structural analyses that show how conformational changes regulate substrate specificity and tightly control enzymatic activity [2].

Several DUBs have been identified to target histones and this interaction has great impact on chromatin structure and downstream DNA-based processes. The ubiquitination of histones has been described for several decades, as H2A was the first protein shown to be ubiquitinated in cells [3], and the number of enzymes and their interacting partners that regulate the addition or removal of ubiquitin from histones increased rapidly. Histone ubiquitination has been primarily linked to transcriptional activation or repression, depending on the chromatin context, but the addition of ubiquitin to chromatin components affects many more DNA-based processes, like cell cycle progression, DNA damage repair, X chromosome inactivation and gene silencing. In this review we will discuss recent advances on deubiquitinating enzymes that have been identified to play a role in the regulation of histone deubiquitination, their role in establishing a fine balance of chromatin ubiquitination-deubiquitination, and how the deregulation of this balance may contribute to cancer development.

1. Histone ubiquitination and deubiquitination in transcriptional regulation

H2B ubiquitination was the first histone modification found to be required for addition of a second modification on a different histone, indicative of regulatory “crosstalk” between histone modifications. H2B ubiquitination is a prerequisite for H3K4 and -K79 tri-methylation, but not for H3K36 methylation [4]. Intriguingly, the absence of H3K4 methylation, in yeast cells expressing a H3K4R mutant, does not affect H2B ubiquitination, demonstrating that this regulatory pathway is unidirectional [4]. The pathway connecting H2B ubiqutination to H3K4 methylation is best defined in yeast, and it consists of a highly orchestrated set of events. The methyltransferase Set1 is recruited as part of the COMPASS complex to the transcription machinery through another complex, the Paf complex, and the resulting H3K4 methylation has been suggested to a provide a memory of recent transcription [5]. The yeast Paf complex is also required for H2B ubiquitination [6,7] and deletion of either the Rtf1 subunit or the Paf1 subunit results in drastic loss of H2B ubiquitination. Furthermore, Rtf1 enables the Bre1-Rad6 E2/E3 complex to associate with RNA Polymerase II (RNA Pol II) during transcription elongation. However, unlike Bre1, neither Rtf1 nor Paf1 are required for the recruitment of the Rad6 E2 enzyme to the promoter of active genes [8]. The ubiquitination of H2B occurs only after the Rad6-Bre1 complex associates with the elongating form of PolII that is phosphorylated at Ser 5 of the C-terminal Domain (CTD) (Fig.1A). Human homologues of Bre1, RNF20 and RNF40, associate with a human RAD6 homologue, to form an E2-E3 complex that is required for H2B monoubiquitination both in vivo and in vitro [9]. Kim and colleagues found that the knockdown of RNF20 in HEK 293T cells recapitulated the cross talk between histone ubiquitination and tri-methylation that has been observed in yeast [10], however Shema et al did not observe significant changes in the global levels of H3K4me3 in HeLa cells depleted for RNF20 [11], bringing the conservation of the trans-histone pathway between species or cell types into question.

Figure 1.

Ubiquitination and deubiquitination of histones H2A and H2B regulate chromatin during transcription. A. Schematic representation of the sequential events in ubiquitination /deubiquitination of H2B in yeast. Rad6-Bre1 E2-E3 complex is recruited to activated genes by the Paf1 transcription elongation complex, which associates with transcribing RNA PolII. H2B is ubiquitinated at lysine 123 by the Bre1 E3 ubiquitin ligase and the Set1 methyltransferase is subsequently recruited to the active promoters where it tri-methylates H3K4. In order for transcriptional elongation to proceed, ubiquitin needs to be removed from H2B via the enzymatic activity of Ubp8, which is part of the SAGA complex.

B. Schematic representation of the ubiquitination /deubiquitination of H2A in mammals. H2A is ubiquitinated by RNF20/40 E3 ubiquitin ligase and blocks RNA polII before the elongation stage. For transcription elongation to proceed, ubiquitin needs to be removed from H2A by a deubiquitinating enzyme, such as USP21. Whether the trans-regulation of trimethylation of H3K4 by H2B ubiquitination is conserved in mammalian cells is controversial, as described in the text.

Although the regulation of Set1-mediated H3K4 methylation and Dot1-mediated H3K79 methylation by H2BK123 ubiquitination has been extensively studied, there are still questions about how uH2B impacts chromatin structure and Set1/Dot1 functions. One possibility is that ubiquitin binds proteins and physically acts as a bridge between uH2B and Set1/Dot1. Another possibility is that ubiquitin acts as a “wedge” that facilitates access by dissociation of the nucleosome. However, more recent discoveries favor a role for the ubiquitin molecule in stabilizing the nucleosome as it prevents the eviction of H2A-H2B [12]. Cps35/Swd2, a component of yeast COMPASS, which contains Set1, appears to mediate crosstalk between ubiquitinated H2B and H3K4 methylation [13]. Proteasome components are also recruited to active genes via H2B ubiquitination, possibly to reconfigure chromatin for access of Set1 and Dot1 during transcription[14]. However, H2BK123 ubiquitination does not appear to regulate recruitment of the methyltransferase complexes but instead controls their processivity [15].

H2B deubiquitination mediated by Ubp8, the yeast ortholog of USP22 within the SAGA complex has a role in transcriptional activation of GAL genes and positively regulates the levels of H3K4 trimethylation, highlighting the role of the deubiquitinating enzymes in transcriptional regulation [16]. On the contrary Henry et al. found that both H2B monoubiquitination and deubiquitination are involved in gene activation, as Rad6 and Ubp8 have opposing functional roles in histone ubiquitination during transcriptional activation of the GAL genes [17]. A more complex regulation of H3K4 methylation has been described in vivo when different SAGA-regulated genes were studied in a Ubp8 mutant background [18], complicating even further our understanding of the function of histone ubiquitination as an activating or a repressive mark. In mammalian cells, ubiquitinated H2B was found to associate with the transcribed region of highly expressed genes [19], suggesting a positive role in transcription regulation, but the knockdown of RNF20, an E3 ubiquitin ligase responsible for H2B ubiquitination, affected the basal expression of only a subset of genes [11]. H2B ubiquitination appears to be required for initial stages of transcription in yeast, but creates a barrier for transcriptional elongation of SAGA-regulated genes by blocking Ctk1 recruitment to the PolII CTD, and this barrier needs to be removed for successful transcription to proceed [20]. However, in vitro transcription elongation assays using a highly reconstituted chromatin system established a role for H2B monoubiquitination in facilitating FACT function, thereby stimulating transcript elongation and the generation of longer transcripts [21].

H2A is the preferred ubiquitination histone substrate in mammalian cells. Ubiquitinated H2A has been estimated to comprise between 5–15% of total H2A, compared to about 1% of overall H2B ubiquitination. Monoubiquitination of H2A by Ring1B E3 ubiquitin ligase, which is a subunit of the transcripitional repressor PRC1 Polycomb-group complex, linked histone ubiquitination with gene silencing and X chromosome inactivation [22-24]. uH2A levels at Polycomb-repressed promoters decrease in RING1A/B deficient cells, followed by increased expression of the normally repressed targets [22,25,26]. Further analysis of promoters at derepressed genes after knock down of Ring1a/b and global loss of H2A ubiquitination in mouse ES cells shows association of PolII with regions downstream of the promoters, supporting a model where H2A ubiquitination hinders transcription at the stage of elongation but not initiation [27]. The finding that ubiquitinated H2A may act as a restraint for poised RNA PolII on gene promoters and a block for transcriptional elongation is further supported by the observation that 2A-HUB, an E3 ubiquitin ligase that is recruited by the repressive complex N-CoR/HDAC1/3 to promoters of chemokine genes of macrophages, monoubiquitinates H2A and blocks RNA polII release at the elongation stage [28]. However, a more recent study challenges the causative link between H2A ubiquitination and blockage of RNA polII activity by showing that Ring1B-regulated chromatin compaction and gene repression is not dependent on a functional RING domain of the ligase and thus is independent of histone H2A ubiquitination [29]. The functional analysis of the deubiquitinating enzymes that have been shown to regulate the levels of ubiquitinated H2A like 2A-DUB, USP21 and USP22, favor a repressive rather than an activating role for H2A ubiquitination on transcription. 2A-DUB has been characterized as an androgen receptor (AR) coactivator, as it deubiquitinates H2A at the promoter of AR target genes, facilitates the dissociation of H1 and activates transcription [30]. Similarly, the deubiquitinating enzyme USP22 is required with ATXN7L3 and ENY2 for the full transcriptional activity of the androgen receptor [31]. In contrast to H2B ubiquitination that is required for H3K4 trimethylation in yeast, H2A ubiquitination needs to be removed by deubiquitinating enzyme for H3K4 di and tri-methylation to occur in mammalian cells [32]. USP21, specifically, relieves ubH2A-dependent repression and facilitates transcription initiation by allowing H3K4 trimethylation in regenerating mouse liver (Fig. 1B) [32].

An inverse cross-talk mechanism has been observed between H3K27 methylation by methyltransferase EZH2 and H2AK119 ubiquitination by Ring1B E3 ligase [26]. PRC2 functions as a histone methyltransferase that trimethylates histone H3 on lysine 27 (H3K27me3). This mark acts as a recruiting platform for PRC1, which catalyzes the ubiquitination of histone H2A on K119 [22,25,33] and is required for homeobox (Hox) gene silencing. Evidence for another level of cross talk between histone H2A ubiquitination and histone acetylation arises from the association of deubiquitinating enzyme 2A-DUB and PCAF [30]. Hyperacetylated nucleosomes enhance the deubiquitination of H2A by 2A-DUB. The deubiquitinating enzyme is recruited to the activated promoter through its interaction with the acetyltransferase PCAF, a mechanism reminiscent of the cooperation between the deubiquitinating enzyme Ubp8 and the acetyltransferase Gcn5 for the transcriptional activation of target genes. The interacting partners 2A-DUB and PCAF, are recruited to the promoter region, remove the uH2A repressive mark and facilitate the dissociation of linker proteins, creating an open chromatin environment.

2. Deubiquitinating enzymes (DUBs) involved in chromatin regulation

Several deubiquitinating enzymes have been identified so far to remove ubiquitin moieties from chromatin templates in mammals, mainly deubiquitinating histones H2A and H2B. Among these histone deubiquitinating enzymes are: 2A-DUB/MYSM1, USP3, USP7, Ubp-M/USP16, USP21, USP22 and BRCC36 [30-32,34-38] (Table 1). All of these enzymes exert higher deubiquitination (DUB) activity toward ubiquitinated histone H2A (uH2A) compared to their activity toward ubiquitinated histone H2B (uH2B) in vitro and in vivo. This observation is in line with the fact that ubiquitination of H2A in higher eukaryotes appears to be the predominant histone mark, compared to the abundance of H2B ubiquitination [39,40]. However, all of the enzymes mentioned above are capable of H2A and/or H2B deubiquitination, raising questions about the regulation of DUB activity in time and space. There are several possible mechanisms by which the specific activity of these enzymes might be regulated, (1) integration into multisubunit complexes and interaction with different partners, (2) different expression levels during the cell cycle and different tissues.

Table 1.

Deubiquitinating enzymes implicated in chromatin regulation

Enzyme	Enzyme Family	Complex association	Substrates	Process
2A-DUB	JAMM	2A DUB	uH2A	Regulation of transcription
USP3	USP	none	uH2A and uH2B	DNA Repair: Regulation of the cell cycle progression
USP7	USP	PRC1	uH2A and uH2B, MEL18, BMI1	Regulation of gene expression, Protein stability
USP16	USP	none	H2A	DNA repair, Regulation of the cell cycle progression.
USP21	USP	none	H2A	Regulation of gene expression, Capable of removing NEDD8 from NEDD8 conjugates
USP22	USP	SAGA	uH2A, uH2B, TRF1	Regulation of gene expression; Cell proliferation; Protein stability
BRCC36	JAMM	BRCA1-A	K63 poly uH2A, uH2A.X	DNA repair, Capable of removing K63 linked polyubiquitin

List of DUBs with histone H2A and H2B deubiquitination activity implicated in regulation of chromatin related processes. DUBs are listed by enzymatic family, the complexes by which they are associated, their known substrates and processes they regulate

2.1 Complex integration and regulation of the DUB activity by interacting partners

Some DUBs work as a part of multisubunit complexes (i.e 2A-DUB, USP22, USP7). USP22 and 2A-DUB are enzymatically active as part of the SAGA and 2A-DUB complexes respectively [30,31,41]. The importance of interacting partners for the regulation of DUB activity is especially valid for USP22, where interactions with its complex partners ENY2 and ATXN7L3 are required for enzyme function. The three proteins are part of a sub domain in the complex known as the SAGA deubiquitination module [31,41,42]. More evidence about the mechanism of how the interacting partners regulate the DUB activity came from the recently published crystal structures of the yeast SAGA DUB module [43,44]. The authors of these studies demonstrated that Sus1 and Sgf11 and Sgf73 (yeast homologues of ENY2 and ATXN7L3 and ATXN7) maintain the “active conformation” of the enzyme. ATXN7 which is another bona fide SAGA component was also shown to anchor the DUB module to the complex [31] and to further facilitate histone deubiquitination by providing interactions with nucleosomes [45]. Both the 2A-DUB and SAGA complexes also harbor histone acetyltransferase (HAT) subunits, PCAF and its homologue GCN5, respectively. How and whether the presence of HATs in these complexes regulates their DUB activity remains elusive. However, the in vitro activity of 2A-DUB is significantly higher toward uH2A when incorporated in acetylated nucleosomes and in addition, the depletion of PCAF leads to increased levels of uH2A in vivo [30]. Experimental evidence addressing whether the USP22 activity is also impaired by the acetylation level of the targeted nucleosomes is not yet available. It is worth mentioning however, that ablation of GCN5 in mammalian cells diminishes the DUB activity of the SAGA complex, at least towards non-histone substrates [46]. Although, the exact mechanism by which acetylation and the presence of PCAF in the 2A-DUB complex enhances the DUB activity is unclear. Besides the HAT domain, both PCAF and GCN5 also contain bromodomains, which recognize acetylated lysines [47,48]. It is tempting to speculate that the bromodomains of these enzymes are involved in substrate recognition and targeting of the DUB activity of the complexes towards ubiquitinated lysines adjacent to acetylated residues.

Very recently the human polycomb repressive complex 1 (PRC1) was found to co-purify with two DUBs, USP7 and USP11 [34]. This finding was surprising since the PRC1 complex is mostly known for its role in gene repression conferred by H2A monoubiquitination. A deubiquitinating enzyme with uH2A specificity, called Calipso, was also found in the Drosophila Polycomb complex. The function of this DUB is important for Hox gene regulation in fruit flies [49]. Interestingly, the enzymatic activity of this DUB is also regulated by ASX, which is a Calipso interacting partner within the complex. From the USPs that co-purify with hPRC1 complex however, only USP7 displays activity toward uH2A and uH2B in vitro and overexpression of USP7 impacts uH2A levels in vivo. USP11, in contrast, does not seem to alter H2A/H2B ubiquitination levels either invitro or in vivo [34].

BRCC36 provides another example of modulation of DUB activity by its interacting partners. This enzyme functions as a part of the BRCA1-A complex and is mainly associated with deubiquitination of H2A/H2AX K63-specific polyubiquitination during DNA repair [38,50,51]. While the activity of this enzyme in the nucleus appears to be regulated by its interaction with CCDC98 within the BRCA1-A complex, association with the cytosolic protein (KIAA0157) activate the enzyme in the cytoplasm [38].

USP3 and USP16 are not known to be associated with any complexes to date. Since these DUBs possess activity independent of other interacting partners or complex integration [36,37], they are different from the DUBs described above. Both USP3 and USP16 are also associated with H2A deubiquitination following DNA damage processing [35,52]. It is possible that the interaction with some factors involved in DNA repair further facilitates their specific activity.

2.2 Connections between cell cycle and regulation of USP function

Some USPs may also be regulated during the cell cycle. Both USP16 and USP22, for example, have different cellular localizations at different stages of the cell cycle. USP16 localizes mostly in the cytoplasm during interphase and associates with chromosomes during mitosis [36]. USP22 on the other hand, is mostly nuclear during interphase but does not localize to mitotic chromosomes during mitosis [53]. UPS16 is phosphorylated at the onset of mitosis and dephosphorylated during the metaphase/anaphase transition. Thus, USP16 localization and activity may be controlled by posttranslational modifications as well. USP22 and USP7 are also posttranslationaly modified. USP22 is acetylated [54], and USP7 is phosphorylated [55]. Interestingly, the USP7 phosphorylation sites are located close to protein-protein interaction domains [55], suggesting that the activity and cellular localization of these enzymes might also be controlled via changes in posttranslational modifications.

Lastly, USP3 and USP22 have completely opposite tissue expression patterns. USP22 appears to be ubiquitously expressed in human tissues, whereas its expression in mouse is most abundant in brain and greatly diminished in liver [56]. USP3 on the other hand is almost absent in human brain and is relatively well expressed in liver [57]. The distinct expression pattern of these enzymes suggests that deubiquitination of H2A and H2B and other chromatin substrates is targeted by diverse DUBs in different tissues and during specific stages of the cell cycle. Redistribution of these enzymes in different cellular compartments suggests additional levels of control of their function (Fig. 2). The functional meaning of this DUB diversity remains to be uncovered, but it seems plausible that these enzymes and the complexes they form are engaged in the regulation of different subset of substrates and/or genomic loci. Clearly, more studies on interacting partners, expression levels and posttranslational modifications are needed to elucidate fully how these chromatin DUBs are regulated.

Figure 2.

Regulation of chromatin related DUBs during cell cycle.

The activity of several H2A/H2B deubiquitinases are associated with specific stages of the cell cycle. USP22, acting as a part of the SAGA complex, regulates the G1-S phase transition, perhaps by altering the expression level of different cell cycle related loci. It is possible that SAGA also regulates the ubiquitination state of different transcriptional regulators. Other H2A DUBs such as USP21 and 2A-DAB contribute to the regulation of cell proliferation, since USP21 function is required for hepatocyte cell cycle reentry during liver regeneration. Depletion of USP3 in cells leads to aberrant S-phase progression and accumulation of double-strand breaks (DSBs) suggesting a function of this DUB in the DNA replication and repair. BRCC36 modulates the chromatin K63 linked polyubiquitination in response of DNA damage. The DUB localize to DSBs and removes ubiquitin, antagonizing the RNF8-dependent ubiquitination at these foci. The regulation of USP16 appears to be more complicated. This DUB is mostly cytoplasmic during interphase of the unperturbed cell cycle and localizes to chromosomes during mitosis. This transition is regulated by phosphorylation and dephosphorylation throughout the cell cycle. USP16 is also engaged in the transcriptional reactivation after DNA repair. Perhaps the enzyme gets phosphorylated in response of DNA damage and relocalizes to the nucleus where it alters the ubiquitination state on the affected chromatin loci. Regulation of the DUBs activity by their postranslational modification during the cell cycle might be a common mechanism since USP22 was also found to be modified by acetylation and its localization during the mitosis differ from the localization during the interphase. It remains to be determined whether and what kind of post-translational modifications govern the activity of USP3, BRCC36 other chromatin associated DUBs

3. Impact of deubiquitinating enzymes activity on nuclear processes

The details of how H2A/H2B deubiquitination impacts gene expression in mammalian cells remains largely unknown. Although all of the above mentioned DUBs target uH2A and uH2B for deubiquitination it seems that the different DUBs have quite distinct impacts on cell cycle progression. Depletion of USP22, for example, leads to increased cell population in the G1 phase, demonstrating that USP22 is necessary for proper cell cycle progression. [41]. USP3 is required for proper S-phase progression, as its depletion leads to a delay in S-phase, whereas knockdown of USP16 results in defective mitosis [36,37]. The different roles of these enzymes in cell cycle control suggest that: (1) these DUBs control the deubiquitination of different chromatin loci altering the expression levels of different subset of genes and (2) the DUB activity of these enzymes is not restricted to histone substrates. Indeed, Maertens and colleagues demonstrated that USP7 and USP11 control the expression levels of p16^IKN4a through regulation of the ubiquitination level and protein turnover of PCR1 complex components, rather than by modulating the uH2A/H2B levels at this locus [34]. USP3 and BRCC36 are more clearly involved in DNA repair [35], and BRCC36 is associated with removal of K63-linked H2A polyubiquitination as well as K63-linked deubiquitination of other unspecified yet non-histone substrates during DNA repair [50,51,58]. The activity of USP3 is crucial for H2A/H2B mono deubiquitination and γ-H2A.X dephosphorylation during the repair process [35]. This observation agrees with the fact that ubiquitination of chromatin surrounding DNA lesions signals the DNA repair machinery [50,59]. Deubiquitination of these loci, then, will be needed to deactivate checkpoints and for transcriptional reactivation after the repair process is completed [52]. It is also possible that active deubquitination is associated with nucleosomal eviction and exchange of H2A/H2B during DNA repair. This possibility stems from the fact that USP3 depletion leads to S-phase delay and accumulation of DNA breaks, which might reflect aberrant nucleosomal exchange at the active sites during DNA repair and replication. Indeed, a functional link between H2A deubiquitination and H1 phosphorylation followed by its release from the chromatin has been reported [30]. The details of how deubiquitination of histones and other chromatin templates regulate cell cycle progression and DNA repair remains to be elucidated. It is highly anticipated that a substantial number of non-histone proteins engaged in the DNA metabolism have yet to be identified as substrates of the histone deubiquitinating enzymes as well.

4. Non-histone chromatin substrates of deubiquitinating enzymes

A growing list of evidence suggests that the regulation of gene expression occurs not only through ubiquitination and deubiquitination of histones but also of other chromatin related substrates. Recently, USP22, acting as a part of the mammalian SAGA complex, was shown to regulate the ubiquitination levels and protein stability of TRF1, one of the main components of the telomere protective complex called shelterin [46]. This finding was one of the first to show that a deubiquitinating enzyme, mostly known from its function on histones, targets another chromatin associated protein. TRF1 is ubiquitinated after its release from the telomeres [60] in human cells and is further targeted for proteasomal degradation. It is likely that USP22 “recycles” TRF1 by deubiquitination, making it available for reincorporation into telomeres. Perhaps this function is one of the reasons why USP22 is overexpressed in rapidly proliferating cancer cells [61].

USP7 (which is capable of H2A and H2B deubiquitination) and USP11 regulate the ubiquitination levels and turnover of chromatin bound PRC1 complex components MEL18 and BMI, which in turn impacts the transcriptional regulation of p16^INK4a [34]. USP7 is also known to regulate p53 function by altering its ubiquitination levels [62], as well as the ubiquitination levels and the turnover of Mdm2 [63,64]. It is worth mentioning that DAXX (death-domain-associated protein), which was recently identified as a histone H3.3 chaperone [65,66], was also found as a USP7 interacting protein [63,64]. It will be interesting to determine if USP7 also deubiquinates and modulates DAXX function, impacting its ability to facilitate histone H3.3 incorporation at telomeric regions. Another DUB, USP21, which is important for uH2A deubiquitination and transcriptional activation during liver regeneration [32] was recently shown to control the ubiquitination levels of receptor-interacting protein 1 (RIP1), event important for the proper functioning of the TNF alpha pathway [67].

Besides K63-linked polyubiquitination of H2A, several non-histone proteins are ubiquitinated when DNA damage occurs. The polyubiquitin chains of these substrates are proposed to serve as an anchor for DNA repair machinery, and their deubiquitination is needed after the repair process is completed. What these substrates might be, however, still need to be discovered. So far the only DUB capable of processing K63-linked polyubiquitin chains during the DNA repair is BRCC36. It remains to be seen if some of the other chromatin associated DUBs exert K63 specificity toward histone or non-histone substrates.

5. Chromatin regulation by deubiquitinating enzymes and cancer development

Although the role of DUBs involved in chromatin deubiquitination and transcriptional regulation has been extensively analyzed in yeast, the precise role of these enzymes in mammalian cells ant their contribution to the tumorigenesis is still elusive. Advances in the transcriptional profiling of tumor cells identified an 11-gene signature that characterizes patients with poor prognosis and likely to suffer death after treatment [68]. This signature has been described in multiple different cancer types, suggesting the presence of a possibly conserved mechanism that drives tumorigenesis that may reflect a cancer stem cell phenotype. Three of the identified genes in the 11 gene signature, Bmi1, Ring1B and USP22, are involved in regulation of histone ubiquitination or deubiquitination. Bmi1 and Ring1B are subunits of the mammalian PRC1 complex and the fact that Ring1B specifically represses p16Ink4a [69] may explain how Ring1B overexpression is linked to aggressive human tumors [68]. Although the simultaneous activity of Ring1B E3 ligase and USP22 deubiquitinating enzyme has not been addressed in different types of cancers, an alternative model for cancer progression has been suggested involving the fine tuning of histone ubiquitination levels by Ring1B and USP22 [41]. USP22 knockdown produces an accumulation of cells in the G1 phase of the cell cycle and leads to a reduction in transcription of several Myc and p53 regulated genes [41]. These results suggest that USP22 positively regulates MYC-dependent transcription, which may at least partially explain its oncogenic properties. Interestingly, Zhang and colleagues [41] found that the depletion of USP22 does not affect the activation of PUMA, a p53 target gene, indicating that USP22 has a gene-specific role in regulating the p53 transcriptional network. Zhao et al., [31] on the other hand, reported a critical role for USP22 in AR-mediated transcactivation, in both Drosophila and human cells. According to this study, the oncogenic potential of USP22 overexpression may arise from the misregulation of AR, as uncontrolled AR activity contributes to the development of prostate cancer [70].

Usp7 has been characterized as the deubiquitinating enzyme for H2B that is involved in epigenetic silencing of homeotic genes in flies [71]. The human ortholog of USP7 can deubiquitinate additional targets besides histone H2A, including Mdm2 and p53, and the deubiquitination of these targets ultimately determines functional p53 levels [62]. Mdm2, rather than p53, appears to be the preferred substrate of USP7, resulting in Mdm2 stabilization as USP7 antagonizes autoubiquitination of Mdm2 and consequently induces the degradation of p53 [72,73]. USP7 has also been shown to deubiquitinate FOXO4, provoking its nuclear export and hence its inactivation [74]. The tumor suppressor PTEN is also deubiquitinated by USP7, resulting in its nuclear export and inactivation. In the same study, USP7 overexpression in prostate cancer was associated with tumor metastasis [63]. A recent work showed that USP7 and USP11 deubiquitinating enzymes play a role in the regulation of INK4a/ARF locus. INK4a/ARF locus encodes two powerful inhibitors of cell cycle progression, p16Ink4a and p19Arf, a negative regulator of p53 [75], and is one of the critical targets of PcG-mediated repression in mammals. Mice lacking particular PcG genes display defects in stem and progenitor cell renewal that is attributable to de-repression of Ink4a/Arf locus [69,76]. Usp7 and Usp11 are associated with chromatin and bind to several PRC1 components in high-molecular-weight complexes. Importantly, knockdown of either USP results in de-repression of INK4a and displacement of PRC1 proteins from the locus. Knockdown of USP7 or USP11 causes increased turnover of chromatin-bound MEL18 and BMI1, whereas over-expression of the USPs reduces the levels of mono- and poly-ubiquitination of these proteins [7].

BAP1 is the largest member of the UCH subfamily of DUBs and it has been found to interact with several proteins like BRCA1 and the transcriptional cofactor host cell factor (HCF-1) [77,78]. Deubiquitinating activity of BAP1 seems to be important in cancer pathogenesis. Missense mutations that abolish the deubiquitinating activity of BAP1 (A95D and G178V) have been identified in cancer cell lines [79,80]. Although the molecular mechanism of BAP1 tumorigenicity is attributed to the regulation of BRCA1 E3 ligase activity or the turnover of HCF-1, a recent study shows that calypso the BAP1 homolog in Drosophila, exists in a complex with the PcG protein Asx and affects HOX genes regulation by regulating the levels of H2A ubiquitination [49]. The DUB activity of BAP1 in mammalian cells was also implicated in the transcriptional regulation [81]. BAP1 is recruited to its target genes via interaction with YY1 to modulates transcriptional initiation. It will be interesting to see if BAP1 regulates the ubiquitination levels of histone H2A at these loci or whether its DUB activity is targeted toward other transcriptional regulators at these sites.

Concluding remarks

It became clear during the recent years that ubiquitination and deubiquitination of chromatin templates regulates major cellular processes such as gene expression, DNA repair and DNA replication. Ubiquitination of chromatin substrates impacts these processes at several levels, directly by altering protein stability and protein localization (monoubiquitination K48 or K63 polyubiquitination), or indirectly by regulating other posttranslational chromatin modifications such as phosphorylation or methylation. Most of the identified DUBs share the same major chromatin substrates, ubiquitinated histones H2A/H2B, and yet their activities appear to have distinct impact on downstream processes. This fact raises questions about the temporal and spatial regulation of these enzymes activities and highlights the importance of studies elucidating their interacting partners and expression patterns. Discovering other, non-histone chromatin related substrates for these enzymes will shed further light on how cell cycle or tissue specific gene expression programs are regulated through both ubiquitination and deubiquitination.

Abbreviations

Ub

ubiquitin

DUBs

deubiquitinating enzymes

OTUs

ovarian tumor proteases

MJDs

Josephin or Machado-Joseph disease protein domain proteases

JAMMs

Jab1/MPN domain-associated metalloisopeptidases

USPs

ubiquitin-specific proteases

UCHs

ubiquitin carboxy-terminal hydrolases

UBPs

ubiquitin-specific processing proteases

Set1

Su(var)3-9/Enhancer of zeste/ trithorax domain protein 1

COMPASS

complex proteins associated with Set1

Paf

polymerase II- associated factor

RNA PolII

RNA polymerase II

CTD

carboxy-terminal domain

Dot1

disruptor of telomeric silencing 1

SAGA

Spt-Ada-Gcn5 acetyltransferase complex

RNF

Ring finger protein

Ctk1

carboxy-terminal domain kinase 1

FACT

facilitates chromatin transcription

PRC

Polycomb repressive complex

N-CoR

Nuclear receptor co-repressor

HDAC

histone deacetylase

AR

androgen receptor

ATXN7L3

ataxin7-like 3

ENY2

human ortholog of enhancer of yellow 2

ATXN7

ataxin7

EZH2

human ortholog of enhancer of zeste 2

P/CAF

p300/CBP-associated factor

GCN5

general control nonderepressible 5

BRCC36

BRCA1-containing complex

Sus1

S1 gene upstream of ySa1

Sgf

SAGA associated factor

HAT

histone acetyltransferase

BRCA1

breast–ovarian cancer-susceptibility 1

CCDC98

coiled-coil domain–containing protein 98

γ-H2A.X

phosphorylated histone variant H2A.X

TRF1

telomeric-repeat binding factor 1

MDM2

murine double minute 2 gene

DAXX

death-domain-associated protein

RIP1

receptor-interacting protein 1

TNF

tumor necrosis factor

PUMA

p53 upregulated modulator of apoptosis

FOXA4

forkhead box-containing transcription factor 4

PTEN

phosphatase and tensin homolog deleted on chromosome ten

INK4/ARF

inhibitor for cyclin-dependent kinase 4/ alternative reading frame

PcG

polycomb group

Bap1

BRCA1-associated protein 1

HCF-1

host cell factor 1