Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：EMBO  

DOI： 10.15252/embj.2022112869

Publishing type：Research paper (scientific journal)   Publisher：Oxford University Press (OUP)  

Summary

Most protein-coding sequences (CDSs) are predicted sequences based on criteria such as a size sufficient to encode a product of at least 100 amino acids and with translation starting at an AUG initiation codon. However, recent studies based on ribosome profiling and mass spectrometry have shown that several RNAs annotated as long noncoding RNAs (lncRNAs) are actually translated to generate polypeptides of fewer than 100 amino acids, and that many proteins are translated from near-cognate initiation codons such as CUG and GUG. Furthermore, studies of genetically engineered mouse models have revealed that such polypeptides and proteins contribute to diverse physiological processes. In this review, we describe the latest methods for the identification of unannotated CDSs and provide examples of their physiological functions.

DOI： 10.1093/jb/mvac064

Language：English   Publishing type：Research paper (scientific journal)  

Although the mammalian intestinal epithelium manifests robust regenerative capacity after various cytotoxic injuries, the underlying mechanism has remained unclear. Here we identify the cyclin-dependent kinase inhibitor p57 as a specific marker for a quiescent cell population located around the +4 position of intestinal crypts. Lineage tracing reveals that the p57+ cells serve as enteroendocrine/tuft cell precursors under normal conditions but dedifferentiate and act as facultative stem cells to support regeneration after injury. Single-cell transcriptomics analysis shows that the p57+ cells undergo a dynamic reprogramming process after injury that is characterized by fetal-like conversion and metaplasia-like transformation. Population-level analysis also detects such spatiotemporal reprogramming widely in other differentiated cell types. In intestinal adenoma, p57+ cells manifest homeostatic stem cell activity, in the context of constitutively activated spatiotemporal reprogramming. Our results highlight a pronounced plasticity of the intestinal epithelium that supports maintenance of tissue integrity in normal and neoplastic contexts.

DOI： 10.1038/s41467-022-29165-z

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

Although several long noncoding RNAs (lncRNAs) have recently been shown to encode small polypeptides, those in testis remain largely uncharacterized. Here we identify two sperm-specific polypeptides, Kastor and Polluks, encoded by a single mouse locus (Gm9999) previously annotated as encoding a lncRNA. Both Kastor and Polluks are inserted in the outer mitochondrial membrane and directly interact with voltage-dependent anion channel (VDAC), despite their different amino acid sequences. Male VDAC3-deficient mice are infertile as a result of reduced sperm motility due to an abnormal mitochondrial sheath in spermatozoa, and deficiency of both Kastor and Polluks also severely impaired male fertility in association with formation of a similarly abnormal mitochondrial sheath. Spermatozoa lacking either Kastor or Polluks partially recapitulate the phenotype of those lacking both. Cooperative function of Kastor and Polluks in regulation of VDAC3 may thus be essential for mitochondrial sheath formation in spermatozoa and for male fertility.

DOI： 10.1038/s41467-022-28677-y

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

Although long noncoding RNAs (lncRNAs) are transcripts that do not encode proteins by definition, some lncRNAs actually contain small open reading frames that are translated. TINCR (terminal differentiation-induced ncRNA) has been recognized as a lncRNA that contributes to keratinocyte differentiation. However, we here show that TINCR encodes a ubiquitin-like protein that is well conserved among species and whose expression was confirmed by the generation of mice harboring a FLAG epitope tag sequence in the endogenous open reading frame as well as by targeted proteomics. Forced expression of this protein promoted cell cycle progression in normal human epidermal keratinocytes, and mice lacking this protein manifested a delay in skin wound healing associated with attenuated cell cycle progression in keratinocytes. We termed this protein TINCR-encoded ubiquitin-like protein (TUBL), and our results reveal a role for TINCR in the regulation of keratinocyte proliferation and skin regeneration that is dependent on TUBL.

DOI： 10.1371/journal.pgen.1009686

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

Although ribosome-profiling and translation initiation sequencing (TI-seq) analyses have identified many noncanonical initiation codons, the precise detection of translation initiation sites (TISs) remains a challenge, mainly because of experimental artifacts of such analyses. Here, we describe a new method, TISCA (TIS detection by translation Complex Analysis), for the accurate identification of TISs. TISCA proved to be more reliable for TIS detection compared with existing tools, and it identified a substantial number of near-cognate codons in Kozak-like sequence contexts. Analysis of proteomics data revealed the presence of methionine at the NH2-terminus of most proteins derived from near-cognate initiation codons. Although eukaryotic initiation factor 2 (eIF2), eIF2A and eIF2D have previously been shown to contribute to translation initiation at near-cognate codons, we found that most noncanonical initiation events are most probably dependent on eIF2, consistent with the initial amino acid being methionine. Comprehensive identification of TISs by TISCA should facilitate characterization of the mechanism of noncanonical initiation.

DOI： 10.1093/nar/gkab549

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

Chromodomain helicase DNA-binding protein 8 (CHD8) is an ATP-dependent chromatin-remodeling factor that is encoded by the most frequently mutated gene in individuals with autism spectrum disorder. CHD8 is expressed not only in neural tissues but also in many other organs; however, its functions are largely unknown. Here, we show that CHD8 is highly expressed in and maintains the stemness of hematopoietic stem cells (HSCs). Conditional deletion of Chd8 specifically in mouse bone marrow induces cell cycle arrest, apoptosis, and a differentiation block in HSCs in association with upregulation of the expression of p53 target genes. A colony formation assay and bone marrow transplantation reveal that CHD8 deficiency also compromises the stemness of HSCs. Furthermore, additional ablation of p53 rescues the impaired stem cell function and differentiation block of CHD8-deficient HSCs. Our results thus suggest that the CHD8-p53 axis plays a key role in regulation of the stemness and differentiation of HSCs.

DOI： 10.1016/j.celrep.2021.108688

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

Ion channel proteins play important roles in various cell functions, making them attractive drug targets. Artificial lipid bilayer recording is a technique used to measure the ion transport activities of channel proteins with high sensitivity and accuracy. However, the measurement efficiency is low. In order to improve the efficiency, we developed a method that allows us to form bilayers on a hydrogel bead and record channel currents promptly. We tested our system by measuring the activities of various types of channels, including gramicidin, alamethicin, α-hemolysin, a voltage-dependent anion channel 1 (VDAC1), a voltage- and calcium-activated large conductance potassium channel (BK channel), and a potassium channel from Streptomyces lividans (KcsA channel). We confirmed the ability for enhanced measurement efficiency and measurement system miniaturizion.

DOI： 10.3390/mi11121070

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

An integrative understanding of nuclear events including transcription in normal and cancer cells requires comprehensive and quantitative measurement of protein dynamics that underlie such events. However, the low abundance of most nuclear proteins hampers their detailed functional characterization. We have now comprehensively quantified the abundance of nuclear proteins with the use of proteomics approaches in both normal and transformed human diploid fibroblasts. We found that subunits of the 26S proteasome complex were markedly down-regulated in the nuclear fraction of the transformed cells compared with that of the wild-type cells. The intranuclear proteasome abundance appeared to be inversely related to the rate of cell cycle progression, with restraint of the cell cycle being associated with an increase in the amount of proteasome subunits in the nucleus, suggesting that the nuclear proteasome content is dependent on the cell cycle. Furthermore, chromatin enrichment for proteomics (ChEP) analysis revealed enrichment of the proteasome in the chromatin fraction of quiescent cells and its apparent dissociation from chromatin in transformed cells. Our results thus suggest that translocation of the nuclear proteasome to chromatin may play an important role in control of the cell cycle and oncogenesis through regulation of chromatin-associated transcription factors.

DOI： 10.1038/s41598-020-62697-2

PubMed

Language：English  

circRNAs arise from back splicing events during mRNA processing, and when deregulated can play an active role in cancer. Here we characterize a new circRNA (circPOK) encoded by the Zbtb7a gene (also kown as POKEMON, LRF) in the context of mesenchymal tumor progression. circPOK functions as a non-coding proto-oncogenic RNA independently and antithetically to its linear transcript counterpart, which acts as a tumor suppressor by encoding the Pokemon transcription factor. We find that circPOK regulates pro-proliferative and pro-angiogenic factors by co-activation of the ILF2/3 complex. Importantly, the expression of Pokemon protein and circRNA is aberrantly uncoupled in cancer through differential post-transcriptional regulation. Thus, we identify a novel type of genetic unit, the iRegulon, that yields biochemically distinct RNA products, circular and linear, with diverse and antithetical functions. Our findings further expand the cellular repertoire towards the control of normal biological outputs, while aberrant expression of such components may underlie disease pathogenesis including cancer.

DOI： 10.1038/s41422-019-0192-1

PubMed

Language：English   Publisher：Japan Society for Cell Biology  

Although the definition of a noncoding RNA (ncRNA) is an RNA molecule that does not encode a protein, recent evidence has revealed that some ncRNAs are indeed translated to give rise to small polypeptides (usually containing fewer than 100 amino acids). Despite their small size, however, these peptides are often biologically relevant in that they are required for a variety of cellular processes. In this review, we summarize the production and functions of peptides that have been recently identified as translation products of putative ncRNAs.

DOI： 10.1247/csf.18005

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PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

Although long non-coding RNAs (lncRNAs) are non-protein-coding transcripts by definition, recent studies have shown that a fraction of putative small open reading frames within lncRNAs are translated(1-3). However, the biological significance of these hidden polypeptides is still unclear. Here we identify and functionally characterize a novel polypeptide encoded by the lncRNA LINC00961. This polypeptide is conserved between human and mouse, is localized to the late endosome/lysosome and interacts with the lysosomal v-ATPase to negatively regulate mTORC1 activation. This regulation of mTORC1 is specific to activation of mTORC1 by amino acid stimulation, rather than by growth factors. Hence, we termed this polypeptide 'small regulatory polypeptide of amino acid response' (SPAR). We show that the SPAR-encoding lncRNA is highly expressed in a subset of tissues and use CRISPR/Cas9 engineering to develop a SPAR-polypeptide-specific knockout mouse while maintaining expression of the host lncRNA. We find that the SPAR-encoding lncRNA is downregulated in skeletal muscle upon acute injury, and using this in vivo model we establish that SPAR downregulation enables efficient activation of mTORC1 and promotes muscle regeneration. Our data provide a mechanism by which mTORC1 activation may be finely regulated in a tissue-specific manner in response to injury, and a paradigm by which lncRNAs encoding small polypeptides can modulate general biological pathways and processes to facilitate tissue-specific requirements, consistent with their restricted and highly regulated expression profile.

DOI： 10.1038/nature21034

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Language：English   Publisher：TAYLOR & FRANCIS INC  

DOI： 10.1080/15384101.2017.1304735

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CURRENT BIOLOGY LTD  

The expansive dimension of non-coding genes is by now a well-recognized feature of eukaryotes genomes. Over the past decades, in vitro functional studies and in vivo manipulation of non-coding genes through Genetically Engineered Mouse Models (GEMMs) have provided compelling evidence that almost every biological phenomenon is regulated, at some level, by non-coding RNA transcripts or by coding RNAs with non-coding functions. In this opinion article, we will discuss how recent discoveries in the field of non-coding RNAs are contributing to advance our understanding of evolution and organismal complexity and its relevance to human diseases.

DOI： 10.1016/j.ceb.2016.10.005

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

Proper development of T cells depends on lineage-specific regulators controlled transcriptionally and posttranslationally to ensure precise levels at appropriate times. Conditional inactivation of F-box protein Fbw7 in mouse T-cell development resulted in reduced thymic CD4 single-positive (SP) and splenic CD4(+) and CD8(+) cell proportions. Fbw7 deficiency skewed CD8 SP lineage differentiation, which exhibited a higher incidence of apoptosis. Similar perturbations during development of CD8-positive cells were reported with transgenic mice, which enforced GATA3 (T-cell differentiation regulator) expression throughout T-cell development. We observed augmented GATA3 in CD4/CD8 double negative (DN) stage 4, CD4 SP, and CD8 SP lineages in Fbw7-deficient thymocytes. Using overexpressed proteins in cultured cells, we demonstrated that Fbw7 bound to, ubiquitylated, and destabilized GATA3. Two Cdc4 phosphodegron (CPD) candidate sequences, consensus Fbw7 recognition domains, were identified in GATA3, and phosphorylation of Thr-156 in CPD was required for Fbw7-mediated ubiquitylation and degradation. Phosphorylation of GATA3 Thr-156 was detected in mouse thymocytes, and cyclin-dependent kinase 2 (CDK2) was identified as a respondent for phosphorylation at Thr-156. These observations suggest that Fbw7-mediated GATA3 regulation with CDK2-mediated phosphorylation of CPD contributes to the precise differentiation of T-cell lineages.

DOI： 10.1128/MCB.01549-13

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC HEMATOLOGY  

T cells are key components of the immune system, playing a central role in cell-mediated immunity. The sequential differentiation of T cells is associated with strict regulation of the cell cycle at each developmental stage. A balance between p53 activity and pre-T cell receptor (TCR) signaling regulates proliferation and differentiation decisions made by these cells. The relation between maintenance of this balance and the function of cell cycle regulators has remained largely unknown, however. We now show that mice with T cell-specific deficiency of the cyclin-dependent kinase inhibitor p57 manifest a differentiation block at the early stageof T cell maturation. Further genetic analysis showed that this defect is attributable to an imbalance between p53 activity and pre-TCR signaling caused by hyperactivation of the E2F-p53 pathway. Moreover, ablation of both p57 and p53 in T cells led to the development of aggressive thymic lymphomas with a reduced latency compared with that apparent for p53-deficient mice, whereas ablation of p57 alone did not confer susceptibility to this hematologic malignancy. Our results thus show that the p57-E2F-p53 axis plays a pivotal role in the proper development of T cells as well as in the prevention of lymphomagenesis.

DOI： 10.1182/blood-2013-10-532390

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：7  

Bisphosphonate-related osteonecrosis of the jaw (BRONJ) is a serious side effect of nitrogen-containing bisphosphonate (NBP) use. Many studies have shown that BRONJ is limited to the jawbone and does not occur in the other bones. We hypothesized that BRONJ is related to local bacterial iections and involves the innate immune system. To examine the relationship between BRONJ and innate immunity, we examined the effects of NBPs on macrophages, one of the important cell types in innate immunity. The expression of toll-like receptor-4 (TLR4) in cells after pretreatment with zoledronic acid (ZOL) did not considerably differ from that in untreated control cells. However, cytokine levels and nitric oxide (NO) production increased after pretreatment with ZOL. Furthermore, ZOL induced NF-κB activation by enhancing IκB-α degradation. Lipopolysaccharide (LPS)-induced apoptosis also increased after pretreatment with ZOL. This effect was mediated by a reduction of suppressor of cytokine signaling-1 (SOCS1), which is a negative regulator of myeloid differentiation primary response gene 88 (MyD 88)-dependent signaling. These results suggest that ZOL induced excessive innate immune response and proinflammatory cytokine production and that these processes may be involved in the bone destruction observed in BRONJ. © 2013 Muratsu et al.

DOI： 10.1371/journal.pone.0067906

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

Throughout life, neural stem cells (NSCs) in the adult hippocampus persistently generate new neurons that modify the neural circuitry. Adult NSCs constitute a relatively quiescent cell population but can be activated by extrinsic neurogenic stimuli. However, the molecular mechanism that controls such reversible quiescence and its physiological significance have remained unknown. Here, we show that the cyclin-dependent kinase inhibitor p57 kip2 (p57) is required for NSC quiescence. In addition, our results suggest that reduction of p57 protein in NSCs contributes to the abrogation of NSC quiescence triggered by extrinsic neurogenic stimuli such as running. Moreover, deletion of p57 in NSCs initially resulted in increased neurogenesis in young adult and aged mice. Long-term p57 deletion, on the contrary, led to NSC exhaustion and impaired neurogenesis in aged mice. The regulation of NSC quiescence by p57 might thus have important implications for the short-term (extrinsic stimuli-dependent) and long-term (age-related) modulation of neurogenesis. The EMBO Journal (2013) 32, 970-981; doi:10.1038/emboj.2013.50; Published online 12 March 2013

DOI： 10.1038/emboj.2013.50

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CELL PRESS  

Imatinib eradicates dividing progenitor cells of chronic myeloid leukemia (CML) but does not effectively target nondividing leukemia-initiating cells (LICs); thus, the disease often relapse after its discontinuation. We now show that Fbxw7 plays a pivotal role in maintenance of quiescence in LICs of CML by reducing the level of c-Myc. Abrogation of quiescence in LICs by Fbxw7 ablation increased their sensitivity to imatinib, and the combination of Fbxw7 ablation with imatinib treatment resulted in a greater depletion of LICs than of normal hematopoietic stem cells in mice. Purging of LICs by targeting Fbxw7 to interrupt their quiescence and subsequent treatment with imatinib may thus provide the basis for a promising therapeutic approach to CML.

DOI： 10.1016/j.ccr.2013.01.026

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Publisher：2  

DOI： 10.1016/j.bbagen.2012.07.004

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Timely exit of cells from the cell cycle is essential for proper cell differentiation during embryogenesis. Cyclin-dependent kinase (CDK) inhibitors (CKIs) of the Cip/Kip family (p21, p27, and p57) are negative regulators of cell cycle progression and are thought to be essential for development. However, the extent of functional redundancy among Cip/Kip family members has remained largely unknown. We have now generated mice that lack all three Cip/Kip CKIs (TKO mice) and compared them with those lacking each possible pair of these proteins (DKO mice). We found that the TKO embryos develop normally until midgestation but die around embryonic day (E) 13.5, slightly earlier than p27/p57 DKO embryos. The TKO embryos manifested morphological abnormalities as well as increased rates of cell proliferation and apoptosis in the placenta and lens that were essentially indistinguishable from those of p27/p57 DKO mice. Unexpectedly, the proliferation rate and cell cycle profile of mouse embryonic fibroblasts (MEFs) lacking all three Cip/Kip CKIs did not differ substantially from those of control MEFs. The abundance and kinase activity of CDK2 were markedly increased, whereas CDK4 activity and cyclin D1 abundance were decreased, in both p27/p57 DKO and TKO MEFs during progression from G(0) to S phase compared with those in control MEFs. The extents of the increase in CDK2 activity and the decrease in CDK4 activity and cyclin D1 abundance were greater in TKO MEFs than in p27/p57 DKO MEFs. These results suggest that p27 and p57 play an essential role in mouse development after midgestation, and that p21 plays only an auxiliary role in normal development (although it is thought to be a key player in the response to DNA damage). (C) 2012 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2012.09.041

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Induced pluripotent stem cells (iPSCs) share many biological properties with embryonic stem cells (ESCs), and are generated from somatic cells by expression of some transcription factors such as Oct3/4, Sox2, Klf4 and c-Myc. Among these factors, the abundance of c-Myc is strictly regulated by Fbxw7, a subunit of Skp1-Cul1-F-box protein-type ubiquitin ligase. We have now shown that the expression of Fbxw7 was increased as ESCs differentiated. To investigate the role of Fbxw7 in the ESCs/iPSCs, we examined the impact of Fbxw7 ablation in the efficiency in iPSC generation. The frequency of iPSC generation from mouse embryonic fibroblasts (MEFs) lacking Fbxw7 was markedly greater than that from control MEFs. Depletion of Fbxw7 also resulted in promotion of iPSC generation. Morphology of iPSC clonies from Fbxw7-depleted MEFs appeared more undifferentiated than that from MEFs overexpressing c-Myc. Additional depletion of c-Myc did not abrogate the effect of Fbxw7 depletion, suggesting that c-Myc accumulation is not necessarily required for the increased efficiency in iPSC generation by Fbxw7 ablation. Substrates of Fbxw7 other than c-Myc might therefore play a key role in iPSC generation. These results suggest that transient inhibition of Fbxw7 would be a more promising approach to efficient generation of iPSCs than c-Myc over-expression.

DOI： 10.1111/j.1365-2443.2012.01626.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CELL PRESS  

The NF kappa B/Rel family of proteins play critical roles in a variety of cellular processes. Thus, their physiological activation is tightly controlled. Recently, the NF kappa B2/p100 precursor has been characterized as the fourth I kappa B type of suppressor for NF kappa B. However, the molecular mechanism(s) underlying regulated destruction of NF kappa B2 remains largely unknown. Here, we report that, unlike other I kappa Bs, ubiquitination and destruction of NF kappa B2 are governed by SCFFbw7 in a GSK3-dependent manner. In Fbw7(-/-) cells, elevated expression of NF kappa B2/p100 leads to a subsequent reduction in NF kappa B signaling pathways and elevated sensitivity to TNF alpha-induced cell death. Reintroducing wild-type Fbw7, but not disease-derived mutant forms of Fbw7, rescues NF kappa B activity. Furthermore, T cell-specific depletion of Fbw7 also leads to reduced NF kappa B activity and perturbed T cell differentiation. Therefore, our work identifies Fbw7 as a physiological E3 ligase controlling NF kappa B2's stability. It further implicates that Fbw7 might exert its tumor-suppressor function by regulating NF kappa B activity.

DOI： 10.1016/j.celrep.2012.04.002

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

D-type cyclins play a pivotal role in G(1)-S progression of the cell cycle, and their expression is frequently deregulated in cancer. Cyclin D1 has a half-life of only similar to 30 min as a result of its ubiquitylation and proteasomal degradation, with various F-box proteins, including Fbxo4, Fbxw8, Skp2, and Fbxo31, having been found to contribute to its ubiquitylation. We have now generated Fbxo4-deficient mice and found no abnormalities in these animals. Cyclin D1 accumulation was thus not observed in Fbxo4(-/-) mouse tissues. The half-life of cyclin D1 in mouse embryonic fibroblasts (MEFs) prepared from Fbxo4(-/-), Fbxw8(-/-), and Fbxo4(-/-); Fbxw8(-/-) mice also did not differ from that in wild-type MEFs. Additional depletion of Skp2 and Fbxo31 in Fbxo4(-/-); Fbxw8(-/-) MEFs by RNA interference did not affect cyclin D1 stability. Although Fbxo31 depletion in MEFs increased cyclin D1 abundance, this effect appeared attributable to upregulation of cyclin D1 mRNA. Furthermore, abrogation of the function of the Skp1-Cul1-F-box protein (SCF) complex or the anaphase-promoting complex/cyclosome (APC/C) complexes did not alter the half-life of cyclin D1, whereas cyclin D1 degradation was dependent largely on proteasome activity. Our genetic analyses thus do not support a role for any of the four F-box proteins examined in cyclin D1 degradation during normal cell cycle progression. They suggest the existence of other ubiquitin ligases that target cyclin D1 for proteolysis.

DOI： 10.1128/MCB.06570-11

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

The cyclin-dependent kinase inhibitor (CKI) p57(Kip2) plays a pivotal role in cell cycle arrest during development, in particular, in the regulation of the entry of proliferating progenitors into quiescence. The gene encoding p57 undergoes genomic imprinting, and impairment of the regulation of p57 expression results in various developmental anomalies in humans and mice. We now show that p57 is expressed predominantly in the subcommissural organ and cerebellar interneurons in the mouse brain and that mice with brain-specific deletion of the p57 gene (Kip2) manifest prominent nonobstructive hydrocephalus as well as cerebellar malformation associated with the loss of Pax2-positive interneuron precursors and their descendants, including Golgi cells and gamma-aminobutyric acid-containing neurons of the deep cerebellar nuclei. These abnormalities were found to be attributable to massive apoptosis of precursor cells in the developing brain. The morphological defects of the p57-deficient mice were corrected by knock-in of the gene for the related CKI p27(Kip1) at the Kip2 locus. The abnormalities were also prevented by additional genetic ablation of p53 or E2F1. Our results thus implicate p57 in cell cycle arrest in the subcommissural organ and Pax2-positive interneuron precursors, with the lack of p57 resulting in induction of p53-dependent apoptosis due to hyperactivation of E2F1.

DOI： 10.1128/MCB.05370-11

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CELL PRESS  

Quiescence is required for the maintenance of hematopoietic stem cells (HSCs). Members of the Cip/Kip family of cyclin-dependent kinase (CDK) inhibitors (p21, p27, p57) have been implicated in HSC quiescence, but loss of p21 or p27 in mice affects HSC quiescence or functionality only under conditions of stress. Although p57 is the most abundant family member in quiescent HSCs, its role has remained un-characterized. Here we show a severe defect in the self-renewal capacity of p57-deficient HSCs and a reduction of the proportion of the cells in G(0) phase. Additional ablation of p21 in a p57-null background resulted in a further decrease in the colony-forming activity of HSCs. Moreover, the HSC abnormalities of p57-deficient mice were corrected by knocking in the p27 gene at the p57 locus. Our results therefore suggest that, among Cip/Kip family CDK inhibitors, p57 plays a predominant role in the quiescence and maintenance of adult HSCs.

DOI： 10.1016/j.stem.2011.06.014

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Control of the growth and differentiation of neural stem cells is fundamental to brain development and is largely dependent on the Notch signaling pathway. The mechanism by which the activity of Notch is regulated during brain development has remained unclear, however. Fbxw7 (also known as Fbw7, SEL-10, hCdc4, or hAgo) is the F-box protein subunit of an Skp1-Cul1-F-box protein (SCF)-type ubiquitin ligase complex that plays a central role in the degradation of Notch family members. We now show that mice with brain-specific deletion of Fbxw7 (Nestin-Cre/Fbxw7(F/F) mice) die shortly after birth with morphological abnormalities of the brain and the absence of suckling behavior. The maintenance of neural stern cells was sustained in association with the accumulation of Notch! and Notch3, as well as up-regulation of Notch target genes in the mutant mice. Astrogenesis was also enhanced in the mutant mice in vivo, and the differentiation of neural progenitor cells was skewed toward astrocytes rather than neurons in vitro, with the latter effect being reversed by treatment of the cells with a pharmacological inhibitor of the Notch signaling pathway. Our results thus implicate Fbxw7 as a key regulator of the maintenance and differentiation of neural stein cells in the brain.

DOI： 10.1074/jbc.M110.194936

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Oxidative stress has been implicated in cancer initiation and progression. Fbxw7 (also known as Fbw7, SEL-10, hCdc4, or hAgo) is the F-box protein subunit of an Skp1-Cul1-F-box (SCF)-type ubiquitin ligase complex that plays a central role in the degradation of oncoproteins such as c-Myc, c-Jun, Notch, and cyclin E. Fbxw7 is therefore thought to function as a tumor suppressor, and indeed the Fbxw7 gene is frequently mutated in many human malignancies. The Fbxw7 gene locus encodes three protein isoforms: Fbxw7 alpha, Fbxw7 beta, and Fbxw7 gamma. Whereas Fbxw7 alpha and Fbxw7 gamma are resident in the nucleus, Fbxw7 beta shows a cytoplasmic distribution suggestive of localization to the endoplasmic reticulum (ER). The specific function of Fbxw7 beta has remained unknown, however. We now show that Fbxw7 beta contains a putative transmembrane domain near its NH2-terminus, and topological analysis revealed that Fbxw7 beta is inserted in the ER membrane. Fbxw7 beta assembled with Skp1, Cul1, and Rbx1 to form an SCF complex, although the efficiency of this process appeared lower than that for Fbxw7 alpha or Fbxw7 gamma. To explore the physiological role of Fbxw7 beta, we generated mice specifically lacking this isoform of Fbxw7. Although these animals did not exhibit any apparent abnormalities in development, primary cultures of neurons prepared from the mutant mice were more vulnerable to oxidative stress than were those prepared from wild-type mice. Conversely, overexpression of Fbxw7 beta rendered cells resistant to oxidative stress, without affecting sensitivity to ER stress or other apoptosis-inducing agents. Our results thus suggest that Fbxw7 beta contributes to the protection of cells from oxidative stress. (Cancer Sci 2011; 102: 749-755)

DOI： 10.1111/j.1349-7006.2011.01851.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC CLINICAL INVESTIGATION INC  

E3 ubiquitin ligase complexes of the SCF type consist of ring-box 1 (Rbx1), cullin 1 (Cull), S-phase kinase-associated protein 1 (Skp1), and a member of the F-box family of proteins The identity of the F-box protein determines the substrate specificity of the complex The F-box family member F-box- and WD repeat domain-containing 7 (Fbxw7, also known as Fbw7, SEL-10, hCdc4, and hAgo) targets for degradation proteins with wide-ranging functions, and uncovering its in vivo role has been difficult, because Fbzw7(-/-) embryos die m utero Using two different Cre-loxP systems (Mx1-Cre and Alb-Cre), we generated mice with liver-specific null mutations of Fbxw7 Hepatic ablation of Fbxw7 resulted m hepatomegaly and steatohepatitis, with massive deposition of triglyceride, a phenotype similar to that observed in humans with nonalcoholic steatohepatitis Both cell proliferation and the abundance of Fbxw7 substrates were increased in the Fbxw7-deficient liver Long-term Fbxw7 deficiency resulted m marked proliferation of the biliary system and the development of hamartomas Fbxw7 deficiency also skewed the differentiation of liver stem cells toward the cholangiocyte lineage rather than the hepatocyte lineage m vitro This bias was corrected by additional loss of the Notch cofactor RBP-J, suggesting that Notch accumulation triggered the abnormal proliferation of the biliary system Together, our results suggest that Fbxw7 plays key roles, regulating lipogenesis and cell proliferation and differentiation in the liver

DOI： 10.1172/JCI40725

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PubMed

The strict regulation of protein degradation by the ubiquitin-proteasome pathway is pivotal for cell cycle progression. Skp2 and Fbw7 are F-box proteins that are substrate-recognition subunits of the SCF-type ubiquitin ligase complex and involved in cell cycle entry and exit. Skp2 promotes the degradation of negative regulators of the cell cycle, such as p27, and facilitates the cell cycle entry. On the other hand, Fbw7 promotes the degradation of positive regulators of the cell cycle, such as c-Myc, and induces the cell cycle exit. We have demonstrated the in vivo roles of Skp2 and Fbw7 in cell cycle regulation using gene-targeting technology in mice. Consistent with our animal models, clinical studies have suggested Skp2 as an oncogene and Fbw7 as an oncosuppressor gene. These accumulating lines of evidence support the notion that Skp2 and Fbw7 are involved in cell cycle regulation and tumorigenesis.

Scopus

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ROCKEFELLER UNIV PRESS  

Cell proliferation is strictly controlled during differentiation. In T cell development, the cell cycle is normally arrested at the CD4(+)CD8(+) stage, but the mechanism underlying such differentiation-specific exit from the cell cycle has been unclear. Fbxw7 ( also known as Fbw7, Sel-10, hCdc4, or hAgo), an F-box protein subunit of an SCF-type ubiquitin ligase complex, induces the degradation of positive regulators of the cell cycle, such as c-Myc, c-Jun, cyclin E, and Notch. FBXW7 is often mutated in a subset of human cancers. We have now achieved conditional inactivation of Fbxw7 in the T cell lineage of mice and found that the cell cycle is not arrested at the CD4(+)CD8(+) stage in the homozygous mutant animals. The mutant mice manifested thymic hyperplasia as a result of c-Myc accumulation and eventually developed thymic lymphoma. In contrast, mature T cells of the mutant mice failed to proliferate in response to mitogenic stimulation and underwent apoptosis in association with accumulation of c-Myc and p53. These latter abnormalities were corrected by deletion of p53. Our results suggest that Fbxw7 regulates the cell cycle in a differentiation-dependent manner, with its loss resulting in c- Myc accumulation that leads to hyper-proliferation in immature T cells but to p53-dependent cell-cycle arrest and apoptosis in mature T cells.

DOI： 10.1084/jem.20062299

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Fbxw7 is the F-box protein component of an SCF-type ubiquitin ligase that contributes to the ubiquitin-dependent degradation of cell cycle activators and oncoproteins. Three isoforms (alpha, beta, and gamma) of Fbxw7 are produced from mRNAs with distinct 5' exons. We have now investigated regulation of Fbxw7 expression in mouse tissues. Fbxw7 alpha mRNA was present in all tissues examined, whereas Fbxw7 beta mRNA was detected only in brain and testis, and Fbxw7 gamma mRNA in heart and skeletal muscle. The amount of Fbxw7 alpha mRNA was high during quiescence (G(0) phase) in mouse embryonic fibroblasts (MEFs) and T cells, but it decreased markedly as these cells entered the cell cycle. The abundance of Fbxw7 alpha mRNA was unaffected by cell irradiation or p53 status. In contrast, X-irradiation increased the amount of Fbxw7 beta mRNA in wild-type MEFs but not in those from p53-deficient mice, suggesting that radiation-induced up-regulation of p53 leads to production of Fbxw7 beta mRNA. Our results thus indicate that expression of Fbxw7 isoforms is differentially regulated in a cell cycle- or p53-dependent manner. (c) 2006 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2006.09.003

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL PUBLISHING  

Fbxw7 (also known as Sel-10, hCdc4 or hAgo) is the F-box protein component of a Skp1-Cul1-F-box protein (SCF) ubiquitin ligase. Fbxw7 contributes to the ubiquitin-mediated degradation of cyclin E, c-Myc, Aurora-A, Notch and c-Jun, all of which appear to function as cell-cycle promoters and oncogenic proteins. Loss of Fbxw7 results in elevated expression of its substrates, which may lead to oncogenesis. However, it remains largely unclear which accumulating substrate is most related to cancer development in Fbxw7-mutant cancer cells. In the present study, we examined the abundance of cyclin E, c-Myc and Aurora-A in seven cancer cell lines, which harbor wild-type (three lines) or mutant (four lines) Fbxw7. Although these three substrates accumulated in the Fbxw7-mutant cells, the extent of increase in the expression of these proteins varied in each line. Forced expression of Fbxw7 reduced the levels of cyclin E, c-Myc and Aurora-A in the Fbxw7-mutant cells. In contrast, a decrease in the expression of cyclin E, c-Myc or Aurora-A by RNA interference significantly suppressed the rate of proliferation and anchorage-independent growth of the Fbxw7-mutant cells. These findings thus suggest that the loss of Fbxw7 results in accumulation of cyclin E, c-Myc and Aurora-A, all of which appear to be required for growth promotion of cancer cells. Fbxw7 seems to regulate the levels of multiple targets to suppress cancer development.

DOI： 10.1111/j.1349-7006.2006.00239.x

Web of Science

PubMed