RESUMEN
The NRF2 transcription factor controls a cell stress program that is implicated in cancer and there is great interest in targeting NRF2 for therapy. We show that NRF2 activity depends on Fructosamine-3-kinase (FN3K)-a kinase that triggers protein de-glycation. In its absence, NRF2 is extensively glycated, unstable, and defective at binding to small MAF proteins and transcriptional activation. Moreover, the development of hepatocellular carcinoma triggered by MYC and Keap1 inactivation depends on FN3K in vivo. N-acetyl cysteine treatment partially rescues the effects of FN3K loss on NRF2 driven tumor phenotypes indicating a key role for NRF2-mediated redox balance. Mass spectrometry reveals that other proteins undergo FN3K-sensitive glycation, including translation factors, heat shock proteins, and histones. How glycation affects their functions remains to be defined. In summary, our study reveals a surprising role for the glycation of cellular proteins and implicates FN3K as targetable modulator of NRF2 activity in cancer.
Asunto(s)
Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Animales , Carcinoma Hepatocelular/patología , Femenino , Técnicas de Silenciamiento del Gen , Glucosa/metabolismo , Glicosilación , Células HEK293 , Células Hep G2 , Xenoinjertos , Humanos , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Neoplasias Hepáticas/patología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones Desnudos , Ratones SCID , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Transducción GenéticaRESUMEN
Recombination is crucial for chromosome pairing and segregation during meiosis. SPATA22, along with its direct binding partner and functional collaborator, MEIOB, is essential for the proper repair of double-strand breaks (DSBs) during meiotic recombination. Here, we describe a novel point-mutated allele (shani) of mouse Spata22 that we isolated in a forward genetic screen. shani mutant mice phenocopy Spata22-null and Meiob-null mice: mutant cells appear to form DSBs and initiate meiotic recombination, but are unable to complete DSB repair, leading to meiotic prophase arrest, apoptosis and sterility. shani mutants show precocious loss of DMC1 foci and improper accumulation of BLM-positive recombination foci, reinforcing the requirement of SPATA22-MEIOB for the proper progression of meiotic recombination events. The shani mutation lies within a Spata22 coding exon and molecular characterization shows that it leads to incorrect splicing of the Spata22 mRNA, ultimately resulting in no detectable SPATA22 protein. We propose that the shani mutation alters an exonic splicing enhancer element (ESE) within the Spata22 transcript. The affected DNA nucleotide is conserved in most tetrapods examined, suggesting that the splicing regulation we describe here may be a conserved feature of Spata22 regulation.
Asunto(s)
Proteínas de Ciclo Celular/genética , Recombinación Homóloga , Meiosis/genética , Mutación , Alelos , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cruzamiento , Conectoma , Femenino , Gametogénesis/genética , Homocigoto , Masculino , Ratones , Ratones Transgénicos , Linaje , Espermatocitos/citología , Espermatocitos/metabolismoRESUMEN
Activation of oncogenes by mechanisms other than genetic aberrations such as mutations, translocations, or amplifications is largely undefined. Here we report a novel isoform of the anaplastic lymphoma kinase (ALK) that is expressed in â¼11% of melanomas and sporadically in other human cancer types, but not in normal tissues. The novel ALK transcript initiates from a de novo alternative transcription initiation (ATI) site in ALK intron 19, and was termed ALK(ATI). In ALK(ATI)-expressing tumours, the ATI site is enriched for H3K4me3 and RNA polymerase II, chromatin marks characteristic of active transcription initiation sites. ALK(ATI) is expressed from both ALK alleles, and no recurrent genetic aberrations are found at the ALK locus, indicating that the transcriptional activation is independent of genetic aberrations at the ALK locus. The ALK(ATI) transcript encodes three proteins with molecular weights of 61.1, 60.8 and 58.7 kilodaltons, consisting primarily of the intracellular tyrosine kinase domain. ALK(ATI) stimulates multiple oncogenic signalling pathways, drives growth-factor-independent cell proliferation in vitro, and promotes tumorigenesis in vivo in mouse models. ALK inhibitors can suppress the kinase activity of ALK(ATI), suggesting that patients with ALK(ATI)-expressing tumours may benefit from ALK inhibitors. Our findings suggest a novel mechanism of oncogene activation in cancer through de novo alternative transcription initiation.
Asunto(s)
Regulación Neoplásica de la Expresión Génica/genética , Neoplasias/enzimología , Neoplasias/genética , Proteínas Tirosina Quinasas Receptoras/genética , Iniciación de la Transcripción Genética , Alelos , Quinasa de Linfoma Anaplásico , Animales , Línea Celular Tumoral , Proliferación Celular , Transformación Celular Neoplásica , Femenino , Células HEK293 , Histonas/química , Histonas/metabolismo , Humanos , Intrones/genética , Isoenzimas/antagonistas & inhibidores , Isoenzimas/biosíntesis , Isoenzimas/química , Isoenzimas/genética , Lisina/metabolismo , Metilación , Ratones , Datos de Secuencia Molecular , Peso Molecular , Células 3T3 NIH , Neoplasias/tratamiento farmacológico , Oncogenes/genética , Estructura Terciaria de Proteína/genética , ARN Polimerasa II/metabolismo , ARN Mensajero/análisis , ARN Mensajero/genética , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Proteínas Tirosina Quinasas Receptoras/biosíntesis , Proteínas Tirosina Quinasas Receptoras/química , Transducción de SeñalRESUMEN
Transcriptional silencing by heritable cytosine-5 methylation is an ancient strategy to repress transposable elements. It was previously thought that mammals possess four DNA methyltransferase paralogs-Dnmt1, Dnmt3a, Dnmt3b and Dnmt3l-that establish and maintain cytosine-5 methylation. Here we identify a fifth paralog, Dnmt3c, that is essential for retrotransposon methylation and repression in the mouse male germline. From a phenotype-based forward genetics screen, we isolated a mutant mouse called 'rahu', which displays severe defects in double-strand-break repair and homologous chromosome synapsis during male meiosis, resulting in sterility. rahu is an allele of a transcription unit (Gm14490, renamed Dnmt3c) that was previously mis-annotated as a Dnmt3-family pseudogene. Dnmt3c encodes a cytosine methyltransferase homolog, and Dnmt3crahu mutants harbor a non-synonymous mutation of a conserved residue within one of its cytosine methyltransferase motifs, similar to a mutation in human DNMT3B observed in patients with immunodeficiency, centromeric instability and facial anomalies syndrome. The rahu mutation lies at a potential dimerization interface and near the potential DNA binding interface, suggesting that it compromises protein-protein and/or protein-DNA interactions required for normal DNMT3C function. Dnmt3crahu mutant males fail to establish normal methylation within LINE and LTR retrotransposon sequences in the germline and accumulate higher levels of transposon-derived transcripts and proteins, particularly from distinct L1 and ERVK retrotransposon families. Phylogenetic analysis indicates that Dnmt3c arose during rodent evolution by tandem duplication of Dnmt3b, after the divergence of the Dipodoidea and Muroidea superfamilies. These findings provide insight into the evolutionary dynamics and functional specialization of the transposon suppression machinery critical for mammalian sexual reproduction and epigenetic regulation.
Asunto(s)
ADN (Citosina-5-)-Metiltransferasas/genética , Represión Epigenética , Células Germinativas/metabolismo , Meiosis/genética , Alelos , Secuencia de Aminoácidos , Animales , Mapeo Cromosómico , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Roturas del ADN de Doble Cadena , Metilación de ADN/genética , Reparación del ADN , Células Germinativas/citología , Infertilidad Masculina/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Mutación , Filogenia , Conformación Proteica , Retroelementos/genética , Análisis de Secuencia de ARN , Regulación hacia ArribaRESUMEN
H1 linker histones facilitate higher-order chromatin folding and are essential for mammalian development. To achieve high-resolution mapping of H1 variants H1d and H1c in embryonic stem cells (ESCs), we have established a knock-in system and shown that the N-terminally tagged H1 proteins are functionally interchangeable to their endogenous counterparts in vivo. H1d and H1c are depleted from GC- and gene-rich regions and active promoters, inversely correlated with H3K4me3, but positively correlated with H3K9me3 and associated with characteristic sequence features. Surprisingly, both H1d and H1c are significantly enriched at major satellites, which display increased nucleosome spacing compared with bulk chromatin. While also depleted at active promoters and enriched at major satellites, overexpressed H1(0) displays differential binding patterns in specific repetitive sequences compared with H1d and H1c. Depletion of H1c, H1d, and H1e causes pericentric chromocenter clustering and de-repression of major satellites. These results integrate the localization of an understudied type of chromatin proteins, namely the H1 variants, into the epigenome map of mouse ESCs, and we identify significant changes at pericentric heterochromatin upon depletion of this epigenetic mark.
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Cromatina/genética , Células Madre Embrionarias , Heterocromatina/genética , Histonas/genética , Animales , Ensamble y Desensamble de Cromatina/genética , Mapeo Cromosómico , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Epigénesis Genética , Técnicas de Sustitución del Gen , N-Metiltransferasa de Histona-Lisina , RatonesRESUMEN
Protein methyltransferases (PMTs) have emerged as important epigenetic regulators in myriad biological processes in both normal physiology and disease conditions. However, elucidating PMT-regulated epigenetic processes has been hampered by ambiguous knowledge about in vivo activities of individual PMTs particularly because of their overlapping but nonredundant functions. To address limitations of conventional approaches in mapping chromatin modification of specific PMTs, we have engineered the chromatin-modifying apparatus and formulated a novel technology, termed clickable chromatin enrichment with parallel DNA sequencing (CliEn-seq), to probe genome-wide chromatin modification within living cells. The three-step approach of CliEn-seq involves in vivo synthesis of S-adenosyl-L-methionine (SAM) analogues from cell-permeable methionine analogues by engineered SAM synthetase (methionine adenosyltransferase or MAT), in situ chromatin modification by engineered PMTs, subsequent enrichment and sequencing of the uniquely modified chromatins. Given critical roles of the chromatin-modifying enzymes in epigenetics and structural similarity among many PMTs, we envision that the CliEn-seq technology is generally applicable in deciphering chromatin methylation events of individual PMTs in diverse biological settings.
Asunto(s)
Cromatina/metabolismo , Metilación de ADN , Ingeniería Genética , Péptido 1 Similar al Glucagón/metabolismo , Antígenos de Histocompatibilidad/metabolismo , N-Metiltransferasa de Histona-Lisina/metabolismo , Procesamiento Proteico-Postraduccional , Cromatina/genética , Epigenómica , Péptido 1 Similar al Glucagón/genética , Péptido 1 Similar al Glucagón/aislamiento & purificación , Células HEK293 , Antígenos de Histocompatibilidad/genética , Antígenos de Histocompatibilidad/aislamiento & purificación , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/aislamiento & purificación , Humanos , Modelos Moleculares , S-Adenosilmetionina/biosíntesis , S-Adenosilmetionina/químicaRESUMEN
The organization of mammalian DNA replication is poorly understood. We have produced high-resolution dynamic maps of the timing of replication in human erythroid, mesenchymal, and embryonic stem (ES) cells using TimEX, a method that relies on gaussian convolution of massive, highly redundant determinations of DNA copy-number variations during S phase to produce replication timing profiles. We first obtained timing maps of 3% of the genome using high-density oligonucleotide tiling arrays and then extended the TimEX method genome-wide using massively parallel sequencing. We show that in untransformed human cells, timing of replication is highly regulated and highly synchronous, and that many genomic segments are replicated in temporal transition regions devoid of initiation, where replication forks progress unidirectionally from origins that can be hundreds of kilobases away. Absence of initiation in one transition region is shown at the molecular level by single molecule analysis of replicated DNA (SMARD). Comparison of ES and erythroid cells replication patterns revealed that these cells replicate about 20% of their genome in different quarters of S phase. Importantly, we detected a strong inverse relationship between timing of replication and distance to the closest expressed gene. This relationship can be used to predict tissue-specific timing of replication profiles from expression data and genomic annotations. We also provide evidence that early origins of replication are preferentially located near highly expressed genes, that mid-firing origins are located near moderately expressed genes, and that late-firing origins are located far from genes.
Asunto(s)
Momento de Replicación del ADN , Replicación del ADN , Células Madre Embrionarias , Células Eritroides , Perfilación de la Expresión Génica , Células Madre Mesenquimatosas , Fase S , Diferenciación Celular , ADN/biosíntesis , ADN/genética , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Células Eritroides/citología , Células Eritroides/metabolismo , Dosificación de Gen , Humanos , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Distribución NormalRESUMEN
The eIF4E translation initiation factor has oncogenic properties and concordantly, the inhibitory eIF4E-binding protein (4EBP1) is considered a tumor suppressor. The exact molecular effects of 4EBP1 activation in cancer are still unknown. Surprisingly, 4EBP1 is a target of genomic copy number gains (Chr. 8p11) in breast and lung cancer. We noticed that 4EBP1 gains are genetically linked to gains in neighboring genes, including WHSC1L1 and FGFR1. Our results show that FGFR1 gains act to attenuate the function of 4EBP1 via PI3K-mediated phosphorylation at Thr37/46, Ser65, and Thr70 sites. This implies that not 4EBP1 but instead FGFR1 is the genetic target of Chr. 8p11 gains in breast and lung cancer. Accordingly, these tumors show increased sensitivity to FGFR1 and PI3K inhibition, and this is a therapeutic vulnerability through restoring the tumor-suppressive function of 4EBP1. Ribosome profiling reveals genes involved in insulin signaling, glucose metabolism, and the inositol pathway to be the relevant translational targets of 4EBP1. These mRNAs are among the top 200 translation targets and are highly enriched for structure and sequence motifs in their 5'UTR, which depends on the 4EBP1-EIF4E activity. In summary, we identified the translational targets of 4EBP1-EIF4E that facilitate the tumor suppressor function of 4EBP1 in cancer.
RESUMEN
Inhibition of the eIF4A RNA helicase with silvestrol and related compounds is emerging as a powerful anti-cancer strategy. We find that a synthetic silvestrol analogue (CR-1-31 B) has nanomolar activity across many cancer cell lines. It is especially active against aggressive MYC+/BCL2+ B cell lymphomas and this likely reflects the eIF4A-dependent translation of both MYC and BCL2. We performed a genome-wide CRISPR/Cas9 screen and identified mechanisms of resistance to this new class of therapeutics. We identify three negative NRF2 regulators (KEAP1, CUL3, CAND1) whose inactivation is sufficient to cause CR1-31-B resistance. NRF2 is known to alter the oxidation state of translation factors and cause a broad increase in protein production. We find that NRF2 activation particularly increases the translation of some eIF4A-dependent mRNAs and restores MYC and BCL2 production. We know that NRF2 functions depend on removal of sugar adducts by the frutosamine-3-kinase (FN3K). Accordingly, loss of FN3K results in NRF2 hyper-glycation and inactivation and resensitizes cancer cells to eIF4A inhibition. Together, our findings implicate NRF2 in the translation of eIF4A-dependent mRNAs and point to FN3K inhibition as a new strategy to block NRF2 functions in cancer.
RESUMEN
Mechanisms regulating mammalian meiotic progression are poorly understood. Here we identify mouse YTHDC2 as a critical component. A screen yielded a sterile mutant, 'ketu', caused by a Ythdc2 missense mutation. Mutant germ cells enter meiosis but proceed prematurely to aberrant metaphase and apoptosis, and display defects in transitioning from spermatogonial to meiotic gene expression programs. ketu phenocopies mutants lacking MEIOC, a YTHDC2 partner. Consistent with roles in post-transcriptional regulation, YTHDC2 is cytoplasmic, has 3'â5' RNA helicase activity in vitro, and has similarity within its YTH domain to an N6-methyladenosine recognition pocket. Orthologs are present throughout metazoans, but are diverged in nematodes and, more dramatically, Drosophilidae, where Bgcn is descended from a Ythdc2 gene duplication. We also uncover similarity between MEIOC and Bam, a Bgcn partner unique to schizophoran flies. We propose that regulation of gene expression by YTHDC2-MEIOC is an evolutionarily ancient strategy for controlling the germline transition into meiosis.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Células Germinativas/fisiología , Meiosis , ARN Helicasas/metabolismo , Animales , Proteínas de Ciclo Celular/genética , Regulación de la Expresión Génica , Pruebas Genéticas , Infertilidad , Masculino , Ratones , Mutación Missense , ARN Helicasas/genéticaRESUMEN
Ovarian cancer is a deadly gynecologic malignancy for which novel biomarkers and therapeutic targets are imperative for improving survival. Previous studies have suggested the expression pattern of linker histone variants as potential biomarkers for ovarian cancer. To investigate the role of histone H1 in ovarian cancer cells, we characterize individual H1 variants and overexpress one of the major somatic H1 variants, H1.3, in the OVCAR-3 epithelial ovarian cancer cell line. We find that overexpression of H1.3 decreases the growth rate and colony formation of OVCAR-3 cells. We identify histone H1.3 as a specific repressor for the noncoding oncogene H19. Overexpression of H1.3 suppresses H19 expression, and knockdown of H1.3 increases its expression in multiple ovarian epithelial cancer cell lines. Furthermore, we demonstrate that histone H1.3 overexpression leads to increased occupancy of H1.3 at the H19 regulator region encompassing the imprinting control region (ICR), concomitant with increased DNA methylation and reduced occupancy of the insulator protein CTCF at the ICR. Finally, we demonstrate that H1.3 overexpression and H19 knockdown synergistically decrease the growth rate of ovarian cancer cells. Our findings suggest that H1.3 dramatically inhibits H19 expression, which contributes to the suppression of epithelial ovarian carcinogenesis.
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Proliferación Celular , Histonas/fisiología , Neoplasias Ováricas/patología , ARN Largo no Codificante/fisiología , Línea Celular Tumoral , Metilación de ADN , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , ARN Largo no Codificante/antagonistas & inhibidores , ARN Largo no Codificante/genéticaRESUMEN
Phased genome maps are important to understand genetic and epigenetic regulation and disease mechanisms, particularly parental imprinting defects. Phasing is also critical to assess the functional consequences of genetic variants, and to allow precise definition of haplotype blocks which is useful to understand gene-flow and genotype-phenotype association at the population level. Transmission phasing by analysis of a family quartet allows the phasing of 95% of all variants as the uniformly heterozygous positions cannot be phased. Here, we report a phasing method based on a combination of transmission analysis, physical phasing by pair-end sequencing of libraries of staggered sizes and population-based analysis. Sequencing of a healthy Caucasians quartet at 120x coverage and combination of physical and transmission phasing yielded the phased genotypes of about 99.8% of the SNPs, indels and structural variants present in the quartet, a phasing rate significantly higher than what can be achieved using any single phasing method. A false positive SNP error rate below 10*E-7 per genome and per base was obtained using a combination of filters. We provide a complete list of SNPs, indels and structural variants, an analysis of haplotype block sizes, and an analysis of the false positive and negative variant calling error rates. Improved genome phasing and family sequencing will increase the power of genome-wide sequencing as a clinical diagnosis tool and has myriad basic science applications.
Asunto(s)
Mapeo Cromosómico/métodos , Genoma Humano , Estudio de Asociación del Genoma Completo/métodos , Patrón de Herencia , Análisis de Secuencia de ADN/estadística & datos numéricos , Algoritmos , Mapeo Cromosómico/estadística & datos numéricos , Familia , Estudio de Asociación del Genoma Completo/estadística & datos numéricos , Haplotipos , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Mutación INDEL , Polimorfismo de Nucleótido SimpleRESUMEN
Experiments with somatic cell nuclear transfer, inter-cellular hybrid formation_ENREF_3, and ectopic expression of transcription factors have clearly demonstrated that cell fate can be dramatically altered by changing the epigenetic state of cell nuclei. Here we demonstrate, using chemical fusion, direct reprogramming of the genome of human embryonic fibroblasts (HEF) into the state of human fetal liver hFL CD34+ (hFL) hematopoietic progenitors capable of proliferating and differentiating into multiple hematopoietic lineages. We show that hybrid cells retain their ploidy and can differentiate into several hematopoietic lineages. Hybrid cells follow transcription program of differentiating hFL cells as shown by genome-wide transcription profiling. Using whole-genome single nucleotide polymorphism (SNP) profiling of both donor genomes we demonstrate reprogramming of HEF genome into the state of hFL hematopoietic progenitors. Our results prove that it is possible to convert the fetal somatic cell genome into the state of fetal hematopoietic progenitors by fusion. This suggests a possibility of direct reprogramming of human somatic cells into tissue specific progenitors/stem cells without going all the way back to the embryonic state. Direct reprogramming of terminally differentiated cells into the tissue specific progenitors will likely prove useful for the development of novel cell therapies.