RESUMO
HMCES (5hmC binding, embryonic stem cell-specific-protein), originally identified as a protein capable of binding 5-hydroxymethylcytosine (5hmC), an epigenetic modification generated by TET proteins, was previously reported to covalently crosslink to DNA at abasic sites via a conserved cysteine. We show here that Hmces-deficient mice display normal hematopoiesis without global alterations in 5hmC. HMCES specifically enables DNA double-strand break repair through the microhomology-mediated alternative-end-joining (Alt-EJ) pathway during class switch recombination (CSR) in B cells, and HMCES deficiency leads to a significant defect in CSR. HMCES mediates Alt-EJ through its SOS-response-associated-peptidase domain (SRAPd), a function that requires DNA binding but is independent of its autopeptidase and DNA-crosslinking activities. We show that HMCES is recruited to switch regions of the immunoglobulin locus and provide a potential structural basis for the interaction of HMCES with long DNA overhangs generated by Alt-EJ during CSR. Our studies provide further evidence for a specialized role for HMCES in DNA repair.
Assuntos
Linfócitos B/fisiologia , Reparo do DNA por Junção de Extremidades/genética , Proteínas de Ligação a DNA/genética , DNA/genética , Switching de Imunoglobulina/genética , Animais , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla , Dano ao DNA/genética , Camundongos , Camundongos Endogâmicos C57BL , Translocação Genética/genéticaRESUMO
Adenine N6 methylation in DNA (6mA) is widespread among bacteria and phage and is detected in mammalian genomes, where its function is largely unexplored. Here we show that 6mA deposition and removal are catalyzed by the Mettl4 methyltransferase and Alkbh4 dioxygenase, respectively, and that 6mA accumulation in genic elements corresponds with transcriptional silencing. Inactivation of murine Mettl4 depletes 6mA and causes sublethality and craniofacial dysmorphism in incross progeny. We identify distinct 6mA sensor domains of prokaryotic origin within the MPND deubiquitinase and ASXL1, a component of the Polycomb repressive deubiquitinase (PR-DUB) complex, both of which act to remove monoubiquitin from histone H2A (H2A-K119Ub), a repressive mark. Deposition of 6mA by Mettl4 triggers the proteolytic destruction of both sensor proteins, preserving genome-wide H2A-K119Ub levels. Expression of the bacterial 6mA methyltransferase Dam, in contrast, fails to destroy either sensor. These findings uncover a native, adversarial 6mA network architecture that preserves Polycomb silencing.
Assuntos
Adenina/análogos & derivados , Homólogo AlkB 4 da Lisina Desmetilase/genética , Anormalidades Craniofaciais/genética , DNA/genética , Metiltransferases/genética , Proteínas Repressoras/genética , Adenina/metabolismo , Homólogo AlkB 4 da Lisina Desmetilase/metabolismo , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Anormalidades Craniofaciais/metabolismo , Anormalidades Craniofaciais/patologia , DNA/metabolismo , Metilação de DNA , Enzimas Desubiquitinantes/genética , Enzimas Desubiquitinantes/metabolismo , Feminino , Inativação Gênica , Genes Letais , Histonas/genética , Histonas/metabolismo , Endogamia , Masculino , Metiltransferases/deficiência , Camundongos , Camundongos Knockout , Proteólise , Proteínas Repressoras/metabolismo , Transdução de Sinais , DNA Metiltransferases Sítio Específica (Adenina-Específica)/genética , DNA Metiltransferases Sítio Específica (Adenina-Específica)/metabolismo , Transcrição Gênica , Ubiquitina/genética , Ubiquitina/metabolismoRESUMO
BACKGROUND & AIMS: Obesity and alcohol consumption contribute to steatohepatitis, which increases the risk for hepatitis C virus (HCV)-associated hepatocellular carcinomas (HCCs). Mouse hepatocytes that express HCV-NS5A in liver up-regulate the expression of Toll-like receptor 4 (TLR4), and develop liver tumors containing tumor-initiating stem-like cells (TICs) that express NANOG. We investigated whether the TLR4 signals to NANOG to promote the development of TICs and tumorigenesis in mice placed on a Western diet high in cholesterol and saturated fat (HCFD). METHODS: We expressed HCV-NS5A from a transgene (NS5A Tg) in Tlr4-/- (C57Bl6/10ScN), and wild-type control mice. Mice were fed a HCFD for 12 months. TICs were identified and isolated based on being CD133+, CD49f+, and CD45-. We obtained 142 paraffin-embedded sections of different stage HCCs and adjacent nontumor areas from the same patients, and performed gene expression, immunofluorescence, and immunohistochemical analyses. RESULTS: A higher proportion of NS5A Tg mice developed liver tumors (39%) than mice that did not express HCV NS5A after the HCFD (6%); only 9% of Tlr4-/- NS5A Tg mice fed HCFD developed liver tumors. Livers from NS5A Tg mice fed the HCFD had increased levels of TLR4, NANOG, phosphorylated signal transducer and activator of transcription (pSTAT3), and TWIST1 proteins, and increases in Tlr4, Nanog, Stat3, and Twist1 messenger RNAs. In TICs from NS5A Tg mice, NANOG and pSTAT3 directly interact to activate expression of Twist1. Levels of TLR4, NANOG, pSTAT3, and TWIST were increased in HCC compared with nontumor tissues from patients. CONCLUSIONS: HCFD and HCV-NS5A together stimulated TLR4-NANOG and the leptin receptor (OB-R)-pSTAT3 signaling pathways, resulting in liver tumorigenesis through an exaggerated mesenchymal phenotype with prominent Twist1-expressing TICs.
Assuntos
Transformação Celular Neoplásica/metabolismo , Proteínas de Homeodomínio/metabolismo , Neoplasias Hepáticas/metabolismo , Fígado/metabolismo , Células-Tronco Neoplásicas/metabolismo , Hepatopatia Gordurosa não Alcoólica/metabolismo , Proteínas Nucleares/metabolismo , Fator de Transcrição STAT3/metabolismo , Receptor 4 Toll-Like/metabolismo , Proteína 1 Relacionada a Twist/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Apolipoproteínas E/genética , Linhagem Celular , Movimento Celular , Autorrenovação Celular , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Dieta Hiperlipídica , Modelos Animais de Doenças , Transição Epitelial-Mesenquimal , Feminino , Regulação Neoplásica da Expressão Gênica , Proteínas de Homeodomínio/genética , Humanos , Fígado/patologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Proteína Homeobox Nanog , Células-Tronco Neoplásicas/patologia , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/patologia , Proteínas Nucleares/genética , Fenótipo , Fosforilação , Regiões Promotoras Genéticas , Fator de Transcrição STAT3/genética , Transdução de Sinais , Fatores de Tempo , Receptor 4 Toll-Like/deficiência , Receptor 4 Toll-Like/genética , Proteína 1 Relacionada a Twist/genética , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismoRESUMO
UNLABELLED: Stem cell populations are maintained through self-renewing divisions in which one daughter cell commits to a particular fate whereas the other retains the multipotent characteristics of its parent. The NUMB, a tumor suppressor, in conjunction with another tumor-suppressor protein, p53, preserves this property and acts as a barrier against deregulated expansion of tumor-associated stem cells. In this context, NUMB-p53 interaction plays a crucial role to maintain the proper homeostasis of both stem cells, as well as differentiated cells. Because the molecular mechanism governing the assembly and stability of the NUMB-p53 interaction/complex are poorly understood, we tried to identify the molecule(s) that govern this process. Using cancer cell lines, tumor-initiating cells (TICs) of liver, the mouse model, and clinical samples, we identified that phosphorylations of NUMB destabilize p53 and promote self-renewal of TICs in a pluripotency-associated transcription factor NANOG-dependent manner. NANOG phosphorylates NUMB by atypical protein kinase C zeta (aPKCζ), through the direct induction of Aurora A kinase (AURKA) and the repression of an aPKCζ inhibitor, lethal (2) giant larvae. By radioactivity-based kinase activity assays, we showed that NANOG enhances kinase activities of both AURKA and aPKCζ, an important upstream process for NUMB phosphorylation. Phosphorylation of NUMB by aPKCζ destabilizes the NUMB-p53 interaction and p53 proteolysis and deregulates self-renewal in TICs. CONCLUSION: Post-translational modification of NUMB by the NANOG-AURKA-aPKCζ pathway is an important event in TIC self-renewal and tumorigenesis. Hence, the NANOG-NUMB-p53 signaling axis is an important regulatory pathway for TIC events in TIC self-renewal and liver tumorigenesis, suggesting a therapeutic strategy by targeting NUMB phosphorylation. Further in-depth in vivo and clinical studies are warranted to verify this suggestion.
Assuntos
Proteínas de Homeodomínio/fisiologia , Neoplasias Hepáticas/patologia , Proteínas de Membrana/metabolismo , Células-Tronco Neoplásicas/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Proteína Supressora de Tumor p53/fisiologia , Antígeno AC133 , Animais , Antígenos CD/análise , Aurora Quinase A/genética , Glicoproteínas/análise , Células Hep G2 , Humanos , Camundongos , Proteína Homeobox Nanog , Peptídeos/análise , Fosforilação , Proteína Quinase C/fisiologia , Processamento de Proteína Pós-Traducional , Estabilidade Proteica , Proteína Supressora de Tumor p53/químicaRESUMO
Alcohol abuse predisposes individuals to the development of hepatocellular carcinoma (HCC) and synergistically heightens the HCC risk in patients infected with hepatitis C virus (HCV). The mechanisms of this synergism have been elusive until our recent demonstration of the obligatory role of ectopically expressed TLR4 in liver tumorigenesis in alcohol-fed HCV Ns5a or Core transgenic mice. CD133+/CD49f+ tumor-initiating stem cell-like cells (TICs) isolated from these models are tumorigenic in a manner dependent on TLR4 and NANOG. TICs' tumor-initiating activity and chemoresistance are causally associated with inhibition of TGF-ß tumor suppressor pathway due to NANOG-mediated expression of IGF2BP3 and YAP1. TLR4/NANOG activation causes p53 degradation via phosphorylation of the protective protein NUMB and its dissociation from p53 by the oncoprotein TBC1D15. Nutrient deprivation reduces overexpressed TBC1D15 in TICs via autophagy-mediated degradation, suggesting a possible role of this oncoprotein in linking metabolic reprogramming and self-renewal.
Assuntos
Carcinoma Hepatocelular/induzido quimicamente , Etanol/toxicidade , Neoplasias Hepáticas/induzido quimicamente , Células-Tronco Neoplásicas/fisiologia , Receptor 4 Toll-Like/fisiologia , Animais , Carcinogênese , Proteínas Ativadoras de GTPase/fisiologia , Humanos , Camundongos , Proto-Oncogenes , Fator de Crescimento Transformador beta/fisiologiaRESUMO
Programmed cell suicide of infected bacteria, known as abortive infection (Abi), serves as an immune defense strategy to prevent the propagation of bacteriophage viruses. Many Abi systems utilize bespoke cyclic nucleotide immune messengers generated upon infection to mobilize cognate death effectors. Here, we identify a family of bacteriophage nucleotidyltransferases (NTases) that synthesize competitor cyclic dinucleotide (CDN) ligands and inhibit TIR NADase effectors activated via a linked STING CDN sensor domain (TIR-STING). Through a functional screen of NTase-adjacent phage genes, we uncover candidate inhibitors of cell suicide induced by heterologous expression of tonically active TIR-STING. Among these, we demonstrate that a virus MazG-like nucleotide pyrophosphohydrolase, Atd1, depletes the starvation alarmone (p)ppGpp, revealing a potential role for the alarmone-activated host toxin MazF as an executioner of TIR-driven Abi. Phage NTases and counterdefenses like Atd1 preserve host viability to ensure virus propagation and represent tools to modulate TIR and STING immune responses.
Assuntos
Bacteriófagos , Guanosina Pentafosfato , Bactérias/metabolismo , Bactérias/virologia , Bacteriófagos/fisiologia , Fosfatos de Dinucleosídeos/metabolismo , Imunidade , Nucleotídeos , Nucleotidiltransferases/metabolismoRESUMO
RNA-binding protein Musashi 2 (MSI2) is elevated in several cancers and is linked to poor prognosis. Here, we tested if MSI2 promotes MYC and viral mRNA translation to induce self-renewal via an internal ribosome entry sequence (IRES). We performed RIP-seq using anti-MSI2 antibody in tumor-initiating stem-like cells (TICs). MSI2 binds the internal ribosome entry site (IRES)-containing oncogene mRNAs including MYC, JUN and VEGFA as well as HCV IRES to increase their synthesis and promote self-renewal and tumor-initiation at the post-transcriptional level. MSI2 binds a lncRNA to interfere with processing of a miRNA that reduced MYC translation in basal conditions. Deregulation of this integrated MSI2-lncRNA-MYC regulatory loop drives self-renewal and tumorigenesis through increased IRES-dependent translation of MYC mRNA. Overexpression of MSI2 in TICs promoted their self-renewal and tumor-initiation properties. Inhibition of MSI2-RNA binding reduced HCV IRES activity, viral replication and liver hyperplasia in humanized mice predisposed by virus infection and alcohol high-cholesterol high-fat diet. Together MSI2, integrating the MYC oncogenic pathway, can be employed as a therapeutic target in the treatment of HCC patients. A hypothetical model shows that MSI2 binds and activates cap-independent translation of MYC, c-JUN mRNA and HCV through MSI2-binding to Internal Ribosome Entry Sites (IRES) resulting in upregulated MYC, c-JUN and viral protein synthesis and subsequent liver oncogenesis. Inhibitor of the interaction between MYC IRES and MSI2 reduces liver hyperplasia, viral mRNA translation and tumor formation.
RESUMO
Enzymatic oxidation of 5-methylcytosine (5mC) in DNA by the Tet dioxygenases reprograms genome function in embryogenesis and postnatal development. Tet-oxidized derivatives of 5mC such as 5-hydroxymethylcytosine (5hmC) act as transient intermediates in DNA demethylation or persist as durable marks, yet how these alternative fates are specified at individual CpGs is not understood. Here, we report that the SOS response-associated peptidase (SRAP) domain protein Srap1, the mammalian ortholog of an ancient protein superfamily associated with DNA damage response operons in bacteria, binds to Tet-oxidized forms of 5mC in DNA and catalyzes turnover of these bases to unmodified cytosine by an autopeptidase-coupled nuclease. Biallelic inactivation of murine Srap1 causes embryonic sublethality associated with widespread accumulation of ectopic 5hmC. These findings establish a function for a class of DNA base modification-selective nucleases and position Srap1 as a determinant of 5mC demethylation trajectories during mammalian embryonic development.
Assuntos
5-Metilcitosina/análogos & derivados , Metilação de DNA , Proteínas de Ligação a DNA/metabolismo , 5-Metilcitosina/metabolismo , Animais , Células Cultivadas , Proteínas de Ligação a DNA/genética , Células-Tronco Embrionárias/metabolismo , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ligação ProteicaRESUMO
Hypoxia is a physiologically important endoplasmic reticulum (ER) stress that is present in all solid tumors. Numerous clinical studies have shown that tumor hypoxia predicts for decreased local control, increased distant metastases, and decreased overall survival in a variety of human tumors. Hypoxia selects for tumors with an increased malignant phenotype and increases the metastatic potential of tumor cells. Tumor cells respond to hypoxia and ER stress through the activation of the unfolded protein response (UPR). The UPR is an adaptive response to increase cell survival during ER stress. XBP-1 is a critical transcriptional regulator of this process and is required for tumor growth. Pancreatic ER kinase (PKR-like ER kinase) regulates the translational branch of the UPR and is also important in the growth of tumors. Although the exact mechanism has yet to be elucidated, recent data suggest that the UPR affects tumor growth through protection from apoptosis and may influence angiogenic signaling pathways. Targeting various components of the UPR is a promising therapeutic strategy. Understanding the relationship between hypoxia, the UPR, and tumor growth is crucial to improving current cancer therapies.
Assuntos
Retículo Endoplasmático/química , Retículo Endoplasmático/metabolismo , Hipóxia/metabolismo , Neoplasias/metabolismo , Humanos , Dobramento de ProteínaRESUMO
Hypoxia activates all components of the unfolded protein response (UPR), a stress response initiated by the accumulation of unfolded proteins within the endoplasmic reticulum (ER). Our group and others have shown previously that the UPR, a hypoxia-inducible factor-independent signaling pathway, mediates cell survival during hypoxia and is required for tumor growth. Identifying new genes and pathways that are important for survival during ER stress may lead to the discovery of new targets in cancer therapy. Using the set of 4,728 homozygous diploid deletion mutants in budding yeast, Saccharomyces cerevisiae, we did a functional screen for genes that conferred resistance to ER stress-inducing agents. Deletion mutants in 56 genes showed increased sensitivity under ER stress conditions. Besides the classic UPR pathway and genes related to calcium homeostasis, we report that two additional pathways, including the SLT2 mitogen-activated protein kinase (MAPK) pathway and the osmosensing MAPK pathway, were also required for survival during ER stress. We further show that the SLT2 MAPK pathway was activated during ER stress, was responsible for increased resistance to ER stress, and functioned independently of the classic IRE1/HAC1 pathway. We propose that the SLT2 MAPK pathway is an important cell survival signaling pathway during ER stress. This study shows the feasibility of using the yeast deletion pool to identify relevant mammalian orthologues of the UPR.
Assuntos
Retículo Endoplasmático/fisiologia , Sistema de Sinalização das MAP Quinases/fisiologia , Saccharomyces cerevisiae/fisiologia , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Sinalização do Cálcio/fisiologia , Sobrevivência Celular , Ditiotreitol/farmacologia , Retículo Endoplasmático/efeitos dos fármacos , Glicoproteínas de Membrana/metabolismo , Mercaptoetanol/farmacologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mutação , Fases de Leitura Aberta , Dobramento de Proteína , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Repressoras/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Tunicamicina/farmacologiaRESUMO
Stem cell populations are maintained through self-renewing divisions in which one daughter cell commits to a specific fate while the other retains the multipotent characteristics of its parent. The p53 tumor suppressor, in conjunction with its interacting partner protein Numb, preserves this asymmetry and functions as a vital barrier against the unchecked expansion of tumor stem cell pools; however, little is known about the biological control of the Numb-p53 interaction. We show here that Numb and p53 are the constituents of a high molecular mass complex, which is disintegrated upon activation of aPKCζ, a Numb kinase. Using large-scale affinity purification and tandem mass spectrometry, we identify TBC1D15 as a Numb-associated protein and demonstrate that its amino-terminal domain disengages p53 from Numb, triggering p53 proteolysis and promoting self-renewal and pluripotency. Cellular levels of TBC1D15 are diminished upon acute nutrient deprivation through autophagy-mediated degradation, indicating that TBC1D15 serves as a conduit through which cellular metabolic status is linked to self-renewal. The profound deregulation of TBC1D15 expression exhibited in a diverse array of patient tumors underscores its proposed function as an oncoprotein.
Assuntos
Proteínas Ativadoras de GTPase/fisiologia , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Células-Tronco/citologia , Proteína Supressora de Tumor p53/metabolismo , Sequência de Aminoácidos , Humanos , Proteínas de Membrana/química , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/químicaRESUMO
The eukaryotic chaperonin TRiC/CCT mediates folding of an essential subset of newly synthesized proteins, including the tumor suppressor VHL. Here we show that chaperonin binding is specified by two short hydrophobic beta strands in VHL that, upon folding, become buried within the native structure. These TRiC binding determinants are disrupted by tumor-causing point mutations that interfere with chaperonin association and lead to misfolding. Strikingly, while unable to fold correctly in vivo, some of these VHL mutants can reach the native state when refolded in a chaperonin-independent manner. The specificity of TRiC/CCT for extended hydrophobic beta strands may help explain its role in folding aggregation-prone polypeptides. Our findings reveal a class of disease-causing mutations that inactivate protein function by disrupting chaperone-mediated folding in vivo.