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E3-ubiquitin ligase Cullin3 (Cul3) is a high confidence risk gene for autism spectrum disorder (ASD) and developmental delay (DD). To investigate how Cul3 mutations impact brain development, we generated a haploinsufficient Cul3 mouse model using CRISPR/Cas9 genome engineering. Cul3 mutant mice exhibited social and cognitive deficits and hyperactive behavior. Brain MRI found decreased volume of cortical regions and changes in many other brain regions of Cul3 mutant mice starting from early postnatal development. Spatiotemporal transcriptomic and proteomic profiling of embryonic, early postnatal and adult brain implicated neurogenesis and cytoskeletal defects as key drivers of Cul3 functional impact. Specifically, dendritic growth, filamentous actin puncta, and spontaneous network activity were reduced in Cul3 mutant mice. Inhibition of small GTPase RhoA, a molecular substrate of Cul3 ligase, rescued dendrite length and network activity phenotypes. Our study identified defects in neuronal cytoskeleton and Rho signaling as the primary targets of Cul3 mutation during brain development.

We combined de novo mutation (DNM) data from 10,927 individuals with developmental delay and autism to identify 253 candidate neurodevelopmental disease genes with an excess of missense and/or likely gene-disruptive (LGD) mutations. Of these genes, 124 reach exome-wide significance (P

Summary BTB-Kelch proteins form the largest subfamily of Cullin-RING E3 ligases, yet their substrate complexes are mapped and structurally characterized only for KEAP1 and KLHL3. KLHL20 is a related CUL3-dependent ubiquitin ligase linked to autophagy, cancer, and Alzheimer's disease that promotes the ubiquitination and degradation of substrates including DAPK1, PML, and ULK1. We identified an “LPDLV”-containing motif in the DAPK1 death domain that determines its recruitment and degradation by KLHL20. A 1.1-Å crystal structure of a KLHL20 Kelch domain-DAPK1 peptide complex reveals DAPK1 binding as a loose helical turn that inserts deeply into the central pocket of the Kelch domain to contact all six blades of the β propeller. Here, KLHL20 forms salt-bridge and hydrophobic interactions including tryptophan and cysteine residues ideally positioned for covalent inhibitor development. The structure highlights the diverse binding modes of β-propeller domains versus linear grooves and suggests a new target for structure-based drug design., Graphical Abstract, Highlights • An “LPDLV” motif in DAPK1 determines its recruitment and degradation by KLHL20 • 1.1-Å crystal structure determined of a KLHL20 Kelch domain-DAPK1 peptide complex • A DAPK1 helical turn inserts into the β propeller to contact all six Kelch repeats • KLHL20 shows a hydrophobic binding pocket suitable for inhibitor development, KLHL20 is a BTB-Kelch family E3 ligase linked to autophagy, cancer, and Alzheimer's disease. Chen et al. identify an “LPDLV” motif in DAPK1 that determines its recruitment and degradation by KLHL20. A 1.1-Å crystal structure of a KLHL20-DAPK1 complex reveals a hydrophobic pocket in KLHL20 suitable for inhibitor development.

Metazoan development depends on tightly regulated gene expression programs that instruct progenitor cells to adopt specialized fates. Recent work found that posttranslational modifications, such as monoubiquitylation, can determine cell fate also independently of effects on transcription, yet how monoubiquitylation is implemented during development is poorly understood. Here, we have identified a regulatory circuit that controls monoubiquitylation-dependent neural crest specification by the E3 ligase CUL3 and its substrate adaptor KBTBD8. We found that CUL3KBTBD8 monoubiquitylates its essential targets only after these have been phosphorylated in multiple motifs by CK2, a kinase whose levels gradually increase during embryogenesis. Its dependency on multisite phosphorylation allows CUL3KBTBD8 to convert the slow rise in embryonic CK2 into decisive recognition of ubiquitylation substrates, which in turn is essential for neural crest specification. We conclude that multisite dependency of an E3 ligase provides a powerful mechanism for switch-like cell fate transitions controlled by monoubiquitylation.

Background: Esophageal squamous cell carcinoma (ESCC) is one of the most lethal cancers in China. Recently, a study identified that cullin 3 (CUL3) was significantly mutated and deleted in ESCC. We then hypothesis that germline variants in CUL3 may also associated with the susceptibility of ESCC. Variants in the gene 3'-untranslated region (3'-UTR) may associate with gene expression by altering miRNAs binding. Material and Methods: We systematically searched for variants in the 3'-UTR of CUL3 using the Ensembl database. Taqman SNP Genotyping Assay was performed in 638 ESCC cases and 546 controls to examine the association between the rs2396092 and the risk of ESCC. The eQTL analysis for CUL3 were conducted by using the GTEx database. Results: We identified that the rs2396092 was significantly associated with the susceptibility of ESCC. Compared with the TT genotype carriers, the CT genotype and CC genotype carriers were correlated with risk of ESCC with odds ratio being 1.33 (95% CI: 1.04-1.70, P=0.0222) and 1.63 (95% CI: 1.07-2.50, P=0.0241), respectively. Different genotypes of rs2396092 was also shown to be correlated with altered CUL3 expression. Conclusion: The results emphasize the importance of CUL3 in the development of ESCC and may contribute to the personalized prevention of this cancer in the future.

Gordon syndrome is a rare inherited monogenic form of hypertension, which is associated with hyperkalaemia and metabolic acidosis. Since the recognition of this predominantly autosomal dominant condition in the 1960s, the study of families with Gordon syndrome has revealed four genes WNK1, WNK4, KLHL3, and CUL3 to be implicated in its pathogenesis after a phenotype–genotype correlation was realised. The encoded proteins Kelch-like 3 and Cullin 3 interact to form a ring-like complex to ubiquitinate WNK-kinase 4, which, in normal circumstances, interacts with the sodium chloride co-symporter (NCC), the epithelial sodium channel (ENaC), and the renal outer medullary potassium channel (ROMK) in an inhibitory manner to maintain normokalaemia and normotension. WNK-kinase 1 has an inhibitory action on WNK-kinase 4. Mutations in WNK1, WNK4, KLHL3, and CUL3 all result in the accumulation of WNK-kinase 4 and subsequent hypertension, hyperkalaemia, and metabolic acidosis. This review explains the clinical aspects, disease mechanisms, and molecular genetics of Gordon syndrome.

Oxidative damage has been associated with various neurodegenerative diseases including Parkinson's disease, amyotrophic lateral sclerosis (ALS), and Alzheimer's disease, as well as non-neurodegenerative conditions such as cancer and heart disease. The Keap1-Nrf2 system plays a central role in the protection of cells against oxidative and xenobiotic stress. The Nrf2 transcription function and its degradation by the proteasomal pathway (Keap1-Nrf2-Cul3-Roc1 complex) are regulated by the cytoplasmic repressor protein, Keap1 which possesses BTB, BACK (IVR region) and Kelch domains. The BTB-BACK domains are important for Keap1 homo-dimerization as well as to interact with Cullin-3 for Nrf2 degradation. The crystal structure of the Keap1-Kelch domain is known; however, that of the BTB-BACK domains are not yet determined. We present here, through molecular modeling studies, the analysis of Keap1-BTB dimerization, and of BTB-BACK domains role in complex with Cul3. The electrostatic charge distribution at the BTB dimer interface of Keap1 is significantly different from other known BTB containing protein structures. Another intriguing feature is also observed that the non-conserved residues at the BTB-BACK-Cul3 interface region may play critical role for differentiating Cul3 recognition by Keap1 from other adaptor proteins for their specific substrates proteasomal degradation.

Drosophila melanogaster is a powerful model organism for dissecting the molecular mechanisms that regulate sleep, and numerous studies in the fly have identified genes that impact sleep–wake cycles. Conditional genetic analysis is essential to distinguish the mechanisms by which these genes impact sleep: some genes might exert their effects developmentally, for instance by directing the assembly of neuronal circuits that regulate sleep; other genes may regulate sleep in adulthood; and yet other genes might influence sleep by both developmental and adult mechanisms. Here we have assessed two ligand-inducible expression systems, Geneswitch and the Q-system, for conditional and neuronally restricted manipulations of sleep in Drosophila. While adult-specific induction of a neuronally expressed Geneswitch transgene (elav-GS) is compatible with studies of sleep as shown previously, developmental induction of elav-GS strongly and nonspecifically perturbs sleep in adults. The alterations of sleep in elav-GS animals occur at low doses of Geneswitch agonist and in the presence of transgenes unrelated to sleep, such as UAS-CD8-GFP. Furthermore, developmental elav-GS induction is toxic and reduces brood size, indicating multiple adverse effects of neuronal Geneswitch activation. In contrast, the transgenes and ligand of the Q-system do not significantly impact sleep–wake cycles when used for constitutive, developmental, or adult-specific neuronal induction. The nonspecific effects of developmental elav-GS activation on sleep indicate that such manipulations require cautious interpretation, and suggest that the Q-system or other strategies may be more suitable for conditional genetic analysis of sleep and other behaviors in Drosophila.

Introduction Inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase beta (IKBKB) (IKK-β/IKK-2), which activates NF-κB, is a substrate of the KEAP1-CUL3-RBX1 E3-ubiquitin ligase complex, implicating this complex in NF-κB pathway regulation. We investigated complex component gene disruption as a novel genetic mechanism of NF-κB activation in non-small cell lung cancer. Methods A total of 644 tumor- and 90 cell-line genomes were analyzed for gene dosage status of the individual complex components and IKBKB. Gene expression of these genes and NF-κB target genes were analyzed in 48 tumors. IKBKB protein levels were assessed in tumors with and without complex or IKBKB genetic disruption. Complex component knockdown was performed to assess effects of the E3-ligase complex on IKBKB and NF-κB levels, and phenotypic importance of IKBKB expression was measured by pharmacological inhibition. Results We observed strikingly frequent genetic disruption (42%) and aberrant expression (63%) of the E3-ligase complex and IKBKB in the samples examined. Although both adenocarcinomas and squamous cell carcinomas showed complex disruption, the patterns of gene disruption differed. IKBKB levels were elevated with complex disruption, knockdown of complex components increased activated forms of IKBKB and NF-κB proteins, and IKBKB inhibition detriments cell viability, highlighting the biological significance of complex disruption. NF-κB target genes were overexpressed in samples with complex disruption, further demonstrating the effect of complex disruption on NF-κB activity. Conclusions Gene dosage alteration is a prominent mechanism that disrupts each component of the KEAP1-CUL3-RBX1 complex and its NF-κB stimulating substrate, IKBKB. Herein, we show that, multiple component disruption of this complex represents a novel mechanism of NF-κB activation in non-small cell lung cancer.

Steroid receptor co-activator-3 (SRC-3/AIB1) is an oncogene that is amplified and overexpressed in many human cancers. However, the molecular mechanisms that regulate 'activated SRC-3 oncoprotein' turnover during tumorigenesis remain to be elucidated. Here, we report that speckle-type POZ protein (SPOP), a cullin 3 (CUL3)-based ubiquitin ligase, is responsible for SRC-3 ubiquitination and proteolysis. SPOP interacts directly with an SRC-3 phospho-degron in a phosphorylation-dependent manner. Casein kinase Iɛ phosphorylates the S102 in this degron and promotes SPOP-dependent turnover of SRC-3. Short hairpin RNA knockdown and overexpression experiments substantiated that the SPOP/CUL3/Rbx1 ubiquitin ligase complex promotes SRC-3 turnover. A systematic analysis of the SPOP genomic locus revealed that a high percentage of genomic loss or loss of heterozygosity occurs at this locus in breast cancers. Furthermore, we demonstrate that restoration of SPOP expression inhibited SRC-3-mediated oncogenic signaling and tumorigenesis, thus positioning SPOP as a tumor suppressor.

Metazoan development depends on accurate execution of differentiation programs that allow pluripotent stem cells to adopt specific fates 1. Differentiation requires changes to chromatin architecture and transcriptional networks, yet whether other regulatory events support cell fate determination is less well understood. Here, we have identified the vertebrate-specific ubiquitin ligase CUL3KBTBD8 as an essential regulator of neural crest specification. CUL3KBTBD8 monoubiquitylates NOLC1 and its paralog TCOF1, whose mutation underlies the neurocristopathy Treacher Collins Syndrome 2,3. Ubiquitylation drives formation of a TCOF1-NOLC1 platform that connects RNA polymerase I with ribosome modification enzymes and remodels the translational program of differentiating cells in favor of neural crest specification. We conclude that ubiquitin-dependent regulation of translation is an important feature of cell fate determination.

Summary The cullin-RING family of ubiquitin ligases regulates diverse cellular functions, such as cell cycle control, via ubiquitylation of specific substrates. CUL3 targets its substrates through BTB proteins. Here we show that depletion of CUL3 and the BTB protein KLHL18 causes a delay in mitotic entry. Centrosomal activation of Aurora-A, a kinase whose activity is required for entry into mitosis, is also delayed in depleted cells. Moreover, we identify Aurora-A as a KLHL18-interacting partner. Overexpression of KLHL18 and CUL3 promotes Aurora-A ubiquitylation in vivo, and the CUL3-KLHL18-ROC1 ligase ubiquitylates Aurora-A in vitro. Our study reveals that the CUL3-KLHL18 ligase is required for timely entry into mitosis, as well as for the activation of Aurora-A at centrosomes. We propose that the CUL3-KLHL18 ligase regulates mitotic entry through an Aurora-A-dependent pathway.

1\. EINLEITUNG 1 1.1 Die BTB-Domäne 1 1.2 Die BTB-Superfamilie 3 1.3 BTB Proteine in Arabidopsis thaliana 5 1.4 Der Ubiquitin-Proteasom-Weg 10 1.5 Ubiquitinierung als regulatorisches Prinzip 13 1.5.1 Selektion der Substratproteine durch Pre-Modifikationen 14 1.5.2 Mono-, Multi- und Polyubiquitinierung 14 1.5.3 Modulation von Ubiquitinketten 15 1.6 Cullin Ubiquitin E3-Ligasen 16 1.6.1 Cullin1 - Der SCF Komplex 16 1.6.2 Cullin4 - DNA damage-binding Proteins (DDB) 17 1.6.3 Der Anaphase Promoting Complex (APC) 17 1.6.4 Cullin3 - BTB Proteine 19 1.7 Die Arabidopsis BPM (BTB/POZ-MATH) Proteinfamilie 19 1.8 Zielsetzung der Arbeit 21 2\. MATERIAL UND METHODEN 23 2.1 Materialien 23 2.1.1 Chemikalien und Oligonukleotide 23 2.1.2 Enzyme, Marker, Antikörper 23 2.1.3 Organismen und deren Anzucht 24 2.1.3.1 Bakterien Stämme 24 2.1.3.2 Saccharomyces cerevisiae Stamm 24 2.1.3.3 Pflanzen 25 2.1.4 Nährmedien, Selektion, Plasmide 25 2.1.4.1 Nährmedien und Selektion - Bakterien 25 2.1.4.2 Nährmedien und Selektion - Saccharomyces cerevisiae 26 2.1.4.3 Nährmedien und Selektion - Arabidopsis thaliana 27 2.1.4.4 Plasmide 28 2.2 Allgemeine Methoden 28 2.3 Molekularbiologische Methoden 28 2.3.1 Herstellung und Transformation chemokompetenter E. coli 28 2.3.2 Herstellung und Transformation elektrokompetenter E. coli 29 2.3.3 Herstellung und Transformation elektrokompetenter A. tumefaciens 29 2.3.4 Isolierung von Plasmid-DNA aus E. coli 30 2.3.5 Isolierung von Plasmid-DNA aus A. tumefaciens 30 2.3.6 DNA-Aufreinigung und Quantifizierung von Nukleinsäuren 31 2.3.7 Isolierung genomischer DNA aus A. thaliana 31 2.3.8 RNA Extraktion aus Pflanzenmaterial 32 2.3.9 Semiquantitative RT PCR 32 2.3.10 Northern-Blot Analyse 33 2.3.10.1 Denaturierende Agarosegelelektrophorese zur Trennung von RNA und Northern-Blottin 33 2.3.10.2 Herstellung radioaktiv markierter Sonden und Northern-Hybridisierung 33 2.3.11 Klassische Klonierung und GatewayTM Klonierungen 34 2.3.12 Proteininteraktionstest in S. cerevisiae mit dem Yeast Two-Hybrid-System 34 2.3.12.1 Herstellung und Transformation kompetenter S. cerevisiae 34 2.3.12.2 Y2H- screening einer cDNA Expressionsbibliothek 35 2.3.12.3 Amplifizierung und Aufreinigung einer Expressionsbibliothek 36 2.3.12.4 Isolierung von Plasmid-DNA aus S. cerevisiae 37 2.3.12.5 Analyse der Plasmide aus dem Hefescreen 38 2.3.13 Zielgerichtete Plasmid-Mutagenese 38 2.4 Pflanzenanzucht und -transformation 38 2.4.1 Samensterilisation und Sterilkultur A. thaliana 38 2.4.2 Anzucht von A. thaliana Pflanzen in Erde 39 2.4.3 Stabile Transformation von A. thaliana durch Agrobakterien 39 2.4.4 Kreuzung von A. thaliana Pflanzen 39 2.4.5 Topfanzucht und Transformation von N. benthamiana Pflanzen 39 2.4.6 Qualitativer Nachweis der GUS-Aktivität durch histologische Färbung 40 2.4.7 Mikroskopie, DAPI-Färbung 41 2.5 Proteinbiochemische Methoden 41 2.5.1 Proteinextraktion aus Pflanzen 41 2.5.2 Expression und Aufreinigung von GST-Fusionsproteinen 42 2.5.3 Proteinbestimmung und SDS-PAGE 42 2.5.4 Western-Blot Analyse 43 2.5.5 DNA- Protein Interaktionen 43 2.5.6 Protein-Protein Interaktionen 44 2.5.6.1 In vitro Transkription/Translation 44 2.5.6.2 in vitro - Interaktionstest 44 2.5.6.3 Interaktionstests in Pflanzenextrakt 45 3\. ERGEBNISSE 47 3.1 Sequenzanalysen der BPM Familie 47 3.2 Interaktionsanalyse innerhalb der BPM Proteinfamilie und mit CUL3 Proteinen 50 3.3. Suche nach BPM- Interaktoren mittels Y2H cDNA library screen 53 3.3.1 Auswahl und Erstellung der Y2H bait- Konstrukte 53 3.3.2 Vorversuche und Durchführung des Hefe-screens 54 3.4. BPM Proteine interagieren mit Proteinen der AP2/ERF Familie 59 3.4.1 RAP2.4 interagiert spezifisch mit den sechs BPM Proteinen 60 3.4.2 BPM Proteine binden spezifisch verschiedene AP2-Proteine 61 3.4.3 RAP2.4 bildet Homo- und Heterodimere 63 3.4.4 Die BPM/RAP2.4 Interaktion erfolgt zwischen der MATH- Domäne und einem N-terminalen Bereich von RAP2.4 64 3.5 Herstellung von Überexpressionspflanzen 67 3.5.1 Interaktionsstudien mit in planta exprimierten Hybridproteinen 70 3.5.1.1 Pulldown-Experimente mit 35S:GFP:BPM4 Pflanzenextrakt (A.thaliana) 70 3.5.1.2 Pulldown-Experimente mit PDX1.3:myc:CUL3A Pflanzenextrakt (A. thaliana) 71 3.5.1.3 Pulldown-Experimente in 35S:RAP2.4:myc Pflanzenextrakt (N. benthamiana) 72 3.5.2 Untersuchungen zur Proteinstabilität von RAP2.4:myc und GFP:BPM4 73 3.6 Subzelluläre Lokalisation 77 3.6.1 Subzelluläre Lokalisation der BPM Proteine 77 3.6.2 Subzelluläre Lokalisation des BPM-Interaktors RAP2.4 82 3.6.3 Subzelluläre Lokalisation des BPM-Interaktors CUL3A 83 3.7 Expressionsanalysen 84 3.7.1 Analyse der BPM Expression anhand von Promotor:GUS Pflanzen 84 3.7.1.1 Expression BPM1 85 3.7.1.2 Expression BPM2 86 3.7.1.3 Expression BPM3 88 3.7.1.4 Expression BPM4 89 3.7.1.5 Expression BPM5 90 3.7.1.6 Expression BPM6 91 3.7.1.7 Zusammenfassung der Ergebnisse der Promotor:GUS Analysen der BPM Gene 93 3.7.1.8 Expression RAP2.4 94 3.7.2 RT-PCR Analyse 96 3.8 Untersuchungen von Insertionsmutanten für BPM2, BPM5, BPM6 und RAP2.4 98 3.8.1 Identifizierung von bpm Mutanten 98 3.8.2 Ergänzender Versuchsansatz zum Ausschalten von BPM1 und BPM4 durch antisense Konstrukte 100 3.8.3 Identifizierung einer rap2.4 Mutante 103 3.8.4 Vergleich von asbpm4, bpm5-2, rap2.4-1 und Col0 unter verschiedenen physiologischen Bedingungen 103 3.9 Induktionsanalysen durch RT- PCR 107 3.10 EMSA Analysen 110 3.10.1 GST:RAP2.4 bindet in vitro an ein Fragment des rd29A Promotors 110 3.10.2 Die Mutagenese der AP2 Domäne führt zum Verlust der DNA-Bindung 111 3.10.3 Die Interaktion BPM/RAP2.4 hat in vitro keinen Einfluß auf die DNA Bindung durch RAP2.4 113 4\. DISKUSSION 115 4.1 Die Interaktoren der BPM Proteine 116 4.2 Kartierung der BPM1/RAP2.4 Interaktion 121 4.3 Stabilitätsanalysen RAP2.4 und BPM4 122 4.4 Die subzelluläre Lokalisation 123 4.5 Die gewebe- und entwicklungsspezifische Expression 124 4.6 Die Wirkung von ABA, ACC, NaCl, Sorbitol und Trockenstress 125 4.7 Eigenschaften der RAP2.4 DNA Bindung 128 4.8 Schlußfolgerungen und Ausblick 129 5\. ZUSAMMENFASSUNG 131 6\. SUMMARY 133 7\. LITERATURVERZEICHNIS 135 8\. ERFOLGTE PUBLIKATIONEN 149 9\. DANKSAGUNG 150 10\. ANHANG 151, BTB-Proteine sind definiert durch das hochgradig konservierte Protein-Protein- Interaktionsmotiv BTB/POZ (Bric-a-Brac/Tramtrack/Broad complex/POX Virus and Zink finger), welches oft in Kombination mit anderen Protein- oder DNA- Bindedomänen auftritt. In Pflanzen und Tieren wurde die Assoziation von BTB- Proteinen mit Cullin3 Proteinen demonstriert. Als Untereinheiten multimerer Ubiquitin-Ligasen vermitteln CUL3-BTB Komplexe die Ubiquitinierung von Substratproteinen, wodurch sie regulierend in biologische Prozesse, wie Entwicklung, Zellzyklus und Pathogenantwort eingreifen. Das Arabidopsis Genom kodiert für zwei CUL3 Proteine und etwa 80 BTB-Proteine. Letztere werden nach ihrer Domänen-Komposition in zehn Familien unterteilt. Die im Rahmen dieser Arbeit untersuchte Arabidopsis BPM (BTB-MATH) Familie umfaßt sechs Mitglieder (61-89 % Identität der Aminosäuresequenzen), die die Fähigkeit zur Homo- und Heterodimerisation besitzen. Neben Interaktionen innerhalb der Familie können BPM1-5 zusätzlich mit CUL3 assoziieren. Diese Ergebnisse führten zu der Hypothese, daß die BPM Proteine Substratadapter von modularen Ubiquitin E3-Ligasen mit CUL3 als zentrale Untereinheit sind. Die Aktivität solcher Komplexe mit CUL3- und BPM-Homologen wurde bereits für Säugetiere und Ceanorhabditis elegans gezeigt. Mittels Promotor:GUS Fusionskonstrukten und RT-PCR Analyse wurde die Expression der BPM Gene in verschiedenen Geweben dokumentiert, und die Induktion der Expression einiger Mitglieder durch die Applikation von Salz, Osmotika bzw. Trockenstress aufgezeigt. Lokalisationsanalysen mit GFP Fusionsproteinen ergaben eine Beschränkung der subzellulären Lokalisation der GFP:BPM Proteine auf den Nukleus und/oder das Cytoplasma. Auf der Suche nach Bindungspartnern und potentiellen Substratproteinen von CUL3-BPM E3-Ligasen wurden in zwei Y2H-screens eine Reihe von Transkriptionsregulatoren der ERF/AP2 (Ethylene Response Factor/APETALA2) Genfamilie als Interaktoren der BPM MATH-Domäne identifiziert. Einer dieser ERF/AP2-Transkriptionsfaktoren, RAP2.4 (At1g78080), wurde im Detail analysiert. RAP2.4 bindet spezifisch die MATH- Domäne der BPM Proteine, innerhalb der Familie aber unspezifisch alle Mitglieder. Eine weitere Kartierung der Bindemotive grenzt die interagierenden Proteinbereiche ein, allerdings wurde weder eine Beteiligung der AP2-Domäne an der BPM1/RAP2.4 Bindung ausgeschlossen, noch ein Konsensusmotiv für mehrere BPM Interaktoren identifiziert. Die Assoziation der BPM Proteine mit Vertretern verschiedener AP2-Familien, bei gleichzeitiger Bindungsspezifität innerhalb der RAP2.4-Unterfamilie, spricht jedoch für ein unabhängiges BPM Bindemotiv. EMSA-Experimente ergaben eine Bindung des RD29A Promotors durch RAP2.4 in vitro, wobei kein Einfluß einer Interaktion mit den BPM Proteinen beobachtet werden konnte. Experimente zur Stabilität des Transkriptionsfaktors zeigten außerdem, daß ein schneller Abbau des RAP2.4 Proteins in planta in Abhängigkeit vom 26S Proteasom erfolgt. Beschränkt durch fehlende Nullmutanten wurden rap2.4-1, bpm5-2, sowie während dieser Arbeit generierte asbpm4 antisense Pflanzen mit dem Wildtyp verglichen. Es wurden keine grundsätzlichen Veränderungen in Morphologie oder Entwicklung beobachtet. Die Ergebnisse der Behandlungen mit Phytohormonen, Salz, Osmotika und Trockenstress gaben Hinweise auf einen funktionalen Zusammenhang von BPMs und RAP2.4 im Bereich der Stresstoleranz. In ihrer Gesamtheit unterstützen die erzielten Ergebnisse die Annahme, daß die Funktion der BPM/RAP2.4 Assoziation die Rekrutierung des RAP2.4 Proteins in eine CUL3 E3-Ligase mit anschließender Ubiquitinierung von RAP2.4 ist. CUL3-BPM E3-Ligasen mit ERF/AP2 Transkriptionsfaktoren als Substratproteine würden einen neuen Regulationsmechanismus transkriptioneller Stressantworten darstellen., The BTB proteins are defined by a highly conserved protein-protein-interaction motif BTB/POZ (Bric-a-Brac/Tramtrack/Broad complex/POX Virus and Zink finger), often combined with a secondary protein- or DNA-binding domain. In plants and animals the association of BTB proteins with Cullin3 proteins was demonstrated. As subunits of multimeric Ubiquitin-ligases CUL3-BTB complexes mediate ubiquitination and subsequent degradation of substrate proteins, and by this regulating diverse biological processes like development, cell cycle and response to pathogens. The Arabidopsis genome encodes two putative redundant CUL3 proteins, called CUL3A and CUL3B, and approximately 80 BTB proteins that are subdivided into 10 families by the composition of their domains. The BTB-MATH (BPM) family, analysed in this work, contains six members (61-89% identity in amino acid sequences) that are capable of forming homo- and heterodimers. Beside the interactions in between the family BPM1-5 can additionally assemble with the CUL3 proteins. These results led to the hypothesis that BPM proteins are substrate adaptors for modular ubiquitin ligases with CUL3 as the central scaffolding subunit. The activity of complexes with CUL3 and BPM-homologes was already shown in mammalia and Ceanorhabditis elegans. Using promoter:GUS fusion constructs and RT-PCR analysis the expression of BPM genes in all tested tissues was documented, and additionally the induction of some of the members by application of salt, osmotics and drought was uncovered. Localisation analysis with GFP fusion proteins showed a restriction of the subcellular localisation of BPM proteins to the nucleus and/or cytoplasm. To search for binding partners and potential substrate proteins of CUL3-BPM E3-ligases two Y2H screens were performed, identifying several transcription regulators of the ERF/AP2 (Ethylene Response Factor/APETALA2) gene family as interactors of the BPM MATH-domain. One of these ERF/AP2 transcription factors, RAP2.4 (At1g78080), was analysed in detail. The protein binds specifically the MATH domain of BPM proteins, but within the BPM family with all members. By further mapping the binding motifs, the interacting areas of the proteins could be narrowed down. However, neither it could be excluded the participation of the AP2 domain, nor a clear consensus sequence for BPM interactors was identified. The assembling of BPM proteins with members of different AP2 families, along with the shown high binding specificity within the subfamily of RAP2.4, argues for an independent BPM binding motif. Using EMSA assays it was demonstrated that RAP2.4 is able to bind the RD29A promoter in vitro and that this interaction is not influenced by assembling with BPM proteins. Moreover the analysis of protein stability of the transcription factor revealed a rapid degradation of RAP2.4 by the 26S proteasome in planta. Restricted by the lack of null mutants in the common seed collections rap2.4-1, bpm5-2 and asbpm4 antisense plants, generated during this project, were compared to wild type plants. No fundamental alterations in development or morphology were observed. Results of diverse treatments with phytohormones, salt, osmotics and drought gave hint to a functional link of BPMs and RAP2.4 in stress tolerance. In summary, the achieved results support the assumption that the function of BPM/RAP2.4 assembling is the recruitment of RAP2.4 to a CUL3-based E3-ligase with subsequent ubiquitination of RAP2.4. CUL3-BPM E3-ligases would constitute a novel regulatory mechanism of transcriptional stress responses.

Summary TP53 deficiency is the most common alteration in cancer; however, this alone is typically insufficient to drive tumorigenesis. To identify genes promoting tumorigenesis in combination with TP53 deficiency, we perform genome-wide CRISPR-Cas9 knockout screens coupled with proliferation and transformation assays in isogenic cell lines. Loss of several known tumor suppressors enhances cellular proliferation and transformation. Loss of neddylation pathway genes promotes uncontrolled proliferation exclusively in TP53-deficient cells. Combined loss of CUL3 and TP53 activates an oncogenic transcriptional program governed by the nuclear factor κB (NF-κB), AP-1, and transforming growth factor β (TGF-β) pathways. This program maintains persistent cellular proliferation, induces partial epithelial to mesenchymal transition, and increases DNA damage, genomic instability, and chromosomal rearrangements. Our findings reveal CUL3 loss as a key event stimulating persistent proliferation in TP53-deficient cells. These findings may be clinically relevant, since TP53-CUL3-deficient cells are highly sensitive to ataxia telangiectasia mutated (ATM) inhibition, exposing a vulnerability that could be exploited for cancer treatment., Graphical Abstract, Highlights • Mixed-effect models with MEMcrispR applied to CRISPR screen analyses • Knockout of neddylation genes increases persistent proliferation in TP53−/− cells • TP53−/−,CUL3−/− cells exhibit persistent proliferation and partial EMT phenotype • TP53−/−,CUL3−/− cells show increased DNA damage and display sensitivity to ATM inhibition, Drainas et al. show that inactivation of genes in the neddylation pathway increases persistent proliferation in TP53-deficient cells. TP53- and CUL3-deficient cells induce an oncogenic transcriptional program, leading to partial EMT and heightened genomic instability. These cells show increased vulnerability to ATM inhibitors.