Eukaryotic transcription is one of the most complex cellular processes and constitutes the first step in protein synthesis. Ubiquitination and subsequent degradation by the 26 S proteasome, on the other hand, represents the final chapter in the life of a protein. Intriguingly, ubiquitin and the ubiquitin– proteasome system play vital roles in the regulation of transcription. Ubiquitin has dual modus operandi: firstly, ubiquitin functions via the 26 S proteasome — it is tagged to components of the transcription machinery, marking them for degradation via the proteasome, which results in the proper exchange of complexes during transcription and the prompt removal of activators after each round of transcription; and secondly, ubiquitin can function independently of the proteasome — histone ubiquitination results in heterochromatin relaxation and assembly of transcription complexes on the promoter, and ubiquitination of transcription factors enhances their transcriptional-activation function. Although ubiquitin and the ubiquitin–proteasome system were initially perceived as a graveyard for proteins, recent advances in molecular biological techniques have redefined their role as a regulatory system that influences the fate of many cellular processes, such as apoptosis, transcription and cell cycle progression.
Ubiquitination is a post-translational protein modification resulting in the covalent linkage of a 76-amino-acid polypeptide, ubiquitin, to lysine residues of substrate proteins. Ubiquitination of a substrate protein is a multi-step process and requires three different sets of enzymes that facilitate the transfer of ubiquitin to the substrate. These are the ubiquitin-activating (E1), ubiquitin-conjugating (E2) and ubiquitin-ligase (E3) enzymes. A combination of specific E2–E3 or E3 alone confers substrate specificity. Recent evidence suggests the existence of an additional factor, E4 (polyubiquitin chain conjugation factor), that could contribute to polyubiquitination of the substrate protein targeted for degradation [1–3]. The ubiquitination process is involved in the regulation of many cellular processes, such as cell-cycle progression, signal transduction, differentiation, apoptosis and transcription .
Eukaryotic transcription is a co-ordinated process that requires the assembly of RNA polymerase II (RNA pol II) and a multitude of protein cofactors on the promoter of a target gene [5–7]. These cofactors are transcriptional activators, chromatin-modifying enzymes, transcriptional co-activators and GTFs (general transcription factors). GTFs are multi-protein complexes that assemble at the gene promoter and facilitate transcription. These complexes comprise RNA pol II, TBP (TATA-box binding protein), TFIIB (transcription factor IIB) and TFIID [8,9]. It has been suggested that TFIID contains multiple activities associated with the formation of the pre-initiation complex. Of these various factors, transcriptional activators are vital because they interact directly with DNA sequences and are essential for the subsequent assembly of other factors for transcription. Transcriptional activators generally contain two distinct domains: a DNA-binding domain that tethers the protein to the specific DNA sequences and a TAD (transcriptional activation domain) that mediates interaction with GTFs and recruitment of transcriptional co-activators. Although these transcriptional proteins, together with the RNA pol II, are necessary and sufficient for transcription, research in recent years has shown that a functional interplay exists between the protein-making (transcription and translation) and the protein-degrading [ubiquitin–proteasome system (UPS)] machinery. In this review, we discuss the role of ubiquitination in the regulation of eukaryotic transcription.
Proteasome-dependent ubiquitin functions
Regulation of levels of transcription factors
The most well-understood characteristic of the UPS is its ability to target proteins for degradation via the 26 S proteasome . This provides a high level of control because the majority of proteins, including transcription factors that are synthesized in the cell, are destined for destruction by this pathway. Transcription factors are especially vulnerable to rapid degradation because a potent transcription factor, when active, can sequester the limited pool of transcription machinery, which is deleterious to the cell. In this section, we discuss a few examples of transcription factors that are regulated by ubiquitin and the UPS.
Regulation of β-catenin is one of the most well-understood examples of UPS-mediated regulation of a transcription factor. β-Catenin is involved in Wnt and other signalling pathways that result in the activation of p53 by genotoxic stress. In the absence of Wnt signalling, β-catenin is phosphorylated by GSK3β (glycogen synthase kinase 3β) . Phosphorylated β-catenin is then ubiquitinated by a SCFbeta;TrCP [Skp1(S-phase associated protein-1)–Cdc(cell-division cycle)53/Cullin-1–F-box β-transducin repeat-containing protein] complex, which specifically targets phosphorylated β-catenin for ubiquitination . Wnt signalling leads to rapid inactivation of GSK3β, and consequently β-catenin stabilizes and enters the nucleus, where it dimerizes with the TCF/LEF (T-cell factor/lymphoid enhancer factor) family of transcription factors and activates its transcription programme . β-Catenin has also been shown to be ubiquitinated and degraded in a GSK3β-independent mechanism by RING (really interesting new gene)-finger E3 Siah-1, which is also implicated in the degradation of oncogenic transcription factor c-myb . Interestingly, Siah-1 protein expression is triggered by p53 signalling, thus allowing both Wnt and p53 signalling pathways to converge on the regulation of β-catenin via the UPS .
Oncogenic transcription factor c-myc is a prominent regulator of cancer as it can collaborate with other oncogenes to transform normal cells into cancerous cells. The UPS is implicated in the modulation of protein levels of c-myc. It is aberrantly expressed in many cancers (adenocarcinomas, colon, breast etc.) and approx. 30% of all human cancers have dysregulated c-myc signalling. Skp2, an F-box protein with E3 activity, promotes c-myc's ubiquitination and degradation. Skp2 has also been shown to be a transcriptional co-activator of c-myc signalling. It is perceived that during S-phase transformation, Skp2 levels are increased, which results in the ubiquitination and activation of c-myc [15,16].
The other major group of transcription factors that is regulated by the UPS is NHRs (nuclear hormone receptors). This transcription-factor superfamily includes steroid hormone receptors such as androgen, progesterone, oestrogen, glucocorticoid and mineralocorticoid receptors. The NHR family also contains receptors for thyroid hormone, vitamin D and retinoids, and some orphan receptors with unknown ligands . All members of the NHR family have similarities in structure and signalling. NHRs have a global effect on gene transcription, from cell-cycle progression to inflammation and apoptosis. Their activity is finely tuned and regulated by a limited pool of proteins called co-activators. These co-activators consist of an array of proteins with various enzymatic activities, and are known to provide the link between NHRs and GTFs.
The UPS regulates the functions of NHRs by modulating the receptor, as well as the co-activator, protein levels. For certain NHRs, UPS-mediated degradation is essential for their transcriptional activities [18–23]. It has also been shown that NHRs cycle their responsive promoters on and off and this cycling is essential for the proper functioning of NHR. The UPS is a critical component of this cycling process [24,25]. These observations suggest that the UPS plays a vital role in the regulation of eukaryotic transcription by modulating the protein levels of many transcription factors in the cell.
Ubiquitin-mediated cofactor exchange
Transcription factors can be repressive or transactivating, depending on the type of cofactor that it interacts with. In this way, cofactor exchange can cause the activity state of transcription factors to change. This type of regulation is observed in the LIM-HD (LIM homeodomain) transcription factor and NHRs. LIM-HD can interact with its repressor, RLIM (RING-finger LIM domain-binding protein), and with its cofactor, CLIM (cofactor of LIM-HD proteins). Interaction of LIM-HD with RLIM represses its activity, whereas interaction with CLIM results in its activation. The UPS controls LIM-HD's transcriptional activity by exchanging CLIM with RLIM (which is also an E3 that targets CLIM for degradation by the 26 S proteasome) . In the case of NHRs, the UPS modulates their transcriptional activity by the recruitment of the components of the ubiquitin–proteasome pathway to the promoter. These components regulate receptor function by exchanging co-repressors and co-activators . Taken together, a clear-cut relationship emerges between transcription activation and protein ubiquitination and degradation.
Ubiquitin–proteasome-pathway enzymes and components as co-regulators
The UPS can also regulate the transactivation function of the transcription factors because components of the UPS function as transcriptional co-activators. It has been shown that E3s, such as Rsp5 and its human homologue RPF1 (receptor potentiation factor 1)/NEDD4 (neural precursor cell expressed developmentally down-regulated 4)  and E6-AP (E6-associated protein) , act as transcriptional co-activators of NHRs. E6-AP is also implicated in the congenital human disease, Angelman syndrome, and is essential for p53 degradation in HPV (human papillomavirus)-induced carcinogenesis . Interestingly, the E3 activity of both proteins is not necessary for their co-activation function. A well-characterized co-activator, p300, has been shown to catalyse the polyubiquitination of p53, suggesting that p300 is a dual-functioning protein that modulates gene transcription and ubiquitination [28,29]. In addition to E3, the E2 UbcH7 has also been shown to act as a co-activator; however, its enzymatic activity is required for its co-activation function .
It has been demonstrated that UPS enzymes as well as the proteasome subunits are recruited to the promoter region of target genes during transcription. SUG1 (supressor of Gal4D lesions1) is an ATPase subunit of the proteasome and is involved in the unfolding of protein while proteins are entering the proteasome. SUG-1 has also been characterized as a co-activator of NHRs . Similarly, other ATPase subunits, TRIP1 (thyroid-hormone-receptor-interacting protein 1) and TBP1 (TATA-box-binding protein 1), have been shown to interact with NHRs and regulate their function . Although the precise molecular role of the ATPase subunits is not fully understood, it is fascinating to comprehend that the proteasome is physically present on the promoter , where various modifications and exchange of cofactor proteins occurs. The proximity of the proteasome to the promoter is also critical for the timely removal of transcriptional repressors during pre-initiation-complex formation and the degradation of activators after initiation, followed by the rapid recycling of the RNA pol II itself, upon completion of transcription (Figure 1). This interesting scenario, if true, will facilitate the recycling and regulation of all the factors involved in the transcription process after each round of transcription, making the process more processive and precise.
Proteasome-independent ubiquitin functions
Regulation of RNA pol II by ubiquitination
One of the profound mechanisms of transcription control is the control of RNA pol II recruitment and subsequent degradation, which influences the transcriptional outcome. Our current understanding of this control mechanism is limited to TCR (transcription-coupled repair), a process by which transcriptionally active genes are preferentially repaired following DNA damage . DNA lesions in the strand that is being transcribed can arrest movement of the transcription complex. Evidence from normal cells, as well as cells from xeroderma pigmentosum patients, indicate that the ubiquitinated form of RNA pol II is hyperphosphorylated at the C-terminal domain of its largest subunit that is involved in transcription elongation . The yeast E3 Rsp5 ubiquitinates RNA pol II's C-terminal domain but is not required for TCR. This does not preclude a role for the 26 S proteasome in TCR; it is conceivable that the proteasome may act as a molecular chaperone and protect the stalled RNA pol II, leading to the assembly of the DNA-repair machinery. Previous reports demonstrate that Rsp5 also acts as a co-activator of steroid hormone receptors  and the finding that Def1, a component of the Rad26 TCR DNA-repair complex, is an essential factor required for transcription elongation  suggests that components of the TCR and proteins of the UPS may have a regulatory role in transcription elongation by RNA pol II [36,37].
Ubiquitin controls the localization of activator proteins
Although our understanding of the functional regulation of RNA pol II is limited, our understanding of the impact of the UPS on transcriptional activators is more extensive. There are numerous studies describing in detail the complex regulation of transcriptional activators by the UPS. Transcription factors have to be within the nucleus to function as activators; various processes, such as phosphorylation, tightly regulate their cellular localization . The UPS has been implicated in this mechanism, as ubiquitin is used for controlling the nuclear import/export of transcription factors. The UPS regulates nuclear factor κB (NF-κB), an essential transcription factor involved in the inflammatory response. NF-κB, under unstimulated conditions, is held in the cytoplasm by an inhibitory κB (IκB) protein. During inflammation, IκB is rapidly degraded via the 26 S proteasome, which releases NF-κB to translocate into the nucleus, which is essential for NF-κB function .
Oncoprotein Mdm2 (murine double minute clone 2 oncoprotein) is an E3 involved in the regulation of the tumour suppressor p53. It has long been known that p53 is ubiquitinated and degraded via the UPS and that Mdm2 is the specific E3 that is involved in this process. Recently, it has been suggested that p53 exists in two different states of ubiquitination (mono and poly), depending on the expression level of Mdm2. Mdm2 at low levels preferentially monoubquitinates p53, which leads to its nuclear exclusion, whereas at high levels of Mdm2, p53 is polyubiquitinated and marked for degradation by the 26 S proteasome [40,41].
A ubiquitin-mediated regulatory mechanism is also observed in the control of the yeast transcriptional activator Spt23, which is involved in fatty acid biosynthesis. Spt23 is synthesized as an endoplasmic reticulum outer-membrane protein following a reduction of fatty acid levels in the cell. It is then clipped precisely by the UPS and moves into the nucleus, accompanied by a ubiquitin-specific chaperone.
Ubiquitin regulates the activities of activator proteins
Apart from controlling activator localization in the cell, the UPS also modulates the activator function of transcriptional activators. One of the best examples available is the ubiquitin-mediated regulation of VP16, a potent viral transcriptional activator. Work by Salghetti et al.  suggests a link between the UPS and transcription activation. They showed that the VP16 TAD signals for ubiquitination via SCF/Met30 (an E3) and that ubiquitination is essential for its transcriptional activation . This process of ubiquitin-mediated activation is also shared by other activators, such as HIV-1 Tat protein. HIV-1 codes for a potent transcriptional activator Tat that hijacks cellular transcriptional co-activators, such as p300, PCAF [p300/CREB(cAMP/Ca2+-responsive element-binding protein)-binding protein-associated factor] and P-TEFb (positive transcription elongation factor b), to promote viral gene expression. Since Tat binds to a short leader RNA, known as the transactivation responsive element, its regulation is critical for HIV-1. Hdm2 (human homologue of Mdm2) is a RING-finger E3 that has been shown to interact with Tat, and this interaction leads to Tat ubiquitination. Interestingly, Tat ubiquitination by Hdm2 positively stimulates its transcriptional activation function [45–47]. Taken together, this suggests a fundamental link between activator potency and protein degradation .
Although this mechanism of TAD regulation is simple, there are more complex and intriguing aspects to this process, as observed in the case of the yeast methionine/cysteine biosynthesis gene activator Met4. SCF/Met30 is an E3 that specifically oligo-ubiquitinates Met4. This ubiquitination event results in dual regulation of Met4: one degradation-dependent and the other degradation-independent . In the degradation-dependent mechanism, Met4 ubiquitination by SCF/Met30 leads to its degradation via the 26 S proteasome only in minimal nutrient conditions. In the degradation-independent mechanism, however, regulation of Met4 occurs in nutrient-rich media, and Met4 is excluded from the MET gene promoters via a ubiquitin-dependent, but unknown, mechanism. A plausible explanation for this degradation-independent mechanism of Met4 regulation is that the SWI/SNF chromatin-remodelling machinery specifically depresses MET genes in rich, but not minimal, nutrient conditions and that this excludes oligo-ubiquitinated Met4 from the promoter region .
Ubiquitin promotes chromatin remodelling by modifying histones
Eukaryotic chromatin consists of individual nucleosomes, comprising DNA wound around histone proteins H2A, H2B, H3 and H4; histone H1 is the linker that sits between two adjacent nucleosomes . There are two classes of chomatin-modifying enzymes: the ATP-dependent nucleosome-remodelling enzymes, such as SW1/SNF, RCS, NURF etc. ; and histone-modifying enzymes, such as histone acetyletransferase, histone deacetylase and methylases. These chromatin modifications lead to the unwinding of the DNA, which aids the assembly of the general transcription machinery that is required for transcription .
Histone proteins undergo multiple post-translational changes (methylation, acetylation, phosphorylation and ubiquitination) that regulate their function by relaxing or condensing chromatin. The UPS has been linked to histone modifications (H2A was the first ubiquitinated protein to be described). Later publications have shown that H2A and H2B ubiquitination is closely linked to actively transcribed genes. Ubiquitination of H2B by Rad6 has been demonstrated to control H3 methylation at Lys4, which leads to gene silencing in Saccharomyces cerevisiae . Furthermore, Dot1-mediated methylation of H3 at Lys79 is also regulated by ubiquitination of H2B by Rad6 ; however, RAD6-deleted strains failed to methylate Lys4 and Lys 79 but could methylate Lys36 . Taken together, these studies show that ubiqutination of H2B acts as a master controller of gene silencing, because it regulates the methylation of Lys4 and Lys79 (which leads to gene silencing) but not Lys36, even though these lysine residues are methylated by different enzymes . Recently, histone H1 (linker histone) was shown to be monoubiquitinated by TAFII250; it was shown that H1s have ubiquitin-activating and -conjugating activities . One school of thought is that monoubiquitination of H1 may result in the destabilization of the higher-order folding of nucleosomes that is mediated by H1 . Considering all the observations described above, a clear relationship emerges between transcription activation and protein ubiquitination and degradation. Although the molecular role played by the ubiquitin modification is not yet clear, a model is definitely appearing. By the judicious use of ubiquitin, the UPS is able to regulate transcription activators at three levels: by regulating activator location within the cell, by activating the transcriptional activators, and, ultimately, by limiting the protein level and stability.
Ubiquitin, until recently, was thought to act solely as a tag for proteins destined to be degraded by the UPS. Research in the last two decades has led us to appreciate its profound influence, directly or indirectly, on one of the most critically regulated cellular processes, transcription. In this review, we have discussed the different modes by which ubiquitin and the UPS regulate transcription, both with and without their closely related signalling partner, the proteasome. Taken together, these observations seem to present the UPS as a complex, yet flexible, system that the cell utilizes to regulate transcription at different stages, such as: pre-initiation complex formation; activator availability; assembly and exchange of co-regulators; transcription elongation; and ultimately, degradation of the entire transcription machinery. Even with the vast amount of literature available about the regulation of transcription by the UPS, we have barely explored the complexity of gene regulation and the functions of the UPS in this regulation; compared with what we know so far, there is much more to learn.
Transcription is tightly regulated from initiation to termination. Ubiquitin and the UPS play critical roles in this process.
Ubiquitination and subsequent degradation is essential for transcription-factor function and activity, as well as the exchange of complexes during transcription that is essential for transcription efficiency.
Regulation of transcription-factor function and translocation are classic examples of ubiquitin-mediated control of transcription via the UPS.
Ubiquitin–proteasome components and enzymes have additional roles as co-activators, facilitating the assembly and turnover of the transcription machinery.
Ubiquitination of histone proteins leads to other modifications, such as methylation, which increases the heterochromatin-facilitating assembly of transcription complexes.
The UPS represents an ultimate tool for the regulation and control of eukaryotic transcription.
This work was supported by a grant from the National Institutes of Health (DK56833) to Z.N.
- © 2005 The Biochemical Society