ATR activity controls stem cell quiescence via the cyclin F-SCF complex.

A key property of adult stem cells is their ability to persist in a quiescent state for prolonged periods of time. The quiescent state is thought to contribute to stem cell resilience by limiting accumulation of DNA replication­associated mutations. Moreover, cellular stress response factors are thought to play a role in maintaining quiescence and stem cell integrity. We utilized muscle stem cells (MuSCs) as a model of quiescent stem cells and find that the replication stress response protein, ATR (Ataxia Telangiectasia and Rad3-Related), is abundant and active in quiescent but not activated MuSCs. Concurrently, MuSCs display punctate RPA (replication protein A) and R-loop foci, both key triggers for ATR activation. To discern the role of ATR in MuSCs, we generated MuSC-specific ATR conditional knockout (ATRcKO) mice. Surprisingly, ATR ablation results in increased MuSC quiescence exit. Phosphoproteomic analysis of ATRcKO MuSCs reveals enrichment of phosphorylated cyclin F, a key component of the Skp1­Cul1­F-box protein (SCF) ubiquitin ligase complex and regulator of key cell-cycle transition factors, such as the E2F family of transcription factors. Knocking down cyclin F or inhibiting the SCF complex results in E2F1 accumulation and in MuSCs exiting quiescence, similar to ATR-deficient MuSCs. The loss of ATR could be counteracted by inhibiting casein kinase 2 (CK2), the kinase responsible for phosphorylating cyclin F. We propose a model in which MuSCs express cell-cycle progression factors but ATR, in coordination with the cyclin F­SCF complex, represses premature stem cell quiescence exit via ubiquitin­proteasome degradation of these factors.

Ferritin heavy chain controls the HIF-driven hypoxic response by activating the asparaginyl hydroxylase FIH.

Hypoxia-inducible factor 1 (HIF-1) is a key player in cellular response to hypoxia. The stability and transcriptional activity of this protein are oxygen-dependently regulated by the prolyl hydroxylases PHD1-3 and the asparaginyl hydroxylase FIH. Recently, ferritin heavy chain (FTH1) has been characterized to reinforce the HIF-1 signaling pathway in an indirect way through the inhibition of PHD activity by depleting the free iron pool in the cytoplasm. In the present study, we addressed the role of FTH1 in the FIH control of HIF-1 activity. Unexpectedly, immunoprecipitation analyses revealed that FTH1 directly interacted with FIH. In an in vitro hydroxylation assay, FTH1 was found to facilitate the FIH-mediated Asn803 hydroxylation in HIF-1α. As expected, FTH1 prevented the recruitment of p300 to HIF-1α through the Asn803 hydroxylation. In luciferase reporter analyses, FTH1 was found to repress the transcriptional activity of HIF-1α in HCT116 cells under either normoxic or hypoxic conditions. Consequently, FTH1 downregulated the expression of the HIF-1 target genes, such as VEGF, CA9 and GLUT1. Our results suggest a new role of FTH1 as a co-regulator for the FIH-mediated oxygen sensing pathway. Since HIF-1α is involved in pathogenesis of diverse hypoxia-associated diseases, we propose that FTH1 be a potential target in developing new therapeutic strategies against such diseases.

A novel HIF1AN substrate KANK3 plays a tumor-suppressive role in hepatocellular carcinoma.

The KN motif and ankyrin repeat domain-containing protein (KANK) family is involved in actin cytoskeleton organization and cell motility. Compared with other KANK members, the biological function of KANK3 is not clear. Here, we identified KANK3 as a new substrate for the oxygen sensor hypoxia-inducible factor 1-alpha inhibitor (HIF1AN), which hydroxylates HIF-1/2α and other ankyrin repeat domain-containing proteins at asparagine residues. An in vitro hydroxylation assay clearly demonstrated asparaginyl hydroxylation of KANK3 by HIF1AN, and mass spectroscopic analysis revealed that KANK3 is hydroxylated at three asparagine residues within the ankyrin repeat domain. Bioinformatics analysis revealed that KANK3 downregulation is correlated with a poor prognosis in several types of cancers, including hepatocellular carcinoma (HCC). In HCC cells, KANK3 knockdown enhanced cell migration and invasion, while its overexpression inhibited these cell behaviors. Interestingly, such effects of KANK3 were not observed under hypoxic conditions, suggesting oxygen-dependent activity of KANK3. Based on these data, we propose that KANK3 acts as a tumor suppressor to control cancer behavior in an oxygen-dependent manner.

The F-box protein FBXO7 positively regulates bone morphogenetic protein-mediated signaling through Lys-63-specific ubiquitination of neurotrophin receptor-interacting MAGE (NRAGE).

Parkinson's disease (PD) is characterized by progressive midbrain dopaminergic neuron degeneration and the formation of intracellular protein aggregates, referred to as Lewy bodies. F-box only protein 7 (FBXO7) gene mutations are closely associated with progression of the autosomal recessive form of familial PD. FBXO7 encodes a component of Skp1, cullin, F-box ubiquitin ligase complexes; however, its cellular targets, including substrates and regulators, are not yet clarified. To identify potential substrates of FBXO7, we performed a yeast two-hybrid screen of a human fetal brain library and identified neurotrophin receptor-interacting MAGE protein (NRAGE) as a novel FBXO7-binding partner. We found that FBXO7 interacts with NRAGE and mediates Lys-63-linked poly-ubiquitination of NRAGE in mammalian cells. FBXO7 overexpression accelerates formation of NRAGE-TAK1-TAB1 complexes, whereas FBXO7 knockdown correspondingly decreases complex formation. In addition, BMP4 stimulation enhances NRAGE ubiquitination through FBXO7 and facilitates endogenous NRAGE-TAK1-TAB1 complex formation. Furthermore, FBXO7 positively regulates formation of the BMP receptor-NRAGE-TAK1-TAB1 complex, and up-regulates NF-κB activity. Taken together, our results suggest that FBXO7 affects BMP4-mediated signaling through proteasome-independent ubiquitination of NRAGE and augments formation of downstream signaling components.

Depletion of SAM leading to loss of heterochromatin drives muscle stem cell ageing.

The global loss of heterochromatin during ageing has been observed in eukaryotes from yeast to humans, and this has been proposed as one of the causes of ageing. However, the cause of this age-associated loss of heterochromatin has remained enigmatic. Here we show that heterochromatin markers, including histone H3K9 di/tri-methylation and HP1, decrease with age in muscle stem cells (MuSCs) as a consequence of the depletion of the methyl donor S-adenosylmethionine (SAM). We find that restoration of intracellular SAM in aged MuSCs restores heterochromatin content to youthful levels and rejuvenates age-associated features, including DNA damage accumulation, increased cell death, and defective muscle regeneration. SAM is not only a methyl group donor for transmethylation, but it is also an aminopropyl donor for polyamine synthesis. Excessive consumption of SAM in polyamine synthesis may reduce its availability for transmethylation. Consistent with this premise, we observe that perturbation of increased polyamine synthesis by inhibiting spermidine synthase restores intracellular SAM content and heterochromatin formation, leading to improvements in aged MuSC function and regenerative capacity in male and female mice. Together, our studies demonstrate a direct causal link between polyamine metabolism and epigenetic dysregulation during murine MuSC ageing.

Multiomics reveals glutathione metabolism as a driver of bimodality during stem cell aging.

With age, skeletal muscle stem cells (MuSCs) activate out of quiescence more slowly and with increased death, leading to defective muscle repair. To explore the molecular underpinnings of these defects, we combined multiomics, single-cell measurements, and functional testing of MuSCs from young and old mice. The multiomics approach allowed us to assess which changes are causal, which are compensatory, and which are simply correlative. We identified glutathione (GSH) metabolism as perturbed in old MuSCs, with both causal and compensatory components. Contrary to young MuSCs, old MuSCs exhibit a population dichotomy composed of GSHhigh cells (comparable with young MuSCs) and GSHlow cells with impaired functionality. Mechanistically, we show that antagonism between NRF2 and NF-κB maintains this bimodality. Experimental manipulation of GSH levels altered the functional dichotomy of aged MuSCs. These findings identify a novel mechanism of stem cell aging and highlight glutathione metabolism as an accessible target for reversing MuSC aging.

Exercise reprograms the inflammatory landscape of multiple stem cell compartments during mammalian aging.

Exercise has the ability to rejuvenate stem cells and improve tissue regeneration in aging animals. However, the cellular and molecular changes elicited by exercise have not been systematically studied across a broad range of cell types in stem cell compartments. We subjected young and old mice to aerobic exercise and generated a single-cell transcriptomic atlas of muscle, neural, and hematopoietic stem cells with their niche cells and progeny, complemented by whole transcriptome analysis of single myofibers. We found that exercise ameliorated the upregulation of a number of inflammatory pathways associated with old age and restored aspects of intercellular communication mediated by immune cells within these stem cell compartments. Exercise has a profound impact on the composition and transcriptomic landscape of circulating and tissue-resident immune cells. Our study provides a comprehensive view of the coordinated responses of multiple aged stem cells and niche cells to exercise at the transcriptomic level.

Fasting induces a highly resilient deep quiescent state in muscle stem cells via ketone body signaling.

Short-term fasting is beneficial for the regeneration of multiple tissue types. However, the effects of fasting on muscle regeneration are largely unknown. Here, we report that fasting slows muscle repair both immediately after the conclusion of fasting as well as after multiple days of refeeding. We show that ketosis, either endogenously produced during fasting or a ketogenic diet or exogenously administered, promotes a deep quiescent state in muscle stem cells (MuSCs). Although deep quiescent MuSCs are less poised to activate, slowing muscle regeneration, they have markedly improved survival when facing sources of cellular stress. Furthermore, we show that ketone bodies, specifically ß-hydroxybutyrate, directly promote MuSC deep quiescence via a nonmetabolic mechanism. We show that ß-hydroxybutyrate functions as an HDAC inhibitor within MuSCs, leading to acetylation and activation of an HDAC1 target protein p53. Finally, we demonstrate that p53 activation contributes to the deep quiescence and enhanced resilience observed during fasting.

Ketohexokinase-A acts as a nuclear protein kinase that mediates fructose-induced metastasis in breast cancer.

Harmful effects of high fructose intake on health have been widely reported. Although fructose is known to promote cancer, little is known about the underlying mechanisms. Here, we found that fructose triggers breast cancer metastasis through the ketohexokinase-A signaling pathway. Molecular experiments showed that ketohexokinase-A, rather than ketohexokinase-C, is necessary and sufficient for fructose-induced cell invasion. Ketohexokinase-A-overexpressing breast cancer was found to be highly metastatic in fructose-fed mice. Mechanistically, cytoplasmic ketohexokinase-A enters into the nucleus during fructose stimulation, which is mediated by LRRC59 and KPNB1. In the nucleus, ketohexokinase-A phosphorylates YWHAH at Ser25 and the YWHAH recruits SLUG to the CDH1 promoter, which triggers cell migration. This study provides the effect of nutrition on breast cancer metastasis. High intake of fructose should be restricted in cancer patients to reduce the risk of metastasis. From a therapeutic perspective, the ketohexokinase-A signaling pathway could be a potential target to prevent cancer metastasis.

FIH permits NAA10 to catalyze the oxygen-dependent lysyl-acetylation of HIF-1α.

The N-terminal acetyltransferase A (NatA) complex, which is composed of NAA10 and NAA15, catalyzes N-terminal acetylation of many proteins in a co-translational manner. Structurally, the catalytic subunit NAA10 was believed to have no activity toward an internal lysine residue because the gate of its catalytic pocket is too narrow. However, several studies have demonstrated that the monomeric NAA10 can acetylate the internal lysine residues of several substrates including hypoxia-inducible factor 1α (HIF-1α). How NAA10 acetylates lysine residues has been an unsolved question. We here found that human FIH (factor inhibiting HIF) hydroxylates human NAA10 at W38 oxygen-dependently and this permits NAA10 to express the lysyl-acetyltransferase activity. The hydroxylated W38 forms a new hydrogen-bond with A67 and widens the gate at the catalytic pocket, which allows the entrance of a lysine residue to the site. Since the FIH-dependent hydroxylation of NAA10 occurs oxygen-dependently, NAA10 acetylates HIF-1α under normoxia but does not under hypoxia. Consequently, the acetylation promotes the pVHL binding to HIF-1α, and in turn HIF-1α is destructed via the ubiquitin-proteasome system. This study provides a novel oxygen-sensing process that determines the substrate specificity of NAA10 depending on an ambient oxygen tension.

Aberrant expression of CITED2 promotes prostate cancer metastasis by activating the nucleolin-AKT pathway.

Despite many efforts to develop hormone therapy and chemotherapy, no effective strategy to suppress prostate cancer metastasis has been established because the metastasis is not well understood. We here investigate a role of CBP/p300-interacting transactivator with E/D-rich carboxy-terminal domain-2 (CITED2) in prostate cancer metastasis. CITED2 is highly expressed in metastatic prostate cancer, and its expression is correlated with poor survival. The CITED2 gene is highly activated by ETS-related gene that is overexpressed due to chromosomal translocation. CITED2 acts as a molecular chaperone to guide PRMT5 and p300 to nucleolin, thereby activating nucleolin. Informatics and experimental data suggest that the CITED2-nucleolin axis is involved in prostate cancer metastasis. This axis stimulates cell migration through the epithelial-mesenchymal transition and promotes cancer metastasis in a xenograft mouse model. Our results suggest that CITED2 plays a metastasis-promoting role in prostate cancer and thus could be a target for preventing prostate cancer metastasis.

FIH Is an Oxygen Sensor in Ovarian Cancer for G9a/GLP-Driven Epigenetic Regulation of Metastasis-Related Genes.

The prolyl hydroxylase domain-containing proteins (PHD1-3) and the asparaginyl hydroxlyase factor inhibiting HIF (FIH) are oxygen sensors for hypoxia-inducible factor-driven transcription of hypoxia-induced genes, but whether these sensors affect oxygen-dependent epigenetic regulation more broadly is not known. Here, we show that FIH exerts an additional role as an oxygen sensor in epigenetic control by the histone lysine methyltransferases G9a and GLP. FIH hydroxylated and inhibited G9a and GLP under normoxia. When the FIH reaction was limited under hypoxia, G9a and GLP were activated and repressed metastasis suppressor genes, thereby triggering cancer cell migration and peritoneal dissemination of ovarian cancer xenografts. In clinical specimens of ovarian cancer, expression of FIH and G9a were reciprocally associated with patient outcomes. We also identified mutations of FIH target motifs in G9a and GLP, which exhibited excessive H3K9 methylation and facilitated cell invasion. This study provides insight into a new function of FIH as an upstream regulator of oxygen-dependent chromatin remodeling. It also implies that the FIH-G9a/GLP pathway could be a potential target for inhibiting hypoxia-induced cancer metastasis.Significance: These findings deepen understanding of oxygen-dependent gene regulation and cancer metastasis in response to hypoxia. Cancer Res; 78(5); 1184-99. ©2017 AACR.