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The F-box proteins (FBP), substrate recognition subunit of the SCF (Skp1-Cullin1-F-box protein complex) E3 ligase, play important roles in the ubiquitylation and subsequent degradation of the target proteins from several cellular processes. Disorders of F-box protein-mediated proteolysis lead to human malignancies. FBP plays an important role in many cellular processes, including cell proliferation, cell cycle, apoptosis, migration, invasion, and metastasis, suggesting that it can be associated with tumorigenesis, cancer development and progression. However, the expression and function of FBXO9 (F-box only protein 9) differ in various types of human cancer. Due to the ability to regulate the stability and activity of oncogenes and tumor-suppressor genes, and the physiological functions of many of the F-box proteins remain subtle, further genetic and mechanistic studies will elaborate and help define FBXO9's role. Targeting F-box protein or F-box protein signaling pathways could be an effective strategy for preventing or treating human cancer. This review is presented to summarize the part of FBXO9 in different types of human cancer and its regulation mechanism, and to pave the way to design FBXO9-targeting anticancer therapies., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

Preconditioning with a mild stressor such as fasting is a promising way to reduce severe side effects from subsequent chemo- or radiotherapy. However, the underlying mechanisms have been largely unexplored. Here, we demonstrate that the TP53/p53-FBXO22-TFEB (transcription factor EB) axis plays an essential role in this process through upregulating basal macroautophagy/autophagy. Mild stress-activated TP53 transcriptionally induced FBXO22, which in turn ubiquitinated KDM4B (lysine-specific demethylase 4B) complexed with MYC-NCOR1 suppressors for degradation, leading to transcriptional induction of TFEB. Upregulation of autophagy-related genes by increased TFEB dramatically enhanced autophagic activity and cell survival upon following a severe stressor. Mitogen-induced AKT1 activation counteracted this process through the phosphorylation of KDM4B, which inhibited FBXO22-mediated ubiquitination. Additionally, fbxo22 -/- mice died within 10 h of birth, and their mouse embryonic fibroblasts (MEFs) showed a lowered basal autophagy, whereas FBXO22-overexpressing mice were resistant to chemotherapy. Taken together, these results suggest that TP53 upregulates basal autophagy through the FBXO22-TFEB axis, which governs the hormetic effect in chemotherapy. Abbreviations : BBC3/PUMA: BCL2 binding component 3; CDKN1A/p21: cyclin dependent kinase inhibitor 1A; ChIP-seq: chromatin immunoprecipitation followed by sequencing; DDB2: damage specific DNA binding protein 2; DRAM: DNA damage regulated autophagy modulator; ESR/ER: estrogen receptor 1; FMD: fasting mimicking diet; HCQ: hydroxychloroquine; KDM4B: lysine-specific demethylase 4B; MAP1LC3/LC3: microtubule associated protein 1 light chain 3 alpha; MEFs: mouse embryonic fibroblasts; MTOR: mechanistic target of rapamycin kinase; NCOR1: nuclear receptor corepressor 1; SCF: SKP1-CUL-F-box protein; SQSTM1: sequestosome 1; TFEB: transcription factor EB.

Purpose: To uncover the role of FBXL19-AS1 in aggravating the progression of hepatocellular cancer (HCC) by downregulating kruppel-likefactor2 (KLF2)., Methods: FBXL19-AS1 level in HCC tissues and adjacent normal tissues were firstly determined. Its level in HCC with different tumor sizes (≤ 5 cm or > 5 cm) and different tumor stages (stage I-II or III-IV) was examined as well. Subcellular distribution of FBXL19-AS1 was detected. The regulatory effect of FBXL19-AS1 on viability, apoptosis and cell cycle progression of HCC cells was assessed. RNA immunoprecipitation (RIP) assay was conducted to explore the interaction between FBXL19-AS1 with EZH2 and SUZ12. Moreover, chromatin immunoprecipitation (ChIP) assay was carried out to identify the recruitment ability of FBXL19-AS1 on EZH2 and H3K27me3. Finally, the potential role of KLF2 in FBXL19-AS1-mediated HCC proliferation was investigated., Results: FBXL19-AS1 was highly expressed in HCC tissues, especially in those larger than 5 cm in tumor size and worse tumor stage. FBXL19-AS1 was mainly distributed in nucleus and interacted with EZH2 and SUZ12. Knockdown of FBXL19-AS1 suppressed proliferation, cell cycle progression and induced apoptosis of HCC cells. Moreover, silence of FBXL19-AS1 attenuated the recruitment ability of EZH2 on KLF2. Knockdown of KLF2 reversed the regulatory effect of FBXL19-AS1 on proliferative ability of HCC cells., Conclusions: Long non-coding RNA (lncRNA) FBXL19-AS1 is upregulated in HCC. It accelerates proliferative ability, cell cycle progression and suppresses apoptosis of tumor cells through interacting with KLF2, thus aggravating the progression of HCC.

The C2H2-type zinc finger transcription factor sensitive to proton rhizotoxicity 1 (STOP1) is crucial for aluminum (Al) resistance in Arabidopsis. The F-box protein Regulation of AtALMT1 Expression 1 (RAE1) was recently reported to regulate the stability of STOP1. There is a unique homolog of RAE1, RAH1 (RAE1 homolog 1), in Arabidopsis, but the biological function of RAH1 is still not known. In this study, we characterize the role of RAH1 and/or RAE1 in the regulation of Al resistance and plant growth. We demonstrate that RAH1 can directly interact with STOP1 and promote its ubiquitination and degradation. RAH1 is preferentially expressed in root caps and various vascular tissues, and its expression is induced by Al and controlled by STOP1. Mutation of RAH1 in rae1 but not the wild-type (WT) background increases the level of STOP1 protein, leading to increased expression of STOP1-regulated genes and enhanced Al resistance. Interestingly, the rah1rae1 double mutant shows reduced plant growth compared with the WT and single mutants under normal conditions, and introduction of stop1 mutation into the double mutant background can rescue its reduced plant growth phenotype. Our results thus reveal that RAH1 plays an unequally redundant role with RAE1 in the modulation of STOP1 stability and plant growth, and dynamic regulation of the STOP1 level is critical for the balance of Al resistance and normal plant growth., (© 2021 Society for Experimental Biology and John Wiley & Sons Ltd.)

In Caenorhabditis elegans , axon regeneration is activated by a signaling cascade through the receptor tyrosine kinase (RTK) SVH-2. Axonal injury induces svh-2 gene expression by degradation of the Mad-like transcription factor MDL-1. In this study, we identify the svh-24 / sdz-33 gene encoding a protein containing F-box and F-box-associated domains as a regulator of axon regeneration in motor neurons. We find that sdz-33 is required for axon injury-induced svh-2 expression. SDZ-33 targets MDL-1 for poly-ubiquitylation and degradation. Furthermore, we demonstrate that SDZ-33 promotes axotomy-induced nuclear degradation of MDL-1, resulting in the activation of svh-2 expression in animals. These results suggest that the F-box protein is required for RTK signaling in the control of axon regeneration. SIGNIFICANCE STATEMENT In Caenorhabditis elegans , axon regeneration is positively regulated by the growth factor SVH-1 and its receptor tyrosine kinase SVH-2. Expression of the svh-2 gene is induced by axonal injury via the Ets-like transcription factor ETS-4, whose transcriptional activity is inhibited by the Mad-like transcription factor MDL-1. Axon injury leads to the degradation of MDL-1, and this is linked to the activation of ETS-4 transcriptional activity. In this study, we identify the sdz-33 gene encoding a protein containing an F-box domain as a regulator of axon regeneration. We demonstrate that MDL-1 is poly-ubiquitylated and degraded through the SDZ-33-mediated 26S proteasome pathway. These results reveal that an F-box protein promotes axon regeneration by degrading the Mad transcription factor., (Copyright © 2021 Shimizu, Pastuhov et al.)

Macroautophagy/autophagy is a highly conserved eukaryotic molecular process that facilitates the recycling of superfluous cytoplasmic materials, damaged organelles, and invading pathogens, resulting in proper cellular homeostasis and survival during stress conditions. Autophagy is stringently regulated at multiple stages, including control at transcriptional, translational, and posttranslational levels. In this work, we identified a mechanism by which regulation of autophagy is achieved through the posttranslational modification of Atg9. Here, we show that, in order to limit autophagy to a low, basal level during normal conditions, Atg9 is ubiquitinated and subsequently targeted for degradation in a proteasome-dependent manner through the action of the E3 ligase Met30. When cells require increased autophagy flux to respond to nutrient deprivation, the proteolysis of Atg9 is significantly reduced. Overall, this work reveals an additional layer of mechanistic regulation that allows cells to further maintain appropriate levels of autophagy and to rapidly induce this process in response to stress., Competing Interests: The authors declare no competing interest.

Germline specification is a fundamental step for human reproduction and this biological phenomenon possesses technical challenges to study in vivo as it occurs immediately after blastocyst implantation. The establishment of in vitro human primordial germ cell-like cells (hPGCLCs) induction system allows sophisticated characterization of human primordial germ cells (hPGCs) development. However, the underlying molecular mechanisms of hPGCLC specification are not fully elucidated. Here, we observed particularly high expression of the histone demethylase KDM2B in male fetal germ cells (FGCs) but not in male somatic cells. Besides, KDM2B shared similar expression pattern with hPGC marker genes in hPGCLCs, suggesting an important role of KDM2B in germ cell development. Although deletion of KDM2B had no significant effects on human embryonic stem cell (hESC)'s pluripotency, loss of KDM2B dramatically impaired hPGCLCs differentiation whereas ectopically expressed KDM2B could efficiently rescue such defect, indicating this defect was due to KDM2B 's loss in hPGCLC specification. Mechanistically, as revealed by the transcriptional profiling, KDM2B suppressed the expression of somatic genes thus inhibited somatic differentiation during hPGCLC specification. These data collectively indicate that KDM2B is an indispensable epigenetic regulator for hPGCLC specification, shedding lights on how epigenetic regulations orchestrate transcriptional events in hPGC development for future investigation., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Improving plant water-use efficiency (WUE) is important to plant survival and crop yield in the context of water limitation. In this study, SlTLFP8 (Tubby-like F-box protein 8) was identified as an osmotic-induced gene in tomato. Transgenic tomato with up-regulated expression of SlTLFP8 showed enhanced water-deficient resistance, whereas knockout mutants generated by CRISPR/Cas9 were more sensitive to water deficit. SlTLFP8 overexpression significantly enhanced WUE by suppressing transpiration under both water-sufficient and water-deficient conditions. Further study showed that overexpressing SlTLFP8 significantly increased leaf epidermal cell size and thereby decreased stomatal density 10-20%, conversely SlTLFP8 knockout resulted in decreased cell size and thereby increased stomatal density 20-50%. SlTLFP8 overexpression and knockout modulated ploidy levels in leaf cells. Changes in expression of cell cycle related genes also indicated that SlTLFP8 affected cell size and stomatal density through endocycle transition. Despite changes in stomata density and transpiration, altering the expression of SlTLFP8 did not change photosynthesis. Additionally, biomass was not altered and there was little difference in fruit yield for transgenic and wild type lines under water-sufficient and water-deficient conditions. Our results demonstrate the effect of SlTLFP8 on endoreduplication and the potential of SlTLFP8 for improvement of WUE. BRIEF SUMMERY: This work found a new mechanism of TLP (Tubby like protein) response to water-deficient stress. SlTLFP8, a member of TLP family, regulates water-deficient resistance by modulating water loss via affecting stomatal density. Expression of SlTLFP8 was induced by osmotic stress. Transgenic tomato lines with SlTLFP8 overexpression or SlTLFP8 knockout showed significantly differences in water-use efficiency (WUE) and water-deficient resistance. The difference of leaf water loss caused by transpiration is the main explanation of the difference in WUE and water-deficient resistance. Additionally, overexpressing SlTLFP8 significantly decreased stomatal density, while SlTLFP8 knockout resulted in increased stomatal density, and SlTLFP8 affected stomatal density through endoreduplication and altered epidermal cell size. Despite changes in stomata density, altering the expression of SlTLFP8 did not result in distinct changes in photosynthesis, biomass and yield of tomato., (© 2020 John Wiley & Sons Ltd.)

Metformin, which is suggested to have anti-cancer effects, activates KDM2A to reduce rRNA transcription and proliferation of cancer cells. Thus, the specific activation of KDM2A may be applicable to the treatment of cancers. In this study, we screened a food-additive compound library to identify compounds that control cell proliferation. We found that gallic acid activated KDM2A to reduce rRNA transcription and cell proliferation in breast cancer MCF-7 cells. Gallic acid accelerated ROS production and activated AMPK. When ROS production or AMPK activity was inhibited, gallic acid did not activate KDM2A. These results suggest that both ROS production and AMPK activation are required for activation of KDM2A by gallic acid. Gallic acid did not reduce the succinate level, which was required for KDM2A activation by metformin. Metformin did not elevate ROS production. These results suggest that the activation of KDM2A by gallic acid includes mechanisms distinct from those by metformin. Therefore, signals from multiple intracellular conditions converge in KDM2A to control rRNA transcription. Gallic acid did not induce KDM2A-dependent anti-proliferation activity in non-tumorigenic MCF10A cells. These results suggest that the mechanism of KDM2A activation by gallic acid may be applicable to the treatment of breast cancers.

Accumulating studies have indicated that long non-coding RNAs (lncRNAs) are crucial modulators in cancer biology. In this work, we investigated the function and related mechanisms of LINC01436 in the progression of gastric cancer (GC). We demonstrated that LINC01436 was significantly up-regulated in cancerous tissues of GC samples, and its overexpression was correlated with a worse prognosis for the patients. In the GC cell line BGC823 cells, LINC01436 knockdown repressed the proliferation and metastasis of cancer cells; conversely, in GC cell line AGS cells, overexpression of LINC01436 showed the opposite effects. We then demonstrated that miR-585, a tumor suppressor, could bind to both LINC01436 and the 3'-UTR of F-box protein 11 (FBOX11), and LINC01436 was proved to sponge miR-585 and repress it, and indirectly promoted the expression of FBOX11. Collectively, these results suggested that LINC01436 was an oncogenic lncRNA in GC and promoted proliferation and metastasis of GC cell via regulating miR-585 and FBOX11., (© 2019 International Federation for Cell Biology.)

The mechanisms underlying spatial and temporal control of cortical neurogenesis of the brain are largely elusive. Long non-coding RNAs (lncRNAs) have emerged as essential cell fate regulators. Here we found LncKdm2b (also known as Kancr), a lncRNA divergently transcribed from a bidirectional promoter of Kdm2b, is transiently expressed during early differentiation of cortical projection neurons. Interestingly, Kdm2b's transcription is positively regulated in cis by LncKdm2b, which has intrinsic-activating function and facilitates a permissive chromatin environment at the Kdm2b's promoter by associating with hnRNPAB. Lineage tracing experiments and phenotypic analyses indicated LncKdm2b and Kdm2b are crucial in proper differentiation and migration of cortical projection neurons. These observations unveiled a lncRNA-dependent machinery in regulating cortical neuronal differentiation.

The TIR1/AFB auxin co-receptors mediate diverse responses to the plant hormone auxin. The Arabidopsis genome encodes six TIR1/AFB proteins representing three of the four clades that were established prior to angiosperm radiation. To determine the role of these proteins in plant development we performed an extensive genetic analysis involving the generation and characterization of all possible multiply-mutant lines. We find that loss of all six TIR1/AFB proteins results in early embryo defects and eventually seed abortion, and yet a single wild-type allele of TIR1 or AFB2 is sufficient to support growth throughout development. Our analysis reveals extensive functional overlap between even the most distantly related TIR1/AFB genes except for AFB1 . Surprisingly, AFB1 has a specialized function in rapid auxin-dependent inhibition of root growth and early phase of root gravitropism. This activity may be related to a difference in subcellular localization compared to the other members of the family., Competing Interests: MP, MP, NK, YZ, KG, WS, BP, RB, MB, WB, ME No competing interests declared, (© 2020, Prigge et al.)

Objective: The aim of the study was to investigate the mechanism and effect of FBXL10 in myocardial ischemia reperfusion injury in vivo and in vitro., Methods: The myocardial ischemia reperfusion (I/R) model was established by 30 min of coronary occlusion followed by 2 h of reperfusion in rats. Western blot and TUNEL assay were used to measure the apoptosis during I/R. The expression levels of endoplasmic reticulum related proteins in myocardial tissues and H9c2 cells were detected by immunohistochemistry staining and immunofluorescence staining. Flow cytometry and CCK-8 were used to detect the apoptosis and viability of H9c2 cells., Results: The results revealed that FBXL10 significantly reduced myocardial infarction, improved the pathological morphology of myocardium, markedly reduced inflammatory response in the myocardial ischemia reperfusion rats. Moreover the expressions of endoplasmic reticulum stress key proteins were caused by I/R were suppressed significantly by FBXL10 treatment, including CHOP, GRP78, ATF4 and p-PERK. Additionally FBXL10 inhibited the expression of endoplasmic reticulum stress key proteins in H/R H9c2 cells. Furthermore, FBXL10 reduced the levels of apoptotic cells and inflammatory response compared with I/R and H/R group., Conclusion: Taken together, we found that FBXL10 could attenuate I/R injury through inhibiting endoplasmic reticulum stress (ERs)., Competing Interests: Declaration of competing interest All authors declare that there is no any conflict of interest., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

During meiosis, telomere attachment to the inner nuclear envelope is required for proper pairing of homologous chromosomes and recombination. Here, we identified F-box protein 47 (FBXO47) as a regulator of the telomeric shelterin complex that is specifically expressed during meiotic prophase I. Knockout of Fbxo47 in mice leads to infertility in males. We found that the Fbxo47 deficient spermatocytes are unable to form a complete synaptonemal complex. FBXO47 interacts with TRF1/2, and the disruption of Fbxo47 destabilizes TRF2, leading to unstable telomere attachment and slow traversing through the bouquet stage. Our findings uncover a novel mechanism of FBXO47 in telomeric shelterin subunit stabilization during meiosis., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Inhibition of apoptosis signal-regulating kinase 1 (ASK1) activation has emerged as a promising target for the treatment of nonalcoholic steatohepatitis (NASH). Multiple forms of posttranslational modifications determine the activity of ASK1. In addition to phosphorylation, recent studies revealed that ubiquitination is essential for ASK1 activation. However, the endogenous factor that regulates ASK1 ubiquitination and activation remains poorly defined. In this study, we identified the E3 ligase Skp1-Cul1-F-box (SCF) protein F-box/WD repeat-containing protein 5 (FBXW5) as a key endogenous activator of ASK1 ubiquitination. FBXW5 is the central component of the SCF complex (SCF Fbxw5 ) that directly interacts with and ubiquitinates ASK1 in hepatocytes during NASH development. An in vivo study showed that hepatocyte-specific overexpression of FBXW5 exacerbated diet-induced systemic and hepatic metabolic disorders, as well as the activation of ASK1-related mitogen-activated protein kinase (MAPK) signaling in the liver. Conversely, hepatocyte-specific deletion of FBXW5 significantly prevented the progression of these abnormalities. Mechanically, FBXW5 facilitated the addition of Lys63-linked ubiquitin to ASK1 and thus exacerbated ASK1-c-Jun N-terminal kinase/p38 MAPK signaling, inflammation, and lipid accumulation. Furthermore, we demonstrated that the N-terminus (S1) and C-terminus (S3) of FBXW5 respectively and competitively ablate the function of FBXW5 on ASK1 activation and served as effective inhibitors of NASH progression. Conclusion: This evidence strongly suggests that SCF Fbxw5 is an important activator of ASK1 ubiquitination in the context of NASH. The development of FBXW5(S1) or FBXW5(S3)-mimicking drugs and screening of small-molecular inhibitors specifically abrogating ASK1 ubiquitination-dependent activation are viable approaches for NASH treatment., (© 2019 by the American Association for the Study of Liver Diseases.)

As the most common cancer and one of the leading causes of cancer-associated mortality, breast cancer continues to need more key molecules to regulate its progression. F-box and leucine-rich repeat protein 19 antisense RNA 1 (known as FBXL19-AS1) is a long non-coding RNA (lncRNA) which has been reported as an oncogene in several types of human cancers. However, the specific downstream targets of FBXL19-AS1 remain unknown. In this study, we set out to find more reliable downstream molecules of FBXL19-AS1 in breast cancer. FBXL19-AS1 was expressed at a high level in breast cancer cells. Loss-of-function experiments revealed that silencing FBXL19-AS1 could impair cell proliferation and induce cell apoptosis in breast cancer. In addition, the location of FBXL19-AS1 in the cytoplasm was detected by fluorescent in situ hybridization assay, while FBXL19-AS1 regulated the expression of Forkhead box M1 (FOXM1) by directly absorbing miR-876-5p. Through rescue assays, it was observed that FOXM1 overexpression recovered the inhibited tumor growth caused by FBXL19-AS1 downregulation. We affirmed the function of FBXL19-AS1 in breast cancer and described the mechanism of the FBXL19-AS1/miR-876-5p/FOXM1 axis. The current work presents the molecular mechanism which underlies FBXL19-AS1 in breast cancer and suggests a comprehensive, feasible FBXL19-AS1-mediated therapeutic approach for treating breast cancer., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

SOX2 is a key transcription factor that plays critical roles in maintaining stem cell property and conferring drug resistance. However, the underlying mechanisms by which SOX2 level is precisely regulated remain elusive. Here we report that MLN4924, also known as pevonedistat, a small-molecule inhibitor of neddylation currently in phase II clinical trials, down-regulates SOX2 expression via causing accumulation of MSX2, a known transcription repressor of SOX2 expression. Mechanistic characterization revealed that MSX2 is a substrate of FBXW2 E3 ligase. FBXW2 binds to MSX2 and promotes MSX2 ubiquitylation and degradation. Likewise, FBXW2 overexpression shortens the protein half-life of MSX2, whereas FBXW2 knockdown extends it. We further identified hypoxia as a stress condition that induces VRK2 kinase to facilitate MSX2-FBXW2 binding and FBXW2-mediated MSX2 ubiquitylation and degradation, leading to SOX2 induction via derepression. Biologically, expression of FBXW2 or SOX2 promotes tumor sphere formation, which is blocked by MSX2 expression. By down-regulating SOX2 through inactivation of FBXW2 E3 ligase, MLN4924 sensitizes breast cancer cells to tamoxifen in both in vitro and in vivo cancer cell models. Thus, a negative cascade of the FBXW2-MSX2-SOX2 axis was established, which regulates stem cell property and drug resistance. Finally, an inverse correlation of expression was found between FBXW2 and MSX2 in lung and breast cancer tissues. Collectively, our study revealed an anticancer mechanism of MLN4924. By inactivating FBXW2, MLN4924 caused MSX2 accumulation to repress SOX2 expression, leading to suppression of stem cell property and sensitization of breast cancer cells to tamoxifen., Competing Interests: Conflict of interest statement: R.S. has received research funding from AstraZeneca, GlaxoSmithKline, Gilead Sciences, and PUMA Biotechnology and is a consulting/advisory committee member for Macrogenics and Eli Lilly. These funds, however, had no impact on the content of this study.

CpG islands (CGIs) are associated with the majority of mammalian gene promoters and function to recruit chromatin modifying enzymes. It has therefore been proposed that CGIs regulate gene expression through chromatin-based mechanisms, however in most cases this has not been directly tested. Here, we reveal that the histone H3 lysine 36 (H3K36) demethylase activity of the CGI-binding KDM2 proteins contributes only modestly to the H3K36me2-depleted state at CGI-associated gene promoters and is dispensable for normal gene expression. Instead, we discover that KDM2 proteins play a widespread and demethylase-independent role in constraining gene expression from CGI-associated gene promoters. We further show that KDM2 proteins shape RNA Polymerase II occupancy but not chromatin accessibility at CGI-associated promoters. Together this reveals a demethylase-independent role for KDM2 proteins in transcriptional repression and uncovers a new function for CGIs in constraining gene expression., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

KDM2B together with RING1B, PCGF1, and BCOR or BCORL1 comprise polycomb repressive complex 1.1 (PRC1.1), a noncanonical PRC1 that catalyzes H2AK119ub1. It binds to nonmethylated CpG islands through its zinc finger-CxxC DNA binding domain and recruits the complex to target gene loci. Recent studies identified the loss of function mutations in the PRC1.1 gene, BCOR and BCORL1 in human T-cell acute lymphoblastic leukemia (T-ALL). We previously reported that Bcor insufficiency induces T-ALL in mice, supporting a tumor suppressor role for BCOR. However, the function of BCOR responsible for tumor suppression, either its corepressor function for BCL6 or that as a component of PRC1.1, remains unclear. We herein examined mice specifically lacking the zinc finger-CxxC domain of KDM2B in hematopoietic cells. Similar to Bcor -deficient mice, Kdm2b -deficient mice developed lethal T-ALL mostly in a NOTCH1-dependent manner. A chromatin immunoprecipitation sequence analysis of thymocytes revealed the binding of KDM2B at promoter regions, at which BCOR and EZH2 colocalized. KDM2B target genes markedly overlapped with those of NOTCH1 in human T-ALL cells, suggesting that noncanonical PRC1.1 antagonizes NOTCH1-mediated gene activation. KDM2B target genes were expressed at higher levels than the others and were marked with high levels of H2AK119ub1 and H3K4me3, but low levels of H3K27me3, suggesting that KDM2B target genes are transcriptionally active or primed for activation. These results indicate that PRC1.1 plays a key role in restricting excessive transcriptional activation by active NOTCH1, thereby acting as a tumor suppressor in the initiation of T-cell leukemogenesis., (© 2019 by The American Society of Hematology.)

Chronic nasal sinusitis with nasal polyps (CRSwNP) is a reversible nasal mucosal remodeling disease caused by persistent inflammation and structural changes in chronic nasal mucosa. Although there have been many studies on the inflammation of the nasal mucosa epithelium, the mechanism remains unclear. Our study found that H3K4me3 histone demethylase KDM2B (also known as JHDM1B) and transcriptional regulator Brg1 (also called SNF2-β or Smarca4) were significantly decreased in nasal mucosa of CRSwNP patients, and they were positively correlated. Brg1 and KDM2B co-localize in the epithelial cells of nasal mucosa. We used poly(I:C)-treated nasal mucosal epithelial cells (HNECs) to find that the expression of KDM2B and Brg1 was also decreased, and the main expression position transferred from the nucleus to the nuclear membrane. We used small interfering RNA to knock down the expression of KDM2B and Brg1 in nasal epithelial cells. It was interesting to find that the decreased expression of KDM2B and Brg1 produced similar effects to that of poly(I:C)-treated cells, which could promote inflammatory response of nasal mucosal epithelial cells. And Brg1 appears to play a role in KDM2B regulating gene promoters of IL-6 and TNF-α inflammatory. This study shows that KDM2B and Brg1 may have an inhibitory effect on the development of CRSwNP nasal mucosal epithelial inflammation. This study will provide a new perspective for gene targeting therapy of CRSwNPs.

By binding to the adaptor protein SKP1 and serving as substrate receptors for the SKP1 Cullin, F-box E3 ubiquitin ligase complex, F-box proteins regulate critical cellular processes including cell cycle progression and membrane trafficking. While F-box proteins are conserved throughout eukaryotes and are well studied in yeast, plants, and animals, studies in parasitic protozoa are lagging. We have identified eighteen putative F-box proteins in the Toxoplasma genome of which four have predicted homologs in Plasmodium. Two of the conserved F-box proteins were demonstrated to be important for Toxoplasma fitness and here we focus on an F-box protein, named TgFBXO1, because it is the most highly expressed by replicative tachyzoites and was also identified in an interactome screen as a Toxoplasma SKP1 binding protein. TgFBXO1 interacts with Toxoplasma SKP1 confirming it as a bona fide F-box protein. In interphase parasites, TgFBXO1 is a component of the Inner Membrane Complex (IMC), which is an organelle that underlies the plasma membrane. Early during replication, TgFBXO1 localizes to the developing daughter cell scaffold, which is the site where the daughter cell IMC and microtubules form and extend from. TgFBXO1 localization to the daughter cell scaffold required centrosome duplication but before kinetochore separation was completed. Daughter cell scaffold localization required TgFBXO1 N-myristoylation and was dependent on the small molecular weight GTPase, TgRab11b. Finally, we demonstrate that TgFBXO1 is required for parasite growth due to its function as a daughter cell scaffold effector. TgFBXO1 is the first F-box protein to be studied in apicomplexan parasites and represents the first protein demonstrated to be important for daughter cell scaffold function., Competing Interests: The authors have declared that no competing interests exist.

During cell cycle progression, the activity of the CycE-Cdk2 complex gates S-phase entry. CycE-Cdk2 is inhibited by CDK inhibitors (CKIs) of the Cip/Kip family, which include the human p21 Cip1 and Drosophila Dacapo (Dap) proteins. Both the CycE and Cip/Kip family proteins are under elaborate control via protein degradation, mediated by the Cullin-RING ligase (CRL) family of ubiquitin ligase complexes. The CRL complex SCF Fbxw7/Ago targets phosphorylated CycE, whereas p21 Cip1 and Dap are targeted by the CRL4 Cdt2 complex, binding to the PIP degron. The role of CRL-mediated degradation of CycE and Cip/Kip proteins during CNS development is not well understood. Here, we analyse the role of ago ( Fbxw7 )-mediated CycE degradation, and of Dap and p21 Cip1 degradation during Drosophila CNS development. We find that ago mutants display over-proliferation, accompanied by elevated CycE expression levels. By contrast, expression of PIP degron mutant Dap and p21 Cip1 transgenes inhibit proliferation. However, surprisingly, this is also accompanied by elevated CycE levels. Hence, ago mutation and PIP degron Cip/Kip transgenic expression trigger opposite effects on proliferation, but similar effects on CycE levels., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Key Message: S-RNase was demonstrated to be predominantly recognized by an S locus F-box-like protein and an S haplotype-specific F-box-like protein in compatible pollen tubes of sweet cherry. Self-incompatibility (SI) is a reproductive barrier that rejects self-pollen and inhibits self-fertilization to promote outcrossing. In Solanaceae and Rosaceae, S-RNase-based gametophytic SI (GSI) comprises S-RNase and F-box protein(s) as the pistil and pollen S determinants, respectively. Compatible pollen tubes are assumed to detoxify the internalized cytotoxic S-RNases to maintain growth. S-RNase detoxification is conducted by the Skp1-cullin1-F-box protein complex (SCF) formed by pollen S determinants, S locus F-box proteins (SLFs), in Solanaceae. In Prunus, the general inhibitor (GI), but not pollen S determinant S haplotype-specific F-box protein (SFB), is hypothesized to detoxify S-RNases. Recently, SLF-like proteins 1-3 (SLFL1-3) were suggested as GI candidates, although it is still possible that other proteins function predominantly in GI. To identify the other GI candidates, we isolated four other pollen-expressed SLFL and SFB-like (SFBL) proteins PavSLFL6, PavSLFL7A, PavSFBL1, and PavSFBL2 in sweet cherry. Binding assays with four PavS-RNases indicated that PavSFBL2 bound to PavS 1, 6 -RNase while the others bound to nothing. PavSFBL2 was confirmed to form an SCF complex in vitro. A co-immunoprecipitation assay using the recombinant PavS 6 -RNase as bait against pollen extracts and a mass spectrometry analysis identified the SCF complex components of PavSLFLs and PavSFBL2, M-locus-encoded glutathione S-transferase (MGST), DnaJ-like protein, and other minor proteins. These results suggest that SLFLs and SFBLs could act as predominant GIs in Prunus-specific S-RNase-based GSI.

Avr9/Cf-9-INDUCED F-BOX1 (ACIF1) was first identified during screening of Avr9/Cf-9-elicited genes in tobacco. Further analysis revealed that ACIF1 was required for hypersensitive responses triggered by various elicitors in tobacco and tomato, indicating that it may be involved in various disease resistance. Here, we cloned its cotton (Gossypium hirsutum) homolog GhACIF1, which encodes an F-box protein. We show that GhACIF1 interacts with the putative SKP1-like protein, named GhSKP1. Disease resistance assays show that GhACIF1 enhances resistance to Verticillium dahliae in Arabidopsis plants, while silencing of GhACIF1 confers sensitivity to V. dahliae in cotton. Further analysis show that PevD1 elicitor activates hypersensitive and acquired immune response mediated by GhACIF1. Collectively, these results indicate that GhACIF1 contributes to protection against V. dahliae infection., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The members of the phytochrome (phy) family of bilin-containing photoreceptors are major regulators of plant photomorphogenesis through their unique ability to photointerconvert between a biologically inactive red light-absorbing Pr state and an active far-red light-absorbing Pfr state. While the initial steps in Pfr signaling are unclear, an early event for the phyB isoform after photoconversion is its redistribution from the cytoplasm into subnuclear foci known as photobodies (PBs), which dissipate after Pfr reverts back to Pr by far-red irradiation or by temperature-dependent nonphotochemical reversion. Here we present evidence that PHOTOPERIODIC CONTROL OF HYPOCOTYL 1 (PCH1) functions both as an essential structural component of phyB-containing PBs and as a direct regulator of thermal reversion that is sufficient to stabilize phyB as Pfr in vitro. By examining the genetic interaction between a constitutively active phyB Y276H -YFP allele ( YHB-YFP ) and PCH1, we show that the loss of PCH1 prevents YHB from coalescing into PBs without affecting its nuclear localization, whereas overexpression of PCH1 dramatically increases PB levels. Loss of PCH1, presumably by impacting phyB-PB assembly, compromises a number of events elicited in YHB-YFP plants, including their constitutive photomorphogenic phenotype, red light-regulated thermomorphogenesis, and input of phyB into the circadian clock. Conversely, elevated levels of both phyB and PCH1 generate stable, yet far-red light-reversible PBs that persisted for days. Collectively, our data demonstrate that the assembly of PCH1-containing PBs is critical for phyB signaling to multiple outputs and suggest that altering PB dynamics could be exploited to modulate plant responses to light and temperature., Competing Interests: The authors declare no conflict of interest.

Background: Emerging evidences show that G-protein-coupled estrogen receptor (GPER) can regulate the progression of various cancers, while its roles in the progression of osteosarcoma (OS) are not well illustrated., Methods: The expression of GPER in OS cells and tissues were checked. Its roles in cell migration and expression of Snail was checked by use of its agonist G-1., Results: We found that the expression of GPER in OS cells and tissues were lower than that in their corresponding controls. OS patients with higher levels of GPER showed increased overall survival rate (OS) as compared with the lower ones. The activator of GPER (G-1) or overexpression of GPER can inhibit the migration and invasion of OS cells and downregulate mesenchymal markers. G-1 can rapidly decrease the expression of Snail, one powerful epithelial-mesenchymal transition transcription factor (EMT-TF). Overexpression of Snail can attenuate the suppression effects of G-1 on migration of OS cells, suggesting that Snail was involved in GPER-regulated migration of OS cells. Mechanically, G-1 rapidly decreased the protein of Snail but had no effect on its mRNA expression. This was because G-1 can decrease the protein stability of Snail. Further, G-1 increased the expression of FBXL5, which can trigger the proteasome-mediated degradation of Snail. Knockdown of FBXL5 can reverse G-1-induced downregulation of Snail in OS cells., Conclusion: Activation of GPER can suppress the migration and invasion of OS cells via FBXL5-mediated post-translational down regulation of Snail. It suggested that targeted activation of GPER might be a potent potential therapy approach to overcome the metastasis of OS patients.

The circadian oscillator is a molecular feedback circuit whose orchestration involves posttranslational control of the activity and protein levels of its components. Although controlled proteolysis of circadian proteins is critical for oscillator function, our understanding of the underlying mechanisms remains incomplete. Here, we report that JmjC domain-containing protein 5 (JMJD5) interacts with CRYPTOCHROME 1 (CRY1) in an F-box/leucine-rich repeat protein 3 (FBXL3)-dependent manner and facilitates targeting of CRY1 to the proteasome. Genetic deletion of JMJD5 results in greater CRY1 stability, reduced CRY1 association with the proteasome, and disruption of circadian gene expression. We also report that in the absence of JMJD5, AMP-regulated protein kinase (AMPK)-induced CRY1 degradation is impaired, establishing JMJD5 as a key player in this mechanism. JMJD5 cooperates with CRY1 to repress circadian locomotor output cycles protein kaput (CLOCK)-brain and muscle ARNT-like protein 1 (BMAL1), thus linking CRY1 destabilization to repressive function. Finally, we find that ablation of JMJD5 impacts FBXL3- and CRY1-related functions beyond the oscillator., Competing Interests: The authors have declared that no competing interests exist.

The molecular underpinnings behind malignant progression of breast cancer from a localized lesion to an invasive and ultimately metastatic disease are incompletely understood. Here, we report that F-box only protein 22 (FBXO22) plays a dual role in mammary tumorigenesis and metastasis. FBXO22 was upregulated in primary breast tumors and promoted cell proliferation and colony formation in vitro and xenograft tumorigenicity in vivo Surprisingly, FBXO22 suppressed epithelial-mesenchymal transition (EMT), cell motility, and invasiveness in vitro and metastatic lung colonization in vivo Clinical data showed that expression levels of FBXO22 were associated with favorable clinical outcomes, supporting the notion that metastasis, rather than primary cancer, is the major determinant of the mortality of patients with breast cancer. Mechanistic investigations further revealed that FBXO22 elicits its antimetastatic effects by targeting SNAIL, a master regulator of EMT and breast cancer metastasis, for ubiquitin-mediated proteasomal degradation in a glycogen synthase kinase 3β phosphorylation-dependent manner. Importantly, expression of SNAIL rescued FBXO22-mediated suppression of EMT, cell migration, and invasion. A patient-derived tryptophan-to-arginine mutation at residue 52 (W52R) within the F-box domain impaired FBXO22 binding to the SKP1-Cullin1 complex and blocked FBXO22-mediated SNAIL degradation, thus abrogating the ability of FBXO22 to suppress cell migration, invasion, and metastasis. Collectively, these findings uncover an unexpected dual role for FBXO22 in mammary tumorigenesis and metastatic progression and delineate the mechanism of an oncogenic mutation of FBXO22 in breast cancer progression. Significance: These findings highlight the paradoxical roles of FBXO22 in breast cancer, as it promotes breast tumor cell proliferation but prevents EMT and metastasis. Cancer Res; 78(18); 5274-86. ©2018 AACR ., (©2018 American Association for Cancer Research.)

In mammals, tumor necrosis factor receptor associated factors (TRAFs) are signaling adaptors that regulate diverse physiological processes, including immunity and stress responses. In Arabidopsis, MUSE13 and MUSE14 are redundant TRAF proteins serving as adaptors in the SCF CRP1 complex to facilitate the turnover of nucleotide-binding domain and leucine-rich repeats (NLR) immune receptors. Degradation of MUSE13 is inhibited by proteasome inhibitor, suggesting that the MUSE13 stability is controlled by the 26S proteasome. However, the E3 ligase that regulates MUSE13 level is unknown. Here we report the identification of an F-box protein, SNIPER4 that regulates the turnover of MUSE13 and MUSE14. Protein levels of MUSE13 and MUSE14 are reduced by SNIPER4 overexpression, while higher accumulation of MUSE13 and MUSE14 is observed when dominant-negative SNIPER4 is expressed. Furthermore, SNIPER4 associates with MUSE13 or MUSE14. Taken together, the SCF SNIPER4 complex controls the turnover of TRAF proteins for an optimum immune output., (© 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd.)

Ethylene signaling converges on the ETHYLENE-INSENSITIVE3 (EIN3)/EIN3-like (EIL) transcription factors to regulate a wide range of developmental processes in plants. EBF1/2 (EIN3-binding F-box protein 1 and 2) negatively regulate the ethylene signaling pathway by mediating the degradation of EIN3/EIL proteins. We uncovered previously that SlEBF1 and SlEBF2 are involved in ethylene response, plant senescence, and fruit ripening in tomato. The present study reports on the identification of a novel tomato F-box gene, designated as SlEBF2-like due that its encoded protein is greater similarity to SlEBF2. The SlEBF2-like promoter region contains three ethylene-response elements (EREs). SlEBF2-like is upregulated by ethylene and downregulated by ethylene inhibitors in tomato seedlings. It is dynamically expressed in flowers during bud-to-anthesis and anthesis-to-post-anthesis transitions, and at the onset of fruit ripening, suggesting its role in these situations where ethylene is required for flower opening and fruit ripening. SlEBF2-like overexpression leaded to fruit elongation, caused ripening and color change to start from fruit bottom and expand gradually to the pedicel, and strongly delayed fruit development and ripening in tomato. Our study indicates that the novel EBF gene, SlEBF2-like, is involved in fruit development and ripening via regulating the ethylene response in tomato., (Copyright © 2018 Elsevier B.V. All rights reserved.)

CpG islands are gene regulatory elements associated with the majority of mammalian promoters, yet how they regulate gene expression remains poorly understood. Here, we identify FBXL19 as a CpG island-binding protein in mouse embryonic stem (ES) cells and show that it associates with the CDK-Mediator complex. We discover that FBXL19 recruits CDK-Mediator to CpG island-associated promoters of non-transcribed developmental genes to prime these genes for activation during cell lineage commitment. We further show that recognition of CpG islands by FBXL19 is essential for mouse development. Together this reveals a new CpG island-centric mechanism for CDK-Mediator recruitment to developmental gene promoters in ES cells and a requirement for CDK-Mediator in priming these developmental genes for activation during cell lineage commitment., Competing Interests: ED, TK, AF, MN, YK, RK, BK, HK, RK No competing interests declared, (© 2018, Dimitrova et al.)

Chromatin accessibility is closely associated with chromatin functions such as gene expression, DNA replication, and maintenance of DNA integrity. However, the relationship between chromatin accessibility and plant hormone signaling has remained elusive. Here, based on the correlation between chromatin accessibility and DNA damage, we used the sensitivity to DNA double strand breaks (DSBs) as an indicator of chromatin accessibility and demonstrated that auxin regulates chromatin accessibility through the TIR1/AFBs signaling pathway in proliferative cells. Treatment of proliferating plant cells with an inhibitor of the TIR1/AFBs auxin signaling pathway, PEO-IAA, caused chromatin loosening, indicating that auxin signaling functions to decrease chromatin accessibility. In addition, a transcriptome analysis revealed that several histone H4 genes and a histone chaperone gene, FAS1, are positively regulated through the TIR1/AFBs signaling pathway, suggesting that auxin plays a role in promoting nucleosome assembly. Analysis of the fas1 mutant of Arabidopsis thaliana confirmed that FAS1 is required for the auxin-dependent decrease in chromatin accessibility. These results suggest that the positive regulation of chromatin-related genes mediated by the TIR1/AFBs auxin signaling pathway enhances nucleosome assembly, resulting in decreased chromatin accessibility in proliferative cells.

The ubiquitin system plays a role in nearly every aspect of eukaryotic cell biology. The enzymes responsible for transferring ubiquitin onto specific substrates are the E3 ubiquitin ligases, a large and diverse family of proteins, for which biological roles and target substrates remain largely undefined. Studies using model organisms indicate that ubiquitin signaling mediates key steps in developmental processes and tissue regeneration. Here, we used the freshwater planarian, Schmidtea mediterranea, to investigate the role of Cullin-RING ubiquitin ligase (CRL) complexes in stem cell regulation during regeneration. We identified six S. mediterranea cullin genes, and used RNAi to uncover roles for homologs of Cullin-1, -3 and -4 in planarian regeneration. The cullin-1 RNAi phenotype included defects in blastema formation, organ regeneration, lesions, and lysis. To further investigate the function of cullin-1-mediated cellular processes in planarians, we examined genes encoding the adaptor protein Skp1 and F-box substrate-recognition proteins that are predicted to partner with Cullin-1. RNAi against skp1 resulted in phenotypes similar to cullin-1 RNAi, and an RNAi screen of the F-box genes identified 19 genes that recapitulated aspects of cullin-1 RNAi, including ones that in mammals are involved in stem cell regulation and cancer biology. Our data provides evidence that CRLs play discrete roles in regenerative processes and provide a platform to investigate how CRLs regulate stem cells in vivo., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Due to the apoptosis-prone nature of primary germinal center B (GCB) cells, it remains a huge challenge to dissect signals that guide their differentiation towards memory B cells and plasma cells in vitro. Here we show that the murine lymphoma cell line A20 resembles primary GCB cells in expression of GC-specific surface markers and the master transcription factor BCL6 and may serve as a useful system to model certain GCB cell behaviors in vitro. Using these cells, we found that both CD40 and B cell receptor (BCR) signaling are able to drive BCL6 downregulation, which is a prerequisite of post-GC B-cell differentiation. Under the steady state, BCL6 is constantly and rapidly degraded in A20 cells by the proteasome in a strictly FBXO11-dependent manner. This process can be further enhanced by signals downstream of the BCR. Both CD40 and BCR stimulation can upregulate IRF4, a transcription factor that suppresses BCL6 expression. However, only BCR signaling downregulate PAX5 and BACH2, two transcription factors that help maintain the GCB identity. Together, these results validate the A20 cell line as an experimental system suitable for studying regulation of BCL6 and potentially other transcription factors relevant to post-GC fate determination, and they support that combined signaling from BCR and CD40 receptors would drive termination of the GC program., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Molecular oscillators are important cellular regulators of, for example, circadian clocks, oscillations of immune regulators, and short-period (ultradian) rhythms during embryonic development. The Notch signaling factor HES1 (hairy and enhancer of split 1) is a well-known repressor of proneural genes, and HES1 ultradian oscillation is essential for keeping cells in an efficiently proliferating progenitor state. HES1 oscillation is driven by both transcriptional self-repression and ubiquitin-dependent proteolysis. However, the E3 ubiquitin ligase targeting HES1 for proteolysis remains unclear. Based on siRNA-mediated gene silencing screening, co-immunoprecipitation, and ubiquitination assays, we discovered that the E3 ubiquitin ligase SCF FBXL14 complex regulates HES1 ubiquitination and proteolysis. siRNA-mediated knockdown of the Cullin-RING E3 ubiquitin ligases RBX1 or CUL1 increased HES1 protein levels, prolonged its half-life, and dampened its oscillation. FBXL14, an F-box protein for SCF ubiquitin ligase, associates with HES1. FBXL14 silencing stabilized HES1, whereas FBXL14 overexpression decreased HES1 protein levels. Of note, the SCF FBXL14 complex promoted the ubiquitination of HES1 in vivo , and a conserved WRPW motif in HES1 was essential for HES1 binding to FBXL14 and for ubiquitin-dependent HES1 degradation. HES1 knockdown promoted neuronal differentiation, but FBXL14 silencing inhibited neuronal differentiation induced by HES1 ablation in mES and F9 cells. Our results suggest that SCF FBXL14 promotes neuronal differentiation by targeting HES1 for ubiquitin-dependent proteolysis and that the C-terminal WRPW motif in HES1 is required for this process., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Smoldering-type and chronic-type adult T-cell leukemia/lymphomas (ATLL) patients have relatively indolent clinical courses, but often progress into aggressive lymphoma-type and acute-type disease. We examined the roles of transcription factor C-MYC and its ubiquitin ligase FBXW7 in tumor tissues from 137 patients with ATLL. Immunohistochemical tests showed ≥50% of lymphoma cells in 78.7% (48/61) of lymphoma-type, and 64.9% (24/37) of acute-type samples expressed C-MYC, significantly higher than was seen in smoldering-type (3.6%) and chronic-type (9.1%) samples (P<0.01). Real-time polymerase chain reaction showed C-MYC mRNA expression in lymphoma-type and acute-type samples were significantly higher than in smoldering-type (P<0.01). C-MYC expression was highly correlated with its mRNA levels (ρ=0.65, P<0.0001), chromosomal amplification and duplication (ρ=0.3, P=0.045) and MIB1 labeling index (ρ=0.69, P<0.0001). Expression of FBXW7 protein and mRNA in lymphoma-type samples were significantly lower than those of smoldering-type (P<0.01 for each), and both were inversely correlated with C-MYC (protein: ρ=-0.4, P=0.0002; mRNA: ρ=-0.31, P=0.015). Seven patients with smoldering-type or chronic-type ATLL converted to acute-type, in 4 of whom C-MYC expression increased from <50% to ≥50%. Patients with ≥50% C-MYC or MIB1 had significantly worse prognosis than those with <50% C-MYC (P=0.0004) or MIB1 (P<0.0001), as did those with ≥7.5 C-MYC mRNA scores (P=0.033); whereas significantly better prognosis was associated with ≥50% FBXW7 protein (P=0.0006) or ≥0.17 FBXW7 mRNA (P=0.016). C-MYC and FBXW7 affect ATLL proliferation and progression, and low FBXW7 may increase C-MYC expression. C-MYC was a critical prognostic factor in ATLL patients.

Background: TIR1-like proteins act as auxin receptors and play essential roles in auxin-mediated plant development processes. The number of auxin receptor family members varies among species. While the functions of auxin receptor genes have been widely studied in Arabidopsis, the distinct functions of cucumber (Cucumis sativus L.) auxin receptors remains poorly understood. To further our understanding of their potential role in cucumber development, two TIR1-like genes were identified and designated CsTIR1 and CsAFB2. In the present study, tomato (Sonanum lycopersicum) was used as a model to investigate the phenotypic and molecular changes associated with the overexpression of CsTIR1 and CsAFB2., Results: Differences in the subcellular localizations of CsTIR1 and CsAFB2 were identified and both genes were actively expressed in leaf, female flower and young fruit tissues of cucumber. Moreover, CsTIR1- and CsAFB2-overexpressing lines exhibited pleotropic phenotypes ranging from leaf abnormalities to seed germination and parthenocarpic fruit compared with the wild-type plants. To further elucidate the regulation of CsTIR1 and CsAFB2, the role of the miR393/TIR1 module in regulating cucumber fruit set were investigated. Activation of miR393-mediated mRNA cleavage of CsTIR1 and CsAFB2 was revealed by qPCR and semi-qPCR, which highlighted the critical role of the miR393/TIR1 module in mediating fruit set development in cucumber., Conclusion: Our results provide new insights into the involvement of CsTIR1 and CsAFB2 in regulating various phenotype alterations, and suggest that post-transcriptional regulation of CsTIR1 and CsAFB2 mediated by miR393 is essential for cucumber fruit set initiation. Collectively, these results further clarify the roles of cucumber TIR1 homologs and miR393 in regulating fruit/seed set development and leaf morphogenesis.

Protein degradation by the ubiquitin-26S proteasome pathway is important for the regulation of cellular processes, but the function of most F-box proteins relevant to substrate recognition is unknown. We describe the analysis of the gene Cytokinin-induced F-box encoding (CFB, AT3G44326), identified in a meta-analysis of cytokinin-related transcriptome studies as one of the most robust cytokinin response genes. F-box domain-dependent interaction with the E3 ubiquitin ligase complex component ASK1 classifies CFB as a functional F-box protein. Apart from F-box and transmembrane domains, CFB contains no known functional domains. CFB is expressed in all plant tissues, predominantly in root tissue. A ProCFB:GFP-GUS fusion gene showed strongest expression in the lateral root cap and during lateral root formation. CFB-GFP fusion proteins were mainly localized in the nucleus and the cytosol but also at the plasma membrane. cfb mutants had no discernible phenotype, but CFB overexpressing plants showed several defects, such as a white upper inflorescence stem, similar to the hypomorphic cycloartenol synthase mutant cas1-1. Both CFB overexpressing plants and cas1-1 mutants accumulated the CAS1 substrate 2,3-oxidosqualene in the white stem tissue, the latter even more after cytokinin treatment, indicating impairment of CAS1 function. This suggests that CFB may link cytokinin and the sterol biosynthesis pathway., (© The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Satellite cells attached to skeletal muscle fibers play a crucial role in skeletal muscle regeneration. During regeneration, the satellite cells proliferate, migrate to the damaged region, and fuse to each other. Although it is important to determine the cellular mechanisms controlling myoblast behavior, their regulators are not well understood. In this study, we evaluated the roles of Fbxw7 in primary myoblasts and determined its potential as a therapeutic target for muscle disease. We originally found that Fbxw7β, one of the E3 ubiquitin ligase Fbxw7 subtypes, negatively regulates differentiation, proliferation and migration of myoblasts and satellite cells on muscle fiber. However, these phenomena were not observed in myoblasts expressing a dominant-negative, F-box deleted Fbxw7β, mutant. Our results suggest that myoblast differentiation potential and muscle regeneration can be regulated by Fbxw7β., (© 2016 Japanese Society of Animal Science.)

The DNA replication or S-phase checkpoint monitors the integrity of DNA synthesis. Replication stress or DNA damage triggers fork stalling and checkpoint signaling to activate repair pathways. Recovery from checkpoint activation is critical for cell survival following DNA damage. Recovery from the S-phase checkpoint includes inactivation of checkpoint signaling and restart of stalled replication forks. Previous studies demonstrated that degradation of Mrc1, the Saccharomyces cerevisiae ortholog of human Claspin, is facilitated by the SCFDia2 ubiquitin ligase and is important for cell cycle re-entry after DNA damage-induced S-phase checkpoint activation. Here, we show that degradation of Mrc1 facilitated by the SCFDia2 complex is critical to restart stalled replication forks during checkpoint recovery. Using DNA fiber analysis, we showed that Dia2 functions with the Sgs1 and Mph1 helicases (orthologs of human BLM and FANCM, respectively) in the recombination-mediated fork restart pathway. In addition, Dia2 physically interacts with Sgs1 upon checkpoint activation. Importantly, failure to target Mrc1 for degradation during recovery inhibits Sgs1 chromatin association, but this can be alleviated by induced proteolysis of Mrc1 after checkpoint activation. Together, these studies provide new mechanistic insights into how cells recover from activation of the S-phase checkpoint., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Glioblastoma is the most lethal brain tumor and harbors glioma stem cells (GSCs) with potent tumorigenic capacity. The function of GSCs in tumor propagation is maintained by several core transcriptional regulators including c-Myc. c-Myc protein is tightly regulated by posttranslational modification. However, the posttranslational regulatory mechanisms for c-Myc in GSCs have not been defined. In this study, we demonstrate that the deubiquitinase USP13 stabilizes c-Myc by antagonizing FBXL14-mediated ubiquitination to maintain GSC self-renewal and tumorigenic potential. USP13 was preferentially expressed in GSCs, and its depletion potently inhibited GSC proliferation and tumor growth by promoting c-Myc ubiquitination and degradation. In contrast, overexpression of the ubiquitin E3 ligase FBXL14 induced c-Myc degradation, promoted GSC differentiation, and inhibited tumor growth. Ectopic expression of the ubiquitin-insensitive mutant T58A-c-Myc rescued the effects caused by FBXL14 overexpression or USP13 disruption. These data suggest that USP13 and FBXL14 play opposing roles in the regulation of GSCs through reversible ubiquitination of c-Myc., (© 2017 Fang et al.)

The (V600E)BRAF oncogenic mutation is detected in a wide range of human cancers and induces hyperactivation of the downstream MEK-ERK signalling cascade. Although output of the BRAF-MEK-ERK pathway is regulated by feed-forward RAF activity, feedback control also plays an important role. One such feedback pathway has been identified in Caenorhabditis elegans and involves ERK-mediated phosphorylation of BRAF within a CDC4 phosphodegron (CPD), targeting BRAF for degradation via CDC4 (also known as FBXW7), a component of the SKP1/CUL1/F-box (SCF) E3 ubiquitin ligase complex. Here we investigate this pathway in mammalian cells. Short-term expression of autochthonous (V600E)BRAF in mouse embryonic fibroblasts (MEFs) leads to down-regulation of BRAF protein levels in a proteasome-dependent manner and (V600E)BRAF has a reduced half-life compared to (WT)BRAF in HEK293(T) cells. These effects were reversed by treatment with the MEK inhibitor PD184352. We have identified the equivalent CPD at residues 400-405 in human BRAF and have found that mutation of ERK phosphorylation sites at residues T401 and S405 in (V600E)BRAF increases the half-life of the protein. While BRAF and FBXW7 co-immunoprecipitated, the overexpression of FBXW7 did not influence the half-life of either (WT)BRAF or (V600E)BRAF. Furthermore, disruption of the substrate-binding site of mouse FBXW7 using the R482Q mutation did not affect the interaction with BRAF and the expression levels of (WT)BRAF and (V600E)BRAF were not altered in MEFs derived from mice with the homozygous knockin (R482Q)FBXW7 mutation. Overall these data confirm the existence of a negative feedback pathway by which BRAF protein stability is regulated by ERK. However, unlike the situation in C. elegans, FBXW7 does not play a unique role in mediating subsequent BRAF degradation., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

The development of the hematopoietic system is a dynamic process that is controlled by the interplay between transcriptional and epigenetic networks to determine cellular identity. These networks are critical for lineage specification and are frequently dysregulated in leukemias. Here, we identified histone demethylase KDM2B as a critical regulator of definitive hematopoiesis and lineage commitment of murine hematopoietic stem and progenitor cells (HSPCs). RNA sequencing of Kdm2b-null HSPCs and genome-wide ChIP studies in human leukemias revealed that KDM2B cooperates with polycomb and trithorax complexes to regulate differentiation, lineage choice, cytokine signaling, and cell cycle. Furthermore, we demonstrated that KDM2B exhibits a dichotomous role in hematopoietic malignancies. Specifically, we determined that KDM2B maintains lymphoid leukemias, but restrains RAS-driven myeloid transformation. Our study reveals that KDM2B is an important mediator of hematopoietic cell development and has opposing roles in tumor progression that are dependent on cellular context.

The F-box proteins (FBPs) in esophageal tumorigenesis are pivotal as they govern a broad array of basic physiological responses including cell growth, cell death and DNA damage repair. Esophageal cancer (EC) is a common and highly aggressive cancer worldwide. Aberrant stabilization of crucial proteins participates in esophageal tumorigenesis. Recently, growing evidence has shown that FBPs play a critical role in oncogenesis, invasion, metastasis and prognosis assessment of EC. In this review we summarized published data on the roles of known FBPs, their respective substrates and the key signaling pathways, in the development of EC, aiming to uncover new ways for the rational design of targeted therapies in EC.

F-box and WD repeat domain-containing7 (Fbxw7), a member of the F-box family of proteins, which are components of an E3 ubiquitin ligase complex, plays an important role as a general tumor suppressor in regulating the effects of various oncoproteins. Recently, accumulating studies have shown that Fbxw7 plays an important role in tumor cell motility, invasion and cancer metastasis. However, little is known about the signaling mechanisms that regulate tumor apoptosis, growth arrest and the epithelial-to-mesenchymal transition (EMT) in gastric cancer. In our study, we confirmed that Fbxw7 expression was decreased in gastric cancer tissues, and that Fbxw7 inhibited gastric cancer progression by inducing apoptosis and growth arrest. Furthermore, gastric cancer migration and invasion were decreased or increased following Fbxw7 overexpression or knockdown, respectively, and the expressions of various EMT markers, such as E-cadherin, N-cadherin and vimentin, were altered after Fbxw7 inhibition or overexpression. Furthermore, we demonstrated that Fbxw7 inhibits the EMT via the down-regulation of Snail 1 and ZEB 1, which are upstream transcription factors that promote this process. Additionally, RhoA showed higher expression in the same gastric cancer tissues than in normal tumor-adjacent samples. We found that Fbxw7 expression was negatively correlated with RhoA protein expression in gastric cancer tissues based on Pearson's correlation coefficient analysis. Moreover, we found that RhoA protein abundance was regulated by Fbxw7 via ubiquitination and proteasomal degradation in gastric cancer. We further demonstrated the effects of RhoA re-expression or inhibition on stable Fbxw7-overexpressing or Fbxw7-silenced cell lines in vitro and in vivo. These results suggest that Fbxw7 induces apoptosis and growth arrest and inhibits the EMT in part by down-regulating the RhoA signaling pathway., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Mesenchymal stem cells (MSCs) are characterized by their self-renewing capacity and differentiation potential into multiple tissues. Thus, management of the differentiation capacities of MSCs is important for MSC-based regenerative medicine, such as craniofacial bone regeneration, and in new treatments for metabolic bone diseases, such as osteoporosis. In recent years, histone modification has been a growing topic in the field of MSC lineage specification, in which the Su(var)3-9, enhancer-of-zeste, trithorax (SET) domain-containing family and the Jumonji C (JmjC) domain-containing family represent the major histone lysine methyltransferases (KMTs) and histone lysine demethylases (KDMs), respectively. In this review, we summarize the current understanding of the epigenetic mechanisms by which SET domain-containing KMTs and JmjC domain-containing KDMs balance the osteogenic and adipogenic differentiation of MSCs.

Emerging evidence has indicated that the pathogenesis of neuropathic pain is mediated by spinal neural plasticity in the dorsal horn, which provides insight for analgesic therapy. Here, we report that the abundance of tumor necrosis factor receptor-associated factor 2 and NcK-interacting kinase (TNIK), a kinase that is presumed to regulate neural plasticity, was specifically enhanced in ipsilateral dorsal horn neurons after spinal nerve ligation (SNL; left L5 and L6). Spinal TNIK-associated allodynia is mediated by downstream TNIK-GluR1 coupling and the subsequent phosphorylation-dependent trafficking of GluR1 toward the plasma membrane in dorsal horn neurons. Tumor necrosis factor receptor-associated factor 2 (TRAF2), which is regulated by spinal F-box protein 3 (Fbxo3)-dependent F-box and leucine-rich repeat protein 2 (Fbxl2) ubiquitination, contributes to SNL-induced allodynia by modifying TNIK/GluR1 phosphorylation-associated GluR1 trafficking. Although exhibiting no effect on Fbxo3/Fbxl2/TRAF2 signaling, focal knockdown of spinal TNIK expression prevented SNL-induced allodynia by attenuating TNIK/GluR1 phosphorylation-dependent subcellular GluR1 redistribution. In contrast, intrathecal administration of BC-1215 (N1,N2-Bis[[4-(2-pyridinyl)phenyl]methyl]-1,2-ethanediamine) (a novel Fbxo3 inhibitor) prevented SNL-induced Fbxl2 ubiquitination and subsequent TFAF2 de-ubiquitination to ameliorate behavioral allodynia via antagonizing TRAF2/TNIK/GluR1 signaling. By targeting spinal Fbxo3-dependent Fbxl2 ubiquitination and the subsequent TRAF2/TNIK/GluR1 cascade, spinal application of a TNF-α-neutralizing antibody ameliorated SNL-induced allodynia, and, conversely, intrathecal TNF-α injection into naive rats induced allodynia via a spinal Fbxo3/Fbxl2-dependent modification of the TRAF2/TNIK/GluR1 cascade. Together, our results suggest that spinal TNF-α contributes to the development of neuropathic pain by upregulating TRAF2/TNIK/GluR1 signaling via Fbxo3-dependent Fbxl2 ubiquitination and degradation. Thus, we propose a potential medical treatment strategy for neuropathic pain by targeting the F-box protein or TNIK., Significance Statement: TNF-α participates in neuropathic pain development by facilitating the spinal TRAF2-dependent TNIK-GluR1 association, which drives GluR1-containing AMPA receptor trafficking toward the plasma membrane. In addition, F-box protein 3 modifies this pathway by inhibiting F-box and leucine-rich repeat protein 2-mediated TRAF2 ubiquitination, suggesting that protein ubiquitination contributes crucially to the development of neuropathic pain. These results provide a novel therapeutic strategy for pain relief., (Copyright © 2015 the authors 0270-6474/15/3516546-16$15.00/0.)

An important characteristic of vertebrate CNS development is the formation of specific amounts of insulating myelin membrane on axons. CNS myelin is produced by oligodendrocytes, glial cells that extend multiple membrane processes to wrap multiple axons. Recent data have shown that signaling mediated by the mechanistic target of rapamycin (mTOR) serine/threonine kinase promotes myelination, but factors that regulate mTOR activity for myelination remain poorly defined. Through a forward genetic screen in zebrafish, we discovered that mutation of fbxw7, which encodes the substrate recognition subunit of a SCF ubiquitin ligase that targets proteins for degradation, causes hypermyelination. Among known Fbxw7 targets is mTOR. Here, we provide evidence that mTOR signaling activity is elevated in oligodendrocyte lineage cells of fbxw7 mutant zebrafish larvae. Both genetic and pharmacological inhibition of mTOR function suppressed the excess myelin gene expression resulting from loss of Fbxw7 function, indicating that mTOR is a functionally relevant target of Fbxw7 in oligodendrocytes. fbxw7 mutant larvae wrapped axons with more myelin membrane than wild-type larvae and oligodendrocyte-specific expression of dominant-negative Fbxw7 produced longer myelin sheaths. Our data indicate that Fbxw7 limits the myelin-promoting activity of mTOR, thereby serving as an important brake on developmental myelination., Significance Statement: Myelin, a specialized, proteolipid-rich membrane that ensheaths and insulates nerve fibers, facilitates the rapid conduction of electrical impulses over long distances. Abnormalities in myelin formation or maintenance result in intellectual and motor disabilities, raising a need for therapeutic strategies designed to promote myelination. The mTOR kinase is a powerful driver of myelination, but the mechanisms that regulate mTOR function in myelination are not well understood. Our studies reveal that Fbxw7, a subunit of a ubiquitin ligase that targets other proteins for degradation, acts as a brake on myelination by limiting mTOR function. These findings suggest that Fbxw7 helps tune the amount of myelin produced during development and raise the possibility that Fbxw7 could be a target of myelin-promoting therapies., (Copyright © 2015 the authors 0270-6474/15/3514861-11$15.00/0.)

How stem cells maintain their stemness or initiate exit from the stem cell state for differentiation remains largely unknown. Aldehyde dehydrogenase (ALDH) activity is a hallmark of stem cells-including embryonic, adult tissue, and cancer stem cells-and is essential for their maintenance. The mechanisms by which ALDH activity is regulated in stem cells have remained poorly understood, however. We now show that the ubiquitin-dependent degradation of ALDH3 mediated by FBXL12 (F box and leucine-rich repeat protein 12) is essential for execution of the differentiation program of trophoblast stem cells (TSCs). FBXL12 is present only in eutherian mammals, and its expression is largely restricted to the placenta during mouse embryogenesis. FBXL12 was found to interact specifically with members of the ALDH3 family and to mediate their polyubiquitylation. Most mice deficient in FBXL12 died during the embryonic or perinatal period probably as a result of abnormal development of the placenta, characterized by impaired formation of the junctional zone. ALDH3 accumulated in the FBXL12-deficient placenta, and forced expression of ALDH3 in wild-type TSCs phenocopied the differentiation defect of FBXL12-deficient TSCs. Conversely, inhibition of ALDH3 activity by gossypol rescued the phenotype of FBXL12 deficiency. Our results suggest that FBXL12 plays a key role in the downregulation of ALDH3 activity in TSCs and thereby initiates trophoblast differentiation during placental development., (© 2015 AlphaMed Press.)

FBW7 (F-box and WD repeat domain containing 7), also known as FBXW7 or hCDC4, is a tumor suppressor gene mutated in a broad spectrum of cancer cell types. As a component of the SCF E3 ubiquitin ligase, FBW7 is responsible for specifically recognizing phosphorylated substrates, many important for tumor progression, and targeting them for ubiquitin-mediated degradation. Although the role of FBW7 as a tumor suppressor is well established, less well studied is how FBW7-mutated cancer cells might be targeted for selective killing. To explore this further, we undertook a genome-wide RNAi screen using WT and FBW7 knockout colorectal cell lines and identified the spindle assembly checkpoint (SAC) protein BUBR1, as a candidate synthetic lethal target. We show here that asynchronous FBW7 knockout cells have increased levels of mitotic APC/C substrates and are sensitive to knockdown of not just BUBR1 but BUB1 and MPS1, other known SAC components, suggesting a dependence of these cells on the mitotic checkpoint. Consistent with this dependence, knockdown of BUBR1 in cells lacking FBW7 results in significant cell aneuploidy and increases in p53 levels. The FBW7 substrate cyclin E was necessary for the genetic interaction with BUBR1. In contrast, the establishment of this dependence on the SAC requires the deregulation of multiple substrates of FBW7. Our work suggests that FBW7 knockout cells are vulnerable in their dependence on the mitotic checkpoint and that this may be a good potential target to exploit in FBW7-mutated cancer cells., (Copyright © 2015 by the Genetics Society of America.)