HECT Domain E3 Ubiquitin Ligase Research Articles

The m6A methyltransferase METTL3 drives neuroinflammation and neurotoxicity through stabilizing BATF mRNA in microglia.

Persistent neuroinflammation and progressive neuronal loss are defining features of acute brain injury including traumatic brain injury (TBI) and cerebral stroke. Microglia, the most abundant type of brain-resident immune cells, continuously surveil the environment and play a central role in shaping the inflammatory state of the central nervous system (CNS). In the study, we discovered that the protein expression of METTL3 (a m6A methyltransferase) was upregulated in inflammatory microglia independent of increased Mettl3 gene transcription following TBI in both human and mouse subjects. Subsequently, we identified TRIP12, a HECT-domain E3 ubiquitin ligase, as a negative regulator of METTL3 protein expression by facilitating METTL3 K48-linked polyubiquitination. Importantly, selective ablation of Mettl3 inhibited microglial pathogenic activities, diminished neutrophil infiltration, rescued neuronal loss and facilitated functional recovery post-TBI. Using MeRIP-seq and CUT&Tag sequencing, we identified that METTL3 promoted the expression of Basic Leucine Zipper Transcriptional Factor ATF-Like (BATF), which in turn directly bound to a cohort of characteristic inflammatory cytokines and chemokine genes. Enhanced activities of BATF in microglia elicited TNF-dependent neurotoxicity and can also promote neutrophil recruitment through releasing CXCL2. Pharmacological inhibition of METTL3 using a BBB-penetrating drug-loaded nano-system showed satisfactory therapeutic effects in both TBI and stroke mouse models. Collectively, our findings identified METTL3-m6A-BATF axis as a potential therapeutic target for terminating detrimental neuroinflammation and progressive neuronal loss following acute brain injury. METTL3 protein is significantly up-regulated in inflammatory microglia due to the decreased proteasomal degradation mediated by TRIP12 and ERK-USP5 pathways. METTL3 stabilized BATF mRNA stability and promoted BATF expression through the m6A-IGF2BP2-dependent mechanism. Elevated expression of BATF elicits a pro-inflammatory gene program in microglia, and aggravates neuroinflammatory response including local immune responses and peripheral immune cell infiltration. Genetic deletion or pharmaceutically targeting METTL3-BATF axis suppressed microglial pro-inflammatory activities and promoted neurological recovery following TBI and stroke.

Protective effects of compound M01 on retinal ganglion cells in experimental anterior ischemic optic neuropathy by inhibiting TXNIP/NLRP3 inflammasome pathway

Apoptotic death of retinal ganglion cells (RGCs) is a common pathologic feature in different types of optic neuropathy, including ischemic optic neuropathy and glaucoma, ultimately leading to irreversible visual function loss. Potent and effective protection against RGC death is determinative in developing a successful treatment for these optic neuropathies. This study evaluated the neuroprotective effect of a HECT domain-E3 ubiquitin ligase inhibitor, M01, on retinal ganglion cells after ischemic injury. Experimental anterior ischemic optic neuropathy (AION) was induced by photothrombotic occlusion of microvessels supplying optic nerve in rats. M01 was administered (100 mg/Kg and 200 mg/Kg) subcutaneously for three consecutive days after AION induction. Administration of M01 (100 mg/Kg) significantly increased RGC survival and preserved visual function after AION induction. The number of TUNEL-positive cells and ED1-positive cells was significantly decreased, and optic disc edema was reduced considerably after ischemic infarction with M01 treatment. Moreover, M01 effectively ameliorated optic nerve demyelination and enhanced M2 microglial polarization after AION induction. M01 enhanced the expression of nuclear factor erythroid 2-related factor (Nrf2); subsequently, downregulated Thioredoxin interacting protein (TXNIP) expression, inhibited NLR family pyrin domain containing 3 (NLRP3) activation, and further decreased inflammatory factors, interleukin (IL)-1β and IL-6 in the retina after ischemic injury. These findings suggested that M01 has therapeutic potential by modulating Nrf2 and TXNIP/NLRP3 inflammasome pathways in the retina and optic nerve ischemic damage-related diseases.

Control of TGFβ signalling by ubiquitination independent function of E3 ubiquitin ligase TRIP12

Transforming growth factor β (TGFβ) pathway is a master regulator of cell proliferation, differentiation, and death. Deregulation of TGFβ signalling is well established in several human diseases including autoimmune disorders and cancer. Thus, understanding molecular pathways governing TGFβ signalling may help better understand the underlying causes of some of those conditions. Here, we show that a HECT domain E3 ubiquitin ligase TRIP12 controls TGFβ signalling in multiple models. Interestingly, TRIP12 control of TGFβ signalling is completely independent of its E3 ubiquitin ligase activity. Instead, TRIP12 recruits SMURF2 to SMAD4, which is most likely responsible for inhibitory monoubiquitination of SMAD4, since SMAD4 monoubiquitination and its interaction with SMURF2 were dramatically downregulated in TRIP12-/- cells. Additionally, genetic inhibition of TRIP12 in human and murine cells leads to robust activation of TGFβ signalling which was rescued by re-introducing wildtype TRIP12 or a catalytically inactive C1959A mutant. Importantly, TRIP12 control of TGFβ signalling is evolutionary conserved. Indeed, genetic inhibition of Drosophila TRIP12 orthologue, ctrip, in gut leads to a reduced number of intestinal stem cells which was compensated by the increase in differentiated enteroendocrine cells. These effects were completely normalised in Drosophila strain where ctrip was co-inhibited together with Drosophila SMAD4 orthologue, Medea. Similarly, in murine 3D intestinal organoids, CRISPR/Cas9 mediated genetic targeting of Trip12 enhances TGFβ mediated proliferation arrest and cell death. Finally, CRISPR/Cas9 mediated genetic targeting of TRIP12 in MDA-MB-231 breast cancer cells enhances the TGFβ induced migratory capacity of these cells which was rescued to the wildtype level by re-introducing wildtype TRIP12. Our work establishes TRIP12 as an evolutionary conserved modulator of TGFβ signalling in health and disease.

Targeting UBR5 inhibits postsurgical breast cancer lung metastases by inducing CDC73 and p53 mediated apoptosis.

UBR5 is a HECT domain E3 ubiquitin ligase that is frequently amplified in breast, ovarian and prostate cancers. Heightened UBR5 expression plays a profound role in tumor growth through immune-dependent mechanisms; however, its mode of action in driving tumor metastasis has not been definitively delineated. Herein, we used a tetracycline (Tet)-inducible RNAi-mediated expression silencing cell system to investigate how UBR5 enables postsurgical mammary tumor metastatic growth in mouse lungs without the continuous influence of the primary lesion. In vitro, Ubr5 knockdown induces morphological and molecular changes characteristic of epithelial-mesenchymal transition (EMT). In vivo, UBR5 promotes lung metastasis in an E3 ubiquitin ligase-dependent manner. Moreover, doxycycline-induced UBR5 expression knockdown in metastatic cells in the lungs, following removing the primary tumors, resulted in increased apoptosis, decreased proliferation and prolonged survival, whereas silencing the expression of cell division cycle 73 (CDC73), a tumor suppressor and E3 ligase substrate of UBR5, reversed these effects. Transcriptome analyses revealed a prominent role of the p53 pathway in dovitinib-induced apoptosis of tumor cells differentially regulated by UBR5 and CDC73. In human triple-negative breast cancer (TNBC) patient specimens, a strong inverse correlation was observed between UBR5 and CDC73 protein levels, with reduced CDC73 expression at metastatic sites compared to primary lesions. Furthermore, a xenograft model of human TNBC recapitulated the metastatic properties and characteristics of the unique UBR5-CDC73 functional antagonism. This study reveals the novel and critical roles and intricate relationships of UBR5, CDC73 and p53 in postsurgical breast cancer metastasis and indicates the potential of targeting this pathway in cancer therapy.

CircUCK2 regulates HECTD1-mediated endothelial-mesenchymal transition inhibition by interacting with FUS and protects the blood-brain barrier in ischemic stroke.

Ischemic stroke (IS) has become a cerebrovascular disease of widespread concern. Overexpression of circUCK2 alleviates neuronal damage in IS. However, the specific regulatory mechanisms of circUCK2 are not fully understood. In this study, we found that circUCK2 and HECT domain E3 ubiquitin ligase 1 (HECTD1) were downregulated in IS models in vitro and in vivo. Overexpression of circUCK2 or HECTD1 inhibited endothelial-mesenchymal transition (EndoMT) and protected the blood-brain barrier (BBB) in transient middle cerebral artery occlusion mice from damage. It was further discovered that circUCK2 regulated HECTD1 expressions by interacting with fused in sarcoma (FUS). Moreover, FUS overexpression partially restored the effect of circUCK2 on EndoMT, and overexpression of HECTD1 weakened the effect of FUS on EndoMT. Collectively, circUCK2 upregulates the expression of HECTD1 by combining with FUS and inhibits EndoMT to alleviate BBB damage in IS both in vivo and in vitro.

UBR5 targets tumor suppressor CDC73 proteolytically to promote aggressive breast cancer

UBR5, a HECT-domain E3 ubiquitin ligase, is an attractive therapeutic target for aggressive breast cancers. Defining the substrates of UBR5 is crucial for scientific understanding and clinical intervention. Here, we demonstrate that CDC73, a component of the RNA polymerase II-associated factor 1 complex, is a key substrate that impedes UBR5’s profound tumorigenic and metastatic activities in triple-negative breast cancer (TNBC) via mechanisms of regulating the expression of β-catenin and E-cadherin, tumor cell apoptosis and CD8+ T cell infiltration. Expression of CDC73 is also negatively associated with the progression of breast cancer patients. Moreover, we show that UBR5 destabilizes CDC73 by polyubiquitination at Lys243, Lys247, and Lys257 in a non-canonical manner that is dependent on the non-phosphorylation state of CDC73 at Ser465. CDC73 could serve as a molecular switch to modulate UBR5’s pro-tumor activities and may provide a potential approach to developing breast cancer therapeutic interventions.

Proteasomal degradation of the tumour suppressor FBW7 requires branched ubiquitylation by TRIP12

The tumour suppressor FBW7 is a substrate adaptor for the E3 ubiquitin ligase complex SKP1-CUL1-F-box (SCF), that targets several oncoproteins for proteasomal degradation. FBW7 is widely mutated and FBW7 protein levels are commonly downregulated in cancer. Here, using an shRNA library screen, we identify the HECT-domain E3 ubiquitin ligase TRIP12 as a negative regulator of FBW7 stability. We find that SCFFBW7-mediated ubiquitylation of FBW7 occurs preferentially on K404 and K412, but is not sufficient for its proteasomal degradation, and in addition requires TRIP12-mediated branched K11-linked ubiquitylation. TRIP12 inactivation causes FBW7 protein accumulation and increased proteasomal degradation of the SCFFBW7 substrate Myeloid Leukemia 1 (MCL1), and sensitizes cancer cells to anti-tubulin chemotherapy. Concomitant FBW7 inactivation rescues the effects of TRIP12 deficiency, confirming FBW7 as an essential mediator of TRIP12 function. This work reveals an unexpected complexity of FBW7 ubiquitylation, and highlights branched ubiquitylation as an important signalling mechanism regulating protein stability.

The LARGE2-APO1/APO2 regulatory module controls panicle size and grain number in rice.

Panicle size and grain number are important agronomic traits and influence grain yield in rice (Oryza sativa), but the molecular and genetic mechanisms underlying panicle size and grain number control remain largely unknown in crops. Here we report that LARGE2 encodes a HECT-domain E3 ubiquitin ligase OsUPL2 and regulates panicle size and grain number in rice. The loss of function large2 mutants produce large panicles with increased grain number, wide grains and leaves, and thick culms. LARGE2 regulates panicle size and grain number by repressing meristematic activity. LARGE2 is highly expressed in young panicles and grains. Biochemical analyses show that LARGE2 physically associates with ABERRANT PANICLE ORGANIZATION1 (APO1) and APO2, two positive regulators of panicle size and grain number, and modulates their stabilities. Genetic analyses support that LARGE2 functions with APO1 and APO2 in a common pathway to regulate panicle size and grain number. These findings reveal a novel genetic and molecular mechanism of the LARGE2-APO1/APO2 module-mediated control of panicle size and grain number in rice, suggesting that this module is a promising target for improving panicle size and grain number in crops.

HECTD1 regulates the expression of SNAIL: Implications for epithelial‑mesenchymal transition.

As a transcription factor, SNAIL plays a crucial role in embryonic development and cancer progression by mediating epithelial-mesenchymal transition (EMT); however, post-translational modifications, such as ubiquitination, which control the degradation of SNAIL have been observed to affect its functional role in EMT. In a previous study by the authors, it was demonstrated that the HECT domain E3 ubiquitin ligase 1 (HECTD1) regulated the dynamic nature of adhesive structures. In the present study, HECTD1 was observed to interact with SNAIL and regulate its stability through ubiquitination, and the knockdown of HECTD1 increased the expression levels of SNAIL. HECTD1 was discovered to contain putative nuclear localization and export signals that facilitated its translocation between the cytoplasm and nucleus, a process regulated by epidermal growth factor (EGF). Treatment with leptomycin B resulted in the nuclear retention of HECTD1, which was associated with the loss of SNAIL expression. The knockdown of HECTD1 in HeLa cells increased cell migration and induced a mesenchymal phenotype, in addition to through increased phosphorylated ERK expression levels. Under hypoxic conditions, HECTD1 expression levels were decreased by microRNA (miRNA or miR)-210. Upon the observation of genetic abnormalities in the HECTD1 gene in cervical cancer specimens, it was observed that the decreased expression levels of HECTD1 were significantly associated with a poor patient survival. Thus, it was hypothesized that HECTD1 may regulate EMT through the hypoxia/hypoxia inducible factor 1α/miR-210/HECTD1/SNAIL signaling pathway and the EGF/EGF receptor/HECTD1/ERK/SNAIL signaling pathway in cervical cancer. On the whole, the data of the present study indicated that HECTD1 serves as an E3 ubiquitin ligase to mediate the stability of SNAIL proteins.

Multiple regions of E6AP (UBE3A) contribute to interaction with papillomavirus E6 proteins and the activation of ubiquitin ligase activity

The HECT domain E3 ubiquitin ligase E6AP (UBE3A) is critical for the development of human papillomavirus (HPV) associated cancers, the neurodevelopment disorder Angelman Syndrome, and some cases of autism spectrum disorders. How E6AP recognizes its cellular targets and how its ubiquitin ligase activity is triggered remain poorly understood, and HPV E6 proteins are models for these processes. We examined diverse E6 proteins from human and non-human papillomaviruses and identified two different modes of interaction between E6 and E6AP. In Type I interactions, E6 can interact directly with the LXXLL peptide motif alone of E6AP (isolated from the rest of E6AP), and then recruit cellular substrates such as p53. In Type II interactions, E6 proteins require additional auxiliary regions of E6AP in either the amino terminus or in the carboxy-terminal HECT domain to interact with the LXXLL peptide motif of E6AP. A region of E6AP amino-terminal to the LXXLL peptide motif both augments association with E6 proteins and is required for E6 proteins to trigger ubiquitin ligase activity in the carboxy-terminal HECT ubiquitin ligase domain of E6AP. In Type I interactions, E6 can associate with E6AP and recruit p53, but a Type II interaction is required for the degradation of p53 or NHERF1. Interestingly, different E6 proteins varied in E6AP auxiliary regions that contributed to enhanced association, indicating evolutionary drift in the formation of Type II interactions. This classification of E6-E6AP interaction types and identification of a region in the E6AP amino terminus that is important for both E6 association and stimulation of ubiquitin ligase activity will inform future structural data of the E6-E6AP complex and future studies aiming to interfere with the activity of the E6-E6AP complex.

UBR5 is a novel E3 ubiquitin ligase involved in skeletal muscle hypertrophy and recovery from atrophy.

We have recently identified that a HECT domain E3 ubiquitin ligase, named UBR5, is altered epigenetically (via DNA methylation) after human skeletal muscle hypertrophy, where its gene expression is positively correlated with increasing lean leg mass after training and retraining. In the present study we extensively investigate this novel and uncharacterised E3 ubiquitin ligase (UBR5) in skeletal muscle atrophy, recovery from atrophy and injury, anabolism and hypertrophy. We demonstrated that UBR5 was epigenetically altered via DNA methylation during recovery from atrophy. We also determined that UBR5 was alternatively regulated versus well characterised E3 ligases, MuRF1/MAFbx, at the gene expression level during atrophy, recovery from atrophy and hypertrophy. UBR5 also increased at the protein level during recovery from atrophy and injury, hypertrophy and during human muscle cell differentiation. Finally, in humans, genetic variations of the UBR5 gene were strongly associated with larger fast-twitch muscle fibres and strength/power performance versus endurance/untrained phenotypes. We aimed to investigate a novel and uncharacterized E3 ubiquitin ligase in skeletal muscle atrophy, recovery from atrophy/injury, anabolism and hypertrophy. We demonstrated an alternate gene expression profile for UBR5 vs. well characterized E3-ligases, MuRF1/MAFbx, where, after atrophy evoked by continuous-low-frequency electrical-stimulation in rats, MuRF1/MAFbx were both elevated, yet UBR5 was unchanged. Furthermore, after recovery of muscle mass post TTX-induced atrophy in rats, UBR5 was hypomethylated and increased at the gene expression level, whereas a suppression of MuRF1/MAFbx was observed. At the protein level, we also demonstrated a significant increase in UBR5 after recovery of muscle mass from hindlimb unloading in both adult and aged rats, as well as after recovery from atrophy evoked by nerve crush injury in mice. During anabolism and hypertrophy, UBR5 gene expression increased following acute loading in three-dimensional bioengineered mouse muscle in vitro, and after chronic electrical stimulation-induced hypertrophy in rats in vivo, without increases in MuRF1/MAFbx. Additionally, UBR5 protein abundance increased following functional overload-induced hypertrophy of the plantaris muscle in mice and during differentiation of primary human muscle cells. Finally, in humans, genetic association studies (>700,000 single nucleotide polymorphisms) demonstrated that the A alleles of rs10505025 and rs4734621 single nucleotide polymorphisms in the UBR5 gene were strongly associated with larger cross-sectional area of fast-twitch muscle fibres and favoured strength/power vs. endurance/untrained phenotypes. Overall, we suggest that: (i) UBR5 comprises a novel E3 ubiquitin ligase that is inversely regulated to MuRF1/MAFbx; (ii) UBR5 is epigenetically regulated; and (iii) UBR5 is elevated at both the gene expression and protein level during recovery from skeletal muscle atrophy and hypertrophy.

The autism-linked UBE3A T485A mutant E3 ubiquitin ligase activates the Wnt/β-catenin pathway by inhibiting the proteasome

UBE3A is a HECT domain E3 ubiquitin ligase whose dysfunction is linked to autism, Angelman syndrome, and cancer. Recently, we characterized a de novo autism-linked UBE3A mutant (UBE3AT485A) that disrupts phosphorylation control of UBE3A activity. Through quantitative proteomics and reporter assays, we found that the UBE3AT485A protein ubiquitinates multiple proteasome subunits, reduces proteasome subunit abundance and activity, stabilizes nuclear β-catenin, and stimulates canonical Wnt signaling more effectively than wild-type UBE3A. We also found that UBE3AT485A activates Wnt signaling to a greater extent in cells with low levels of ongoing Wnt signaling, suggesting that cells with low basal Wnt activity are particularly vulnerable to UBE3AT485A mutation. Ligase-dead UBE3A did not stimulate Wnt pathway activation. Overexpression of several proteasome subunits reversed the effect of UBE3AT485A on Wnt signaling. We also observed that subunits that interact with UBE3A and affect Wnt signaling are located along one side of the 19S regulatory particle, indicating a previously unrecognized spatial organization to the proteasome. Altogether, our findings indicate that UBE3A regulates Wnt signaling in a cell context-dependent manner and that an autism-linked mutation exacerbates these signaling effects. Our study has broad implications for human disorders associated with UBE3A gain or loss of function and suggests that dysfunctional UBE3A might affect additional proteins and pathways that are sensitive to proteasome activity.

Generation of SMURF2 knockout human cells using the CRISPR/Cas9 system

The HECT domain E3 ubiquitin ligase SMURF2 regulates stability of several key protein targets involved in tumorigenesis, cell proliferation, migration, differentiation, and senescence. While altered levels and aberrant cellular distribution of SMURF2 were reported in different types of cancer, its role in tumorigenesis is far from understood. To elucidate the role of SMURF2 in cancer, appropriate human cancer cell models are needed. Here, we describe approaches that can be used to generate human normal and cancer cell strains knocked-out for SMURF2 using the clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) gene-editing technology.

Resistance to UV-induced apoptosis by β-HPV5 E6 involves targeting of activated BAK for proteolysis by recruitment of the HERC1 ubiquitin ligase.

UV exposure is the main etiological agent in the development of non-melanoma skin cancer (NMSC), but mounting evidence suggests a co-factorial role for β-genus HPV types early in tumor initiation or progression. UV damage initiates an apoptotic response, driven at the mitochondrial level by BCL-2 family proteins, that eliminates damaged cells that may accumulate deleterious mutations and acquire tumorigenic properties. BAK is a pro-apoptotic BCL-2 protein that functions ultimately to form pores that permeabilize the mitochondrial outer membrane, thereby committing a cell to death, a process involving changes in BAK phosphorylation and conformation. The E6 protein of β-type HPV5 signals BAK for proteasomal degradation, a function that confers protection from UV-induced apoptosis. We find that HPV5 E6 does not constitutively target BAK for proteolysis, but targets the latter stages of BAK activation, following changes in phosphorylation and conformation. A mutational analysis identified the lysine residue on BAK required for proteolysis, and a functional siRNA screen identified the HECT domain E3 ubiquitin ligase HERC1 as being required for E6-mediated BAK degradation. We show that HERC1 interacts with BAK in E6-expressing cells that have been damaged by UV, and provide evidence that the interaction of HERC1 with BAK requires access to a hydrophobic surface on BAK that binds BH3 domains of BCL-2 proteins. We also show that HERC1 contains a putative BH3 domain that can bind to BAK. These findings reveal a specific and unique mechanism used by the HPV5 E6 protein to target BAK.

Trip12, a HECT domain E3 ubiquitin ligase, targets Sox6 for proteasomal degradation and affects fiber type-specific gene expression in muscle cells

BackgroundA sophisticated level of coordinated gene expression is necessary for skeletal muscle fibers to obtain their unique functional identities. We have previously shown that the transcription factor Sox6 plays an essential role in coordinating muscle fiber type differentiation by acting as a transcriptional suppressor of slow fiber-specific genes. Currently, mechanisms regulating the activity of Sox6 in skeletal muscle and how these mechanisms affect the fiber phenotype remain unknown.MethodsYeast two-hybrid screening was used to identify binding partners of Sox6 in muscle. Small interfering RNA (siRNA)-mediated knockdown of one of the Sox6 binding proteins, Trip12, was used to determine its effect on Sox6 activity in C2C12 myotubes using quantitative analysis of fiber type-specific gene expression.ResultsWe found that the E3 ligase Trip12, a HECT domain E3 ubiquitin ligase, recognizes and polyubiquitinates Sox6. Inhibiting Trip12 or the 26S proteasome activity resulted in an increase in Sox6 protein levels in C2C12 myotubes. This control of Sox6 activity in muscle cells via Trip12 ubiquitination has significant phenotypic outcomes. Knockdown of Trip12 in C2C12 myotubes led to upregulation of Sox6 protein levels and concurrently to a decrease in slow fiber-specific Myh7 expression coupled with an increased expression in fast fiber-specific Myh4. Therefore, regulation of Sox6 cellular levels by the ubiquitin-proteasome system can induce identity-changing alterations in the expression of fiber type-specific genes in muscle cells.ConclusionsBased on our data, we propose that in skeletal muscle, E3 ligases have a significant role in regulating fiber type-specific gene expression, expanding their importance in muscle beyond their well-established role in atrophy.

Hect E3 ubiquitin ligase Tom1 controls Dia2 degradation during the cell cycle

The ubiquitin proteasome system plays a pivotal role in controlling the cell cycle. The budding yeast F-box protein Dia2 is required for genomic stability and is targeted for ubiquitin-dependent degradation in a cell cycle-dependent manner, but the identity of the ubiquitination pathway is unknown. We demonstrate that the Hect domain E3 ubiquitin ligase Tom1 is required for Dia2 protein degradation. Deletion of DIA2 partially suppresses the temperature-sensitive phenotype of tom1 mutants. Tom1 is required for Dia2 ubiquitination and degradation during G1 and G2/M phases of the cell cycle, whereas the Dia2 protein is stabilized during S phase. We find that Tom1 binding to Dia2 is enhanced in G1 and reduced in S phase, suggesting a mechanism for this proteolytic switch. Tom1 recognizes specific, positively charged residues in a Dia2 degradation/NLS domain. Loss of these residues blocks Tom1-mediated turnover of Dia2 and causes a delay in G1-to-S phase progression. Deletion of DIA2 rescues a delay in the G1-to-S phase transition in the tom1Δ mutant. Together our results suggest that Tom1 targets Dia2 for degradation during the cell cycle by recognizing positively charged residues in the Dia2 degradation/NLS domain and that Dia2 protein degradation contributes to G1-to-S phase progression.

Abstract 2195: Smurf2 is a novel tumor suppressor gene that governs chromatin structure and genome integrity through RNF20

Abstract Cancers are known to harbour alterations in chromatin landscape in addition to allelic mutations. Here, we show that genomic ablation of Smurf2, a HECT-domain E3 ubiquitin ligase, results in dysregulation of global gene expression, altered DNA damage response, and unstable genomes, which culminate to increased susceptibility to various types of cancers in aged mice. We demonstrate that Smurf2 regulates histone H2B monoubiquitination as well as histone H3 tri-methylation at K4 and K79 by targeting RNF20, the major E3 ligase for histone H2B ubiquitination, to proteasomal degradation. We further show that Smurf2 and RNF20 are co-localized at the γ-H2AX foci of double-stranded DNA breaks in the nucleus, and Smurf2 regulates RNF20 stability in human cancer cells. Thus, our data indicate that Smurf2 has a tumor suppression function that normally maintains genomic stability by controlling the epigenetic landscape of histone modifications through RNF20. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2195. doi:1538-7445.AM2012-2195

A tumor suppressor function of Smurf2 associated with controlling chromatin landscape and genome stability through RNF20

In addition to allelic mutations, cancers are known to harbor alterations in their chromatin landscape. Here, we show that genomic ablation of Smurf2, a HECT-domain E3 ubiquitin ligase, results in dysregulation of DNA damage response and genomic stability, culminating to increased susceptibility to various types of cancers in aged mice. We demonstrate that Smurf2 regulates histone H2B monoubiquitination as well as histone H3 tri-methylation at K4 and K79 by targeting RNF20 to proteasomal degradation in both mouse and human cells. We further show that Smurf2 and RNF20 are co-localized at the γ-H2AX foci of double-stranded DNA breaks in the nucleus. Thus, Smurf2 has a tumor suppression function that normally maintains genomic stability by controlling the epigenetic landscape of histone modifications through RNF20.

The E3 ubiquitin ligase CTRIP controls CLOCK levels and PERIOD oscillations in Drosophila

In the Drosophila circadian clock, the CLOCK/CYCLE complex activates the period and timeless genes that negatively feedback on CLOCK/CYCLE activity. The 24-h pace of this cycle depends on the stability of the clock proteins. RING-domain E3 ubiquitin ligases have been shown to destabilize PERIOD or TIMELESS. Here we identify a clock function for the circadian trip (ctrip) gene, which encodes a HECT-domain E3 ubiquitin ligase. ctrip expression in the brain is mostly restricted to clock neurons and its downregulation leads to long-period activity rhythms in constant darkness. This altered behaviour is associated with high CLOCK levels and persistence of phosphorylated PERIOD during the subjective day. The control of CLOCK protein levels does not require PERIOD. Thus, CTRIP seems to regulate the pace of the oscillator by controlling the stability of both the activator and the repressor of the feedback loop.

A HECT E3 ubiquitin ligase negatively regulates Arabidopsis leaf senescence through degradation of the transcription factor WRKY53.

WRKY transcription factors play a central role in controlling leaf senescence in Arabidopsis. One important member, WRKY53, is tightly regulated by various mechanisms, and is a convergence node between senescence and pathogen responses. Using WRKY53 in a yeast two-hybrid screen, we isolated the HECT domain E3 ubiquitin ligase UPL5. In contrast to mammals, Arabidopsis contains only seven HECT E3 ubiquitin ligases, whose targets and functions are largely unknown. In yeast cells, UPL5 interacts with WRKY53 via its leucine zipper domain, and this interaction was confirmed in the cytoplasm of plant cells by a bimolecular fluorescence complementation assay. UPL5 was able to use the WRKY53 protein as a substrate for polyubiquitination in an in vitro system, and induction of UPL5 expression by an ethanol-inducible system in upl5 plants led to degradation of the WRKY53 protein. Expression of both genes is regulated antagonistically in response to hydrogen peroxide, jasmonic acid and plant development. Two T-DNA insertion lines (upl5-1 and upl5-2) showed the same senescence phenotype as WRKY53 over-expressers. Over-expression of WRKY53 in the upl5 background enhanced the accelerated senescence phenotype of WRKY53 over-expressers. Therefore, we conclude that UPL5 regulates leaf senescence in Arabidopsis through degradation of WRKY53 and ensures that senescence is executed in the correct time frame.