The transcription factor NRF1 (NFE2L1) activates aggrephagy by inducing p62 and GABARAPL1 after proteasome inhibition to maintain proteostasis.

The ubiquitin‒proteasome system (UPS) and autophagy are the two primary cellular pathways of misfolded or damaged protein degradation that maintain cellular proteostasis. When the proteasome is dysfunctional, cells compensate for impaired protein clearance by activating aggrephagy, a type of selective autophagy, to eliminate ubiquitinated protein aggregates; however, the molecular mechanisms by which impaired proteasome function activates aggrephagy remain poorly understood. Here, we demonstrate that activation of aggrephagy is transcriptionally induced by the transcription factor NRF1 (NFE2L1) in response to proteasome dysfunction. Although NRF1 has been previously shown to induce the expression of proteasome genes after proteasome inhibition (i.e., the proteasome bounce-back response), our genome-wide transcriptome analyses identified autophagy-related p62/SQSTM1 and GABARAPL1 as genes directly targeted by NRF1. Intriguingly, NRF1 was also found to be indispensable for the formation of p62-positive puncta and their colocalization with ULK1 and TBK1, which play roles in p62 activation via phosphorylation. Consistently, NRF1 knockdown substantially reduced the phosphorylation rate of Ser403 in p62. Finally, NRF1 selectively upregulated the expression of GABARAPL1, an ATG8 family gene, to induce the clearance of ubiquitinated proteins. Our findings highlight the discovery of an activation mechanism underlying NRF1-mediated aggrephagy through gene regulation when proteasome activity is impaired.

NRF3 activates mTORC1 arginine-dependently for cancer cell viability.

Cancer cells coordinate the mTORC1 signals and the related metabolic pathways to robustly and rapidly grow in response to nutrient conditions. Although a CNC-family transcription factor NRF3 promotes cancer development, the biological relevance between NRF3 function and mTORC1 signals in cancer cells remains unknown. Hence, we showed that NRF3 contributes to cancer cell viability through mTORC1 activation in response to amino acids, particularly arginine. NRF3 induced SLC38A9 and RagC expression for the arginine-dependent mTORC1 recruitment onto lysosomes, and it also enhanced RAB5-mediated bulk macropinocytosis and SLC7A1-mediated selective transport for arginine loading into lysosomes. Besides, the inhibition of the NRF3-mTORC1 axis impaired mitochondrial function, leading to cancer cell apoptosis. Consistently, the aberrant upregulation of the axis caused tumor growth and poor prognosis. In conclusion, this study sheds light on the unique function of NRF3 in arginine-dependent mTORC1 activation and the pathophysiological aspects of the NRF3-mTORC1 axis in cancer development.

The CNC-family transcription factor Nrf3 coordinates the melanogenesis cascade through macropinocytosis and autophagy regulation.

Melanin is a pigment produced from the amino acid L-tyrosine in melanosomes. The CNC-family transcription factor Nrf3 is expressed in the basal layer of the epidermis, where melanocytes reside, but its melanogenic function is unclear. Here, we show that Nrf3 regulates macropinocytosis and autophagy to coordinate melanogenesis cascade. In response to an exogenous inducer of melanin production, forskolin, Nrf3 upregulates the core melanogenic gene circuit, which includes Mitf, Tyr, Tyrp1, Pmel, and Oca2. Furthermore, Nrf3 induces the gene expression of Cln3, an autophagosome-related factor, for melanin precursor uptake by macropinocytosis. Ulk2 and Gabarapl2 are also identified as Nrf3-target autophagosome-related genes for melanosome formation. In parallel, Nrf3 prompts autolysosomal melanosome degradation for melanocyte survival. An endogenous melanogenic inducer αMSH also activates Nrf3-mediated melanin production, whereas it is suppressed by an HIV-1 protease inhibitor, nelfinavir. These findings indicate the significant role of Nrf3 in the melanogenesis and the anti-melanogenic potential of nelfinavir.

Nrf3 Functions Reversely as a Tumorigenic to an Antitumorigenic Transcription Factor in Obese Mice.

Tumor tissue includes cancer cells and their associated stromal cells, such as adipocytes, myocytes, and immune cells. Obesity modulates tumor microenvironment through the secretion of several inflammatory mediators by inducing adipogenesis and myogenesis. Previously, we indicated that tumor growth is promoted by a transcription factor nuclear factor erythroid 2-related factor 3 (NRF3) in human cancer cells. However, the impact of obesity on NRF3-mediated tumorigenesis remains unknown. Here we show that obesity reprograms the tumorigenic to the antitumorigenic function of Nrf3 using a diet-induced obese mouse model. Nrf3 knockdown decreased tumor growth in mice fed a normal diet (ND), whereas it reversely increased tumor growth in mice fed a high-fat diet (HFD). Then, the tumor tissues derived from Nrf3 knockdown or control cancer cells in ND- or HFD-fed mice were subjected to a DNA microarray-based analysis. Similar to the tumor formation results, the expressions of genes related to adipogenesis, myogenesis, and interferon-alpha response were reversed by obesity, implying an increase or recruitment (or both combined) of adipocytes, myocytes, and immune cells. Among these gene sets, we focused on adipocytes. We showed that Nrf3 knockdown reduced cancer cell growth in the preadipocyte culture medium, while the growth inhibitory effect of Nrf3 knockdown on cancer cells was abolished in the adipocyte culture medium. These results suggest the possibility that cancer-associated adipocytes secrete the potential reprogramming factor from the tumorigenic to the antitumorigenic function of Nrf3 in cancer cells.

Pathophysiological Potentials of NRF3-Regulated Transcriptional Axes in Protein and Lipid Homeostasis.

NRF3 (NFE2L3) belongs to the CNC-basic leucine zipper transcription factor family. An NRF3 homolog, NRF1 (NFE2L1), induces the expression of proteasome-related genes in response to proteasome inhibition. Another homolog, NRF2 (NFE2L2), induces the expression of genes related to antioxidant responses and encodes metabolic enzymes in response to oxidative stress. Dysfunction of each homolog causes several diseases, such as neurodegenerative diseases and cancer development. However, NRF3 target genes and their biological roles remain unknown. This review summarizes our recent reports that showed NRF3-regulated transcriptional axes for protein and lipid homeostasis. NRF3 induces the gene expression of POMP for 20S proteasome assembly and CPEB3 for NRF1 translational repression, inhibiting tumor suppression responses, including cell-cycle arrest and apoptosis, with resistance to a proteasome inhibitor anticancer agent bortezomib. NRF3 also promotes mevalonate biosynthesis by inducing SREBP2 and HMGCR gene expression, and reduces the intracellular levels of neural fatty acids by inducing GGPS1 gene expression. In parallel, NRF3 induces macropinocytosis for cholesterol uptake by inducing RAB5 gene expression. Finally, this review mentions not only the pathophysiological aspects of these NRF3-regulated axes for cancer cell growth and anti-obesity potential but also their possible role in obesity-induced cancer development.

NRF3 upregulates gene expression in SREBP2-dependent mevalonate pathway with cholesterol uptake and lipogenesis inhibition.

Lipids, such as cholesterol and fatty acids, influence cell signaling, energy storage, and membrane formation. Cholesterol is biosynthesized through the mevalonate pathway, and aberrant metabolism causes metabolic diseases. The genetic association of a transcription factor NRF3 with obesity has been suggested, although the molecular mechanisms remain unknown. Here, we show that NRF3 upregulates gene expression in SREBP2-dependent mevalonate pathway. We further reveal that NRF3 overexpression not only reduces lanosterol, a cholesterol precursor, but also induces the expression of the GGPS1 gene encoding an enzyme in the production of GGPP from farnesyl pyrophosphate (FPP), a lanosterol precursor. NRF3 overexpression also enhances cholesterol uptake through RAB5-mediated macropinocytosis process, a bulk and fluid-phase endocytosis pathway. Moreover, we find that GGPP treatment abolishes NRF3 knockdown-mediated increase of neutral lipids. These results reveal the potential roles of NRF3 in the SREBP2-dependent mevalonate pathway for cholesterol uptake through macropinocytosis induction and for lipogenesis inhibition through GGPP production.

NFE2L1 and NFE2L3 Complementarily Maintain Basal Proteasome Activity in Cancer Cells through CPEB3-Mediated Translational Repression.

Proteasomes are protease complexes essential for cellular homeostasis, and their activity is crucial for cancer cell growth. However, the mechanism of how proteasome activity is maintained in cancer cells has remained unclear. The CNC family transcription factor NFE2L1 induces the expression of almost all proteasome-related genes under proteasome inhibition. Both NFE2L1 and its phylogenetically closest homolog, NFE2L3, are highly expressed in several types of cancer, such as colorectal cancer. Here, we demonstrate that NFE2L1 and NFE2L3 complementarily maintain basal proteasome activity in cancer cells. Double knockdown of NFE2L1 and NFE2L3 impaired basal proteasome activity in cancer cells and cancer cell resistance to a proteasome inhibitor anticancer drug, bortezomib, by significantly reducing the basal expression of seven proteasome-related genes: PSMB3, PSMB7, PSMC2, PSMD3, PSMG2, PSMG3, and POMP Interestingly, the molecular basis behind these cellular consequences was that NFE2L3 repressed NFE2L1 translation by the induction of the gene encoding the translational regulator CPEB3, which binds to the NFE2L1 3' untranslated region and decreases polysome formation on NFE2L1 mRNA. Consistent results were obtained from clinical analysis, wherein patients with cancer having tumors expressing higher levels of CPEB3/NFE2L3 exhibit poor prognosis. These results provide the novel regulatory mechanism of basal proteasome activity in cancer cells through an NFE2L3-CPEB3-NFE2L1 translational repression axis.

NRF3-POMP-20S Proteasome Assembly Axis Promotes Cancer Development via Ubiquitin-Independent Proteolysis of p53 and Retinoblastoma Protein.

Proteasomes are essential protease complexes that maintain cellular homeostasis, and aberrant proteasomal activity supports cancer development. The regulatory mechanisms and biological function of the ubiquitin-26S proteasome have been studied extensively, while those of the ubiquitin-independent 20S proteasome system remain obscure. Here, we show that the cap 'n' collar (CNC) family transcription factor NRF3 specifically enhances 20S proteasome assembly in cancer cells and that 20S proteasomes contribute to colorectal cancer development through ubiquitin-independent proteolysis of the tumor suppressor p53 and retinoblastoma (Rb) proteins. The NRF3 gene is highly expressed in many cancer tissues and cell lines and is important for cancer cell growth. In cancer cells, NRF3 upregulates the assembly of the 20S proteasome by directly inducing the gene expression of the 20S proteasome maturation protein POMP. Interestingly, NRF3 knockdown not only increases p53 and Rb protein levels but also increases p53 activities for tumor suppression, including cell cycle arrest and induction of apoptosis. Furthermore, protein stability and cell viability assays using two distinct proteasome inhibitor anticancer drugs, the 20S proteasome inhibitor bortezomib and the ubiquitin-activating enzyme E1 inhibitor TAK-243, show that the upregulation of the NRF3-POMP axis leads to ubiquitin-independent proteolysis of p53 and Rb and to impaired sensitivity to bortezomib but not TAK-243. More importantly, the NRF3-POMP axis supports tumorigenesis and metastasis, with higher NRF3/POMP expression levels correlating with poor prognoses in patients with colorectal or rectal adenocarcinoma. These results suggest that the NRF3-POMP-20S proteasome assembly axis is significant for cancer development via ubiquitin-independent proteolysis of tumor suppressor proteins.

The inducible amphisome isolates viral hemagglutinin and defends against influenza A virus infection.

The emergence of drug-resistant influenza type A viruses (IAVs) necessitates the development of novel anti-IAV agents. Here, we target the IAV hemagglutinin (HA) protein using multivalent peptide library screens and identify PVF-tet, a peptide-based HA inhibitor. PVF-tet inhibits IAV cytopathicity and propagation in cells by binding to newly synthesized HA, rather than to the HA of the parental virus, thus inducing the accumulation of HA within a unique structure, the inducible amphisome, whose production from the autophagosome is accelerated by PVF-tet. The amphisome is also produced in response to IAV infection in the absence of PVF-tet by cells overexpressing ABC transporter subfamily A3, which plays an essential role in the maturation of multivesicular endosomes into the lamellar body, a lipid-sorting organelle. Our results show that the inducible amphisomes can function as a type of organelle-based anti-viral machinery by sequestering HA. PVF-tet efficiently rescues mice from the lethality of IAV infection.

New addiction to the NRF2-related factor NRF3 in cancer cells: Ubiquitin-independent proteolysis through the 20S proteasome.

Accumulating evidence has revealed that human cancers develop by sequentially mutating pivotal genes, including driver genes, and acquiring cancer hallmarks. For instance, cancer cells are addicted to the transcription factor NRF2 (NFE2L2), which is a driver gene that utilizes the cellular cytoprotection system against oxidative stress and metabolic pathway reprogramming for sustaining high growth. Our group has recently discovered a new addiction to the NRF2-related factor NRF3 (NFE2L3) in cancer. For many years, the physiological function of NRF3 remained obscure, in part because Nrf3-deficient mice do not show apparent abnormalities. Nevertheless, human cancer genome databases suggest critical roles of NRF3 in cancer because of high NRF3 mRNA induction in several cancer types, such as colorectal cancer and pancreatic adenocarcinoma, with a poor prognosis. We found that NRF3 promotes tumor growth and malignancy by activating ubiquitin-independent 20S proteasome assembly through inducing the expression of the proteasome maturation protein (POMP) chaperone and thereby degrading the tumor suppressors p53 and Rb. The NRF3-POMP-20S proteasome axis has an entirely different effect on cancer than NRF2. In this review, we describe recent research advances regarding the new cancer effector NRF3, including unclarified ubiquitin-independent proteolysis by the NRF3-POMP-20S proteasome axis. The expected development of cancer therapeutic interventions for this axis is also discussed.

ß-Catenin/TCF4 Complex-Mediated Induction of the NRF3 (NFE2L3) Gene in Cancer Cells.

Remarkable upregulation of the NRF2 (NFE2L2)-related transcription factor NRF3 (NFE2L3) in several cancer tissues and its correlation with poor prognosis strongly suggest the physiological function of NRF3 in tumors. Indeed, we had recently uncovered the function of NRF3, which promotes cancer cell proliferation by p53 degradation via the 20S proteasome. Nevertheless, the molecular mechanism underlying the induction of NRF3 gene expression in cancer cells is highly elusive. We herein describe that NRF3 upregulation is induced by the ß-catenin/TCF4 complex in colon cancer cells. We first confirmed high NRF3 mRNA expression in human colon cancer specimens. The genome database indicated that the human NRF3 gene possesses a species-conserved WRE sequence (TCF/LEF consensus element), implying that the ß-catenin/TCF complex activates NRF3 expression in colon cancer. Consistently, we observed that the ß-catenin/TCF4 complex mediates NRF3 expression by binding directly to the WRE site. Furthermore, inducing NRF3 activates cell proliferation and the expression of the glucose transporter GLUT1. The existence of the ß-catenin/TCF4-NRF3 axis was also validated in the intestine and organoids of Apc-deficient mice. Finally, the positive correlation between NRF3 and ß-catenin target gene expression strongly supports our conclusion. Our findings clearly demonstrate that NRF3 induction in cancer cells is controlled by the Wnt/ß-catenin pathway.

Klf5 suppresses ERK signaling in mouse pluripotent stem cells.

Mouse embryonic stem cells (ESCs) are pluripotent stem cells, which have the ability to differentiate into all three germ layers: mesoderm, endoderm, and ectoderm. Proper levels of phosphorylated extracellular signal-regulated kinase (pERK) are critical for maintaining pluripotency, as elevated pERK evoked by fibroblast growth factor (FGF) receptor activation results in differentiation of ESCs, while, conversely, reduction of pERK by a MEK inhibitor maintains a pluripotent ground state. However, mechanisms underlying proper control of pERK levels in mouse ESCs are not fully understood. Here, we find that Klf5, a Krüppel-like transcription factor family member, is a component of pERK regulation in mouse ESCs. We show that ERK signaling is overactivated in Klf5-KO ESCs and the overactivated ERK in Klf5-KO ESCs is suppressed by the introduction of Klf5, but not Klf2 or Klf4, indicating a unique role for Klf5 in ERK suppression. Moreover, Klf5 regulates Spred1, a negative regulator of the FGF-ERK pathway. Klf5 also facilitates reprogramming of EpiSCs into a naïve state in combination with a glycogen synthase kinase 3 inhibitor and LIF, and in place of a MEK inhibitor. Taken together, these results show for the first time that Klf5 has a unique role suppressing ERK activity in mouse ESCs.

Multiple regulatory mechanisms of the biological function of NRF3 (NFE2L3) control cancer cell proliferation.

Accumulated evidence suggests a physiological relationship between the transcription factor NRF3 (NFE2L3) and cancers. Under physiological conditions, NRF3 is repressed by its endoplasmic reticulum (ER) sequestration. In response to unidentified signals, NRF3 enters the nucleus and modulates gene expression. However, molecular mechanisms underlying the nuclear translocation of NRF3 and its target gene in cancer cells remain poorly understood. We herein report that multiple regulation of NRF3 activities controls cell proliferation. Our analyses reveal that under physiological conditions, NRF3 is rapidly degraded by the ER-associated degradation (ERAD) ubiquitin ligase HRD1 and valosin-containing protein (VCP) in the cytoplasm. Furthermore, NRF3 is also degraded by ß-TRCP, an adaptor for the Skp1-Cul1-F-box protein (SCF) ubiquitin ligase in the nucleus. The nuclear translocation of NRF3 from the ER requires the aspartic protease DNA-damage inducible 1 homolog 2 (DDI2) but does not require inhibition of its HRD1-VCP-mediated degradation. Finally, NRF3 mediates gene expression of the cell cycle regulator U2AF homology motif kinase 1 (UHMK1) for cell proliferation. Collectively, our study provides us many insights into the molecular regulation and biological function of NRF3 in cancer cells.

Klf5 maintains the balance of primitive endoderm versus epiblast specification during mouse embryonic development by suppression of Fgf4.

The inner cell mass of the mouse blastocyst gives rise to the pluripotent epiblast (EPI), which forms the embryo proper, and the primitive endoderm (PrE), which forms extra-embryonic yolk sac tissues. All inner cells coexpress lineage markers such as Nanog and Gata6 at embryonic day (E) 3.25, and the EPI and PrE precursor cells eventually segregate to exclusively express Nanog and Gata6, respectively. Fibroblast growth factor (FGF)-extracellular signal-regulated kinase (ERK) signalling is involved in segregation of the EPI and PrE lineages; however, the mechanism involved in Fgf4 regulation is poorly understood. Here, we identified Klf5 as an upstream repressor of Fgf4Fgf4 was markedly upregulated in Klf5 knockout (KO) embryos at E3.0, and was downregulated in embryos overexpressing Klf5 Furthermore, Klf5 KO and overexpressing blastocysts showed skewed lineage specification phenotypes, similar to FGF4-treated preimplantation embryos and Fgf4 KO embryos, respectively. Inhibitors of the FGF receptor (Fgfr) and ERK pathways reversed the skewed lineage specification of Klf5 KO blastocysts. These data demonstrate that Klf5 suppresses Fgf4-Fgfr-ERK signalling, thus preventing precocious activation of the PrE specification programme.

Possible roles of the transcription factor Nrf1 (NFE2L1) in neural homeostasis by regulating the gene expression of deubiquitinating enzymes.

The transcription factor Nrf1 (NFE2L1) maintains protein homeostasis (proteostasis) by regulating the gene expression of proteasome subunits in response to proteasome inhibition. The deletion of the Nrf1 gene in neural stem/progenitor cells causes severe neurodegeneration due to the accumulation of ubiquitinated proteins in Purkinje cells and motor neurons (Nrf1 NKO mice). However, the molecular mechanisms governing this neurodegenerative process remain unclear. We demonstrate herein that the loss of Nrf1 leads to the reduced gene expression of the deubiquitinating enzymes (DUBs) but not proteasome subunits in Nrf1 NKO mice between P7 and P18. First, we show that K48-linked polyubiquitinated proteins accumulate in Nrf1-deficient Purkinje cells and cerebral cortex neurons. Nevertheless, loss of Nrf1 does not alter the expression and proteolytic activity of proteasome. A significantly reduced expression of deubiquitinating enzymes was also demonstrated in Nrf1-deficient cerebellar tissue using microarray analysis. The genome database further reveals species-conserved ARE, a Nrf1 recognition element, in the regulatory region of certain DUB genes. Furthermore, we show that Nrf1 can activate Usp9x gene expression related to neurodegeneration. Altogether these findings suggest that neurodegeneration in Nrf1 NKO mice may stem from the dysfunction of the ubiquitin-mediated regulation of neuronal proteins.

Hypermethylation of the CaSR and VDR genes in the parathyroid glands in chronic kidney disease rats with high-phosphate diet.

Chronic kidney disease (CKD) disrupts mineral homeostasis and its representative pathosis is defined as secondary hyperparathyroidism (SHPT). SHPT occurs during the early course of progressive renal insufficiency, and is associated with mortality and cardiovascular events. SHPT results in reduction of calcium-sensing receptor (CaSR) and vitamin D receptor (VDR) in the parathyroid glands during CKD. However, the precise mechanism of CaSR and VDR reduction is largely unknown. CKD was induced through two-step 5/6 nephrectomy, and then CKD rats and sham-operated rats were maintained for 8 weeks on diets containing 0.7 % phosphorus (normal phosphate) or 1.2 % phosphorus (high phosphate). In gene expression analysis, TaqMan probes were used for quantitative real-time polymerase chain reaction. Finally, CaSR and VDR protein expressions were analyzed using immunohistochemistry. DNA methylation analysis was performed using a restriction digestion and quantitative PCR. CaSR and VDR mRNA were reduced only in CKD rats fed the high-phosphorus diets (CKD HP), then CaSR and VDR immunohistochemical expressions were compatible with gene expression assay. SHPT was then confirmed only in CKD HP rats. Furthermore, sole CKD HP rats showed the hypermethylation in CaSR and VDR genes; however, the percentage methylation of both genes was low. Although CaSR and VDR hypermethylation was demonstrated in PTGs of CKD HP rats, the extent of hypermethylation was insufficient to support the relevance between hypermethylation and down-regulation of gene expression because of the low percentage of methylation. Consequently, our data suggest that mechanisms, other than DNA hypermethylation, were responsible for the reduction in mRNA and protein levels of CaSR and VDR in PTGs of CKD HP rats.

Regulation of the vitamin D receptor by vitamin D lactam derivatives.

The active metabolite of vitamin D3 , 1α,25-dihydroxyvitamin D3 , acts as a ligand for the vitamin D receptor (VDR) and activates VDR-mediated gene expression. Recently, we characterized 1α,25-dihydroxyvitamin D3 -26,23-lactams (DLAMs), which mimic vitamin D3 metabolites, as noncalcemic VDR ligands that barely activate the receptor. In this study, we present structural insights onto the regulation of VDR function by DLAMs. X-ray crystallographic analysis revealed that DLAMs induced a large conformational change in the loop region between helices H6 and H7 in the VDR ligand-binding domain. Our structural analysis suggests that targeting of the loop region may be a new mode of VDR regulation.

USP15 stabilizes the transcription factor Nrf1 in the nucleus, promoting the proteasome gene expression.

The transcriptional factor Nrf1 (NF-E2-related factor 1) sustains protein homeostasis (proteostasis) by regulating the expression of proteasome genes. Under physiological conditions, the transcriptional activity of Nrf1 is repressed by its sequestration into the endoplasmic reticulum (ER) and furthermore by two independent ubiquitin-proteasome pathways, comprising Hrd1 and ß-TrCP in the cytoplasm and nucleus, respectively. However, the molecular mechanisms underlying Nrf1 activation remain unclear. Here, we report that USP15 (Ubiquitin-Specific Protease 15) activates Nrf1 in the nucleus by stabilizing it through deubiquitination. We first identified USP15 as an Nrf1-associated factor through proteome analysis. USP15 physically interacts with Nrf1, and it markedly stabilizes Nrf1 by removing its ubiquitin moieties. USP15 activates the Nrf1-mediated expression of a proteasome gene luciferase reporter and endogenous proteasome activity. The siRNA-mediated knockdown of USP15 diminishes the Nrf1-induced proteasome gene expression in response to proteasome inhibition. These results uncover a new regulatory mechanism that USP15 activates Nrf1 against the ß-TrCP inhibition to maintain proteostasis.

NML-mediated rRNA base methylation links ribosomal subunit formation to cell proliferation in a p53-dependent manner.

Ribosomal RNAs (rRNAs) act as scaffolds and ribozymes in ribosomes, and these functions are modulated by post-transcriptional modifications. However, the biological role of base methylation, a well-conserved modification of rRNA, is poorly understood. Here, we demonstrate that a nucleolar factor, nucleomethylin (NML; also known as RRP8), is required for the N(1)-methyladenosine (m(1)A) modification in 28S rRNAs of human and mouse cells. NML also contributes to 60S ribosomal subunit formation. Intriguingly, NML depletion increases 60S ribosomal protein L11 (RPL11) levels in the ribosome-free fraction and protein levels of p53 through an RPL11-MDM2 complex, which activates the p53 pathway. Consequently, the growth of NML-depleted cells is suppressed in a p53-dependent manner. These observations reveal a new biological function of rRNA base methylation, which links ribosomal subunit formation to p53-dependent inhibition of cell proliferation in mammalian cells.

Comprehensive Identification of Krüppel-Like Factor Family Members Contributing to the Self-Renewal of Mouse Embryonic Stem Cells and Cellular Reprogramming.

Pluripotency is maintained in mouse embryonic stem (ES) cells and is induced from somatic cells by the activation of appropriate transcriptional regulatory networks. Krüppel-like factor gene family members, such as Klf2, Klf4 and Klf5, have important roles in maintaining the undifferentiated state of mouse ES cells as well as in cellular reprogramming, yet it is not known whether other Klf family members exert self-renewal and reprogramming functions when overexpressed. In this study, we examined whether overexpression of any representative Klf family member, such as Klf1-Klf10, would be sufficient for the self-renewal of mouse ES cells. We found that only Klf2, Klf4, and Klf5 produced leukemia inhibitory factor (LIF)-independent self-renewal, although most KLF proteins, if not all, have the ability to occupy the regulatory regions of Nanog, a critical Klf target gene. We also examined whether overexpression of any of Klf1-Klf10 would be sufficient to convert epiblast stem cells into a naïve pluripotent state and found that Klf5 had such reprogramming ability, in addition to Klf2 and Klf4. We also delineated the functional domains of the Klf2 protein for LIF-independent self-renewal and reprogramming. Interestingly, we found that both the N-terminal transcriptional activation and C-terminal zinc finger domains were indispensable for this activity. Taken together, our comprehensive analysis provides new insight into the contribution of Klf family members to mouse ES self-renewal and cellular reprogramming.