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1.
Nucleic Acids Res ; 52(3): 1498-1511, 2024 Feb 09.
Artículo en Inglés | MEDLINE | ID: mdl-38180813

RESUMEN

A 'genomically' humanized animal stably maintains and functionally expresses the genes on human chromosome fragment (hCF; <24 Mb) loaded onto mouse artificial chromosome (MAC); however, cloning of hCF onto the MAC (hCF-MAC) requires a complex process that involves multiple steps of chromosome engineering through various cells via chromosome transfer and Cre-loxP chromosome translocation. Here, we aimed to develop a strategy to rapidly construct the hCF-MAC by employing three alternative techniques: (i) application of human induced pluripotent stem cells (hiPSCs) as chromosome donors for microcell-mediated chromosome transfer (MMCT), (ii) combination of paclitaxel (PTX) and reversine (Rev) as micronucleation inducers and (iii) CRISPR/Cas9 genome editing for site-specific translocations. We achieved a direct transfer of human chromosome 6 or 21 as a model from hiPSCs as alternative human chromosome donors into CHO cells containing MAC. MMCT was performed with less toxicity through induction of micronucleation by PTX and Rev. Furthermore, chromosome translocation was induced by simultaneous cleavage between human chromosome and MAC by using CRISPR/Cas9, resulting in the generation of hCF-MAC containing CHO clones without Cre-loxP recombination and drug selection. Our strategy facilitates rapid chromosome cloning and also contributes to the functional genomic analyses of human chromosomes.


Asunto(s)
Clonación Molecular , Animales , Cricetinae , Humanos , Ratones , Cromosomas Artificiales , Clonación Molecular/métodos , Cricetulus , Sistemas CRISPR-Cas , ADN , Edición Génica , Células Madre Pluripotentes Inducidas , Translocación Genética
2.
J Transl Med ; 20(1): 517, 2022 11 08.
Artículo en Inglés | MEDLINE | ID: mdl-36348371

RESUMEN

BACKGROUND: Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant muscular disorder characterized by asymmetric muscle wasting and weakness. FSHD can be subdivided into two types: FSHD1, caused by contraction of the D4Z4 repeat on chromosome 4q35, and FSHD2, caused by mild contraction of the D4Z4 repeat plus aberrant hypomethylation mediated by genetic variants in SMCHD1, DNMT3B, or LRIF1. Genetic diagnosis of FSHD is challenging because of the complex procedures required. METHODS: We applied Nanopore CRISPR/Cas9-targeted resequencing for the diagnosis of FSHD by simultaneous detection of D4Z4 repeat length and methylation status at nucleotide level in genetically-confirmed and suspected patients. RESULTS: We found significant hypomethylation of contracted 4q-D4Z4 repeats in FSHD1, and both 4q- and 10q-D4Z4 repeats in FSHD2. We also found that the hypomethylation in the contracted D4Z4 in FSHD1 is moderately correlated with patient phenotypes. CONCLUSIONS: Our method contributes to the development for the diagnosis of FSHD using Nanopore long-read sequencing. This finding might give insight into the mechanisms by which repeat contraction causes disease pathogenesis.


Asunto(s)
Distrofia Muscular Facioescapulohumeral , Humanos , Distrofia Muscular Facioescapulohumeral/genética , Distrofia Muscular Facioescapulohumeral/diagnóstico , Proteínas de Homeodominio/genética , Metilación de ADN/genética , Cromosomas/metabolismo , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo
3.
Hum Mol Genet ; 27(4): 716-731, 2018 02 15.
Artículo en Inglés | MEDLINE | ID: mdl-29281018

RESUMEN

In humans, a copy of the DUX4 retrogene is located in each unit of the D4Z4 macrosatellite repeat that normally comprises 8-100 units. The D4Z4 repeat has heterochromatic features and does not express DUX4 in somatic cells. Individuals with facioscapulohumeral muscular dystrophy (FSHD) have a partial failure of somatic DUX4 repression resulting in the presence of DUX4 protein in sporadic muscle nuclei. Somatic DUX4 derepression is caused by contraction of the D4Z4 repeat to 1-10 units (FSHD1) or by heterozygous mutations in genes responsible for maintaining the D4Z4 chromatin structure in a repressive state (FSHD2). One of the FSHD2 genes is the structural maintenance of chromosomes hinge domain 1 (SMCHD1) gene. SMCHD1 mutations have also been identified in FSHD1; patients carrying a contracted D4Z4 repeat and a SMCHD1 mutation are more severely affected than relatives with only a contracted repeat or a SMCHD1 mutation. To evaluate the modifier role of SMCHD1, we crossbred mice carrying a contracted D4Z4 repeat (D4Z4-2.5 mice) with mice that are haploinsufficient for Smchd1 (Smchd1MommeD1 mice). D4Z4-2.5/Smchd1MommeD1 mice presented with a significantly reduced body weight and developed skin lesions. The same skin lesions, albeit in a milder form, were also observed in D4Z4-2.5 mice, suggesting that reduced Smchd1 levels aggravate disease in the D4Z4-2.5 mouse model. Our study emphasizes the evolutionary conservation of the SMCHD1-dependent epigenetic regulation of the D4Z4 repeat array and further suggests that the D4Z4-2.5/Smchd1MommeD1 mouse model may be used to unravel the function of DUX4 in non-muscle tissues like the skin.


Asunto(s)
Proteínas Cromosómicas no Histona/metabolismo , Haploinsuficiencia/fisiología , Animales , Western Blotting , Células Cultivadas , Proteínas Cromosómicas no Histona/genética , Metilación de ADN/genética , Metilación de ADN/fisiología , Fibroblastos/metabolismo , Citometría de Flujo , Haploinsuficiencia/genética , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Queratinocitos/metabolismo , Ratones , Ratones Transgénicos , Músculo Esquelético/metabolismo , Distrofia Muscular Facioescapulohumeral/genética , Distrofia Muscular Facioescapulohumeral/metabolismo , Fenotipo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Piel , Timocitos
4.
FASEB J ; 33(7): 8094-8109, 2019 07.
Artículo en Inglés | MEDLINE | ID: mdl-30939245

RESUMEN

Skeletal muscle satellite cells (SMSCs), the major stem cells responsible for the regeneration of skeletal muscle, are normally cell cycle arrested but differentiate to generate myocytes upon muscle damage, forming new myofibers along with self-renewing stem cells in preparation for subsequent injury. In this study, we investigated which factors stimulate the proliferation and differentiation of SMSCs and found that pyruvate, the end product of glycolysis, stimulates their differentiation. Pyruvate antagonizes the effects of hypoxia on preferential self-renewal of SMSCs through dephosphorylation or activation of pyruvate dehydrogenase (PDH), which mediates opening of the gateway from glycolysis to the tricarboxylic acid (TCA) cycle by producing acetyl coenzyme A from pyruvate. PDH kinase 1, highly expressed under hypoxia, is down-regulated under normoxic conditions, leading to an increase in dephosphorylated PDH. Conditional deletion of PDH in SMSCs affects cell divisions generating myocytes and subsequent myotube formation, inefficient skeletal muscle regeneration upon injury, and aggravated pathogenesis of a dystrophin-deficient mouse model of Duchenne muscular dystrophy. Thus, the flow from glycolysis to the TCA cycle mediated by PDH plays a pivotal role in the differentiation of SMSCs, which is critical for the progression of skeletal muscle regeneration.-Hori, S., Hiramuki, Y., Nishimura, D., Sato, F., Sehara-Fujisawa, A. PDH-mediated metabolic flow is critical for skeletal muscle stem cell differentiation and myotube formation during regeneration in mice.


Asunto(s)
Diferenciación Celular , Cetona Oxidorreductasas/metabolismo , Fibras Musculares Esqueléticas/fisiología , Regeneración , Células Satélite del Músculo Esquelético/enzimología , Animales , Línea Celular , Ciclo del Ácido Cítrico , Eliminación de Gen , Glucólisis , Cetona Oxidorreductasas/genética , Ratones , Ratones Noqueados , Fibras Musculares Esqueléticas/citología , Células Satélite del Músculo Esquelético/citología
5.
Sci Rep ; 13(1): 4360, 2023 03 16.
Artículo en Inglés | MEDLINE | ID: mdl-36928364

RESUMEN

Dystrophin maintains membrane integrity as a sarcolemmal protein. Dystrophin mutations lead to Duchenne muscular dystrophy, an X-linked recessive disorder. Since dystrophin is one of the largest genes consisting of 79 exons in the human genome, delivering a full-length dystrophin using virus vectors is challenging for gene therapy. Human artificial chromosome is a vector that can load megabase-sized genome without any interference from the host chromosome. Chimeric mice carrying a 2.4-Mb human dystrophin gene-loaded human artificial chromosome (DYS-HAC) was previously generated, and dystrophin expression from DYS-HAC was confirmed in skeletal muscles. Here we investigated whether human dystrophin expression from DYS-HAC rescues the muscle phenotypes seen in dystrophin-deficient mice. Human dystrophin was normally expressed in the sarcolemma of skeletal muscle and heart at expected molecular weights, and it ameliorated histological and functional alterations in dystrophin-deficient mice. These results indicate that the 2.4-Mb gene is enough for dystrophin to be correctly transcribed and translated, improving muscular dystrophy. Therefore, this technique using HAC gives insight into developing new treatments and novel humanized Duchenne muscular dystrophy mouse models with human dystrophin gene mutations.


Asunto(s)
Cromosomas Artificiales Humanos , Distrofina , Distrofia Muscular de Duchenne , Animales , Humanos , Ratones , Cromosomas Artificiales Humanos/genética , Modelos Animales de Enfermedad , Distrofina/genética , Distrofina/metabolismo , Ratones Endogámicos mdx , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , Distrofia Muscular de Duchenne/metabolismo , Sarcolema/metabolismo
6.
Mol Ther Nucleic Acids ; 33: 444-453, 2023 Sep 12.
Artículo en Inglés | MEDLINE | ID: mdl-37588685

RESUMEN

Mammalian artificial chromosomes have enabled the introduction of extremely large amounts of genetic information into animal cells in an autonomously replicating, nonintegrating format. However, the evaluation of human artificial chromosomes (HACs) as novel tools for curing intractable hereditary disorders has been hindered by the limited efficacy of the delivery system. We generated dystrophin gene knockout (DMD-KO) pigs harboring the HAC bearing the entire human DMD via a somatic cell cloning procedure (DYS-HAC-cloned pig). Restored human dystrophin expression was confirmed by immunofluorescence staining in the skeletal muscle of the DYS-HAC-cloned pigs. Viability at the first month postpartum of the DYS-HAC-cloned pigs, including motor function in the hind leg and serum creatinine kinase level, was improved significantly when compared with that in the original DMD-KO pigs. However, decrease in systemic retention of the DYS-HAC vector and limited production of the DMD protein might have caused severe respiratory impairment with general prostration by 3 months postpartum. The results demonstrate that the use of transchromosomic cloned pigs permitted a straightforward estimation of the efficacy of the DYS-HAC carried in affected tissues/organs in a large-animal disease model, providing novel insights into the therapeutic application of exogenous mammalian artificial chromosomes.

7.
Skelet Muscle ; 8(1): 24, 2018 08 02.
Artículo en Inglés | MEDLINE | ID: mdl-30071896

RESUMEN

BACKGROUND: SMCHD1 is a disease modifier and a causative gene for facioscapulohumeral muscular dystrophy (FSHD) type 1 and type 2, respectively. A large variety of different mutations in SMCHD1 have been identified as causing FSHD2. In many cases, it is unclear how these mutations disrupt the normal function of SMCHD1. METHODS: We made and analyzed lenti-viral vectors that express Flag-tagged full-length or different mutant SMCHD1 proteins to better understand the functional domains of SMCHD1 in muscle cells. RESULTS: We identified regions necessary for nuclear localization, dimerization, and cleavage sites. Moreover, we confirmed that some mutants increased DUX4 expression in FSHD1 myoblasts. CONCLUSIONS: These findings provide an additional basis for understanding the molecular consequences of SMCHD1 mutations.


Asunto(s)
Núcleo Celular/metabolismo , Proteínas Cromosómicas no Histona/química , Distrofia Muscular Facioescapulohumeral/genética , Multimerización de Proteína , Señales de Clasificación de Proteína , Transporte Activo de Núcleo Celular , Células Cultivadas , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Células HEK293 , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Mutación , Mioblastos/metabolismo , Proteolisis
8.
Dev Neurobiol ; 77(9): 1086-1100, 2017 09.
Artículo en Inglés | MEDLINE | ID: mdl-28371543

RESUMEN

In adult Xenopus eyes, when the whole retina is removed, retinal pigmented epithelial (RPE) cells become activated to be retinal stem cells and regenerate the whole retina. In the present study, using a tissue culture model, it was examined whether upregulation of matrix metalloproteinases (Mmps) triggers retinal regeneration. Soon after retinal removal, Xmmp9 and Xmmp18 were strongly upregulated in the tissues of the RPE and the choroid. In the culture, Mmp expression in the RPE cells corresponded with their migration from the choroid. A potent MMP inhibitor, 1,10-PNTL, suppressed RPE cell migration, proliferation, and formation of an epithelial structure in vitro. The mechanism involved in upregulation of Mmps was further investigated. After retinal removal, inflammatory cytokine genes, IL-1ß and TNF-α, were upregulated both in vivo and in vitro. When the inflammation inhibitors dexamethasone or Withaferin A were applied in vitro, RPE cell migration was severely affected, suppressing transdifferentiation. These results demonstrate that Mmps play a pivotal role in retinal regeneration, and suggest that inflammatory cytokines trigger Mmp upregulation, indicating a direct link between the inflammatory reaction and retinal regeneration. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 1086-1100, 2017.


Asunto(s)
Diferenciación Celular/fisiología , Metaloproteinasas de la Matriz/metabolismo , Retina/citología , Epitelio Pigmentado de la Retina/fisiología , Regulación hacia Arriba/fisiología , Xenopus laevis/anatomía & histología , Animales , Antiinflamatorios/farmacología , Adhesión Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Dexametasona/farmacología , Metaloproteinasas de la Matriz/genética , Regeneración Nerviosa/efectos de los fármacos , Técnicas de Cultivo de Órganos , Fenantrolinas/farmacología , Inhibidores de Proteasas/farmacología , Factores de Tiempo , Activación Transcripcional/efectos de los fármacos , Tubulina (Proteína)/metabolismo , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Witanólidos/farmacología , Witanólidos/toxicidad
9.
Skelet Muscle ; 7(1): 16, 2017 09 04.
Artículo en Inglés | MEDLINE | ID: mdl-28870238

RESUMEN

BACKGROUND: Facioscapulohumeral dystrophy (FSHD) is a progressive muscle disease caused by mutations that lead to epigenetic derepression and inappropriate transcription of the double homeobox 4 (DUX4) gene in skeletal muscle. Drugs that enhance the repression of DUX4 and prevent its expression in skeletal muscle cells therefore represent candidate therapies for FSHD. METHODS: We screened an aggregated chemical library enriched for compounds with epigenetic activities and the Pharmakon 1600 library composed of compounds that have reached clinical testing to identify molecules that decrease DUX4 expression as monitored by the levels of DUX4 target genes in FSHD patient-derived skeletal muscle cell cultures. RESULTS: Our screens identified several classes of molecules that include inhibitors of the bromodomain and extra-terminal (BET) family of proteins and agonists of the beta-2 adrenergic receptor. Further studies showed that compounds from these two classes suppress the expression of DUX4 messenger RNA (mRNA) by blocking the activity of bromodomain-containing protein 4 (BRD4) or by increasing cyclic adenosine monophosphate (cAMP) levels, respectively. CONCLUSIONS: These data uncover pathways involved in the regulation of DUX4 expression in somatic cells, provide potential candidate classes of compounds for FSHD therapeutic development, and create an important opportunity for mechanistic studies that may uncover additional therapeutic targets.


Asunto(s)
Agonistas de Receptores Adrenérgicos beta 2/farmacología , Proteínas de Homeodominio/metabolismo , Distrofia Muscular Facioescapulohumeral/metabolismo , Proteínas Nucleares/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Factores de Transcripción/metabolismo , Proteínas de Ciclo Celular , Células Cultivadas , AMP Cíclico/metabolismo , Ensayos Analíticos de Alto Rendimiento , Proteínas de Homeodominio/genética , Humanos , Mioblastos/efectos de los fármacos , Mioblastos/metabolismo
10.
PLoS One ; 10(6): e0130436, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-26098312

RESUMEN

When skeletal muscle fibers are injured, they regenerate and grow until their sizes are adjusted to surrounding muscle fibers and other relevant organs. In this study, we examined whether Mest, one of paternally expressed imprinted genes that regulates body size during development, and miR-335 located in the second intron of the Mest gene play roles in muscle regeneration. We generated miR-335-deficient mice, and found that miR-335 is a paternally expressed imprinted microRNA. Although both Mest and miR-335 are highly expressed during muscle development and regeneration, only Mest+/- (maternal/paternal) mice show retardation of body growth. In addition to reduced body weight in Mest+/-; DMD-null mice, decreased muscle growth was observed in Mest+/- mice during cardiotoxin-induced regeneration, suggesting roles of Mest in muscle regeneration. Moreover, expressions of H19 and Igf2r, maternally expressed imprinted genes were affected in tibialis anterior muscle of Mest+/-; DMD-null mice compared to DMD-null mice. Thus, Mest likely mediates muscle regeneration through regulation of imprinted gene networks in skeletal muscle.


Asunto(s)
MicroARNs/genética , Desarrollo de Músculos/genética , Músculo Esquelético/crecimiento & desarrollo , Proteínas/genética , Regeneración/genética , Animales , Distrofina/genética , Regulación del Desarrollo de la Expresión Génica , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , MicroARNs/biosíntesis , Músculo Esquelético/metabolismo , Proteínas/metabolismo , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Receptor IGF Tipo 2/genética , Receptor IGF Tipo 2/metabolismo , Regeneración/fisiología
11.
Dev Cell ; 17(6): 800-10, 2009 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-20059950

RESUMEN

The serine threonine kinase Akt is a core survival factor that underlies a variety of human diseases. Although regulatory phosphorylation and dephosphorylation have been well documented, the other posttranslational mechanisms that modulate Akt activity remain unclear. We show here that tetratricopeptide repeat domain 3 (TTC3) is an E3 ligase that interacts with Akt. TTC3 contains a canonical RING finger motif, a pair of tetratricopeptide motifs, a putative Akt phosphorylation site, and nuclear localization signals, and is encoded by a gene within the Down syndrome (DS) critical region on chromosome 21. TTC3 is an Akt-specific E3 ligase that binds to phosphorylated Akt and facilitates its ubiquitination and degradation within the nucleus. Moreover, DS cells exhibit elevated TTC3 expression, reduced phosphorylated Akt, and accumulation in the G(2)M phase, which can be reversed by TTC3 siRNA or Myr-Akt. Thus, interaction between TTC3 and Akt may contribute to the clinical symptoms of DS.


Asunto(s)
Síndrome de Down/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Línea Celular , Células Cultivadas , Humanos , Inmunoprecipitación , Fosforilación , Complejo de la Endopetidasa Proteasomal/metabolismo , Mapeo de Interacción de Proteínas , Ubiquitinación
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