How ubiquitin and
SUMOs control trx
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Why this?
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Ubiquitylation of proteins not only
targets them for destruction, but is
also a regulatory event in the
nucleus
In recent years, important
connections between
ubiquitylation, chromatin structure,
signaling pathways and
transcriptional control have
emerged.
The Ub-proteasome system is
ideally suited to controlling the
distribution, abundance and
activity of components of the
transcriptional machinery.
The life of a protein
Ubiquitin and the
protesome
.. A reminder
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Ubiquitin-family proteins
and the proteasome
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Ub covalently linked to targets

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Ubiquitin is the defining member
of this class,
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The ubiquitin (Ub) system defines a family
of related modifier proteins that are linked
covalently to target proteins.
butat least nine other related proteins with
this function have been described (see
figure).
Degron recognition
Ubiquitylation is a specific process that is
signalled by an element — a degradation
signal (degron) — in the substrate protein.
 The degron is recognized by a Ub-ligase
(Ubl), E3,which in turn recruits a Ubconjugating (Ubc) enzyme, E2, to the
substrate. The E3 then catalyses the transfer
of Ub groups to a lysine (K) residue that is
somewhere in the target protein.

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Ubiquitin-family proteins
and the proteasome
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Multi-Ub = Targeted for proteosomal
destruction
The exact nature of ubiquitylation determines the
fate of the substrate protein. If a multi-Ub chain
— linked by lysine 48 (K48) in Ub itself —
forms, the substrate is targeted for destruction
by a large, self-compartmentalized, protease
known as the 26S proteasome.
 The 19S subcomplex of the proteasome
recognizes the multi-ubiquitylated substrate,
removes the Ub groups, unfolds the substrate
and feeds it into the core of the 20S subcomplex
where it is destroyed.
 If, however, the multi-Ub chain is linked by
lysine 63 (K63), or if it has less than four Ub
chains, proteolysis does not occur.

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A family of Ubi-related small
proteins can also be conjugated
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Ubiquitin conjugation to substrates
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Nobel Prize in Chemistry for 2004
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"for the discovery of ubiquitin-mediated
protein degradation”

Aaron Ciechanover
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Avram Hershko
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Technion – Israel Institute of Technology, Haifa, Israel,
Technion – Israel Institute of Technology, Haifa, Israel and
Irwin Rose

University of California, Irvine, USA
Oslo
Jan 06
Ubiquitin and
chromatin
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Ubiquitylation of histones
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Histone H2A and H2B Ubiquitylation - one of
the first recognized markers of trx active
chromatin
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The first ubiquitylated protein to be described was histone H2A
ubiquitylated forms of histones H2A and H2B were associated
specifically with actively transcribed genes
Later also H1 and H3 reported to be ubiquitylated
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Ubiquitylation and the histone code
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Ubiquitylation of chromatin
The ubiquitin (Ub)-conjugating enzyme
Rad6 ubiquitylates K123 in the core of
histone H2B. This modification
promotes the methylation of another
histone, H3, at two positions, K4 and
K79. These modifications, in turn, are
required for telomeric-gene silencing.
 TAFII250 (TFIID component) can
ubiquitylate the linker histone H1; might
relate to the role of this TAF in
transcriptional activation.

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Ubiquitylation = an integral
part of the histone code
SEMINAR Inger Louise Bones: Wang et al. (2004) Role of histone
H2A ubiquitination in Polycomb silencing. Nature, 431, 873-878.
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Mechanism?
Direct structural role by
loosening chromatin structure
 Or as ”tag” recognized by
proteins such as the proteasome
or HDAC6

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A coming role for De-ubiquitylation?
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Ubps - possible novel regulators?

studies have identified Ub-specific proteases (Ubps) associated with
components of both the SIR4 silencing and the SAGA chromatin
remodelling complexes.
Regulating RNAPII by
ubiquitylation
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DNA damage - use of RNAPII to
direct repair to active genes
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Regulation of trx-coupled repair
(TCR) by ubiquitylation of RNA
polymerase II.
Transcription-coupled repair (TCR) is the
mechanism through which mutations in
actively transcribed genes are preferentially
repaired.
 Elongating RNAPII, with a unique pattern of
CTD phosphorylation, encounters a damaged
DNA segment. Here the stalled polymerase
recruits the Ub-ligase Rsp5, which in turn
ubiquitylates the largest subunit of pol II.
 Ubiquitylation is followed by the proteasomal
destruction of at least one subunit of
polymerase, recruitment of the repair
machinery and restoration of DNA integrity.

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Rsp5 is also a co-activator for the
steroid hormone receptors
Regulating TFs by
ubiquitylation
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Three strategies
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Controlling the localization of the TF
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Controlling the activity of the TF
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Controlling the abundance of the TF
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Regulating TFs by the ubiquitylation
- 3 strategies and 4 models
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Regulating location.
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As with NFkB, the TF can be maintained outside the nucleus by interactions
with an inhibitor (IkB) that is destroyed by the Ub–proteasome system.
Another Ub-family member SUMO (S) can directly conjugate to activators and
sequester them into nuclear bodies.
Regulating activity.

Ubiquitylation can regulate the association of activators with co-activator
proteins either directly, by blocking the association of an activator with its
essential cofactor, or indirectly, by facilitating the exchange of cofactors with
an activator.
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Regulating TF abundance - 1. model
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Regulating abundance I - constitutive turnover.
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By maintaining an activator in a constitutively unstable form, cells are primed
for a transcriptional response when appropriate. In this model, a signal from
outside the nucleus leads to a transient stabilization of the activator, which
elicits a rapid induction of target genes.
Examples
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p53 - later lecture
Wnt-signalling
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Destroying TFs when not needed
- shutting off proteolysis then gives a rapid response
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Beta-catenin and wntsignalling
Signalling
Inactivation of GSKb
Stabilization of
b-catenin
Phosphorylation
ubiquitylation
Degradation
Rapid
Accumulation
To the nucleus
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Regulating TF abundance - 2. model
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Regulating abundance II - trx-coupled
destruction
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In this model, activators are destroyed during the act of transcriptional
activation as a way of limiting uncontrolled activation by any one DNAbound transcription factor.
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Link: trx activation  degradation
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TFs often unstable
TAD overlaps closely with degrons
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Strong activators = rapidly degraded
Weak activators = more stable

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Degron = domain that signals ubiquitination
Myc and many others
Q-rich, N-rich
Mutant TADs with activation lost = stabilized
Link:
activation - degradation
TAD ≈ degron
Strong TAD = highly unstable
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TADs and degrons overlap
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Marking and destroying
active TFs are part of into the
activation process itself.
A functional relationship
between Trx Activation
Domain (TADs) and
degradation signals
(DEGRONS).
The transcriptional activation
domains (TADs) and
degradation signals
(degrons) overlap in 19
unstable transcription
factors
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Transcriptional activation
- risky business?
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Evidence indicates that
marking and destroying
active TFs are part of
into the activation
process itself.
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Kamikaze activators
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Srb10 (Cdk8) also targets activator
(Gcn4p) sentencing it for destruction
CTD
Ubiquitinated
Degraded
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Gcn4 = targeted by Srb10, on the
way to destruction
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Gcn4 is phosphorylated by
Srb10
Phospho-Gcn4p is
recognized by Ub-ligase
complex SCFCdc4
WD40 repeats mediates
substrate recognition
Ubiquitinylation of Gcn4p
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A ”black widow” model
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Why should Srb10 destroy
the activator?
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= Activators are destroyed
as a direct consequence of
recruiting the basal trx
machinery to a promoter
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Basal trx machinery can mark the
activators it has encountered,
sentencing them to an early death
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Cyclic processes in trx
- role of the Proteasome (Gannon version)
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3. conclusion
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VP16 TAD signals ubiquitination through
Met30 ubi-ligase
Met30 is required for VP16 TAD to activate
This requirement circumvented by Ub-fusion
Activator ubiquitination is essential for trx
activation
Ubiquitination = dual signal for activation and
activator destruction
A unified model
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A unified model?
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In this model, the ubiquitin (Ub)–proteasome system regulates
transcription at numerous levels.
Interactions of a TF (activator) with the general trx machinery (green) functions to recruit
ubiquitin ligase(s) to the site of transcription and ubiquitylates many factors, including the
activator, RNAPII and histones.
 These ubiquitylation events in turn recruit the 26S proteasome, which simultaneously
destroys the activator and promotes elongation of transcription by pol II.

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A unified model?
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Limiting uncontrolled trx
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Importantly, this proposed mechanism limits uncontrolled transcription in two
ways - by destroying the activator at each cycle of promoter ‘firing’ and by
ensuring that interactions between pol II and the proteasome are made in an
activator- and promoter-dependent manner.
A cousin - SUMO-1
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What is SUMO-1 ?
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SUMO-1 (small ubiquitin-related modifier)
peptide of 101 residues
 function ≠ ubiquitin.
 NOT tagged for degradation
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Rather stabilized or ”targeted” to subnuclear structures
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The SUMO-1 protein
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Small Ubiquitin MOdifier
Link: isopeptide bond
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GG
between the C-terminal glycine of
SUMO and the e-amino group of a
lysine residue in the target protein.
Structure

characteristic ubiquitin-fold + unique
unstructured N-terminal extension of
up to 22 residues - possible protein
interaction site?
XKE
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Many nuclear
targets
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Largely nuclear target
proteins
NLS + KxE
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A short peptide that contains
the KxE motif and a NLS
suffices to produce a SUMO
conjugate in vivo.
Mutated NLS abolish
Sumoylation

SP100, HDAC4, MDM2
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Sumoylation - functional roles
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Altering the subcellular localization of the
protein
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Antagonizing other modifications
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Sumoylation causes the relocalization of the nuclear import factor
RanGAP1 from the cytoplasm to the nuclear pore complex (NPC).
Many Trx factors associated with PML nuclear bodies.
SUMO modification of IkBa stabilizes this NF-kB inhibitor by blocking
ubiquitylation at the same acceptor site.
Important for Repressor functions?
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PML and
PML nuclear bodies - SUMO required
Zhong et al. (2000) Nature Cell Biol. 2:E85-E90
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Many trx regulators found ass with
PML nuclear bodies
c-Myb
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Sumoylation
and nuclear import
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NPC passage
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A substrate that contains a NLS might be sumoylated at the nuclear pore by the
E3 ligase activity of RanBP2, after which it might be de-modified by a Ulp1type SUMO protease that resides at the nucleoplasmic face of the nuclear pore
complex (NPC), or by a Ulp2-type, nucleoplasmic protease.
Nuclear dynamic modification

Once inside the nucleus, substrates might undergo SUMO modification that is
mediated by PIAS or Pc2 E3 ligases.
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Regulating the trx Initiation
Machinery by Lysine Modification