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How ubiquitin and SUMOs control trx How ubiquitin and SUMOs control trx

MBV 4230 Why this? n n n Ubiquitylation of proteins not only targets them MBV 4230 Why this? n n n 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 Ubiquitin and the protesome. . A reminder

MBV 4230 Ubiquitin-family proteins and the proteasome n Ub covalently linked to targets ¨ MBV 4230 Ubiquitin-family proteins and the proteasome n Ub covalently linked to targets ¨ n Ubiquitin is the defining member of this class, ¨ n The ubiquitin (Ub) system defines a family of related modifier proteins that are linked covalently to target proteins. 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), E 3, which in turn recruits a Ubconjugating (Ubc) enzyme, E 2, to the substrate. The E 3 then catalyses the transfer of Ub groups to a lysine (K) residue that is somewhere in the target protein. ¨

MBV 4230 Ubiquitin-family proteins and the proteasome n Multi-Ub = Targeted for proteosomal destruction MBV 4230 Ubiquitin-family proteins and the proteasome n 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 (K 48) in Ub itself — forms, the substrate is targeted for destruction by a large, self-compartmentalized, protease known as the 26 S proteasome. ¨ The 19 S subcomplex of the proteasome recognizes the multi-ubiquitylated substrate, removes the Ub groups, unfolds the substrate and feeds it into the core of the 20 S subcomplex where it is destroyed. ¨ If, however, the multi-Ub chain is linked by lysine 63 (K 63), or if it has less than four Ub chains, proteolysis does not occur. ¨ n A family of Ubi-related small proteins can also be conjugated

MBV 4230 Ubiquitin conjugation to substrates MBV 4230 Ubiquitin conjugation to substrates

MBV 4230 Nobel Prize in Chemistry for 2004 n MBV 4230 Nobel Prize in Chemistry for 2004 n "for the discovery of ubiquitin-mediated protein degradation” n Aaron Ciechanover ¨ n Avram Hershko ¨ n Technion – Israel Institute of Technology, Haifa, Israel, Technion – Israel Institute of Technology, Haifa, Israel and Irwin Rose ¨ University of California, Irvine, USA

Ubiquitin and chromatin Ubiquitin and chromatin

MBV 4230 Ubiquitylation of histones n Histone H 2 A and H 2 B MBV 4230 Ubiquitylation of histones n Histone H 2 A and H 2 B Ubiquitylation - one of the first recognized markers of trx active chromatin The first ubiquitylated protein to be described was histone H 2 A ¨ ubiquitylated forms of histones H 2 A and H 2 B were associated specifically with actively transcribed genes ¨ Later also H 1 and H 3 reported to be ubiquitylated ¨

MBV 4230 Ubiquitylation and the histone ”code” n Ubiquitylation of chromatin The ubiquitin (Ub)-conjugating MBV 4230 Ubiquitylation and the histone ”code” n Ubiquitylation of chromatin The ubiquitin (Ub)-conjugating enzyme Rad 6 ubiquitylates K 123 in the core of histone H 2 B. This modification promotes the methylation of another histone, H 3, at two positions, K 4 and K 79. These modifications, in turn, are required for telomeric-gene silencing. ¨ TAFII 250 (TFIID component) can ubiquitylate the linker histone H 1; might relate to the role of this TAF in n transcriptional activation. ¨ n 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 HDAC 6 ¨

MBV 4230 A coming role for De-ubiquitylation? n Ubps - possible novel regulators? ¨ MBV 4230 A coming role for De-ubiquitylation? n Ubps - possible novel regulators? ¨ studies have identified Ub-specific proteases (Ubps) associated with components of both the SIR 4 silencing and the SAGA chromatin remodelling complexes.

Regulating RNAPII by ubiquitylation Regulating RNAPII by ubiquitylation

MBV 4230 DNA damage - use of RNAPII to direct repair to active genes MBV 4230 DNA damage - use of RNAPII to direct repair to active genes n 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 Rsp 5, 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. ¨ n Rsp 5 is also a co-activator for the steroid hormone receptors ?

Regulating TFs by ubiquitylation Regulating TFs by ubiquitylation

MBV 4230 Three strategies n Controlling the localization of the TF n Controlling the MBV 4230 Three strategies n Controlling the localization of the TF n Controlling the activity of the TF n Controlling the abundance of the TF

MBV 4230 Regulating TFs by the ubiquitylation - 3 strategies and 4 models n MBV 4230 Regulating TFs by the ubiquitylation - 3 strategies and 4 models n Regulating location. As with NFk. B, the TF can be maintained outside the nucleus by interactions with an inhibitor (Ik. B) 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. ¨ n 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.

MBV 4230 Regulating TF abundance - 1. model n Regulating abundance I - constitutive MBV 4230 Regulating TF abundance - 1. model n Regulating abundance I - constitutive turnover. ¨ n 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 p 53 - later lecture ¨ Wnt-signalling ¨

MBV 4230 Destroying TFs when not needed - shutting off proteolysis then gives a MBV 4230 Destroying TFs when not needed - shutting off proteolysis then gives a rapid response n Beta-catenin and wntsignalling Signalling Inactivation of GSKb Stabilization of b-catenin Phosphorylation ubiquitylation Degradation Rapid Accumulation To the nucleus

MBV 4230 Regulating TF abundance - 2. model n Regulating abundance II - trx-coupled MBV 4230 Regulating TF abundance - 2. model n 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 DNAbound transcription factor.

MBV 4230 Link: trx activation degradation n n TFs often unstable TAD overlaps closely MBV 4230 Link: trx activation degradation n n TFs often unstable TAD overlaps closely with degrons Degron = domain that signals ubiquitination ¨ Myc and many others ¨ n n Strong activators = rapidly degraded Weak activators = more stable ¨ n Q-rich, N-rich Mutant TADs with activation lost = stabilized n. Link: activation - degradation n. TAD ≈ degron n. Strong TAD = highly unstable

MBV 4230 TADs and degrons overlap n n n Marking and destroying active TFs MBV 4230 TADs and degrons overlap n n n 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

MBV 4230 Transcriptional activation - risky business? n Evidence indicates that marking and destroying MBV 4230 Transcriptional activation - risky business? n Evidence indicates that marking and destroying active TFs are part of into the activation process itself. n Kamikaze activators

MBV 4230 Srb 10 (Cdk 8) also targets activator (Gcn 4 p) sentencing it MBV 4230 Srb 10 (Cdk 8) also targets activator (Gcn 4 p) sentencing it for destruction CTD Ubiquitinated Degraded

MBV 4230 Gcn 4 = targeted by Srb 10, on the way to destruction MBV 4230 Gcn 4 = targeted by Srb 10, on the way to destruction n n Gcn 4 is phosphorylated by Srb 10 Phospho-Gcn 4 p is recognized by Ub-ligase complex SCFCdc 4 WD 40 repeats mediates substrate recognition Ubiquitinylation of Gcn 4 p

MBV 4230 A ”black widow” model n Why should Srb 10 destroy the activator? MBV 4230 A ”black widow” model n Why should Srb 10 destroy the activator? n = Activators are destroyed as a direct consequence of recruiting the basal trx machinery to a promoter n Basal trx machinery can mark the activators it has encountered, sentencing them to an early death

MBV 4230 Cyclic processes in trx - role of the Proteasome (Gannon version) MBV 4230 Cyclic processes in trx - role of the Proteasome (Gannon version)

MBV 4230 3. conclusion n n VP 16 TAD signals ubiquitination through Met 30 MBV 4230 3. conclusion n n VP 16 TAD signals ubiquitination through Met 30 ubi-ligase Met 30 is required for VP 16 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 A unified model

MBV 4230 A unified model? n In this model, the ubiquitin (Ub)–proteasome system regulates MBV 4230 A unified model? n 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 26 S proteasome, which simultaneously destroys the activator and promotes elongation of transcription by pol II. ¨

MBV 4230 A unified model? n Limiting uncontrolled trx ¨ Importantly, this proposed mechanism MBV 4230 A unified model? n 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 A cousin - SUMO-1

MBV 4230 What is SUMO-1 ? n SUMO-1 (small ubiquitin-related modifier) peptide of 101 MBV 4230 What is SUMO-1 ? n SUMO-1 (small ubiquitin-related modifier) peptide of 101 residues / mature polypeptide 98 residues ¨ function ≠ ubiquitin ¨ NOT tagged for degradation ¨ n Rather stabilized or ”targeted” to subnuclear structures

MBV 4230 The SUMO-1 protein n Small Ubiquitin MOdifier n Link: isopeptide bond ¨ MBV 4230 The SUMO-1 protein n Small Ubiquitin MOdifier n Link: isopeptide bond ¨ n 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? GG XKE

MBV 4230 Many nuclear targets n n Largely nuclear target proteins NLS + Kx. MBV 4230 Many nuclear targets n n Largely nuclear target proteins NLS + Kx. E ¨ n A short peptide that contains the Kx. E motif and a NLS suffices to produce a SUMO conjugate in vivo. Mutated NLS abolish Sumoylation ¨ SP 100, HDAC 4, MDM 2

MBV 4230 Sumoylation - functional roles n Antagonizing other modifications ¨ n SUMO modification MBV 4230 Sumoylation - functional roles n Antagonizing other modifications ¨ n SUMO modification of Ik. Ba stabilizes this NF-k. B inhibitor by blocking ubiquitylation at the same acceptor site. Confer new interactions Conformational change ¨ New interaction surface – enhance or inhibit interactions ¨ n Altering the subcellular localization of the protein Sumoylation causes the relocalization of the nuclear import factor Ran. GAP 1 from the cytoplasm to the nuclear pore complex (NPC). ¨ Many Trx factors associated with PML nuclear bodies ¨

MBV 4230 Sumoylation - consequences MBV 4230 Sumoylation - consequences

MBV 4230 Sumoylation – co-repressor recruitment n Sumoylated TF promotes binding of corepressor. MBV 4230 Sumoylation – co-repressor recruitment n Sumoylated TF promotes binding of corepressor.

MBV 4230 Sumoylation and nuclear import n NPC passage ¨ n A substrate that MBV 4230 Sumoylation and nuclear import n NPC passage ¨ n A substrate that contains a NLS might be sumoylated at the nuclear pore by the E 3 ligase activity of Ran. BP 2, after which it might be de-modified by a Ulp 1 type SUMO protease that resides at the nucleoplasmic face of the nuclear pore complex (NPC), or by a Ulp 2 -type, nucleoplasmic protease. Nuclear dynamic modification ¨ Once inside the nucleus, substrates might undergo SUMO modification that is mediated by PIAS or Pc 2 E 3 ligases.

MBV 4230 PML and PML nuclear bodies - SUMO required Zhong et al. (2000) MBV 4230 PML and PML nuclear bodies - SUMO required Zhong et al. (2000) Nature Cell Biol. 2: E 85 -E 90

MBV 4230 Many trx regulators found ass with PML nuclear bodies c-Myb MBV 4230 Many trx regulators found ass with PML nuclear bodies c-Myb

MBV 4230 MBV 4230

MBV 4230 Regulating the trx Initiation Machinery by Lysine Modification MBV 4230 Regulating the trx Initiation Machinery by Lysine Modification