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1.
Genes Dev ; 33(5-6): 348-364, 2019 03 01.
Artículo en Inglés | MEDLINE | ID: mdl-30808657

RESUMEN

RNAi and Polycomb repression play evolutionarily conserved and often coordinated roles in transcriptional silencing. Here, we show that, in the protozoan Tetrahymena thermophila, germline-specific internally eliminated sequences (IESs)-many related to transposable elements (TEs)-become transcriptionally activated in mutants deficient in the RNAi-dependent Polycomb repression pathway. Germline TE mobilization also dramatically increases in these mutants. The transition from noncoding RNA (ncRNA) to mRNA production accompanies transcriptional activation of TE-related sequences and vice versa for transcriptional silencing. The balance between ncRNA and mRNA production is potentially affected by cotranscriptional processing as well as RNAi and Polycomb repression. We posit that interplay between RNAi and Polycomb repression is a widely conserved phenomenon, whose ancestral role is epigenetic silencing of TEs.


Asunto(s)
Elementos Transponibles de ADN/genética , Proteínas del Grupo Polycomb/genética , Proteínas Protozoarias/genética , Interferencia de ARN , Tetrahymena thermophila/genética , Activación Transcripcional/genética , Epigénesis Genética , Silenciador del Gen , Mutación , ARN Mensajero/genética , ARN no Traducido/genética
2.
Anal Bioanal Chem ; 2024 Sep 13.
Artículo en Inglés | MEDLINE | ID: mdl-39269501

RESUMEN

Per- and polyfluoroalkyl substances (PFAS) are widely used in industry, residential, and consumer products. Studies have shown associations between high PFAS exposure and adverse health effects. In 2022, the National Academies of Science, Engineering, and Medicine (NASEM) published Guidance on PFAS Exposure, Testing, and Clinical Follow-up providing laboratory and clinical direction. The Guidance suggests nine PFAS should be measured in serum or plasma specimens and summed to provide a total PFAS concentration using a NASEM-recommended method. Follow-up clinical recommendations are based on the calculated PFAS NASEM summation. We developed and validated a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method in accordance with NASEM recommendations but distinguished by the ability to separate closely related structural isomers. As part of our validation, PFAS prevalence was evaluated in a population survey comprised of clinical donor and remnant specimens (n = 1023 in total). In this study, 82.2% of the specimens had PFAS NASEM summations of 2 to < 20 ng/mL and 2.5% had a summation ≥ 20 ng/mL. The median PFAS NASEM summation was 4.65 ng/mL in this study, lower than the 7.74 ng/mL median observed in the 2017-2020 Centers for Disease Control and Prevention, National Health and Nutrition Examination Survey (n = 3072). This lower median PFAS NASEM summation may reflect a decline in PFAS population levels over time or sample population exposure differences.

3.
Nucleic Acids Res ; 45(16): 9481-9502, 2017 Sep 19.
Artículo en Inglés | MEDLINE | ID: mdl-28934495

RESUMEN

Developmentally programmed genome rearrangement accompanies differentiation of the silent germline micronucleus into the transcriptionally active somatic macronucleus in the ciliated protozoan Tetrahymena thermophila. Internal eliminated sequences (IES) are excised, followed by rejoining of MAC-destined sequences, while fragmentation occurs at conserved chromosome breakage sequences, generating macronuclear chromosomes. Some macronuclear chromosomes, referred to as non-maintained chromosomes (NMC), are lost soon after differentiation. Large NMC contain genes implicated in development-specific roles. One such gene encodes the domesticated piggyBac transposase TPB6, required for heterochromatin-dependent precise excision of IES residing within exons of functionally important genes. These conserved exonic IES determine alternative transcription products in the developing macronucleus; some even contain free-standing genes. Examples of precise loss of some exonic IES in the micronucleus and retention of others in the macronucleus of related species suggest an evolutionary analogy to introns. Our results reveal that germline-limited sequences can encode genes with specific expression patterns and development-related functions, which may be a recurring theme in eukaryotic organisms experiencing programmed genome rearrangement during germline to soma differentiation.


Asunto(s)
Proteínas Protozoarias/metabolismo , Tetrahymena thermophila/genética , Transposasas/metabolismo , Cromosomas/genética , Exones , Reordenamiento Génico , Heterocromatina/genética , Secuencias Invertidas Repetidas , Macronúcleo/genética , Micronúcleo Germinal , Proteínas Protozoarias/genética , Interferencia de ARN , Transposasas/genética
4.
Nucleic Acids Res ; 44(21): 10091-10105, 2016 12 01.
Artículo en Inglés | MEDLINE | ID: mdl-27488188

RESUMEN

The ciliate protozoan Tetrahymena thermophila contains two types of structurally and functionally differentiated nuclei: the transcriptionally active somatic macronucleus (MAC) and the transcriptionally silent germ-line micronucleus (MIC). Here, we demonstrate that MAC features well-positioned nucleosomes downstream of transcription start sites and flanking splice sites. Transcription-associated trans-determinants promote nucleosome positioning in MAC. By contrast, nucleosomes in MIC are dramatically delocalized. Nucleosome occupancy in MAC and MIC are nonetheless highly correlated with each other, as well as with in vitro reconstitution and predictions based upon DNA sequence features, revealing unexpectedly strong contributions from cis-determinants. In particular, well-positioned nucleosomes are often matched with GC content oscillations. As many nucleosomes are coordinately accommodated by both cis- and trans-determinants, we propose that their distribution is shaped by the impact of these nucleosomes on the mutational and transcriptional landscape, and driven by evolutionary selection.


Asunto(s)
Cromatina/genética , Macronúcleo/genética , Nucleosomas/genética , Tetrahymena thermophila/genética , Cromatina/metabolismo , ADN Polimerasa II/genética , ADN Polimerasa II/metabolismo , Macronúcleo/metabolismo , Nucleasa Microcócica/genética , Nucleasa Microcócica/metabolismo , Micronúcleo Germinal/genética , Nucleosomas/metabolismo , Sitios de Empalme de ARN , Sitio de Iniciación de la Transcripción
5.
BMC Biol ; 11: 12, 2013 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-23374633

RESUMEN

BACKGROUND: Skeletal muscle undergoes rapid atrophy upon denervation and the underlying mechanisms are complicated. FOXO3a has been implicated as a major mediator of muscle atrophy, but how its subcellular location and activity is controlled during the pathogenesis of muscle atrophy remains largely unknown. MST1 (Mammalian Sterile 20-like kinase 1) is identified as a central component of the Hippo signaling pathway. MST1 has been shown to mediate phosphorylation of FOXO3a at Ser207. Whether this MST1-FOXO signaling cascade exerts any functional consequence on cellular homeostasis remains to be investigated. RESULT: We identified that MST1 kinase was expressed widely in skeletal muscles and was dramatically up-regulated in fast- but not slow-dominant skeletal muscles immediately following denervation. The results of our histological and biochemical studies demonstrated that deletion of MST1 significantly attenuated denervation-induced skeletal muscle wasting and decreased expression of Atrogin-1 and LC3 genes in fast-dominant skeletal muscles from three- to five-month-old adult mice. Further studies indicated that MST1, but not MST2, remarkably increased FOXO3a phosphorylation level at Ser207 and promoted its nuclear translocation in atrophic fast-dominant muscles. CONCLUSIONS: We have established that MST1 kinase plays an important role in regulating denervation-induced skeletal muscle atrophy. During the early stage of muscle atrophy, the up-regulated MST1 kinase promoted progression of neurogenic atrophy in fast-dominant skeletal muscles through activation of FOXO3a transcription factors.


Asunto(s)
Músculo Esquelético/fisiopatología , Atrofia Muscular/fisiopatología , Proteínas Serina-Treonina Quinasas/fisiología , Animales , Animales Recién Nacidos , Proteína Forkhead Box O3 , Factores de Transcripción Forkhead/metabolismo , Ratones , Proteínas Musculares/genética , Músculo Esquelético/enzimología , Atrofia Muscular/enzimología , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Ligasas SKP Cullina F-box/genética , Transducción de Señal , Transcripción Genética , Regulación hacia Arriba
6.
Cell Res ; 25(1): 93-109, 2015 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-25522691

RESUMEN

Deregulation of the evolutionarily conserved Hippo pathway has been implicated in abnormal development of animals and in several types of cancer. One mechanism of Hippo pathway regulation is achieved by controlling the stability of its regulatory components. However, the executive E3 ligases that are involved in this process, and how the process is regulated, remain poorly defined. In this study, we identify, through a genetic candidate screen, the SCF(Slmb) E3 ligase as a novel negative regulator of the Hippo pathway in Drosophila imaginal tissues via mediation of the degradation of Expanded (Ex). Mechanistic study shows that Slmb-mediated degradation of Ex is inhibited by the Hippo signaling. Considering the fact that Hippo signaling suppresses the transcription of ex, we propose that the Hippo pathway employs a double security mechanism to ensure fine-tuned homeostasis during development.


Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Ligasas SKP Cullina F-box/metabolismo , Transducción de Señal , Animales , Línea Celular , Proteínas de la Membrana/metabolismo , Mapas de Interacción de Proteínas , Proteínas Quinasas/metabolismo , Proteolisis , Ubiquitinación
7.
Mol Biol Cell ; 24(11): 1676-87, S1-7, 2013 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-23552694

RESUMEN

Cell cycle progression is controlled by a complex regulatory network consisting of interacting positive and negative factors. In humans, the positive regulator Skp2, an F-box protein, has been a subject of intense investigation in part because of its oncogenic activity. By contrast, the molecular and developmental functions of its Drosophila homologue, dSkp2, are poorly understood. Here we investigate the role of dSkp2 by focusing on its functional relationship with Dacapo (Dap), the Drosophila homologue of the cyclin-dependent kinase inhibitors p21(cip1)/p27(kip1)/p57(kip2). We show that dSkp2 interacts physically with Dap and has a role in targeting Dap for ubiquitination and proteasome-mediated degradation. We present evidence that dSkp2 regulates cell cycle progression by antagonizing Dap in vivo. dSkp2 knockdown reduces cell density in the wing by prolonging the cell doubling time. In addition, the wing phenotype caused by dSkp2 knockdown resembles that caused by dap overexpression and can be partially suppressed by reducing the gene dose of dap. Our study thus documents a conserved functional relationship between dSkp2 and Dap in their control of cell cycle progression, suggesting the possibility of using Drosophila as a model system to study Skp2-mediated tumorigenesis.


Asunto(s)
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Macrófagos/metabolismo , Proteínas Nucleares/genética , Proteínas Quinasas Asociadas a Fase-S/genética , Animales , Animales Modificados Genéticamente , Ciclo Celular , Línea Celular , Proliferación Celular , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/metabolismo , Ojo/metabolismo , Ojo/patología , Regulación del Desarrollo de la Expresión Génica , Humanos , Macrófagos/citología , Proteínas Nucleares/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteínas Quinasas Asociadas a Fase-S/antagonistas & inhibidores , Proteínas Quinasas Asociadas a Fase-S/metabolismo , Homología de Secuencia de Aminoácido , Transducción de Señal , Ubiquitinación , Alas de Animales/metabolismo , Alas de Animales/patología
8.
J Genet Genomics ; 39(8): 397-413, 2012 Aug 20.
Artículo en Inglés | MEDLINE | ID: mdl-22884096

RESUMEN

F-box proteins are components of the SCF (SkpA-Cullin 1-F-box) E3 ligase complexes, acting as the specificity-determinants in targeting substrate proteins for ubiquitination and degradation. In humans, at least 22 out of 75 F-box proteins have experimentally documented substrates, whereas in Drosophila 12 F-box proteins have been characterized with known substrates. To systematically investigate the genetic and molecular functions of F-box proteins in Drosophila, we performed a survey of the literature and databases. We identified 45 Drosophila genes that encode proteins containing at least one F-box domain. We collected publically available RNAi lines against these genes and used them in a tissue-specific RNAi-based phenotypic screen. Here, we present our systematic phenotypic dataset from the eye, the wing and the notum. This dataset is the first of its kind and represents a useful resource for future studies of the molecular and genetic functions of F-box genes in Drosophila. Our results show that, as expected, F-box genes in Drosophila have regulatory roles in a diverse array of processes including cell proliferation, cell growth, signal transduction, and cellular and animal survival.


Asunto(s)
Proteínas de Drosophila/genética , Drosophila/genética , Proteínas F-Box/genética , Interferencia de ARN , Animales , Drosophila/crecimiento & desarrollo , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Ojo/crecimiento & desarrollo , Ojo/metabolismo , Proteínas F-Box/metabolismo , Datos de Secuencia Molecular , Especificidad de Órganos , Fenotipo , Alas de Animales/crecimiento & desarrollo , Alas de Animales/metabolismo
9.
J Genet Genomics ; 38(6): 225-34, 2011 Jun 20.
Artículo en Inglés | MEDLINE | ID: mdl-21703546

RESUMEN

CoQ is an essential electron carrier in the mitochondrial respiratory chain of both eukaryotes and prokaryotes. It consists of a benzoquinone head group and a hydrophobic polyisoprenoid tail. The genes (COQ1-9) involved in CoQ biosynthesis have been characterized in yeast. In this study, we generated and molecularly characterized a mutant allele of a novel Drosophila gene, sbo, which encodes a protein that is predicted to catalyze the prenylation of p-hydroxybenzoate with the isoprenoid chain during the process of CoQ synthesis. Expression of sbo in yeast rescues the lethality of ∆COQ2 mutant cells, indicating that sbo is a functional homolog of COQ2. HPLC results show that the levels of CoQ(9) and CoQ(10) were significantly reduced in sbo heterozygous adult flies. Furthermore, the mean lifespans of males and females heterozygous for sbo are extended by 12.5% and 30.8%, respectively. Homozygous sbo animals exhibit reduced activities of the insulin/insulin-like growth factor signaling (IIS) pathway. Taken together, we conclude that sbo is an essential gene for Drosophila development, mutation of which leads to an extension of lifespan most likely by altering endogenous CoQ biosynthesis.


Asunto(s)
Transferasas Alquil y Aril/fisiología , Proteínas de Drosophila/fisiología , Drosophila melanogaster/crecimiento & desarrollo , Longevidad/fisiología , Ubiquinona/biosíntesis , Transferasas Alquil y Aril/genética , Transferasas Alquil y Aril/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Femenino , Humanos , Larva/genética , Longevidad/genética , Masculino , Mitocondrias/genética , Mitocondrias/metabolismo , Datos de Secuencia Molecular , Mutación , Parabenos/metabolismo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Homología de Secuencia , Ubiquinona/genética
10.
Protein Cell ; 1(5): 478-90, 2010 May.
Artículo en Inglés | MEDLINE | ID: mdl-21203963

RESUMEN

RecQ5 in mammalian cells has been suggested to suppress inappropriate homologous recombination. However, the specific pathway(s) in which it is involved and the underlining mechanism(s) remain poorly understood. We took advantage of genetic tools in Drosophila to investigate how Drosophila RecQ5 (dRecQ5) functions in vivo in homologous recombination-mediated double strand break (DSB) repair. We generated null alleles of dRecQ5 using the targeted recombination technique. The mutant animals are homozygous viable, but with growth retardation during development. The mutants are sensitive to both exogenous DSB-inducing treatment, such as gamma-irradiation, and endogenously induced double strand breaks (DSBs) by I-Sce I endonuclease. In the absence of dRecQ5, single strand annealing (SSA)-mediated DSB repair is compromised with compensatory increases in either inter-homologous gene conversion, or non-homologous end joining (NHEJ) when inter-chromosomal homologous sequence is unavailable. Loss of function of dRecQ5 also leads to genome instability in loss of heterozygosity (LOH) assays. Together, our data demonstrate that dRecQ5 functions in SSA-mediated DSB repair to achieve its full efficiency and in suppression of LOH in Drosophila.


Asunto(s)
Reparación del ADN/genética , ADN de Cadena Simple/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Pérdida de Heterocigocidad/genética , RecQ Helicasas/metabolismo , Animales , ADN Helicasas , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , RecQ Helicasas/genética
11.
PLoS One ; 4(7): e6107, 2009 Jul 02.
Artículo en Inglés | MEDLINE | ID: mdl-19572017

RESUMEN

BACKGROUND: The family of RecQ DNA helicases plays an important role in the maintenance of genomic integrity. Mutations in three of the five known RecQ family members in humans, BLM, WRN and RecQ4, lead to disorders that are characterized by predisposition to cancer and premature aging. METHODOLOGY/PRINCIPAL FINDINGS: To address the in vivo functions of Drosophila RecQ4 (dRecQ4), we generated mutant alleles of dRecQ4 using the targeted gene knock-out technique. Our data show that dRecQ4 mutants are homozygous lethal with defects in DNA replication, cell cycle progression and cell proliferation. Two sets of experiments suggest that dRecQ4 also plays a role in DNA double strand break repair. First, mutant animals exhibit sensitivity to gamma irradiation. Second, the efficiency of DsRed reconstitution via single strand annealing repair is significantly reduced in the dRecQ4 mutant animals. Rescue experiments further show that both the N-terminal domain and the helicase domain are essential to dRecQ4 function in vivo. The N-terminal domain is sufficient for the DNA repair function of dRecQ4. CONCLUSIONS/SIGNIFICANCE: Together, our results show that dRecQ4 is an essential gene that plays an important role in not only DNA replication but also DNA repair and cell cycle progression in vivo.


Asunto(s)
Proliferación Celular , Replicación del ADN/fisiología , RecQ Helicasas/fisiología , Animales , Secuencia de Bases , Aberraciones Cromosómicas , Reparación del ADN , Drosophila , Técnicas de Inactivación de Genes
12.
PLoS One ; 2(7): e629, 2007 Jul 18.
Artículo en Inglés | MEDLINE | ID: mdl-17637844

RESUMEN

Recent studies have shown that neurodegeneration is closely related to misfolding and aggregation of neuronal tau. Our previous results show that neuronal tau aggregates in formaldehyde solution and that aggregated tau induces apoptosis of SH-SY5Y and hippocampal cells. In the present study, based on atomic force microscopy (AFM) observation, we have found that formaldehyde at low concentrations induces tau polymerization whilst acetaldehyde does not. Neuronal tau misfolds and aggregates into globular-like polymers in 0.01-0.1% formaldehyde solutions. Apart from globular-like aggregation, no fibril-like polymerization was observed when the protein was incubated with formaldehyde for 15 days. SDS-PAGE results also exhibit tau polymerizing in the presence of formaldehyde. Under the same experimental conditions, polymerization of bovine serum albumin (BSA) or alpha-synuclein was not markedly detected. Kinetic study shows that tau significantly misfolds and polymerizes in 60 minutes in 0.1% formaldehyde solution. However, presence of 10% methanol prevents protein tau from polymerization. This suggests that formaldehyde polymerization is involved in tau aggregation. Such aggregation process is probably linked to the tau's special "worm-like" structure, which leaves the epsilon-amino groups of Lys and thiol groups of Cys exposed to the exterior. Such a structure can easily bond to formaldehyde molecules in vitro and in vivo. Polymerizing of formaldehyde itself results in aggregation of protein tau. Immunocytochemistry and thioflavin S staining of both endogenous and exogenous tau in the presence of formaldehyde at low concentrations in the cell culture have shown that formaldehyde can induce tau into amyloid-like aggregates in vivo during apoptosis. The significant protein tau aggregation induced by formaldehyde and the severe toxicity of the aggregated tau to neural cells may suggest that toxicity of methanol and formaldehyde ingestion is related to tau misfolding and aggregation.


Asunto(s)
Acetaldehído/farmacología , Amiloide/metabolismo , Formaldehído/farmacología , Neuronas/metabolismo , Proteínas tau/metabolismo , Amiloide/efectos de los fármacos , Animales , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Humanos , Microscopía de Fuerza Atómica , Neuroblastoma/metabolismo , Neuronas/efectos de los fármacos , Ratas , Compuestos de Sulfhidrilo/metabolismo , Proteínas tau/química , Proteínas tau/efectos de los fármacos
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