RESUMEN
Cytosine methylation of DNA is a widespread modification of DNA that plays numerous critical roles. In the yeast Cryptococcus neoformans, CG methylation occurs in transposon-rich repeats and requires the DNA methyltransferase Dnmt5. We show that Dnmt5 displays exquisite maintenance-type specificity in vitro and in vivo and utilizes similar in vivo cofactors as the metazoan maintenance methylase Dnmt1. Remarkably, phylogenetic and functional analysis revealed that the ancestral species lost the gene for a de novo methylase, DnmtX, between 50-150 mya. We examined how methylation has persisted since the ancient loss of DnmtX. Experimental and comparative studies reveal efficient replication of methylation patterns in C. neoformans, rare stochastic methylation loss and gain events, and the action of natural selection. We propose that an epigenome has been propagated for >50 million years through a process analogous to Darwinian evolution of the genome.
Asunto(s)
Cryptococcus neoformans/genética , Metilación de ADN/genética , Metiltransferasas/genética , Evolución Biológica , Cryptococcus neoformans/metabolismo , ADN/metabolismo , ADN (Citosina-5-)-Metiltransferasa 1/genética , ADN (Citosina-5-)-Metiltransferasas/genética , Metilación de ADN/fisiología , Metilasas de Modificación del ADN/genética , Elementos Transponibles de ADN/genética , Epigenómica/métodos , Evolución Molecular , Genoma/genética , Metiltransferasas/metabolismo , FilogeniaRESUMEN
Tools to understand how the spliceosome functions in vivo have lagged behind advances in the structural biology of the spliceosome. Here, methods are described to globally profile spliceosome-bound pre-mRNA, intermediates, and spliced mRNA at nucleotide resolution. These tools are applied to three yeast species that span 600 million years of evolution. The sensitivity of the approach enables the detection of canonical and non-canonical events, including interrupted, recursive, and nested splicing. This application of statistical modeling uncovers independent roles for the size and position of the intron and the number of introns per transcript in substrate progression through the two catalytic stages. These include species-specific inputs suggestive of spliceosome-transcriptome coevolution. Further investigations reveal the ATP-dependent discard of numerous endogenous substrates after spliceosome assembly in vivo and connect this discard to intron retention, a form of splicing regulation. Spliceosome profiling is a quantitative, generalizable global technology used to investigate an RNP central to eukaryotic gene expression.
Asunto(s)
Ribonucleoproteínas Nucleares Pequeñas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Empalmosomas/metabolismo , Adenosina Trifosfato/metabolismo , Teorema de Bayes , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Inmunoprecipitación , Precursores del ARN/metabolismo , Empalme del ARN , Factores de Empalme de ARN/genética , Factores de Empalme de ARN/metabolismo , ARN de Hongos/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Telomerasa/genética , Telomerasa/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Broadly neutralizing antibodies (bnAbs) to the HIV envelope (Env) V2-apex region are important leads for HIV vaccine design. Most V2-apex bnAbs engage Env with an uncommonly long heavy-chain complementarity-determining region 3 (HCDR3), suggesting that the rarity of bnAb precursors poses a challenge for vaccine priming. We created precursor sequence definitions for V2-apex HCDR3-dependent bnAbs and searched for related precursors in human antibody heavy-chain ultradeep sequencing data from 14 HIV-unexposed donors. We found potential precursors in a majority of donors for only two long-HCDR3 V2-apex bnAbs, PCT64 and PG9, identifying these bnAbs as priority vaccine targets. We then engineered ApexGT Env trimers that bound inferred germlines for PCT64 and PG9 and had higher affinities for bnAbs, determined cryo-EM structures of ApexGT trimers complexed with inferred-germline and bnAb forms of PCT64 and PG9, and developed an mRNA-encoded cell-surface ApexGT trimer. These methods and immunogens have promise to assist HIV vaccine development.
Asunto(s)
Vacunas contra el SIDA , Infecciones por VIH , VIH-1 , Humanos , Anticuerpos ampliamente neutralizantes , Anticuerpos Anti-VIH , Productos del Gen env del Virus de la Inmunodeficiencia Humana , Anticuerpos Neutralizantes , Regiones Determinantes de Complementariedad/genética , Infecciones por VIH/prevención & controlRESUMEN
Defects in circadian rhythm influence physiology and behavior with implications for the treatment of sleep disorders, metabolic disease, and cancer. Although core regulatory components of clock rhythmicity have been defined, insight into the mechanisms underpinning amplitude is limited. Here, we show that REV-ERBα, a core inhibitory component of clock transcription, is targeted for ubiquitination and subsequent degradation by the F-box protein FBXW7. By relieving REV-ERBα-dependent repression, FBXW7 provides an unrecognized mechanism for enhancing the amplitude of clock gene transcription. Cyclin-dependent kinase 1 (CDK1)-mediated phosphorylation of REV-ERBα is necessary for FBXW7 recognition. Moreover, targeted hepatic disruption of FBXW7 alters circadian expression of core clock genes and perturbs whole-body lipid and glucose levels. This CDK1-FBXW7 pathway controlling REV-ERBα repression defines an unexpected molecular mechanism for re-engaging the positive transcriptional arm of the clock, as well as a potential route to manipulate clock amplitude via small molecule CDK1 inhibition.
Asunto(s)
Ritmo Circadiano , Proteínas F-Box/metabolismo , Hígado/metabolismo , Miembro 1 del Grupo D de la Subfamilia 1 de Receptores Nucleares/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Proteína Quinasa CDC2/metabolismo , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Relojes Circadianos , Proteínas F-Box/genética , Proteína 7 que Contiene Repeticiones F-Box-WD , Técnicas de Inactivación de Genes , Humanos , Metabolismo de los Lípidos , Ratones , Fosforilación , Procesamiento Proteico-Postraduccional , Transcriptoma , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Effective silencing by RNA-interference (RNAi) depends on mechanisms that amplify and propagate the silencing signal. In some organisms, small-interfering RNAs (siRNAs) are amplified from target mRNAs by RNA-dependent RNA polymerase (RdRP). Both RdRP recruitment and mRNA silencing require Argonaute proteins, which are generally thought to degrade RNAi targets by directly cleaving them. However, in C. elegans, the enzymatic activity of the primary Argonaute, RDE-1, is not required for silencing activity. We show that RDE-1 can instead recruit an endoribonuclease, RDE-8, to target RNA. RDE-8 can cleave RNA in vitro and is needed for the production of 3' uridylated fragments of target mRNA in vivo. We also find that RDE-8 promotes RdRP activity, thereby ensuring amplification of siRNAs. Together, our findings suggest a model in which RDE-8 cleaves target mRNAs to mediate silencing, while generating 3' uridylated mRNA fragments to serve as templates for the RdRP-directed amplification of the silencing signal.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Endorribonucleasas/metabolismo , Secuencia de Aminoácidos , Animales , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Gránulos Citoplasmáticos/metabolismo , Endorribonucleasas/química , Endorribonucleasas/genética , Datos de Secuencia Molecular , Interferencia de ARN , ARN Bicatenario , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , ARN Polimerasa Dependiente del ARN/metabolismo , Ribonucleasa III/metabolismo , Alineación de SecuenciaRESUMEN
Histidine phosphorylation (pHis) is well studied in bacteria; however, its role in mammalian signaling remains largely unexplored due to the lack of pHis-specific antibodies and the lability of the phosphoramidate (P-N) bond. Both imidazole nitrogens can be phosphorylated, forming 1-phosphohistidine (1-pHis) or 3-phosphohistidine (3-pHis). We have developed monoclonal antibodies (mAbs) that specifically recognize 1-pHis or 3-pHis; they do not cross-react with phosphotyrosine or the other pHis isomer. Assays based on the isomer-specific autophosphorylation of NME1 and phosphoglycerate mutase were used with immunoblotting and sequencing IgG variable domains to screen, select, and characterize anti-1-pHis and anti-3-pHis mAbs. Their sequence independence was determined by blotting synthetic peptide arrays, and they have been tested for immunofluorescence staining and immunoaffinity purification, leading to putative identification of pHis-containing proteins. These reagents should be broadly useful for identification of pHis substrates and functional study of pHis using a variety of immunological, proteomic, and biological assays.
Asunto(s)
Anticuerpos Monoclonales , Histidina/metabolismo , Animales , Centrosoma , Cromatografía Liquida , Células HeLa , Humanos , Modelos Químicos , Péptidos/análisis , Fosforilación , Polos del Huso , Espectrometría de Masas en TándemRESUMEN
We characterize the Polycomb system that assembles repressive subtelomeric domains of H3K27 methylation (H3K27me) in the yeast Cryptococcus neoformans. Purification of this PRC2-like protein complex reveals orthologs of animal PRC2 components as well as a chromodomain-containing subunit, Ccc1, which recognizes H3K27me. Whereas removal of either the EZH or EED ortholog eliminates H3K27me, disruption of mark recognition by Ccc1 causes H3K27me to redistribute. Strikingly, the resulting pattern of H3K27me coincides with domains of heterochromatin marked by H3K9me. Indeed, additional removal of the C. neoformans H3K9 methyltransferase Clr4 results in loss of both H3K9me and the redistributed H3K27me marks. These findings indicate that the anchoring of a chromatin-modifying complex to its product suppresses its attraction to a different chromatin type, explaining how enzymes that act on histones, which often harbor product recognition modules, may deposit distinct chromatin domains despite sharing a highly abundant and largely identical substrate-the nucleosome.
Asunto(s)
Cryptococcus neoformans/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas del Grupo Polycomb/metabolismo , Secuencia de Aminoácidos , Centrómero/metabolismo , Cryptococcus neoformans/genética , Heterocromatina/metabolismo , Código de Histonas , N-Metiltransferasa de Histona-Lisina/metabolismo , Datos de Secuencia Molecular , Alineación de SecuenciaRESUMEN
Eukaryotic cells regulate 5'-triphosphorylated RNAs (ppp-RNAs) to promote cellular functions and prevent recognition by antiviral RNA sensors. For example, RNA capping enzymes possess triphosphatase domains that remove the γ phosphates of ppp-RNAs during RNA capping. Members of the closely related PIR-1 (phosphatase that interacts with RNA and ribonucleoprotein particle 1) family of RNA polyphosphatases remove both the ß and γ phosphates from ppp-RNAs. Here, we show that C. elegans PIR-1 dephosphorylates ppp-RNAs made by cellular RNA-dependent RNA polymerases (RdRPs) and is required for the maturation of 26G-RNAs, Dicer-dependent small RNAs that regulate thousands of genes during spermatogenesis and embryogenesis. PIR-1 also regulates the CSR-1 22G-RNA pathway and has critical functions in both somatic and germline development. Our findings suggest that PIR-1 modulates both Dicer-dependent and Dicer-independent Argonaute pathways and provide insight into how cells and viruses use a conserved RNA phosphatase to regulate and respond to ppp-RNA species.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/enzimología , Monoéster Fosfórico Hidrolasas/metabolismo , Procesamiento Postranscripcional del ARN , ARN/metabolismo , Animales , Animales Modificados Genéticamente , Caenorhabditis elegans/embriología , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Regulación del Desarrollo de la Expresión Génica , Monoéster Fosfórico Hidrolasas/genética , Fosforilación , ARN/genética , Caperuzas de ARN , ARN Polimerasa Dependiente del ARN/genética , ARN Polimerasa Dependiente del ARN/metabolismo , Ribonucleasa III/genética , Ribonucleasa III/metabolismo , Espermatogénesis , Especificidad por SustratoRESUMEN
Signaling via the inducible costimulator ICOS fuels the stepwise development of follicular helper T cells (TFH cells). However, a signaling pathway unique to ICOS has not been identified. We found here that the kinase TBK1 associated with ICOS via a conserved motif, IProx, that shares homology with the tumor-necrosis-factor receptor (TNFR)-associated factors TRAF2 and TRAF3. Disruption of this motif abolished the association of TBK1 with ICOS, TRAF2 and TRAF3, which identified a TBK1-binding consensus. Alteration of this motif in ICOS or depletion of TBK1 in T cells severely impaired the differentiation of germinal center (GC) TFH cells and the development of GCs, interfered with B cell differentiation and disrupted the development of antibody responses, but the IProx motif and TBK1 were dispensable for the early differentiation of TFH cells. These results reveal a previously unknown ICOS-TBK1 signaling pathway that specifies the commitment of GC TFH cells.
Asunto(s)
Linfocitos B/fisiología , Linfocitos T CD4-Positivos/fisiología , Centro Germinal/inmunología , Proteína Coestimuladora de Linfocitos T Inducibles/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Formación de Anticuerpos/genética , Diferenciación Celular/genética , Células Cultivadas , Proteína Coestimuladora de Linfocitos T Inducibles/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Unión Proteica , Proteínas Serina-Treonina Quinasas/genética , Transducción de Señal , Factor 2 Asociado a Receptor de TNF/genética , Factor 3 Asociado a Receptor de TNF/genéticaRESUMEN
During each life cycle, germ cells preserve and pass on both genetic and epigenetic information. In C. elegans, the ALG-3/4 Argonaute proteins are expressed during male gametogenesis and promote male fertility. Here, we show that the CSR-1 Argonaute functions with ALG-3/4 to positively regulate target genes required for spermiogenesis. Our findings suggest that ALG-3/4 functions during spermatogenesis to amplify a small RNA signal that represents an epigenetic memory of male-specific gene expression. CSR-1, which is abundant in mature sperm, appears to transmit this memory to offspring. Surprisingly, in addition to small RNAs targeting male-specific genes, we show that males also harbor an extensive repertoire of CSR-1 small RNAs targeting oogenesis-specific mRNAs. Together, these findings suggest that C. elegans sperm transmit not only the genome but also epigenetic binary signals in the form of Argonaute/small RNA complexes that constitute a memory of gene expression in preceding generations.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Epigénesis Genética , Proteínas de Unión al ARN/metabolismo , Espermatogénesis , Animales , Caenorhabditis elegans/genética , Femenino , Masculino , ARN Pequeño no Traducido/metabolismo , Transducción de Señal , Espermatozoides , Transcripción GenéticaRESUMEN
Intracellular proteins with long lifespans have recently been linked to age-dependent defects, ranging from decreased fertility to the functional decline of neurons. Why long-lived proteins exist in metabolically active cellular environments and how they are maintained over time remains poorly understood. Here, we provide a system-wide identification of proteins with exceptional lifespans in the rat brain. These proteins are inefficiently replenished despite being translated robustly throughout adulthood. Using nucleoporins as a paradigm for long-term protein persistence, we found that nuclear pore complexes (NPCs) are maintained over a cell's life through slow but finite exchange of even its most stable subcomplexes. This maintenance is limited, however, as some nucleoporin levels decrease during aging, providing a rationale for the previously observed age-dependent deterioration of NPC function. Our identification of a long-lived proteome reveals cellular components that are at increased risk for damage accumulation, linking long-term protein persistence to the cellular aging process. PAPERCLIP:
Asunto(s)
Encéfalo/citología , Senescencia Celular , Proteínas de Complejo Poro Nuclear/metabolismo , Proteoma/metabolismo , Animales , Encéfalo/metabolismo , Neuroglía/metabolismo , Neuronas/metabolismo , Poro Nuclear/metabolismo , Biosíntesis de Proteínas , RatasRESUMEN
Microglia are the resident macrophages of the CNS, and their functions have been extensively studied in various brain pathologies. The physiological roles of microglia in brain plasticity and function, however, remain unclear. To address this question, we generated CX3CR1(CreER) mice expressing tamoxifen-inducible Cre recombinase that allow for specific manipulation of gene function in microglia. Using CX3CR1(CreER) to drive diphtheria toxin receptor expression in microglia, we found that microglia could be specifically depleted from the brain upon diphtheria toxin administration. Mice depleted of microglia showed deficits in multiple learning tasks and a significant reduction in motor-learning-dependent synapse formation. Furthermore, Cre-dependent removal of brain-derived neurotrophic factor (BDNF) from microglia largely recapitulated the effects of microglia depletion. Microglial BDNF increases neuronal tropomyosin-related kinase receptor B phosphorylation, a key mediator of synaptic plasticity. Together, our findings reveal that microglia serve important physiological functions in learning and memory by promoting learning-related synapse formation through BDNF signaling.
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo/metabolismo , Aprendizaje/fisiología , Microglía/fisiología , Sinapsis , Animales , Receptor 1 de Quimiocinas CX3C , Expresión Génica , Ratones , Microglía/citología , Plasticidad Neuronal , Proteínas Quinasas/metabolismo , Receptores de Quimiocina/genética , Receptores de Quimiocina/metabolismo , Transducción de SeñalRESUMEN
Using the yeast Cryptococcus neoformans, we describe a mechanism by which transposons are initially targeted for RNAi-mediated genome defense. We show that intron-containing mRNA precursors template siRNA synthesis. We identify a Spliceosome-Coupled And Nuclear RNAi (SCANR) complex required for siRNA synthesis and demonstrate that it physically associates with the spliceosome. We find that RNAi target transcripts are distinguished by suboptimal introns and abnormally high occupancy on spliceosomes. Functional investigations demonstrate that the stalling of mRNA precursors on spliceosomes is required for siRNA accumulation. Lariat debranching enzyme is also necessary for siRNA production, suggesting a requirement for processing of stalled splicing intermediates. We propose that recognition of mRNA precursors by the SCANR complex is in kinetic competition with splicing, thereby promoting siRNA production from transposon transcripts stalled on spliceosomes. Disparity in the strength of expression signals encoded by transposons versus host genes offers an avenue for the evolution of genome defense.
Asunto(s)
Cryptococcus neoformans/genética , Elementos Transponibles de ADN , Interferencia de ARN , Empalmosomas/metabolismo , Genoma Fúngico , Intrones , Cinética , ARN Mensajero/metabolismo , ARN Nuclear/metabolismo , ARN Interferente Pequeño/metabolismo , ARN Polimerasa Dependiente del ARN/metabolismoRESUMEN
Parkinson's disease (PD) is characterized by loss of A9 dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc). An association has been reported between PD and exposure to mitochondrial toxins, including environmental pesticides paraquat, maneb, and rotenone. Here, using a robust, patient-derived stem cell model of PD allowing comparison of A53T α-synuclein (α-syn) mutant cells and isogenic mutation-corrected controls, we identify mitochondrial toxin-induced perturbations in A53T α-syn A9 DA neurons (hNs). We report a pathway whereby basal and toxin-induced nitrosative/oxidative stress results in S-nitrosylation of transcription factor MEF2C in A53T hNs compared to corrected controls. This redox reaction inhibits the MEF2C-PGC1α transcriptional network, contributing to mitochondrial dysfunction and apoptotic cell death. Our data provide mechanistic insight into gene-environmental interaction (GxE) in the pathogenesis of PD. Furthermore, using small-molecule high-throughput screening, we identify the MEF2C-PGC1α pathway as a therapeutic target to combat PD.
Asunto(s)
Interacción Gen-Ambiente , Mitocondrias/efectos de los fármacos , Paraquat/toxicidad , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/patología , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Factores de Transcripción MEF2 , Mutación/efectos de los fármacos , Neuronas/metabolismo , Estrés Oxidativo , Enfermedad de Parkinson/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Especies de Nitrógeno Reactivo/metabolismo , Sustancia Negra/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismoRESUMEN
Neuronal activity influences genes involved in circuit development and information processing. However, the molecular basis of this process remains poorly understood. We found that HDAC4, a histone deacetylase that shuttles between the nucleus and cytoplasm, controls a transcriptional program essential for synaptic plasticity and memory. The nuclear import of HDAC4 and its association with chromatin is negatively regulated by NMDA receptors. In the nucleus, HDAC4 represses genes encoding constituents of central synapses, thereby affecting synaptic architecture and strength. Furthermore, we show that a truncated form of HDAC4 encoded by an allele associated with mental retardation is a gain-of-function nuclear repressor that abolishes transcription and synaptic transmission despite the loss of the deacetylase domain. Accordingly, mice carrying a mutant that mimics this allele exhibit deficits in neurotransmission, spatial learning, and memory. These studies elucidate a mechanism of experience-dependent plasticity and define the biological role of HDAC4 in the brain.
Asunto(s)
Transporte Activo de Núcleo Celular , Encéfalo/metabolismo , Histona Desacetilasas/metabolismo , Memoria , Plasticidad Neuronal , Neuronas/metabolismo , Sinapsis/metabolismo , Transcripción Genética , Animales , Ratones , Prosencéfalo/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Embryonic stem cell (ESC) pluripotency requires bivalent epigenetic modifications of key developmental genes regulated by various transcription factors and chromatin-modifying enzymes. How these factors coordinate with one another to maintain the bivalent chromatin state so that ESCs can undergo rapid self-renewal while retaining pluripotency is poorly understood. We report that Utf1, a target of Oct4 and Sox2, is a bivalent chromatin component that buffers poised states of bivalent genes. By limiting PRC2 loading and histone 3 lysine-27 trimethylation, Utf1 sets proper activation thresholds for bivalent genes. It also promotes nuclear tagging of messenger RNAs (mRNAs) transcribed from insufficiently silenced bivalent genes for cytoplasmic degradation through mRNA decapping. These opposing functions of Utf1 promote coordinated differentiation. The mRNA degradation function also ensures rapid cell proliferation by blocking the Myc-Arf feedback control. Thus, Utf1 couples the core pluripotency factors with Myc and PRC2 networks to promote the pluripotency and proliferation of ESCs.
Asunto(s)
Células Madre Embrionarias/metabolismo , Proteínas Nucleares/metabolismo , Células Madre Pluripotentes/metabolismo , ARN Mensajero/metabolismo , Transactivadores/metabolismo , Factores de Ribosilacion-ADP/metabolismo , Diferenciación Celular , Células Madre Embrionarias/citología , Epigénesis Genética , Humanos , Células Madre Pluripotentes/citología , Proteínas Proto-Oncogénicas c-myc/metabolismoRESUMEN
Synaptotagmin-11 (Syt11) is a Synaptotagmin isoform that lacks an apparent ability to bind calcium, phospholipids, or SNARE proteins. While human genetic studies have linked mutations in the Syt11 gene to schizophrenia and Parkinson's disease, the localization or physiological role of Syt11 remain unclear. We found that in neurons, Syt11 resides on abundant vesicles that differ from synaptic vesicles and resemble trafficking endosomes. These vesicles recycle via the plasma membrane in an activity-dependent manner, but their exocytosis is slow and desynchronized. Constitutive knockout mice lacking Syt11 died shortly after birth, suggesting Syt11-mediated membrane transport is required for survival. In contrast, selective ablation of Syt11 in excitatory forebrain neurons using a conditional knockout did not affect life span but impaired synaptic plasticity and memory. Syt11-deficient neurons displayed normal secretion of fast neurotransmitters and peptides but exhibited a reduction of long-term synaptic potentiation. Hence, Syt11 is an essential component of a neuronal vesicular trafficking pathway that differs from the well-characterized synaptic vesicle trafficking pathway but is also essential for life.
Asunto(s)
Plasticidad Neuronal/genética , Neuronas/fisiología , Vesículas Sinápticas/metabolismo , Sinaptotagminas/genética , Sinaptotagminas/metabolismo , Animales , Corteza Cerebral/embriología , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Técnicas de Sustitución del Gen , Hipocampo/fisiopatología , Memoria/fisiología , Ratones , Ratones Noqueados , Neurotransmisores/metabolismo , Prosencéfalo/citología , Prosencéfalo/fisiología , Potenciales Sinápticos/genética , Transmisión Sináptica , Vesículas Sinápticas/genética , Sinaptotagminas/deficienciaRESUMEN
Heterochromatin is a highly condensed form of chromatin that silences gene transcription. Although high levels of transcriptional activities disrupt heterochromatin, transcription of repetitive DNA elements and subsequent processing of the transcripts by the RNAi machinery are required for heterochromatin assembly. In fission yeast, a JmjC domain protein, Epe1, promotes transcription of DNA repeats to facilitate heterochromatin formation, but overexpression of Epe1 leads to heterochromatin defects. However, the molecular function of Epe1 is not well understood. By screening the fission yeast deletion library, we found that heterochromatin defects associated with Epe1 overexpression are alleviated by mutations of the SAGA histone acetyltransferase complex. Overexpressed Epe1 associates with SAGA and recruits SAGA to heterochromatin regions, which leads to increased histone acetylation, transcription of repeats, and the disruption of heterochromatin. At its normal expression levels, Epe1 also associates with SAGA, albeit weakly. Such interaction regulates histone acetylation levels at heterochromatin and promotes transcription of repeats for heterochromatin assembly. Our results also suggest that increases of certain chromatin protein levels, which frequently occur in cancer cells, might strengthen relatively weak interactions to affect the epigenetic landscape.
Asunto(s)
Regulación Fúngica de la Expresión Génica/genética , Heterocromatina/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Acetilación , Ensamble y Desensamble de Cromatina/genética , Inestabilidad Cromosómica/genética , Eliminación de Gen , Heterocromatina/metabolismo , Heterocromatina/patología , Histona Acetiltransferasas/genética , Histona Acetiltransferasas/metabolismo , Repeticiones de Microsatélite/genética , Transporte de ProteínasRESUMEN
Analyzing proteins from single cells by tandem mass spectrometry (MS) has recently become technically feasible. While such analysis has the potential to accurately quantify thousands of proteins across thousands of single cells, the accuracy and reproducibility of the results may be undermined by numerous factors affecting experimental design, sample preparation, data acquisition and data analysis. We expect that broadly accepted community guidelines and standardized metrics will enhance rigor, data quality and alignment between laboratories. Here we propose best practices, quality controls and data-reporting recommendations to assist in the broad adoption of reliable quantitative workflows for single-cell proteomics. Resources and discussion forums are available at https://single-cell.net/guidelines .