Investigations on the roles of ubiquitin in the regulation of heat shock gene HSP70B 3

6 1.2 The Mediator Complex 1.2.1 Introduction to the Mediator Complex Along with RNA Polymerase II and the general transcription factors, another critical component of the Eukaryotic tr

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Chapter 1: Introduction

1.1 The Process and Mechanisms of Transcription

1.1.1 Introduction to Transcription and the Transcriptional Machinery

Transcription, the process by which RNA is synthesized from a Deoxyribonucleic Acid (DNA) template, is one of the most fundamental processes in a cell If follows several stages first of which is the assembly of a “preinitiation complex” This complex drives transcription from the initiation stage to elongation stage where most of the preinitiation complex is released from the active complex After elongation is complete, post-processing of the RNA product occurs and it

is exported from the organelle where it was synthesized (Hahn, 2004)

The proteins that comprise a cell’s core transcriptional machinery, the Ribonucleic Acid Polymerase (RNA Polymerase), are strongly conserved within Kingdoms Indeed, five subunits

of RNA polymerase are known to be conserved even between Superkingdoms Prokaryota, Archaea and Eukaryota while the latter two share even an even greater number of conserved subunits These subunits are classified in Eukaryotic RNA Polymerase II as Rpb1, Rpb2, Rpb3, Rpb6 and Rpb11 (Young, 1991)

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In Super Kingdoms Prokaryota and Archaea, there exists only one RNA Polymerase while in Super Kingdom Eukaryota, three RNA Polymerases have been identified RNA Polymerase I transcribes Ribosomal RNA (rRNA) save for 5S rRNA (Russell and Zomerdijk, 2006) which is the purview of RNA Polymerase III RNA Polymerase III also transcribes Transfer RNA (tRNA)

and other Short Nuclear RNAs (snRNA) (Dieci et al., 2007) RNA Polymerase II however is

perhaps the widest studied as it transcribes Messenger RNA (mRNA) in the nucleus (Woychik and Hampsey, 2002) Despite the fact that Eukaryotic RNA Polymerase is comprised of far more subunits than that of Prokaryota and Archaea, the majority of subunits share functional if not structural homology and are thought to share the same basic mechanisms of function and regulation (Ebright, 2000)

1.1.2 The Eukaryotic Transcriptional Machinery

RNA Polymerase II typically comprises of about a dozen subunits although the exact number of which varies from organism to organism Of these subunits, designated as “Rpb”s (Repressor of RNA Polymerase B) in modern parlance, Rpb1 and Rpb2 are usually the core catalytic components of RNA Polymerase II (Cramer, 2004) This catalytic core, while necessary for RNA synthesis, is in and of itself insufficient for transcription to proceed Rather RNA Polymerase II has no actual ability to recognize promoter DNA It relies on the assembly of various other factors to give it specificity and the ability to bind template DNA Specifically the Rpb4/7 complex and general transcription factors are also necessary for the initiation of

transcription from promoter DNA (Edwards et al., 1991)

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As DNA in Eukaryotes is found packed in chromatin and thus inaccessible to the transcriptional machinery, gene-specific transcription factors must bind proximally to the site of initiation and recruit the factors necessary to modify the chromatin structure before transcription can begin (Cosma, 2002) Assembly of this “preinitiation complex” marks the beginning of the transcription process and starts when the TBP (TATA-binding protein) component of TFIID binds with a promoter sequence, such as “TATA” in yeast This is followed by other general transcription factors like TFIIB, TFIIE, TFIIH and TFIIF which is the general transcription

factor directly bound to RNA Polymerase II (Reinberg et al., 1998)

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Despite the fact that the preinitiation complex is fully assembled after this stage, it remains in an inactive state until a conformational change occurs in the template strand to place the coding DNA in the “catalytic cleft” of the RNA polymerase II complex After the synthesis of the first

30 or so bases, RNA polymerase II is thought to release its contacts with the rest of the transcription machinery and leaves the core promoter region to enter the stage of RNA

elongation (Wang et al., 1992)

This “release” of the transcriptional machinery by RNA Polymerase II is mediated by the phosphorylation status of its carboxy-terminal domain (CTD) RNA Polymerase II has a

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repeating Tyr-Ser-Pro-Thr-Ser-Pro-Ser sequence which is unphosphorylated in the initiation stage of transcription Phosphorylation by a kinase of this CTD signals the disassembly of the preinitiation complex and the progress of transcription into the elongation stage Phosphatases recycle phosphorylated RNA Polymerase II after termination phase for use in further rounds of

transcription (Murray et al., 2001) The phosphorylation status of the RNA Polymerase II CTD

also has a role to play in the processing of mRNA It is implicated in several phenomena such as

5’ cap addition, 3’ poly-A tail synthesis and the splicing of introns (Proudfoot et al., 2002)

Many of the released general transcription factors, termed the Scaffold Complex, remain at the site of initiation; a phenomena which can be used to mark transcriptionally active genes This allows the cell to circumvent the arduous process of reassembling the initiation complex for subsequent rounds of transcription by using these general transcription factors to aid in the recruitment of the remaining factors necessary to begin the next round of transcription

(Yudovsky et al., 2000)

The Rpb4/Rpb7 complex is also necessary for proper transcriptional activity Rather than process RNA or binding to DNA, it is thought that the Rpb4/7 complex’s role in RNA Polymerase II is that of a “clamp” to bind RNA and to contribute to the stability of the RNA Polymerase II

complex as a whole (Cramer et al., 2000) However, not all RNA Polymerase II complexes

necessarily contain the Rbp4/7 complex, rather subunit composition of the polymerase is dependent on several factors (Kolodziej et al., 1990) These include growth conditions (Choder and Young, 1993) and the transcription factors associated with a particular promoter (Xue and Lehming, 2008)

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1.2 The Mediator Complex

1.2.1 Introduction to the Mediator Complex

Along with RNA Polymerase II and the general transcription factors, another critical component

of the Eukaryotic transcriptional machinery is the Mediator Complex The first inklings that such

a complex existed originated in biochemical experiments that attempted to reconstitute an active

transcriptional machinery in vitro While the general transcription factors and RNA Polymerase

II were sufficient to drive promoter-targeted gene expression, they could not replicate a cell’s ability to respond to activators or repressors (Hampsey and Reinberg, 1999) Further experiments

identified the Mediator complex in the yeast Saccharomyces cerevisiae (Thompson et al., 1993)

Many of these components had already been identified as transcription factors in various genetic screens and thus many of the designations of yeast mediator bear names that are a legacy of this era For example several yeast mediator components are designated as “Srb”s for “Suppressor of RNA Holoenzyme B”; others were likewise named for the identification methods used (Myers and Kornberg, 2000) or apparent role in cellular regulation such as Gal11 (Carlson, 1997)

The human Mediator Complex subcomponents were thus subsequently isolated in various

biochemical reactions in vitro by identifying the proteins necessary to restore activator driven

transcription of a fully reconstituted RNA Polymerase II and general transcription factors (Sato

et al., 2003) Due to this, Mediator is classified as a coactivator of transcription although recent

evidence is emerging that challenges this classical view and asserts that Mediator is as fully involved in transcription as a traditional general transcription factor (Taatjes, 2010) Unlike RNA

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Polymerase II, Mediator is absent from Prokaryotes and Archaea Furthermore, only eight

Mediator subunits are known to be conserved from Saccharomyces cerevisiae to Homo sapiens (Rachez et al., 2001) This is not too large a surprise as the general transcription factors

themselves are also absent in Prokaryotes and only a few analogues have been identified in Archaea (Taatjes, 2010)

1.2.2 Structure and Function of the Mediator Complex

The Mediator Complex originally isolated in S cerevisiae has 21 subcomponents while mammalian Mediator has a little over 30 (Tomomori-Sato et al., 2004) These subunits form

three distinct modules that undergo significant conformational changes upon binding to RNA Polymerase II or its CTD portion These have been termed the “head”, “middle” and “tail”

modules and each has their own role to play in the function of Mediator (Chadick et al., 2005)

The head module is the most evolutionarily conserved and is chiefly concerned with binding RNA Polymerase II The middle module has two submodules each with a contrary role The Med9 submodule is involved in repression while the Med10 submodule is involved in the activation of genes These allow the middle module to respond to signals even after binding to RNA Polymerase II The tail module mediates binding with DNA-bound transcription factors and thus is the module most associated with Mediator’s classical role in transcription (Woychik,

et al., 2002)

The Cdk8 submodule is known to transiently associate with the Mediator Complex It is comprised of Cdk8, Cdk11, Cyclin C, Med12, Med13 and splice variants of the latter two

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Mediator subunits (Sun et al., 1998) Similar to the middle Mediator module, the Cdk8

submodule can act to both repress and activate transcription Initial studies indicated that containing Mediator could not initiate transcription because Cdk8 can negatively affect Mediator’s ability to both recruit RNA Polymerase II to the promoter and activate RNA

Cdk8-synthesis (Knuesel et al., 2009)

Along with its global effect on transcription, Cdk8 is also implicated in gene-specific repression The Cdk8 submodule, particularly the Med12 subunit, activates the G9a histone methyltransferase This catalyses methylation of H3K9 which typically leads to restricted access

to chromatin However this only represses a particular subset of neuronal genes rather than a

general decrease in transcription (Ding et al., 2008)

The Cdk8 submodule’s positive role in transcription follows a similar pattern Cdk8 can function

as an H3S10 kinase to create a permissive chromatin environment for the assembly of

transcriptional activity (Cheung et al., 2000) A link to cancer has recently been established as Cdk8 is a positive factor for the activation of certain p53 regulated genes such as p21 and HDM2 (Donner et al., 2007)

In contrast to the general transcription factors, Mediator lacks the ability to bind to template or promoter DNA As was alluded to in how it was originally characterized, Mediator has a high affinity for DNA-binding transcription factors and is thus recruited to various regulatory sites in the genome during transcription Indeed, there is evidence in yeast that Mediator subunits

localize to promoters on a genome-wide scale (Zhu et al., 2006) This is the origin of its name as

9 Mediator was proposed to be the bridge that mediated binding between promoter-specific

transcription factors and the general transcription factors (Ryu et al., 1999)

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1.3 The Ubiquitin Proteosome System

1.3.1 The Discovery of the Ubiquitin Proteosome System

With the discovery of the lysosome in the 1950s, contemporary scientists believed that proteolysis of cellular proteins were localized in this organelle (de Duve, 1953) It was logically consistent that proteases were not only sequestered from their substrates by a membrane but that they required a radically different environment from the cytoplasm in order to function Otherwise a protease would indiscriminately degrade its substrates In addition the ability of the

lysosome to perform autophagy (Ashford et al., 1962) on smaller vesicles further strengthened

the notion that while proteins were in a dynamic state of synthesis and degradation (Simpson, 1953), these activities were confined to very particular organelles specifically designed for such

purposes (Mortimore et al., 1987)

This view held for a time but with increasingly sophisticated tools for investigation into cellular phenomena, various observations could not be accounted for solely by the activity of the lysosome For one, cellular proteins have wildly different half-lives and the stability of a given

protein can be altered due to the status of the cell (Goldberg et al., 1976) This is patently

inconsistent with the notion that the lysosome was supposed to be the organelle that indiscriminately degraded proteins through autophagy, a process that did not allow for selective proteolysis Added to that was the observation that different drugs inhibiting protein degradation

did not have a uniform effect across all protein populations (Knowles et al., 1976) Yet another

piece of evidence that contradicted the notion that the lysosome was the sole effector of protein

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degradation was that proteolysis required energy in the form of Adenosine-Triphosphate (ATP) and could be all but completely inhibited by the lack thereof This was not consistent with passive degradation in the acidic confines of the cytoplasm which would not require ATP

expenditure (Segal et al., 1974) The effectiveness of ATP depletion on the reducing the

degradation of proteins also implied that the lysosome was not the chief organelle that degraded proteins as lysosome inhibitors had an inconsistent effect on protein stability To account for this property of lysosome inhibitors, it was theorized that the variation in protein stability in the cell was due to the different binding affinities of proteins for the lysosomal membrane However this

proved to be true only for a relatively small proportion of proteins (Müller et al., 1981)

A strong piece of evidence comes from an experiment that demonstrated a significant difference

in the rate at which intracellular and extracellular proteins were degraded Living macrophages with Tritium-labeled leucine were placed in the same media as dead macrophages labeled with Carbon-14 leucine By using radiation as a proxy measure it was possible to observe the rate of degradation of both populations of proteins Not only did the extracellular proteins localize to the lysosome for destruction, it was found that lysosomotrophic agents which interfered with the activity of the lysosome only impeded the degradation of extracellular proteins From this it can

be concluded that at least two separate forces are at work in the degradation of proteins rather

than the lysosome alone (Poole et al., 1977)

Another significant milestone was an experiment where mutated haemoglobins mimicking known diseased forms were rapidly degraded in rabbit reticulocytes which lacked lysosomes

(Rabinovitz et al., 1964) This led to later experiments where a crude extract of reticulocytes was

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demonstrated to selectively degrade abnormal haemoglobin with the addition of ATP and this degradation occurs at a neutral pH rather than the acidic pH of the lysosome (Etling and Goldberg, 1977)

Initial fractionation of this crude extract through anion-exchange chromatography yielded two fractions, the flow through and the elute It was found that neither fraction alone could catalyze the specific degradation of misfolded haemoglobins observed in the crude extract This suggested that whatever was in the extract, it was a complex of at least two proteins instead of a single protein with protease activity which was commonly thought of at the time as the typical

form of a protease (Ciechanover et al., 1978)

Further purification of the flow through revealed an 8.5kDa, protein that was necessary for the degradation reaction to occur Dubbed “ATP-dependant Proteolytic Factor 1” (Apf-1), chains of several units of this protein was found to be covalently conjugated to substrates of the degradation reaction and that this reaction required the expenditure of ATP The conjugation of

this protein was also shown to be reversible (Hershko et al., 1980) This protein was soon

identified as ubiquitin, a 76 amino acid protein that had already been discovered but heretofore

had no known physiological function (Wilkinson et al., 1980) It was thought to be well

conserved through all Kingdoms of life; ubiquitin is so named due to the fact that it is ubiquitous

to all living tissue and known life forms However later studies found that ubiquitin is present

only in Eukaryotes but is indeed ubiquitous to all eukaryotic cells (Goldstein, et al., 1975)

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With the characterization of ubiquitin, it became possible to use ubiquitin as an immobilized bait protein to fish out the components of the ubiquitin ligase Isolation of the E1 Ubiquitin Activating Enzyme, E2 Ubiquitin Conjugating Enzyme and the E3 Ubiquitin Ligase resulted in the Nobel Prize for Chemistry being awarded to Ciechanover and colleagues in 2004 (Hershko,

et al., 1983) The notion that it is the ubiquitin moiety that signals the destruction of proteins

answered the key criticisms leveled against the Lysosome hypothesis The next question that needed to be answered was what else was there is the elute fraction of the reticulocyte crude extract that was necessary for proteolysis

Various experiments found that ATP hydrolysis was also needed for proteolysis of ubiquinated proteins aside from the already characterized requirement in the process of ubiquitin ligation

(Hershko, et al., 1984) When the 26S proteosome was finally isolated from the rabbit

reticulocyte extract, it was unlike most other characterized proteases at the time It was massive

at around 1.5MDa (Hough et al., 1986)

1.3.2 The Structure and Function of the 26S Proteosome

The 26S proteosome is now recognized as one of the main complexes involved in intracellular degradation of ubiquitinated proteins Following its initial discovery, the full 26S complex was found to have 31 subunits and can be up to 2.5MDa in size depending on the association with its various subcomponents Originally named for the sedimentation coefficient at which it

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precipitates during density-gradient centrifugation, it is now known that this nomenclature is somewhat inaccurate for the 26S proteosome, as mentioned, can vary in size (Tanaka, 2009) It has two main sub-complexes; the 20S Core Particle and the 19S Regulatory Particle which can exist in association with each other as the 26S proteosome or independently in both the cytosol

and nucleus (Peters et al., 1994) The former is an ATP-independent enzyme complex which is

responsible for the catalytic destruction of proteins, able to cleave the carboxy-terminal side of

hydrophic, acidic and basic residues of its substrates (Eytan et al., 1989) The 20S Core Particle

has a structure not unlike a barrel with four heptameric rings with a pair of the structural α rings sandwiching a couple of the catalytically active β rings This arrangement allows the α rings to deny access to the proteases in the β rings, the regulation of which is handled by the 19S

Regulatory Particle (Smith et al., 2007)

The 19S Regulatory Particle has often been likened to a “lid” that keeps the 20S catalytic core safely sequestered from any stray proteins It has 19 subunits divided into two distinct sub-complexes; nine in a “lid” sub-complex and ten in a “base” sub-complex Of these only six subunits in the base have ATPase activity (Rpt1-6), the other four in the lid (Rpn1, Rpn2, Rpn10 and Rpn13) and the nine in the lid sub-complex Rpn3, Rpn 5-9, Rpn11, Rpn12 and Rpn15) do

not (Hoffman et al., 1992) As its name implies, the 19S Regulatory Particle is responsible for

the recognition of ubiquitinated substrates and for unfolding a substrate before opening a channel into the 20S Core Particle for its degradation It is the 19S that has ATP-dependant activity which found in “base” sub-component; presumably its main role is the unfolding of proteins

although there is evidence that this is not its only role (Smith et al., 2005)

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In its native form the N-terminus of the α2, α3 and α4 of the 20S Core particle form a “gate” that restricts access to the β catalytic rings When the 19S Regulatory particle binds with the 20S to form the 26S proteosome, a conformational change occurs in these three α subunits to permit

access to the proteolytic centre of the 20S (Jung et al., 2009) This is facilitated by a conserved

YRD-motif (Tyr8-Asp9-Arg10) in the α subunits that serves as the “hinge” that opens or closes this After recognition by the 19S, proteins tagged for destruction with a poly-ubiquitin chain are targeted by deubiquitinating enzymes (DUB) and then fed into the 20S core particle for proteolysis This has two functions; firstly it recycles ubiquitin for subsequent use by the cell Secondly, the proteolytic chamber is very small at roughly 13 Angstroms and while an unfolded protein chain can pass through this chamber, one modified with a polypeptide moiety like the ubiquitin chain might not This it prevents this chamber from “blockage” by the very signal used

to target its destructive capabilities After processing by the 20S catalytic core, the tagged protein

is degraded into peptides approximately 8-12 amino acids long gate (Hegde, 2004)

This is facilitated by the different subunits of the β-rings that catalyze the degradation of proteins

in the proteosome having different proteolytic capabilities, named after the analogous properties

of classical proteases The β1 subunit possesses “caspase-like” activity, the β2 subunit has

“trypsin-like” activity and the β5 subunit is characterized as having “chymotrypsin-like” activity

(Gaczynska et al., 1993) Under the right conditions, these three subunits can be replaced with

immunoforms to assemble the immunoproteosome This immunoproteosome’s activity favors cleavage that creates peptides not unlike the MHC class I antigens through additional proteolytic

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activity such as BrAAP (cleavage after branched amino acids) and SNAAP (cleavage after small

neutral amino acids) (Orlowski et al., 2000) Aside from the classical 19S Regulatory particle,

the 20S Core particle can also be bound to the 11S activator ring comprised of three PA28α

subunits and either three or four PA28β subunits (Zhang et al., 1999) This configuration

increases the proteolytic capacity of the proteosome by granting greater access to the catalytic

core (Whitby et al., 2000) and is induced by interferon γ to promote antigen presentation (Kloetzel et al., 1999)

1.3.3 The Machinery and Process of Ubiquitination in Brief

The ubiquitination of a single protein requires the concerted action of three separate enzymes with the expenditure of ATP The process is started when the E1 ubiquitin-activating enzyme interacts with the C-terminal glycine residue of a free ubiquitin molecule to form an ubiquitin-adenylate intermediate This releases the pyrophosphate molecule PPi (P2O7 4−

) which can be a proxy measure of ubiquitin activation This intermediate is transferred to a cysteine residue in the E1 through the formation of a thiolester bond which releases Adenosine Monophosphate (AMP)

as a byproduct This activated ubiquitin is subsequently transferred to a cysteine residue on the corresponding E2 ubiquitin conjugating enzyme through another thiolester bond Subsequently the E3 ubiquitin ligase mediates the transfer of this activated ubiquitin from the E2 to the target substrate It is the E3 that confers specificity to the ubiquitination process and catalyses the formation of an isopeptide bond between the ε-amino group of one of the substrate’s internal lysines and the C-terminal glycine residue of ubiquitin (Hochstrasser, 1996)

a cell, even if each E2 can interact with multiple E3s to create various E2 and E3 combinations, several E3s are in and of themselves complexes rather than a single protein Some E3s are

“modular” like the SCF Complex (Skp1 - Cullin - F-box Protein) with one component actually responsible for the recognition of substrates This allows the cells to conserve resources rather than synthesize entire E3s from scratch (Ciechanover, 1994)

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The E3 ubiquitin ligases, with their immense diversity, elicit a certain amount of interest as a topic of scientific research due to their role in ubiquitin and ubiquitination’s various roles in the cell And like any diverse group there has been an effort to classify them into separate groups based on sequence homology and biochemical properties E3s can be loosely divided into two distinct groups The Homologous to E6-AP C-terminus (HECT) possess strong homology to its

eponymous protein E6-AP, the product of gene UBE3A; best known for catalyzing the

destruction of p53 by Human Papillomaviral E6 (HPV E6) In HECT class E3s, the ubiquitin ligase itself forms a direct bond to ubiquitin before it is ligated onto the substrate through a transient covalent bond with a conserved cysteine (Weissman 2001)

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In contrast, the Really Interesting New Gene (RING) type E3s only serve to mediate the transfer

of ubiquitin from the E2 to the substrate This family of E3s where the RING domain serves to recruit the E2 can be further sub-divided into single proteins that contain both the RING domain and the substrate adaptor domains or multi-unit complexes where these are found on different proteins The SCF complex is considered the textbook example of a multi-unit RING ubiquitin ligase Cullin (Cul1) is the central scaffold of the whole complex, binding RING-box protein (Rbx1) and S-phase Kinase Associated Protein 1 (Skp1) to interact with E2s and the substrate recognition subunits respectively These subunits are known as F-box proteins and many possess multiple substrate recognition capabilities Thus while the human genome only encodes for 69 F-box proteins, together with E2/E3 combinations, the SCF complex is capable of specifically targeting a wide range of proteins The human genome also encodes eight different cullin proteins (Cul 1, 2, 3, 4A, 4B, 5, 6, 7 and 9) that also form Cullin-RING ligase complexes much like SCF (Petroski and Deshaies, 2005)

1.3.4 E4 Ubiquitin Ligases in the Elongation of Polyubiquitin Chains

While the E3 ubiquitin ligases mediate the initial ubiquitination of the substrate, a new class of ubiquitin ligases has been proposed based on new evidence whose role it is to elongate existing ubiquitin chains These E4 ubiquitin ligases work in concert with the E1, E2 and E3s to ensure efficient polyubiquitination of substrates Rather than the HECT or RING domains of established

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E3 Ubiquitin ligases, E4s have different motifs and are also classified into different “families” in much the same way The first enzymes classified as E4s are the U-box Containing E4s based on the yeast Ufd2 These have a conserved C-terminal U-box (Ufd2-homology domain) which is about 70 amino acids long and has structural similarities to the RING-finger domains found in conventional E3s The eponymous Ufd2 was identified as an enzyme that conjugated an additional ubiquitin molecule to an existing chain of up to three ubiquitin moieties in the presence of E1, E2 and E3 enzymes This is considered now the textbook example of an E4 and

E4 activity that distinguishes the E4 ubiquitin ligases from E3 ubiquitin ligases (Koegl, et al.,

1999)

A physiological role for Ufd2 has also been found in the regulation of the transcription factor Spt23 This protein is typically anchored to the membranes in an inactive form until mono-ubiquitination and subsequent cleavage of the 26S proteosome results in the active p90 form of

Spt23 (Hoppe et al., 2000) Mono-ubiquitinated p90 Spt23 is a substrate of Ufd2 in the nucleus

and this might have multiubiquitination signaling or proteolytic consequences although the current evidence leans towards the latter Generally yeast Ufd2 is involved in cellular stress

responses and unsaturated fatty acid metabolism (Richly et al., 2005) Ufd2 homologues have been identified in various species including Ufd2a and Ufd2b in Homo sapiens and Mus musculus (Koegl et al., 1999) Ufd2a is known to be involved in the degradation of pathological

forms of ataxin-3 These forms of ataxin-3 are the cause of a neurodegenerative disorder

commonly known as Machado-Joseph disease or spinocellular ataxia type 3 (Matsumoto et al.,

2004)

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Experiments where Ufd2a and other U-box proteins were demonstrably shown to have E3

properties in vitro have cast the notion of a distinct class of E4 ubiquitin ligases into doubt However it is well known that enzymes often lose specificity under in vitro conditions Further work shows that all known E4s require E3s for their physiological functions in vivo thus

justifying a new class of ubiquitin ligases that specifically target other ubiquitin molecules to lengthen an existing chain Given that some E4s cannot interact with E2s and generally lack the ability to ubiquitinate a substrate without existing ubiquitin conjugates, E4s cannot simply be

described as specialist versions of E3 ubiquitin ligases containing a new motif (Matsumoto et al.,

2004) There is some dispute though if all U-box containing proteins are E4s or if they cannot also exhibit E3 activity For example C-terminus of Hsc70-interacting protein (CHIP) has been reported to possess E3 activity that targets proteins bound by chaperones like Hsc70 and Hsp90 CHIP is known to bind to misfolded proteins leading to their polyubiquitination and subsequent

degradation by the 26S proteosome much like a classical E3 ubiquitin ligase (Murata et al.,

2003) but requires the RING-finger E3 Parkin in order to polyubiquitinate the unfolded Pael receptor Accumulation of unfolded Pael receptor is a key cause of juvenile familial Parkinson’s disease When Parkin is inactive, sufficient amounts of unfolded Pael receptor builds up in the endoplasmic reticulum (ER) to cause a stress-induced degeneration of the dopaminergic neurons despite the presence of CHIP While Parkin alone can indeed polyubiquitinate Pael receptor in

biochemical trials, in vivo only the combination of Parkin and CHIP can polyubiquitinate Pael

receptor in coordination with E1 and E2s Thus CHIP can be said to be a U-box protein that

possesses both E3 and E4 activity based on the substrate (Imai et al., 2002) Whether it is the

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exception or the rule is yet to be determined but it does blur the line somewhat between E3 and E4 proteins

An example of a pair of U-box proteins functioning in concert as an E3 and an E4 can be found

in C elegans C-terminus of Hsp70-interacting protein (Chn1), the ortholog of CHIP, and Ufd2

can both function as E3s to polyubiquitinate Unc-45, a myosin chaperone However they are far more efficient when working as an E3 and E4 pair While this does challenge the notion that E4s cannot interact with E2s, it does raise the concept that E4s are a new layer of regulation over the traditional ubiquitin ligases and that the combination of E3 and E4 ubiquitin ligases can determine the stability or rate of ubiquitination of a given protein CHIP can form a homodimer,

it is possible that this allows for a similar mechanism where one molecule each take on the role

of an E3 and an E4 respectively (Hoppe et al., 2004) Outside of the U-box family, Bul1 and

Bul2 served as the E4 for the yeast protein Gap1 with the HECT-domain Rsp5 as the E3 Bul1 and Bul2 are notable as the first E4s observed that must be in a complex with each other in order

to exhibit E4 activity analogous to other ubiquitin ligase complexes such as SCF or APC

(Helliwell et al., 2001) p300 is yet another example of a non-U-box E4 where it polyubiquitinates p53 after mono-ubiquitination by Mdm2 (Grossman et al., 2003) The mono- ubiquitinated species is thought to be a signal for nuclear export of p53 (Brooks et al., 2004)

while the polyubiquitinated species is naturally tagged for destruction by the proteosome

(Kubbutat et al., 1997) It is interesting that p300 is also described as a histone acetyltransferase and transcriptional cofactor (Iyer et al., 2004) While there is very little homology between p300,

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Bul1/Bul2 and U-box proteins it does imply that the process of ubiquitination and the regulation

of transcription may be somewhat more complex than currently understood

1.3.5 Ubiquitin-Proteosome Adaptors

Evidence that ubiquitin can benefit from other proteins during proteosomal degradation comes from several sources The yeast proteins Rad23 and Dsk2 have ubiquitin-like domains and possess ubiquitin binding domains that allow them to act as a connector between the proteosome

and polyubiquitinated cargo tagged for degradation (Elsasser et al., 2005) Strengthening the

notion that such adapters are crucial for proper degradation of at least some proteins is the discovery of ZNF216 Possessing the ability to bind polyubiquitin chains through an N-terminal ubiquitin binding domain, ZNF216 is known to aid in the efficiency of proteosomal degradation The deletion of this gene in mice results in a substantial increase in polyubiquitinated proteins in muscle tissue which also served to protect this tissue from artificially induced muscular atrophy

It is possible that ZNF216 not only has a role as a passive ubiquitin-proteosome adaptor but also

as a regulator of protein degradation rates (Hishiya et al., 2006)

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1.4 The Non-proteolytic Roles of Ubiquitin

1.4.1 An Introduction to the Role of Ubiquitin as a Signaling Molecule

That ubiquitin is a signaling molecule surprises no one, ubiquitin was originally characterized as

a signal for protein degradation by the 26S proteosome after all The fact that ubiquitin can serve

as a signaling molecule outside of its traditional role in the ubiquitin proteosome system (UPS) however is a concept that has also gained sufficient evidence in recent years that it is not in great dispute Even the kind of polyubiquitin chain formed by the ubiquitin ligases can have an effect

on cellular regulation Ubiquitin has seven lysine residues (K6, K11, K27, K29, K33, K48 and K63) that all could theoretically be the link for a polyubiquitin chain A polyubiquitin chain propagated at lysine 48 (K48) on ubiquitin is the classical degradation signal but a similar chain

propagated at lysine 63 (K63) on ubiquitin (Hicke et al., 2005) instead has other physiological

roles; one such role is the regulation of NF-κB

1.4.2 Ubiquitin as a Signaling Molecule in the Regulation of Transcription Factors

NF-κB is among a well characterized transcription factor family that controls a wide variety of cellular processes including proliferation and apoptosis It is under the control of various factors but most famously under the proinflammatory cytokines interleukine 1β (IL1β) and tumor necrosis factor α (TNF-α) (Silverman and Maniatis, 2001) In its native state an inhibitor, IκBα, sequesters NFκB in the cytoplasm denying it access to its target promoters Activation of NF-κB requires the phosphorylation of IκBα by IκB kinase (IKK) This leads to the polyubiquitination

of IKK not only requires the mono-ubiquitination of IKKβ following the phosphorylation of one

of its serine residues (Carter et al., 2004) but also the ligation of a K63 polyubiquitin chain to NEMO (Zhou et al., 2004) TAK1 is likewise found as a complex with three TAK1 binding

proteins, TAB1, TAB2 and TAB3 These proteins, thought to enhance or facilitate TAK1’s role

as a kinase, possess a zinc-finger domain that preferentially binds to K-63 ubiquitin chains and

these domains are necessary for TAK1 activation by TAB2 and TAB3 (Kanayama et al., 2004)

One possible explanation for this is that these domains bind to K-63 ubiquitin modified NEMO and bring the two complexes into proximity so that TAK1 can phosphorylate IKK to continue the signal cascade From this we have some evidence that K-63 ubiquitin chains can serve as a scaffold to bring various components of a signal cascade together

TNF-α is also regulated by Traf6 synthesized K-63 ubiquitin chains The receptor interacting protein (RIP) of the TNF receptor 1 signaling complex is a substrate both of Traf6 as well as a DUB, A20 A20 not only inhibits TNF-α activation by breaking the K-63 ubiquitin-mediated signal cascade but is also an E3 ubiquitin ligase in and of itself that can ligate a K-48 ubiquitin

chain to RIP to signal its destruction by the 26S proteosome (Wertz et al., 2004) Before A20 can

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conjugate a K-48 ubiquitin chain however, it must first remove all K-63 ubiquitin molecules from RIP Thus these K-63 polyubiquitin chains protect RIP from degradation in an ironic reversal of the role classically assigned to ubiquitin NEMO, Traf2 and Traf6 are also targets of another DUB, cylindromatosis (CYLD), a tumor suppressor CYLD removes K-63 polyubiquitin

chains from these proteins which results in interference with NF-κB signaling (Brummelkamp et al., 2003)

1.4.3 An Introduction to Mono-ubiquitination as a Signaling Molecule

Much like any protease, the ubiquitination machinery in the cell is under tight control through various means, including compartmentalization, degradation, oligomerization as well as several

post-translational modifications, and can be induced by signal cascades in the cell (Dikic et al.,

2003) This is one of the factors that gives weight to the concept of ubiquitin as a signaling molecule; ubiquitination can not only be induced but deubiquitinating enzymes (DUBs) can remove a conjugated ubiquitin moiety from proteins While an argument can be made for DUBs

as a means by which a cell can rescind or delay a proteolytic signal, the vast variety of DUBs would imply that they play other roles in the cell as well After all, if the sole function of DUBs

is to prevent recognition by the proteosome, the most efficient functional form would be that of

an enzyme that targets and cleaves only polyubiquitin chains longer than 3 residues, something patently untrue of most DUBs (Hershko and Ciechanover, 1998) Rather it is far more likely that DUBs serve to deactivate a mono-ubiquitination signal or to allow for a switch in the modification of that particular lysine residue

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One modification known to often lead to ubiquitination is phosphorylation Although this has been observed in both cytoplasmic and nuclear proteins; the link between ubiquitination and

phosphorylation is not universally true (Di Fiore et al., 2003) However phosphorylation of many

proteins such as the ubiquitin ligase Cbl or other regulatory proteins such as IκB, Hrs and Eps15

is a prelude to ubiquitination and this occurs often enough that drawing a link between the two is not facetious (Haglund and Dikic, 2005) Both phosphorylation and ubiquitination are recognized by particular domains in proteins and these can be an effective means to propagate a

phosphorylation or ubiquitination event to a downstream effector (Pawson et al., 2001) The key

difference between ubiquitination and phosphorylation is that the former is a far more complex molecule and can be conjugated to proteins as a single molecule or as a polypeptide chain

Mono-ubiquitin of a protein is also recognized as a post-translational modification with important regulatory effects Among the roles played by mono-ubiquitination are the regulation

of a target’s subcellular localization, changes in protein conformation, enzymatic activity and affinity for protein-protein interactions (Hicke and Dune, 2003) This also extends to multiubiquitination, the state where a protein is mono-ubiquitinated on several different residues

(Haglund et al., 2003a) This nomenclature is used to differentiate multiubiquitination from

polyubiquitination where a protein has a chain of two or more ubiquitin molecules ligated together and conjugated to the substrate protein at one residue much like the classical K-48 polyubiquitin chain that signals degradation by the 26S proteosome That is not to say that multi-polyubiquitination does not exist, in light of K-63 polyubiquitin chains such a phenomena is entirely possible

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1.4.4 Ubiquitin as a Mediator of Protein Interaction

One means by which ubiquitination is thought to regulate events in the cell is through altering the affinity of proteins for each other presumably through the nine known ubiquitin binding domains (CUE, GAT, GLUE NZF, PAZ, UBA, UEV, UIM and VHS) to enhance interactions These domains allow ubiquitinated proteins to interact with each other or with ubiquitin itself to regulate various processes in the cell Specifically a hydrophobic patch around isoleucine 44 in ubiquitin is the binding site of these ubiquitin binding domains (UBDs) despite the different structural folds in each UBD resulting in minor variations with their exact interaction surface As

a diverse group, the UBDs have a wide range of binding affinities and tend to have a significantly stronger interaction with polyubiquitin chains than a mono-ubiquitinated protein

(Hicke et al., 2005)

Thus the interaction brought about by mono-ubiquitinated proteins tends to be rather specific and exhibit low affinity This can be remedied by the formation of multimeric complexes of ubiquitin binding proteins In some cases the ubiquitin binding proteins can have additional interactions with the mono-ubiquitinated target through other domains to achieve a

non-similar result (Di Fiore et al., 2003) Another means of conferring specificity is through the use

of the structural differences inherent in the types of ubiquitin chains K-48 chains tend to be more “kinked” in structure while the K-63 chains have a more “extended” conformation It is the dynamic between these interactions of ubiquitinated and ubiquitin-binding proteins that gives

rise to an “ubiquitin network” in the cell (Kanayama et al., 2004) It has been posited that

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ubiquitinated cargo is sorted to the endosomes by certain ubiquitin-binding endocytotic regulators and adaptors

The ubiquitin-binding domains have been implicated in the ubiquitination of their parent proteins

as well Many proteins possessing UBDs are mono-ubiquitinated and their UBDs are required for

this mono-ubiquitination to occur (Polo et al., 2002) One hypothesis is that these UBDs are

necessary to recruit an E2 ubiquitin conjugating enzyme that has already bound a molecule of

ubiquitin in order to facilitate ubiquitination by an E3 ubiquitin ligase (Di Fiore et al., 2003) It is

thought that this mono-ubiquitination serves to regulate the activity or the binding affinity of proteins possessing UBDs to either free ubiquitin in the cell or to other ubiquitinated proteins

One such example where UBDs and the protein-protein interactions they foster are utilized in the regulation of cellular events is the case of Vps9 Functioning as a guanine exchange factor for Vps21, Vps9 is known to have a role in both the endocytotic and biosynthethic pathways of endosomal fusion during cargo transport such as the sorting of ubiquitinated Ste2 to the vacuole

(Shih et al., 2003) This sorting can be abrogated by replacing the ubiquitin in the cell with a

synthetic mutant with an isoleucine 44 mutation or the deletion of Vps9 itself As Vps9 possesses the ubiquitin-binding CUE domain, one possibility is that this UBD mediates intracellular interactions with a conjugated ubiquitin molecule on the same protein and gains sorting activity

for ubiquitinated cargo upon release of this interaction (Donaldson et al., 2003)

1.4.5 Ubiquitin in the Regulation of Endocytosis and Intercellular Trafficking

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There is also growing evidence that mono-ubiquitination and polyubiquitination are crucial signals for receptor endocytosis as well Much like Vps9 and Ste2, the mono-ubiquitination of cell surface receptors serves as an endosomal sorting signal resulting in lysosomal degradation (Hicke and Dune, 2003) This is in contrast with the loss of receptor ubiquitination which results

in increased levels of receptors recycling back to the cell surface (Levkowitz et al., 1998) There

is some dispute about how necessary ubiquitination is to the early stages of receptor endocytosis This stems from the observation that while ubiquitination is enough to trigger the removal of cell surface receptors from their functional location, it is not necessary for the internalization of

several transmembrane receptors in vivo (Hollerand Dikic, 2004) In addition

mono-ubiquitination is critical for the endocytoic sorting of receptors destined for lysosomal

degradation into the inner vesicles that ultimately form the multivescicular body (Katzmann et al., 2002)

For example the ubiquitin ligase Cbl is known to multiubiquitinate active forms of receptor tyrosine kinases which appear to ensure that these proteins are subsequently transported to and

destroyed in the lysosome (Haglund et al., 2003b) Likewise there are quite a few endocytotic

regulators and endocytotic adaptors that possess ubiquitin-binding domains that allow them to interact with mono-ubiquitinated proteins These domains have been proven necessary for the proper endosomal sorting of ubiquitinated cargo and are found in proteins such as epsins, HRS,

Eps15 and tumor susceptibility gene 101 (TSG101) (Railborg et al., 2002) All these proteins are

localized in particular endocytotic compartments where they serve to regulate the endosomal

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sorting of ubiquitinated cargo along with adaptor proteins such as GGA and TOM1 (Scott et al.,

2004)

The assumption that internalized receptors no longer influence cellular events is now considered

to be somewhat fallacious Not only can certain receptors transmit signals from endocytotic compartments, but the nature and effect of these signals are patently different from those

initiated by the same receptor on the plasma membrane (Miacynska et al., 2004) Some receptors

like the neurotrophin receptors only initiate signaling after their internalization while the receptor tyrosine kinase TrkA display this “dual mode” signaling As a cell surface receptor, it responds

to NGF (nuclear growth factor) as a cell survival signal while it promotes cell differentiation

when internalized (Zhang et al., 2000) In additional, the endocytotic routes taken by a receptor

may also alter its signaling properties One such case is that of the Notch receptors which are thought to regulate several different developmental decisions For these receptors, proper receptor activation and signaling are dependent on ubiquitination; the Notch receptor ligand is ubiquitinated as well which is necessary for ligand endocytosis within the signaling cell and

reception of the Notch signal in the receiving cell (Lai et al., 2005)

Further investigation into the mechanisms behind the recognition of ubiquitin by proteins functioning as endocytotic adaptors found that some of these proteins can interact with more than one ubiquitin molecule at a time either through possessing more than one ubiquitin-binding domain or by the auspices of ubiquitin interacting motifs (UIM) that can bind multiple ubiquitin molecules simultaneously The Hrs-UIM found in many receptor tyrosine kinases is one such UIM capable of binding two ubiquitin molecules at once and is thought to be critical for the

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lysosomal degradation of these receptors Other similar UIMs are found in Eps15 and in the ubiquitin-binding domain GAT (GGA and Tom1 domain) It is possible that such UIMs also possess different binding properties to K63 polyubiquitin chains and multiubiquitinated proteins

allowing for differentiation between the two modifications in the same protein (Hirano et al.,

2006)

One endocytotic adaptor that has two ubiquitin-binding domains is Rabex-5 Like its yeast homologue Vsp9, Rabex-5 is a GTPase-exchange factor and is crucial for the activation of the GTPase Rab5 which serves to regulate endosome fusion As with many proteins involved in endocytosis, Rabex-5 is recruited to mono-ubiquitinated cell surface receptors presumably

through one of its two UBDs (Penengo et al., 2006) The first, motif interacting with ubiquitin (MIU), is a conventional UBD that interacts with the Ile44 patch of ubiquitin (Lee et al., 2006),

whilst the more novel A20 zinc finger (ZnF_A20) interacts with a hydrophobic patch of ubiquitin situated around residue aspartic acid 58 (Asp58) In this case Rabex-5 is an example of

an ubiquitin-binding protein that can interact with the same ubiquitin molecule through two

different ubiquitin-binding domains simultaneously (Mattera et al., 2006)

It is also interesting to note that mono-ubiquitination of many endocytotic adaptor proteins can greatly hinder their ability to bind to ubiquitinated cargo This is mainly due to the ubiquitin molecule interacting with their ubiquitin binding domains and “occupying” these domains such that they lose the ability to bind other ubiquitinated targets in a manner analogous to steric

hindrance or in some cases of a competitive inhibitor (Hoeller et al., 2006) Certain endocytotic

proteins are also capable of self-ubiquitination which gives rise to a means of regulating the

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ubiquitin signal independent of the conventional ubiquitination pathway save for the ubiquitin ligase itself For example Eps15 can recruit the mono-ubiquitinated species of NEDD4 and the

Parkin E3 ligases to affect its own mono-ubiquitination (Fallon et al., 2006)

1.4.6 Ubiquitin’s Role in DNA Repair

One of the important roles that ubiquitination as a signal plays in the cell is in the repair of DNA Upon DNA damage, Fancomi anemia protein of subtype 2 (FANCD2) is phosphorylated which,

as is sometimes the case, is a prelude to its mono-ubiquitination This modification subsequently results in interaction between FANCDS2 and Breast Cancer 1 (BRCA1), a tumor suppressor

protein, within the chromatin-associated nuclear foci (Vandenberg et al., 2003) These foci not

only influence DNA repair but are known to be important in the activation of the S-phase checkpoint FANCD2 recycling is likewise triggered by deubiquitination and this signals the cell

to resume the cell cycle after DNA repair (Nijman et al., 2005)

Proliferating cell nuclear antigen (PCNA) is another key protein in the repair process This replicative processivity factor creates a “clamp” around the DNA to allow for the binding of

numerous factors during DNA replication (Haracska et al., 2004) The ubiquitination status of

PCNA determines exactly which DNA polymerases will bind to PCNA and thus what sort of DNA repair they would perpetuate A single ubiquitin moiety at lysine 164 of PCNA promotes error-prone repair while a lysine 63 polyubiquitin chain conjugated at the same residue triggers error-free DNA repair (Stelter and Ulrich, 2003) A model has been put forth where mono-ubiquitination is a repair signal that recruits low-fidelity lesion-bypass polymerases in contrast to

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K63 polyubiquitin chains which are a replicative signal recruiting high fidelity replicases

(Friedberg et al., 2005) Lysine 164 of PCNA can also have a SUMO (small ubiquitin-like

modifier) molecule conjugated at that residue which is a modification that is implicated in proper

DNA synthesis during S-phase (Hoege et al., 2002) These three mutually exclusive

modifications at the same lysine residue would seem to indicate that there can be significant interplay between mono-ubiquitination, polyubiquitination and ubiquitin-like molecules in cellular regulation

A similar mechanism where SUMO and ubiquitin work in concert as regulatory modifications is the activation of NF-κB by DNA damage Rather than activation by cytokines such as interlukine-1β or TNF-α, the NF-κB essential modulator (NEMO) is the key activator of NF-κB

in this situation Sumoylation signals for accumulation of NEMO in the nucleus and where NEMO is desumoylated and subsequently phosphorylated by the DNA damage response kinase ATM This leads to ubiquitination of NEMO at the two residues that were previously modified with SUMO, resulting in the translocation of NEMO out of the nucleus In the cytoplasm NEMO

activates IKK which initiates an NF-κB signal cascade (Huang et al., 2005)

1.4.7 Signaling by Ubiquitin-like Molecules

As one of the first ubiquitin-like molecules to be described, SUMO is unsurprisingly one of the best characterized Much like ubiquitin itself, SUMO is involved in various cellular processes including transcriptional regulation, DNA repair, nuclear transport and cell cycle regulation

(Seeler et al., 2003) In keeping with the motif that SUMO has analogous properties to ubiquitin,

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several SUMO-binding domains (SBDs) have been characterized that allow SUMO to influence

protein-protein interactions as ubiquitin does (Song et al., 2004) One of the key differences is

the binding surface; rather than the isoleucine 44 patch that ubiquitin has, SUMO has several

distinct binding patches that differ from ubiquitin’s (Hecker et al., 2006)

One of SUMO’s best known properties is as an inhibitor of transcription and to date most of the proteins that possess SBDs are found in the nucleus In a similar vein, the conjugation of SUMO

to transcription factors often leads to the recruitment of their co-repressors (Gill et al., 2005)

One line of evidence that points towards the role of SUMO in transcriptional regulation is the sumoylation of MBD1 A methyl-CpG-binding protein, sumoylated MBD1 is thought to have a role in the recruitment of MBD1-containing chromatin-associated factor 1 (MCAF1) to the chromatin through its SBD MCAF1 is part of a chromatin-remodeling complex that also includes the histone methyltransferase SETDB1 linking sumoylation to methylation and

subsequent heterochromatin formation (Uchimura et al., 2006) At this moment this conclusion

is educated conjecture as direct interaction between SETDB1 and sumoylated MBD1 mediated

by MCAF1 cannot be demonstrated This is compounded by the observation that sumoylated MBD1 not only fails to interact with SETDB1 when bound to methylated DNA but in this state also actively inhibits MBD1-SETDB1 complex formation Rather MBD1 binds to SETDB1

under other circumstances regardless of its sumoylation status (Lyst et al., 2006)

As mentioned earlier, sumoylation is thought to have an antagonistic relationship with ubiquitination in the regulation of PCNA, competing for the same lysine residue This is not simple competition however; this sumoylation recruits the Srs2 helicase to replication forks

translesion DNA synthesis polymerases (Ulrich et al., 2005)

Aside from SUMO, various other ubiquitin-like molecules have been described such as SUMO2, SUMO3, NEDD8, FAT10, ISG15, Urm1, Ufm1, FUB1, Hub1, Atg8 and Atg12 which contain the “ubiquitin fold”, a β-grasp fold that allows them to be conjugated to other proteins via their c-

terminal domains (Welchman et al., 2005) Many of these ubiquitin-like molecules have

analogous function to ubiquitin itself and have significant roles as signaling molecules in various

cellular processes (Kerscher et al., 2006) Ubiquitin was first characterized as a proteosomal

degradation signal and it is becoming increasingly clear that some ubiquitin-like molecules have similar roles

FAT10 is an ubiquitin-like molecule characterized as a regulator of apoptosis and the cell cycle Possessing two ubiquitin-like moieties, FAT10 is induced in these pathways by IFN-γ

(Welchman et al., 2005) Its link to the ubiquitin proteosome system comes from NEDD8

ultimate buster-1L (NUB1L) A non-covalently interacting partner of FAT10, NUB1L is known

to interact with the proteosome through its ubiquitin-like domain (Hipp et al., 2004) and can be

induced by interferons much like FAT10 itself As FAT10 can also interact with the proteosome

(Schmidtke et al., 2006) and NUB1L and some of its splice variants like NUB can target NEDD8

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for degradation (Tanaka et al., 2003), it has been proposed that these two proteins function much

like an immune response-triggered version of the conventional ubiquitin proteosome system

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1.5 The Role of Mediator, Ubiquitin and the Proteosome in Transcriptional Regulation

1.5.1 Introduction to Transcriptional Regulation in Eukaryotes

A nucleated cell contains within its genome all of the genes necessary to adapt to a changing environment and to utilize various metabolites In multicellular organisms, each such cell also has all of the information needed to differentiate into specialized tissues (Levine and Tijian, 2003) And yet cells have the ability to regulate not only which genes are expressed but also at what level This regulation of transcription is a complex and critical process in all cells which functions on various levels Aside from regulation stemming from Mediator which has already been mentioned earlier in the text, Eukaryotes have various mechanisms for transcriptional regulation

In Eukaryotes, transcriptional regulation involves chromatin remodeling which includes

complexes such as Swi/Snf which directly affect the nucleosomes (Wang et al., 1996) Another

method the cell uses to regulate transcription is to modify the histones themselves with enzymes like Histone Acetyltransferases (HATs) and Histone Deacetylases (HDACs) to alter the accessibility of DNA to the transcriptional machinery Such modifications can be both positive and repressive for transcription depending on the covalent modification in question For example histone deacetylation and histone methylation are well characterized as repressing markers of

transcription (Margueron et al., 2006) These modifications of histones and the histone tails are

extensive and significant enough to the regulation of transcription that a “histone code” has been

a moiety have all been reported (Cosma, 2002) Of greatest interest to this project is the role of ubiquitin and the proteosome system in the regulation of transcription factors and, subsequently, transcription

1.5.2 Mediator Complex in Transcriptional Regulation

Lately there is a growing body of evidence that Mediator has other roles besides its traditional activator-binding activity In this instance it exerts its influence chiefly by its aforementioned

binding to the unphosphorylated CTD of RNA Polymerase II to form a holoenzyme (Naar et al.,

2002) As a complex itself this has various effects For one it is known that Mediator serves to not only increase the rate of recruitment of RNA Polymerase II to the promoter but it also serves

to stabilize the transcriptional machinery complexes at the promoter (Cantin et al., 2003)

Evidence for this can be drawn from the fact that Mediator can increase basal levels of

transcription even without stimulation from activators (Baek et al., 2006)

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Beyond this, very little else is concretely known about the mechanisms by which Mediator regulates transcription Various pieces of data have suggested different roles of Mediator and its subunits in the regulation of transcription but at this stage, they are more hints of a larger role than substantial evidence That said; enough evidence has accrued that it cannot be dismissed out

of hand that Mediator’s classical role in facilitating transcription is its only role This is borne out

in the relationship between Elk1 and Med23, its specific binding target in mammalian Mediator

Activation of EGR1 is regulated by Elk1 and its recruitment of Mediator components can

disproportionately affect transcription In that study, the authors report a 13-fold increase in transcription following a three-fold increase in promoter occupancy by Mediator components

(Wang et al., 2005) Doubt can be cast on such a conclusion however since promoter occupancy

alone is often not a good proxy measure of transcriptional activity and more evidence is needed before Mediator’s regulatory role in transcription can be fully understood

1.5.3 The Role of Ubiquitin in Transcriptional Regulation

Mono-ubiquitination as a signaling molecule is known to play an important part in the regulation

of transcription The first pieces of evidence to support this notion come from experiments originally performed in yeast The deletion of Met-30, an ubiquitin ligase, could largely abolish

the transactivity of the viral protein 16 (VP-16) transactivational domain (Salghetti, et al., 2001)

The fusion of the DNA binding domain LexA and VP-16 can recruit positive transcription elongation factor to target promoters and this activity is enhanced upon mono-ubiquitination of