How ubiquitin and SUMOs control trx

MBV4230

Odd S. Gabrielsen

Why this?

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

MBV4230

Odd S. Gabrielsen

Ubiquitin-family proteins and the proteasome

Ub covalently linked to targets The ubiquitin (Ub) system defines a family

of related modifier proteins that are linked covalently to target proteins.

Ubiquitin is the defining member of this class, But at 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 Ub-conjugating (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.

MBV4230

Odd S. Gabrielsen

Ubiquitin-family proteins and the proteasome

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.

A family of Ubi-related small proteins can also be conjugated

MBV4230

Odd S. Gabrielsen

Ubiquitin conjugation to substrates

MBV4230

Odd S. Gabrielsen

Nobel Prize in Chemistry for 2004

"for the discovery of ubiquitin-mediated protein degradation”

Aaron Ciechanover Technion – Israel Institute of Technology, Haifa, Israel,

Avram Hershko Technion – Israel Institute of Technology, Haifa, Israel and

Irwin Rose University of California, Irvine, USA

Ubiquitin and chromatin

MBV4230

Odd S. Gabrielsen

Ubiquitylation of histones

Histone H2A and H2B Ubiquitylation - one of the first recognized markers of trx active chromatin 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

MBV4230

Odd S. Gabrielsen

Ubiquitylation and the histone ”code”

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.

Ubiquitylation = an integral part of the histone ”code”

Mechanism? Direct structural role by

loosening chromatin structure Or as ”tag” recognized by

proteins such as the proteasome or HDAC6

MBV4230

Odd S. Gabrielsen

A coming role for De-ubiquitylation?

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

MBV4230

Odd S. Gabrielsen

DNA damage - use of RNAPII to direct repair to active genes 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.

Rsp5 is also a co-activator for the steroid hormone receptors ?

Regulating TFs by ubiquitylation

MBV4230

Odd S. Gabrielsen

Three strategies

Controlling the localization of the TF

Controlling the activity of the TF

Controlling the abundance of the TF

MBV4230

Odd S. Gabrielsen

Regulating TFs by the ubiquitylation - 3 strategies and 4 models Regulating location.

As with NFB, the TF can be maintained outside the nucleus by interactions with an inhibitor (IB) 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.

MBV4230

Odd S. Gabrielsen

Regulating TF abundance - 1. model

Regulating abundance I - constitutive turnover. 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 p53 - later lecture Wnt-signalling

MBV4230

Odd S. Gabrielsen

Destroying TFs when not needed- shutting off proteolysis then gives a rapid response

Beta-catenin and wnt-signalling

PhosphorylationubiquitylationDegradation

SignallingInactivation of GSK

Stabilization of -catenin

Rapid AccumulationTo the nucleus

MBV4230

Odd S. Gabrielsen

Regulating TF abundance - 2. model

Regulating abundance II - trx-coupled destruction In this model, activators are destroyed during the act of transcriptional

activation as a way of limiting uncontrolled activation by any one DNA-bound transcription factor.

MBV4230

Odd S. Gabrielsen

Link: trx activation degradation

TFs often unstable TAD overlaps closely with degrons

Degron = domain that signals ubiquitination Myc and many others

Strong activators = rapidly degraded Weak activators = more stable

Q-rich, N-rich

Mutant TADs with activation lost = stabilized

Link: activation - degradationTAD ≈ degron

Strong TAD = highly unstable

MBV4230

Odd S. Gabrielsen

TADs and degrons overlap

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

MBV4230

Odd S. Gabrielsen

Transcriptional activation- risky business? Evidence indicates that

marking and destroying active TFs are part of into the activation process itself.

Kamikaze activators

MBV4230

Odd S. Gabrielsen

Srb10 (Cdk8) also targets activator (Gcn4p) sentencing it for destruction

UbiquitinatedDegraded

CTD

MBV4230

Odd S. Gabrielsen

Gcn4 = targeted by Srb10, on the way to destruction

Gcn4 is phosphorylated by Srb10

Phospho-Gcn4p is recognized by Ub-ligase complex SCFCdc4

WD40 repeats mediates substrate recognition

Ubiquitinylation of Gcn4p

MBV4230

Odd S. Gabrielsen

A ”black widow” model

Why should Srb10 destroy the activator?

= Activators are destroyed as a direct consequence of recruiting the basal trx machinery to a promoter

Basal trx machinery can mark the activators it has encountered, sentencing them to an early death

MBV4230

Odd S. Gabrielsen

Cyclic processes in trx- role of the Proteasome (Gannon version)

MBV4230

Odd S. Gabrielsen

3. conclusion

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

MBV4230

Odd S. Gabrielsen

A unified model?

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.

MBV4230

Odd S. Gabrielsen

A unified model?

Limiting uncontrolled trx 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

MBV4230

Odd S. Gabrielsen

What is SUMO-1 ?

SUMO-1 (small ubiquitin-related modifier) peptide of 101 residues / mature polypeptide 98 residues function ≠ ubiquitin NOT tagged for degradation

Rather stabilized or ”targeted” to sub-nuclear structures

MBV4230

Odd S. Gabrielsen

The SUMO-1 protein

Small Ubiquitin MOdifier

Link: isopeptide bond between the C-terminal glycine of

SUMO and the -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?

GG

XKE

MBV4230

Odd S. Gabrielsen

Many nuclear targets

Largely nuclear target proteins

NLS + KxE 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

MBV4230

Odd S. Gabrielsen

Sumoylation - functional roles Antagonizing other modifications

SUMO modification of IBstabilizes this NF-B inhibitor by blocking ubiquitylation at the same acceptor site.

Confer new interactions Conformational change New interaction surface – enhance or inhibit interactions

Altering the subcellular localization of the protein 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

MBV4230

Odd S. Gabrielsen

Sumoylation - consequences

MBV4230

Odd S. Gabrielsen

Sumoylation – co-repressor recruitment

Sumoylated TF promotes binding of co-repressor.

MBV4230

Odd S. Gabrielsen

Sumoylation and nuclear import

NPC passage 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 Ulp1-type 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.

MBV4230

Odd S. Gabrielsen

PML and PML nuclear bodies - SUMO required

Zhong et al. (2000) Nature Cell Biol. 2:E85-E90

MBV4230

Odd S. Gabrielsen

Many trx regulators found ass with PML nuclear bodies

c-Myb

MBV4230

Odd S. Gabrielsen

MBV4230

Odd S. Gabrielsen

Regulating the trx InitiationMachinery by Lysine Modification