Ubiquitin and proteasomes in transcription

Abstract

Regulation of gene transcription is vitally important for the maintenance of normal cellular homeostasis. Failure to correctly regulate gene expression, or to deal with problems that arise during the transcription process, can lead to cellular catastrophe and disease. One of the ways cells cope with the challenges of transcription is by making extensive use of the proteolytic and nonproteolytic activities of the ubiquitin-proteasome system (UPS). Here, we review recent evidence showing deep mechanistic connections between the transcription and ubiquitin-proteasome systems. Our goal is to leave the reader with a sense that just about every step in transcription-from transcription initiation through to export of mRNA from the nucleus-is influenced by the UPS and that all major arms of the system--from the first step in ubiquitin (Ub) conjugation through to the proteasome-are recruited into transcriptional processes to provide regulation, directionality, and deconstructive power.

Figure 1

Transcriptional regulation. (a) Activation of transcription. Within a permissive chromatin environment, a transcriptional activator (TA) binds to promoter sequences, often in response to environmental signals. The activator typically requires a coactivator (CA), and together they recruit general transcription factors (GTFs) and RNA polymerase II (Pol II), leading to the initiation of transcription. (b) As Pol II enters the elongation phase, histones are evicted ahead of the transcribing Pol II and redeposited in its wake. At this stage, components of the RNA processing (RnP) machinery join Pol II, carrying out premessenger RNA maturation. This is also a stage when transcription can reinitiate, or the signal to initiate is terminated. (c) Transcription-coupled chromatin marks persist on the recently transcribed gene, and transcription terminates by association of Pol II with the termination machinery (Tm). mRNA is then exported from the nucleus. Blue lines represent the template DNA duplex.

Figure 2

Ubiquitylation. A destruction element in the substrate (a degron) is activated, often by phosphorylation in response to a specific signal. The degron is then bound by a ubiquitin (Ub)-protein ligase (E3), which acts in conjunction with a Ub-activating enzyme (E1) and Ub-conjugating enzyme (E2) to transfer Ub (green circle) to the substrate (SUBS), typically at a lysine residue. Repeated rounds of this process, perhaps catalyzed by a Ub chain elongation factor (E4), give rise to a polyubiquitylated substrate. Ubiquitylation can be opposed by the action of deubiquitylating enzymes (DUbs), which can remove all Ub or trim the length of the Ub chain.

Figure 3

Architecture of the 26S proteasome. The proteasome is a self-compartmentalized protease that consists of three main subcomplexes: a 20S core particle (CP) that houses three distinct proteolytic activities in its inner core, a 19S lid that recognizes and deubiquitylates substrates, and a 19S base structure that uses the energy of ATP hydrolysis to unfold proteins and pass them into the 20S CP for destruction. 26S proteasomes can be capped at one or both ends by a 19S complex.

Figure 4

Roles for ubiquitylation in controlling transcriptional regulators on chromatin. (a) Activation by monoubiquitylation. In this view, monoubiquitylation of DNA-bound activators stimulates their inherent activation properties, a process that is promoted by Ub ligases (E3s) and antagonized by deubiquitylating enzymes (DUbs). (b) The Ub clock model. In this view, the period of activity of a transcriptional regulator is governed by the length of time it takes to transition from the monoubiquitylated and active form to a polyubiquitylated form that is rapidly destroyed by the proteasome. (c) Control of promoter stripping by activator ubiquitylation. In this model, activators are aggressively removed from chromatin by resident ATPases in the 19S base complex (blue ring). The presence of Ub on the activator blocks this activity, allowing the TA to stably associate with its cognate DNA element. (d) Extraction of ubiquitylated transcription factors from chromatin by Ub-selective chaperones. Here, polyubiquitylation of the α2 repressor causes it to be extracted from promoter DNA by the ATPase activities of Cdc48 (p97), a process that allows immediate cessation of function before α2 is subsequently destroyed by the proteasome. Note that these models are not mutually exclusive, and it is possible that multiple mechanisms contribute to transcription factor control at a specific promoter. Abbreviations: GTFs, general transcription factors; Pol II, RNA polymerase II; TA, transcriptional activator. Blue lines represent the template DNA duplex.

Figure 5

Transcriptional activation domains (TADs) often overlap with degrons. The domain structure of 28 transcription factors is shown. Although the overlap of TADs and degrons is not always perfect, it is worth remembering that different studies have typically defined each type of element and that different sets of mutations (which define the boundaries) have been used in these studies.

Figure 6

Two modes of activator regulation by ubiquitin-mediated proteolysis. The figure shows how a transcription factor can be regulated in disparate ways by proteolysis and the consequences of disrupting the ubiquitin-proteasome system (UPS) on those modes of regulation. Mode 1 occurs off chromatin, and the UPS is used to limit the concentration of available activators. When the UPS is disrupted in this case, the transcription activator (TA) accumulates, and transcription is induced. Mode 2 occurs on promoter DNA and during the course of transcriptional activation. We posit that kinases (K) associated with the general transcriptional machinery phosphorylate activators at some point during transcriptional activation. This phosphorylation (P, red circles) has two functions. Its marks the activator as spent and incapable of stimulating further rounds of transcription. Concurrently, it also recruits a Ub ligase that ubiquitylates the transcription activator (TA), allowing it to be destroyed and a fresh activator to reach the promoter. In this mode, if the UPS is perturbed, the inactive TA remains on chromatin, and subsequent rounds of transcription are blocked. Abbreviations: CA, coactivator; E2, ubiquitin-conjugating enzyme; E3, ubiquitin-protein ligase. Blue lines represent the template DNA duplex.

Figure 7

Functions of H2B ubiquitylation. (a) During transcriptional elongation, the ubiquitylation machinery for H2B (Rad6/Bre1) is recruited to transcriptional complexes, where it ubiquitylates H2B, most prominently on lysine 120. This modification does not impact nucleosome structure, but it does relax higher-order chromatin configurations. (b) As transcription proceeds, ubiquitylation of H2B selectively recruits the FACT elongation complex (F), which not only displaces H2A/H2B dimers ahead of RNA polymerase II (Pol II), but also insures that histones are redeposited afterward. (c) H2B ubiquitylation recruits and activates Dot1- and Set1-containing histone methyltransferase complexes (MeT), which promote the di- and trimethylation of histone H3 at lysine residues 4 and 79. These methylation events, in turn, recruit other factors to chromatin and repel binding of transcriptional silencing complexes (not shown). If levels of ubiquitylated H2B accumulate, elongation factors such as TFIIS are excluded from chromatin, preventing the rescue of stalled polymerase molecules. Abbreviations: E2, ubiquitin-conjugating enzyme; E3, ubiquitin-protein ligase. Blue lines represent the DNA duplex. Brown circles represent nucleosomes. Green circles represent ubiquitylated histone H2B.

Figure 8

Ways in which the ubiquitin-proteasome system (UPS) can sense the activity of the transcriptional machinery. (a) By recognizing the specific context of the substrate. This example shows how the ubiquitylation machinery detects RNA polymerase II (Pol II) only when it is engaged in transcription, via a combination of phosphorylation events and structural changes in polymerase that are induced by transcription. (b) By context-specific activation of the ubiquitylation machinery. In this example, the ubiquitin-protein ligase, E3, is not active off chromatin, but it is activated by histone tails, insuring that it ubiquitylates its target only within a transcription setting. (c) By reprogramming Ub ligase selectivity. Here, a transcription factor (TF) serves as a substrate adapter for the ubiquitylation machinery, reprogramming E3 specificity from substrate 1 to substrate 2 and allowing selective modification of the second substrate in a transcriptional context. Abbreviation: E2, ubiquitin-conjugating enzyme. Blue lines represent the DNA duplex. Green circles represent ubiquitin. Brown circles represent nucleosomes. Red circles represent covalent histone modifications.

Figure 9

The Swiss army knife model of proteasome function in transcription. This model posits that the entire 26S proteasome is recruited into transcriptional processes, and its various activities are used depending on the molecular requirements. (a) The proteasome uses a combination of proteolytic and nonproteolytic functions to control activator binding, residency time, and coactivator (CA) exchange on promoter DNAs. (b) We posit that proteasomes enter the open reading frame (ORF) of transcribed genes, where their ATPase activities stimulate elongation, while their proteolytic functions serve to remove terminally stalled RNA polymerase II (Pol II) complexes that may arise. (c) Proteolytic activity of the proteasome is also important for attenuating cryptic, nonauthentic, and transcription complexes as well as for processes required for appropriate termination of the transcription event. Abbreviation: Tm, termination machinery. Blue lines represent the DNA duplex. Brown circles represent nucleosomes. Red circles represent covalent histone modifications present before transcription. Cyan circles represent covalent histone modifications present after transcription. The spool is the 26S proteasome (as in Figure 3).