ABSTRACT
Approximately 30% of human lung cancers acquire mutations in either Keap1 or Nfe2l2, resulting in the stabilization of Nrf2, the Nfe2l2 gene product, which controls oxidative homeostasis. Here, we show that heme triggers the degradation of Bach1, a pro-metastatic transcription factor, by promoting its interaction with the ubiquitin ligase Fbxo22. Nrf2 accumulation in lung cancers causes the stabilization of Bach1 by inducing Ho1, the enzyme catabolizing heme. In mouse models of lung cancers, loss of Keap1 or Fbxo22 induces metastasis in a Bach1-dependent manner. Pharmacological inhibition of Ho1 suppresses metastasis in a Fbxo22-dependent manner. Human metastatic lung cancer display high levels of Ho1 and Bach1. Bach1 transcriptional signature is associated with poor survival and metastasis in lung cancer patients. We propose that Nrf2 activates a metastatic program by inhibiting the heme- and Fbxo22-mediated degradation of Bach1, and that Ho1 inhibitors represent an effective therapeutic strategy to prevent lung cancer metastasis.
Subject(s)
Basic-Leucine Zipper Transcription Factors/metabolism , Lung Neoplasms/pathology , NF-E2-Related Factor 2/metabolism , Animals , Basic-Leucine Zipper Transcription Factors/antagonists & inhibitors , Basic-Leucine Zipper Transcription Factors/genetics , Cell Line, Tumor , Cell Movement , F-Box Proteins/antagonists & inhibitors , F-Box Proteins/genetics , F-Box Proteins/metabolism , Female , Heme Oxygenase-1/antagonists & inhibitors , Heme Oxygenase-1/genetics , Heme Oxygenase-1/metabolism , Humans , Kaplan-Meier Estimate , Kelch-Like ECH-Associated Protein 1/antagonists & inhibitors , Kelch-Like ECH-Associated Protein 1/genetics , Kelch-Like ECH-Associated Protein 1/metabolism , Lung Neoplasms/metabolism , Lung Neoplasms/mortality , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , NF-E2-Related Factor 2/genetics , Neoplasm Metastasis , RNA Interference , RNA, Small Interfering/metabolism , Receptors, Cytoplasmic and Nuclear/antagonists & inhibitors , Receptors, Cytoplasmic and Nuclear/genetics , Receptors, Cytoplasmic and Nuclear/metabolism , Transcriptional ActivationABSTRACT
Proteasome is essential for cell survival, and proteasome inhibition induces proteasomal gene transcription via the activated endoplasmic-reticulum-associated transcription factor nuclear factor erythroid 2-like 1 (Nrf1/NFE2L1). Nrf1 activation requires proteolytic cleavage by DDI2 and N-glycan removal by NGLY1. We previously showed that Nrf1 ubiquitination by SKP1-CUL1-F-box (SCF)FBS2/FBXO6, an N-glycan-recognizing E3 ubiquitin ligase, impairs its activation, although the molecular mechanism remained elusive. Here, we show that SCFFBS2 cooperates with the RING-between-RING (RBR)-type E3 ligase ARIH1 to ubiquitinate Nrf1 through oxyester bonds in human cells. Endo-ß-N-acetylglucosaminidase (ENGASE) generates asparagine-linked N-acetyl glucosamine (N-GlcNAc) residues from N-glycans, and N-GlcNAc residues on Nrf1 served as acceptor sites for SCFFBS2-ARIH1-mediated ubiquitination. We reconstituted the polyubiquitination of N-GlcNAc and serine/threonine residues on glycopeptides and found that the RBR-specific E2 enzyme UBE2L3 is required for the assembly of atypical ubiquitin chains on Nrf1. The atypical ubiquitin chains inhibited DDI2-mediated activation. The present results identify an unconventional ubiquitination pathway that inhibits Nrf1 activation.
Subject(s)
Nuclear Respiratory Factor 1 , Ubiquitination , Humans , HEK293 Cells , Nuclear Respiratory Factor 1/metabolism , Nuclear Respiratory Factor 1/genetics , NF-E2-Related Factor 1/metabolism , NF-E2-Related Factor 1/genetics , Ubiquitin-Protein Ligases/metabolism , Ubiquitin-Protein Ligases/genetics , Acetylglucosamine/metabolism , HeLa Cells , Proteasome Endopeptidase Complex/metabolism , F-Box Proteins/metabolism , F-Box Proteins/geneticsABSTRACT
Proliferation is tightly regulated during T cell development, and is limited to immature CD4-CD8- thymocytes. The major proliferative event is initiated at the 'ß-selection' stage following successful rearrangement of Tcrß, and is triggered by and dependent on concurrent signaling by Notch and the pre-T cell receptor (TCR); however, it is unclear how these signals cooperate to promote cell proliferation. Here, we found that ß-selection-associated proliferation required the combined activity of two Skp-cullin-F-box (SCF) ubiquitin ligase complexes that included as substrate recognition subunits the F-box proteins Fbxl1 or Fbxl12. Both SCF complexes targeted the cyclin-dependent kinase inhibitor Cdkn1b for polyubiquitination and proteasomal degradation. We found that Notch signals induced the transcription of Fbxl1, whereas pre-TCR signals induced the transcription of Fbxl12. Thus, concurrent Notch and pre-TCR signaling induced the expression of two genes, Fbxl1 and Fbxl12, whose products functioned identically but additively to promote degradation of Cdkn1b, cell cycle progression, and proliferation of ß-selected thymocytes.
Subject(s)
F-Box Proteins/metabolism , Receptors, Antigen, T-Cell, alpha-beta/metabolism , Receptors, Notch/metabolism , SKP Cullin F-Box Protein Ligases/metabolism , T-Lymphocytes/physiology , Thymocytes/physiology , Animals , Cell Differentiation , Cell Proliferation , Clonal Selection, Antigen-Mediated , Cyclin-Dependent Kinase Inhibitor p27/metabolism , F-Box Proteins/genetics , Gene Expression Regulation , Genes, T-Cell Receptor beta , Mice , Mice, Inbred C57BL , Receptor Cross-Talk , Signal TransductionABSTRACT
Defects in circadian rhythm influence physiology and behavior with implications for the treatment of sleep disorders, metabolic disease, and cancer. Although core regulatory components of clock rhythmicity have been defined, insight into the mechanisms underpinning amplitude is limited. Here, we show that REV-ERBα, a core inhibitory component of clock transcription, is targeted for ubiquitination and subsequent degradation by the F-box protein FBXW7. By relieving REV-ERBα-dependent repression, FBXW7 provides an unrecognized mechanism for enhancing the amplitude of clock gene transcription. Cyclin-dependent kinase 1 (CDK1)-mediated phosphorylation of REV-ERBα is necessary for FBXW7 recognition. Moreover, targeted hepatic disruption of FBXW7 alters circadian expression of core clock genes and perturbs whole-body lipid and glucose levels. This CDK1-FBXW7 pathway controlling REV-ERBα repression defines an unexpected molecular mechanism for re-engaging the positive transcriptional arm of the clock, as well as a potential route to manipulate clock amplitude via small molecule CDK1 inhibition.
Subject(s)
Circadian Rhythm , F-Box Proteins/metabolism , Liver/metabolism , Nuclear Receptor Subfamily 1, Group D, Member 1/metabolism , Ubiquitin-Protein Ligases/metabolism , Animals , CDC2 Protein Kinase/metabolism , Cell Cycle Proteins/metabolism , Cell Line, Tumor , Circadian Clocks , F-Box Proteins/genetics , F-Box-WD Repeat-Containing Protein 7 , Gene Knockout Techniques , Humans , Lipid Metabolism , Mice , Phosphorylation , Protein Processing, Post-Translational , Transcriptome , Ubiquitin-Protein Ligases/geneticsABSTRACT
Ethylene is a gaseous phytohormone that plays vital roles in plant growth and development. Previous studies uncovered EIN2 as an essential signal transducer linking ethylene perception on ER to transcriptional regulation in the nucleus through a "cleave and shuttle" model. In this study, we report another mechanism of EIN2-mediated ethylene signaling, whereby EIN2 imposes the translational repression of EBF1 and EBF2 mRNA. We find that the EBF1/2 3' UTRs mediate EIN2-directed translational repression and identify multiple poly-uridylates (PolyU) motifs as functional cis elements of 3' UTRs. Furthermore, we demonstrate that ethylene induces EIN2 to associate with 3' UTRs and target EBF1/2 mRNA to cytoplasmic processing-body (P-body) through interacting with multiple P-body factors, including EIN5 and PABs. Our study illustrates translational regulation as a key step in ethylene signaling and presents mRNA 3' UTR functioning as a "signal transducer" to sense and relay cellular signaling in plants. VIDEO ABSTRACT.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , Ethylenes/metabolism , Gene Expression Regulation, Plant , Receptors, Cell Surface/metabolism , Arabidopsis Proteins/genetics , Exoribonucleases/metabolism , F-Box Proteins/genetics , Nucleic Acid Conformation , Plant Proteins/metabolism , Protein Biosynthesis , RNA, Messenger/chemistry , RNA, Messenger/metabolismABSTRACT
The central role of translation in modulating gene activity has long been recognized, yet the systematic exploration of quantitative changes in translation at a genome-wide scale in response to a specific stimulus has only recently become technically feasible. Using the well-characterized signaling pathway of the phytohormone ethylene and plant-optimized genome-wide ribosome footprinting, we have uncovered a molecular mechanism linking this hormone's perception to the activation of a gene-specific translational control mechanism. Characterization of one of the targets of this translation regulatory machinery, the ethylene signaling component EBF2, indicates that the signaling molecule EIN2 and the nonsense-mediated decay proteins UPFs play a central role in this ethylene-induced translational response. Furthermore, the 3'UTR of EBF2 is sufficient to confer translational regulation and required for the proper activation of ethylene responses. These findings represent a mechanistic paradigm of gene-specific regulation of translation in response to a key growth regulator.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , Protein Biosynthesis , Receptors, Cell Surface/metabolism , Signal Transduction , 3' Untranslated Regions , Arabidopsis/genetics , Arabidopsis Proteins/genetics , DNA-Binding Proteins , Ethylenes/metabolism , F-Box Proteins/genetics , Gene Expression Regulation, Plant , Nuclear Proteins/metabolism , RNA, Messenger/metabolism , Ribosomes/metabolism , Transcription Factors/metabolismABSTRACT
Mosaic loss of the X chromosome (mLOX) is the most common clonal somatic alteration in leukocytes of female individuals1,2, but little is known about its genetic determinants or phenotypic consequences. Here, to address this, we used data from 883,574 female participants across 8 biobanks; 12% of participants exhibited detectable mLOX in approximately 2% of leukocytes. Female participants with mLOX had an increased risk of myeloid and lymphoid leukaemias. Genetic analyses identified 56 common variants associated with mLOX, implicating genes with roles in chromosomal missegregation, cancer predisposition and autoimmune diseases. Exome-sequence analyses identified rare missense variants in FBXO10 that confer a twofold increased risk of mLOX. Only a small fraction of associations was shared with mosaic Y chromosome loss, suggesting that distinct biological processes drive formation and clonal expansion of sex chromosome missegregation. Allelic shift analyses identified X chromosome alleles that are preferentially retained in mLOX, demonstrating variation at many loci under cellular selection. A polygenic score including 44 allelic shift loci correctly inferred the retained X chromosomes in 80.7% of mLOX cases in the top decile. Our results support a model in which germline variants predispose female individuals to acquiring mLOX, with the allelic content of the X chromosome possibly shaping the magnitude of clonal expansion.
Subject(s)
Aneuploidy , Chromosomes, Human, X , Clone Cells , Leukocytes , Mosaicism , Adult , Female , Humans , Male , Middle Aged , Alleles , Autoimmune Diseases/genetics , Biological Specimen Banks , Chromosome Segregation/genetics , Chromosomes, Human, X/genetics , Chromosomes, Human, Y/genetics , Clone Cells/metabolism , Clone Cells/pathology , Exome/genetics , F-Box Proteins/genetics , Genetic Predisposition to Disease/genetics , Germ-Line Mutation , Leukemia/genetics , Leukocytes/metabolism , Models, Genetic , Multifactorial Inheritance/genetics , Mutation, Missense/geneticsABSTRACT
A mesenchymal tumor phenotype associates with immunotherapy resistance, although the mechanism is unclear. Here, we identified FBXO7 as a maintenance regulator of mesenchymal and immune evasion phenotypes of cancer cells. FBXO7 bound and stabilized SIX1 co-transcriptional regulator EYA2, stimulating mesenchymal gene expression and suppressing IFNα/ß, chemokines CXCL9/10, and antigen presentation machinery, driven by AXL extracellular ligand GAS6. Ubiquitin ligase SCFFBXW7 antagonized this pathway by promoting EYA2 degradation. Targeting EYA2 Tyr phosphatase activity decreased mesenchymal phenotypes and enhanced cancer cell immunogenicity, resulting in attenuated tumor growth and metastasis, increased infiltration of cytotoxic T and NK cells, and enhanced anti-PD-1 therapy response in mouse tumor models. FBXO7 expression correlated with mesenchymal and immune-suppressive signatures in patients with cancer. An FBXO7-immune gene signature predicted immunotherapy responses. Collectively, the FBXO7/EYA2-SCFFBXW7 axis maintains mesenchymal and immune evasion phenotypes of cancer cells, providing rationale to evaluate FBXO7/EYA2 inhibitors in combination with immune-based therapies to enhance onco-immunotherapy responses.
Subject(s)
F-Box Proteins , F-Box-WD Repeat-Containing Protein 7 , Neoplasms , Animals , Cell Line, Tumor , F-Box Proteins/genetics , F-Box Proteins/metabolism , F-Box-WD Repeat-Containing Protein 7/genetics , Homeodomain Proteins/genetics , Humans , Immune Evasion , Intracellular Signaling Peptides and Proteins/metabolism , Mice , Neoplasms/genetics , Nuclear Proteins/metabolism , Phenotype , Protein Tyrosine Phosphatases/genetics , Ubiquitin/metabolismABSTRACT
Disruption of antagonism between SWI/SNF chromatin remodelers and polycomb repressor complexes drives the formation of numerous cancer types. Recently, an inhibitor of the polycomb protein EZH2 was approved for the treatment of a sarcoma mutant in the SWI/SNF subunit SMARCB1, but resistance occurs. Here, we performed CRISPR screens in SMARCB1-mutant rhabdoid tumor cells to identify genetic contributors to SWI/SNF-polycomb antagonism and potential resistance mechanisms. We found that loss of the H3K36 methyltransferase NSD1 caused resistance to EZH2 inhibition. We show that NSD1 antagonizes polycomb via cooperation with SWI/SNF and identify co-occurrence of NSD1 inactivation in SWI/SNF-defective cancers, indicating in vivo relevance. We demonstrate that H3K36me2 itself has an essential role in the activation of polycomb target genes as inhibition of the H3K36me2 demethylase KDM2A restores the efficacy of EZH2 inhibition in SWI/SNF-deficient cells lacking NSD1. Together our data expand the mechanistic understanding of SWI/SNF and polycomb interplay and identify NSD1 as the key for coordinating this transcriptional control.
Subject(s)
Enhancer of Zeste Homolog 2 Protein , F-Box Proteins , Histone-Lysine N-Methyltransferase , Jumonji Domain-Containing Histone Demethylases , Polycomb-Group Proteins , SMARCB1 Protein , Chromatin/genetics , Chromatin/metabolism , Enhancer of Zeste Homolog 2 Protein/antagonists & inhibitors , Enhancer of Zeste Homolog 2 Protein/genetics , Enhancer of Zeste Homolog 2 Protein/metabolism , F-Box Proteins/genetics , F-Box Proteins/metabolism , Histone-Lysine N-Methyltransferase/genetics , Histone-Lysine N-Methyltransferase/metabolism , Histones/genetics , Histones/metabolism , Humans , Jumonji Domain-Containing Histone Demethylases/genetics , Jumonji Domain-Containing Histone Demethylases/metabolism , Polycomb-Group Proteins/genetics , Polycomb-Group Proteins/metabolism , Rhabdoid Tumor/genetics , Rhabdoid Tumor/metabolism , Rhabdoid Tumor/pathology , SMARCB1 Protein/genetics , SMARCB1 Protein/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Transcriptional Activation/genetics , Tumor Cells, Cultured/metabolismABSTRACT
Innate lymphoid cells (ILCs) communicate with other hematopoietic and nonhematopoietic cells to regulate immunity, inflammation and tissue homeostasis. How ILC lineages develop and are maintained remains largely unknown. In this study we observed that a divergent long noncoding RNA (lncRNA), lncKdm2b, was expressed at high levels in intestinal group 3 ILCs (ILC3s). LncKdm2b deficiency in the hematopoietic system led to reductions in the number and effector functions of ILC3s. LncKdm2b expression sustained the maintenance of ILC3s by promoting their proliferation through activation of the transcription factor Zfp292. Mechanistically, lncKdm2b recruited the chromatin organizer Satb1 and the nuclear remodeling factor (NURF) complex onto the Zfp292 promoter to initiate its transcription. Deletion of Zfp292 or Bptf also abrogated the maintenance of ILC3s, leading to susceptibility to bacterial infection. Therefore, our findings reveal that lncRNAs may represent an additional layer of regulation of ILC development and function.
Subject(s)
Bacterial Infections/genetics , F-Box Proteins/genetics , Immunity, Innate , Jumonji Domain-Containing Histone Demethylases/genetics , Lymphocytes/physiology , RNA, Long Noncoding/genetics , Animals , Antigens, Nuclear/genetics , Cell Differentiation/genetics , Cell Lineage/genetics , Cell Proliferation/genetics , Chromatin Assembly and Disassembly , DNA-Binding Proteins/genetics , Disease Susceptibility , Matrix Attachment Region Binding Proteins/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Nerve Tissue Proteins/genetics , Transcription Factors/genetics , Transcriptional ActivationABSTRACT
Chromatin modifying activities inherent to polycomb repressive complexes PRC1 and PRC2 play an essential role in gene regulation, cellular differentiation, and development. However, the mechanisms by which these complexes recognize their target sites and function together to form repressive chromatin domains remain poorly understood. Recruitment of PRC1 to target sites has been proposed to occur through a hierarchical process, dependent on prior nucleation of PRC2 and placement of H3K27me3. Here, using a de novo targeting assay in mouse embryonic stem cells we unexpectedly discover that PRC1-dependent H2AK119ub1 leads to recruitment of PRC2 and H3K27me3 to effectively initiate a polycomb domain. This activity is restricted to variant PRC1 complexes, and genetic ablation experiments reveal that targeting of the variant PCGF1/PRC1 complex by KDM2B to CpG islands is required for normal polycomb domain formation and mouse development. These observations provide a surprising PRC1-dependent logic for PRC2 occupancy at target sites in vivo.
Subject(s)
Embryonic Stem Cells/metabolism , F-Box Proteins/metabolism , Histones/metabolism , Jumonji Domain-Containing Histone Demethylases/metabolism , Polycomb Repressive Complex 1/metabolism , Polycomb Repressive Complex 2/metabolism , Animals , Bone Development , CpG Islands , F-Box Proteins/chemistry , F-Box Proteins/genetics , Genes, Lethal , Genome-Wide Association Study , Jumonji Domain-Containing Histone Demethylases/chemistry , Jumonji Domain-Containing Histone Demethylases/genetics , Mice , Protein Structure, TertiaryABSTRACT
Period determination in the mammalian circadian clock involves the turnover rate of the repressors CRY and PER. We show that CRY ubiquitination engages two competing E3 ligase complexes that either lengthen or shorten circadian period in mice. Cloning of a short-period circadian mutant, Past-time, revealed a glycine to glutamate missense mutation in Fbxl21, an F-box protein gene that is a paralog of Fbxl3 that targets the CRY proteins for degradation. While loss of function of FBXL3 leads to period lengthening, mutation of Fbxl21 causes period shortening. FBXL21 forms an SCF E3 ligase complex that slowly degrades CRY in the cytoplasm but antagonizes the stronger E3 ligase activity of FBXL3 in the nucleus. FBXL21 plays a dual role: protecting CRY from FBXL3 degradation in the nucleus and promoting CRY degradation within the cytoplasm. Thus, the balance and cellular compartmentalization of competing E3 ligases for CRY determine circadian period of the clock in mammals.
Subject(s)
Cryptochromes/metabolism , F-Box Proteins/metabolism , Animals , CLOCK Proteins/genetics , Cell Nucleus/metabolism , Crosses, Genetic , Cytoplasm/metabolism , F-Box Proteins/genetics , Female , Male , Mice , Mice, Inbred C3H , Mice, Inbred C57BL , ProteolysisABSTRACT
In the mammalian circadian clockwork, CRY1 and CRY2 repressor proteins are regulated by posttranslational modifications for temporally coordinated transcription of clock genes. Previous studies revealed that FBXL3, an F-box-type E3 ligase, ubiquitinates CRYs and mediates their degradation. Here, we found that FBXL21 also ubiquitinates CRYs but counteracts FBXL3. Fbxl21(-/-) mice exhibited normal periodicity of wheel-running rhythms with compromised organization of daily activities, while an extremely long-period phenotype of Fbxl3(-/-) mice was attenuated in Fbxl3/Fbxl21 double-knockout mice. The double knockout destabilized the behavioral rhythms progressively and sometimes elicited arrhythmicity. Surprisingly, FBXL21 stabilized CRYs and antagonized the destabilizing action by FBXL3. Predominantly cytosolic distribution of FBXL21 contrasts with nuclear localization of FBXL3. These results emphasize the physiological importance of antagonizing actions between FBXL21 and FBXL3 on CRYs, and their combined actions at different subcellular locations stabilize oscillation of the circadian clock.
Subject(s)
Circadian Clocks , Cryptochromes/metabolism , F-Box Proteins/metabolism , Amino Acid Sequence , Animals , Embryo, Mammalian/cytology , Embryo, Mammalian/metabolism , F-Box Proteins/genetics , Fibroblasts , Mice , Mice, Knockout , Molecular Sequence Data , Multiprotein Complexes , Sequence Alignment , UbiquitinationABSTRACT
Sequencing efforts led to the identification of somatic mutations that could affect the self-renewal and differentiation of cancer-initiating cells. One such recurrent mutation targets the binding pocket of the ubiquitin ligase Fbxw7. Missense FBXW7 mutations are prevalent in various tumors, including T cell acute lymphoblastic leukemia (T-ALL). To study the effects of such lesions, we generated animals carrying regulatable Fbxw7 mutant alleles. Here, we show that these mutations specifically bolster cancer-initiating cell activity in collaboration with Notch1 oncogenes but spare normal hematopoietic stem cell function. We were also able to show that FBXW7 mutations specifically affect the ubiquitylation and half-life of c-Myc protein, a key T-ALL oncogene. Using animals carrying c-Myc fusion alleles, we connected Fbxw7 function to c-Myc abundance and correlated c-Myc expression to leukemia-initiating activity. Finally, we demonstrated that small-molecule-mediated suppression of MYC activity leads to T-ALL remission, suggesting an effective therapeutic strategy.
Subject(s)
Cell Cycle Proteins/metabolism , F-Box Proteins/metabolism , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Proto-Oncogene Proteins c-myc/metabolism , Ubiquitin-Protein Ligases/metabolism , Animals , Cell Cycle Proteins/genetics , Disease Models, Animal , F-Box Proteins/genetics , F-Box-WD Repeat-Containing Protein 7 , Hematopoietic Stem Cells/metabolism , Humans , Mice , Mice, Knockout , Mutation, Missense , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/genetics , Proto-Oncogene Proteins c-myc/antagonists & inhibitors , Receptor, Notch1/metabolism , Tumor Suppressor Protein p53/metabolism , Ubiquitin-Protein Ligases/genetics , UbiquitinationABSTRACT
The phytohormone auxin is the major coordinative signal in plant development1, mediating transcriptional reprogramming by a well-established canonical signalling pathway. TRANSPORT INHIBITOR RESPONSE 1 (TIR1)/AUXIN-SIGNALING F-BOX (AFB) auxin receptors are F-box subunits of ubiquitin ligase complexes. In response to auxin, they associate with Aux/IAA transcriptional repressors and target them for degradation via ubiquitination2,3. Here we identify adenylate cyclase (AC) activity as an additional function of TIR1/AFB receptors across land plants. Auxin, together with Aux/IAAs, stimulates cAMP production. Three separate mutations in the AC motif of the TIR1 C-terminal region, all of which abolish the AC activity, each render TIR1 ineffective in mediating gravitropism and sustained auxin-induced root growth inhibition, and also affect auxin-induced transcriptional regulation. These results highlight the importance of TIR1/AFB AC activity in canonical auxin signalling. They also identify a unique phytohormone receptor cassette combining F-box and AC motifs, and the role of cAMP as a second messenger in plants.
Subject(s)
Adenylyl Cyclases , Arabidopsis Proteins , Arabidopsis , F-Box Proteins , Indoleacetic Acids , Receptors, Cell Surface , Adenylyl Cyclases/genetics , Adenylyl Cyclases/metabolism , Arabidopsis/enzymology , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , F-Box Proteins/genetics , F-Box Proteins/metabolism , Gene Expression Regulation, Plant , Indoleacetic Acids/metabolism , Indoleacetic Acids/pharmacology , Plant Growth Regulators/pharmacology , Plant Growth Regulators/metabolism , Receptors, Cell Surface/genetics , Receptors, Cell Surface/metabolism , Mutation , Gravitropism , Plant Roots/growth & development , Cyclic AMP/metabolism , Second Messenger SystemsABSTRACT
Both an increased frequency of chromosome missegregation (chromosomal instability, CIN) and the presence of an abnormal complement of chromosomes (aneuploidy) are hallmarks of cancer. To better understand how cells are able to adapt to high levels of chromosomal instability, we previously examined yeast cells that were deleted of the gene BIR1, a member of the chromosomal passenger complex (CPC). We found bir1Δ cells quickly adapted by acquiring specific combinations of beneficial aneuploidies. In this study, we monitored these yeast strains for longer periods of time to determine how cells adapt to high levels of both CIN and aneuploidy in the long term. We identify suppressor mutations that mitigate the chromosome missegregation phenotype. The mutated proteins fall into four main categories: outer kinetochore subunits, the SCFCdc4 ubiquitin ligase complex, the mitotic kinase Mps1, and the CPC itself. The identified suppressor mutations functioned by reducing chromosomal instability rather than alleviating the negative effects of aneuploidy. Following the accumulation of suppressor point mutations, the number of beneficial aneuploidies decreased. These experiments demonstrate a time line of adaptation to high rates of CIN.
Subject(s)
F-Box Proteins , Neoplasms , Saccharomyces cerevisiae Proteins , Saccharomycetales , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomycetales/genetics , Saccharomycetales/metabolism , Aneuploidy , Chromosomal Instability/genetics , Kinetochores/metabolism , Neoplasms/genetics , Chromosome Segregation , Cell Cycle Proteins/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Ubiquitin-Protein Ligases/metabolism , F-Box Proteins/geneticsABSTRACT
Roses are among the most popular ornamental plants cultivated worldwide for their great economic, symbolic, and cultural importance. Nevertheless, rapid petal senescence markedly reduces rose (Rosa hybrida) flower quality and value. Petal senescence is a developmental process tightly regulated by various phytohormones. Ethylene accelerates petal senescence, while gibberellic acid (GA) delays this process. However, the molecular mechanisms underlying the crosstalk between these phytohormones in the regulation of petal senescence remain largely unclear. Here, we identified SENESCENCE-ASSOCIATED F-BOX (RhSAF), an ethylene-induced F-box protein gene encoding a recognition subunit of the SCF-type E3 ligase. We demonstrated that RhSAF promotes degradation of the GA receptor GIBBERELLIN INSENSITIVE DWARF1 (RhGID1) to accelerate petal senescence. Silencing RhSAF expression delays petal senescence, while suppressing RhGID1 expression accelerates petal senescence. RhSAF physically interacts with RhGID1s and targets them for ubiquitin/26S proteasome-mediated degradation. Accordingly, ethylene-induced RhGID1C degradation and RhDELLA3 accumulation are compromised in RhSAF-RNAi lines. Our results demonstrate that ethylene antagonizes GA activity through RhGID1 degradation mediated by the E3 ligase RhSAF. These findings enhance our understanding of the phytohormone crosstalk regulating petal senescence and provide insights for improving flower longevity.
Subject(s)
Ethylenes , F-Box Proteins , Flowers , Gene Expression Regulation, Plant , Gibberellins , Plant Proteins , Rosa , Ethylenes/metabolism , Ethylenes/pharmacology , Gibberellins/metabolism , Gibberellins/pharmacology , F-Box Proteins/metabolism , F-Box Proteins/genetics , Plant Proteins/metabolism , Plant Proteins/genetics , Rosa/genetics , Rosa/drug effects , Rosa/metabolism , Flowers/genetics , Flowers/drug effects , Flowers/growth & development , Gene Expression Regulation, Plant/drug effects , Plant Growth Regulators/metabolism , Plant Growth Regulators/pharmacology , Plant Senescence/genetics , Proteasome Endopeptidase Complex/metabolism , Receptors, Cell Surface/metabolism , Receptors, Cell Surface/geneticsABSTRACT
Pancreatic ductal adenocarcinoma (PDAC) poses a significant threat due to its tendency to evade early detection, frequent metastasis, and the subsequent challenges in devising effective treatments. Processes that govern epithelial-mesenchymal transition (EMT) in PDAC hold promise for advancing novel therapeutic strategies. SAMD1 (SAM domain-containing protein 1) is a CpG island-binding protein that plays a pivotal role in the repression of its target genes. Here, we revealed that SAMD1 acts as a repressor of genes associated with EMT. Upon deletion of SAMD1 in PDAC cells, we observed significantly increased migration rates. SAMD1 exerts its effects by binding to specific genomic targets, including CDH2, encoding N-cadherin, which emerged as a driver of enhanced migration upon SAMD1 knockout. Furthermore, we discovered the FBXO11-containing E3 ubiquitin ligase complex as an interactor and negative regulator of SAMD1, which inhibits SAMD1 chromatin-binding genome-wide. High FBXO11 expression in PDAC is associated with poor prognosis and increased expression of EMT-related genes, underlining an antagonistic relationship between SAMD1 and FBXO11. In summary, our findings provide insights into the regulation of EMT-related genes in PDAC, shedding light on the intricate role of SAMD1 and its interplay with FBXO11 in this cancer type.
Subject(s)
Carcinoma, Pancreatic Ductal , Epithelial-Mesenchymal Transition , F-Box Proteins , Gene Expression Regulation, Neoplastic , Pancreatic Neoplasms , Receptors, LDL , Animals , Humans , Cadherins/metabolism , Cadherins/genetics , Carcinoma, Pancreatic Ductal/genetics , Carcinoma, Pancreatic Ductal/metabolism , Carcinoma, Pancreatic Ductal/pathology , Cell Line, Tumor , Cell Movement/genetics , Epithelial-Mesenchymal Transition/genetics , F-Box Proteins/metabolism , F-Box Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Intracellular Signaling Peptides and Proteins/genetics , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/metabolism , Pancreatic Neoplasms/pathology , Prognosis , Receptors, LDL/genetics , Receptors, LDL/metabolismABSTRACT
The iron-sensing protein FBXL5 is the substrate adaptor for a SKP1-CUL1-RBX1 E3 ubiquitin ligase complex that regulates the degradation of iron regulatory proteins (IRPs). Here, we describe a mechanism of FBXL5 regulation involving its interaction with the cytosolic Fe-S cluster assembly (CIA) targeting complex composed of MMS19, FAM96B, and CIAO1. We demonstrate that the CIA-targeting complex promotes the ability of FBXL5 to degrade IRPs. In addition, the FBXL5-CIA-targeting complex interaction is regulated by oxygen (O2) tension displaying a robust association in 21% O2 that is severely diminished in 1% O2 and contributes to O2-dependent regulation of IRP degradation. Together, these data identify a novel oxygen-dependent signaling axis that links IRP-dependent iron homeostasis with the Fe-S cluster assembly machinery.
Subject(s)
Cell Cycle Proteins/genetics , F-Box Proteins/genetics , Molecular Chaperones/genetics , Multiprotein Complexes/genetics , Ubiquitin-Protein Ligase Complexes/genetics , Cell Cycle Proteins/chemistry , F-Box Proteins/chemistry , HeLa Cells , Homeostasis , Humans , Iron/metabolism , Iron-Regulatory Proteins/genetics , Iron-Sulfur Proteins/chemistry , Iron-Sulfur Proteins/genetics , Molecular Chaperones/chemistry , Multiprotein Complexes/chemistry , Oxygen/metabolism , Proteolysis , Transcription Factors/genetics , Ubiquitin-Protein Ligase Complexes/chemistryABSTRACT
The BRCA1-BRCA2-RAD51 axis is essential for homologous recombination repair (HRR) and is frequently disrupted in breast cancers. PARP inhibitors (PARPis) are used clinically to treat BRCA-mutated breast tumors. Using a genetic screen, we identified EMI1 as a modulator of PARPi sensitivity in triple-negative breast cancer (TNBC) cells. This function requires the F-box domain of EMI1, through which EMI1 assembles a canonical SCF ubiquitin ligase complex that constitutively targets RAD51 for degradation. In response to genotoxic stress, CHK1-mediated phosphorylation of RAD51 counteracts EMI1-dependent degradation by enhancing RAD51's affinity for BRCA2, leading to RAD51 accumulation. Inhibition of RAD51 degradation restores HRR in BRCA1-depleted cells. Human breast cancer samples display an inverse correlation between EMI1 and RAD51 protein levels. A subset of BRCA1-deficient TNBC cells develop resistance to PARPi by downregulating EMI1 and restoring RAD51-dependent HRR. Notably, reconstitution of EMI1 expression reestablishes PARPi sensitivity both in cellular systems and in an orthotopic mouse model.