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1.
Dev Biol ; 459(2): 161-180, 2020 03 15.
Artículo en Inglés | MEDLINE | ID: mdl-31862379

RESUMEN

Animal embryogenesis is initiated by maternal factors, but zygotic genome activation (ZGA) shifts regulatory control to the embryo during blastula stages. ZGA is thought to be mediated by maternally provided transcription factors (TFs), but few such TFs have been identified in vertebrates. Here we report that NF-Y and TALE TFs bind zebrafish genomic elements associated with developmental control genes already at ZGA. In particular, co-regulation by NF-Y and TALE is associated with broadly acting genes involved in transcriptional control, while regulation by either NF-Y or TALE defines genes in specific developmental processes, such that NF-Y controls a cilia gene expression program while TALE controls expression of hox genes. We also demonstrate that NF-Y and TALE-occupied genomic elements function as enhancers during embryogenesis. We conclude that combinatorial use of NF-Y and TALE at developmental enhancers permits the establishment of distinct gene expression programs at zebrafish ZGA.


Asunto(s)
Factor de Unión a CCAAT/metabolismo , Expresión Génica , Genoma , Proteínas de Homeodominio/metabolismo , Activación Transcripcional , Pez Cebra/embriología , Cigoto/metabolismo , Animales , Cilios/genética , Desarrollo Embrionario/genética , Femenino , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/genética , Masculino , Proteínas de Pez Cebra
2.
Elife ; 72018 06 18.
Artículo en Inglés | MEDLINE | ID: mdl-29911973

RESUMEN

TALE factors are broadly expressed embryonically and known to function in complexes with transcription factors (TFs) like Hox proteins at gastrula/segmentation stages, but it is unclear if such generally expressed factors act by the same mechanism throughout embryogenesis. We identify a TALE-dependent gene regulatory network (GRN) required for anterior development and detect TALE occupancy associated with this GRN throughout embryogenesis. At blastula stages, we uncover a novel functional mode for TALE factors, where they occupy genomic DECA motifs with nearby NF-Y sites. We demonstrate that TALE and NF-Y form complexes and regulate chromatin state at genes of this GRN. At segmentation stages, GRN-associated TALE occupancy expands to include HEXA motifs near PBX:HOX sites. Hence, TALE factors control a key GRN, but utilize distinct DNA motifs and protein partners at different stages - a strategy that may also explain their oncogenic potential and may be employed by other broadly expressed TFs.


Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Genes Esenciales/genética , Proteínas de Homeodominio/genética , Proteínas de Pez Cebra/genética , Pez Cebra/genética , Animales , Blástula/embriología , Blástula/metabolismo , Factor de Unión a CCAAT/genética , Factor de Unión a CCAAT/metabolismo , Técnicas de Silenciamiento del Gen , Redes Reguladoras de Genes , Proteínas de Homeodominio/metabolismo , Unión Proteica , Pez Cebra/embriología , Pez Cebra/metabolismo , Proteínas de Pez Cebra/metabolismo
3.
Curr Biol ; 24(8): 845-51, 2014 Apr 14.
Artículo en Inglés | MEDLINE | ID: mdl-24684931

RESUMEN

Argonaute (AGO) proteins are key nuclease effectors of RNAi. Although purified AGOs can mediate a single round of target RNA cleavage in vitro, accessory factors are required for small interfering RNA (siRNA) loading and to achieve multiple-target turnover. To identify AGO cofactors, we immunoprecipitated the C. elegans AGO WAGO-1, which engages amplified small RNAs during RNAi. These studies identified a robust association between WAGO-1 and a conserved Vasa ATPase-related protein RDE-12. rde-12 mutants are deficient in RNAi, including viral suppression, and fail to produce amplified secondary siRNAs and certain endogenous siRNAs (endo-siRNAs). RDE-12 colocalizes with WAGO-1 in germline P granules and in cytoplasmic and perinuclear foci in somatic cells. These findings and our genetic studies suggest that RDE-12 is first recruited to target mRNA by upstream AGOs (RDE-1 and ERGO-1), where it promotes small RNA amplification and/or WAGO-1 loading. Downstream of these events, RDE-12 forms an RNase-resistant (target mRNA-independent) complex with WAGO-1 and may thus have additional functions in target mRNA surveillance and silencing.


Asunto(s)
Proteínas Argonautas/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiología , ARN Helicasas DEAD-box/metabolismo , Interferencia de ARN/fisiología , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Secuencia de Aminoácidos , Animales , Northern Blotting , Proteínas de Caenorhabditis elegans/genética , ARN Helicasas DEAD-box/genética , Immunoblotting , Inmunoprecipitación , Microscopía Fluorescente , Datos de Secuencia Molecular , Proteínas de Unión al ARN/genética
4.
J Ocul Pharmacol Ther ; 29(10): 855-64, 2013 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-24180627

RESUMEN

PURPOSE: The major challenges of developing an RNAi therapeutic include efficient delivery to and entry into the cell type of interest. Conventional ("naked" and chemically stabilized) small interfering RNAs (siRNAs) have been used in the eye in the past but they demonstrated limited clinical efficacy. Here we investigated a recently developed class of small, hydrophobic, asymmetric RNAi compounds. These compounds, termed "self-delivering rxRNAs" (sd-rxRNA(®)), are extensively modified, have a small duplex region of <15 base pairs, contain a fully phosphorothioated single-stranded tail, and readily enter cells and tissues without the requirement for a delivery vehicle. METHODS: We compared sd-rxRNA compounds with stabilized siRNAs in vitro (in ARPE-19 cells) and in vivo (intravitreal injection in mouse and rabbit eyes). Specifically, we investigated the retinal uptake, distribution, efficacy, and preliminary safety of sd-rxRNAs. RESULTS: Treatment with sd-rxRNAs resulted in uniform cellular uptake and full retina penetration in both animal models while no detectable cellular uptake was observed with stabilized siRNAs either in vitro or in vivo. Further, both in vitro and in vivo delivery (without any transfection reagent or formulation) resulted in a significant reduction of the targeted mRNA levels, which lasted 14-21 days in vivo. Retinal morphology and function were unaltered following a single administration of sd-rxRNAs. CONCLUSION: These data support the potential of developing sd-rxRNAs as a therapeutic for ocular disease.


Asunto(s)
Interferencia de ARN , ARN Interferente Pequeño/administración & dosificación , Retina/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Animales , Línea Celular , Oftalmopatías/terapia , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Inyecciones Intravítreas , Ratones , Ratones Endogámicos C57BL , ARN Mensajero/metabolismo , Conejos , Factores de Tiempo
5.
RNA ; 17(6): 1032-7, 2011 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-21493786

RESUMEN

RNA interference (RNAi) has been established as an important tool for functional genomics studies and has great promise as a therapeutic intervention for human diseases. In mammalian cells, RNAi is conventionally induced by 19-27-bp RNA duplexes generated by hybridization of two complementary oligonucleotide strands (oligos). Here we describe a novel class of RNAi molecules composed of a single 25-28-nucleotide (nt) oligo. The oligo has a 16-nt mRNA targeting region, followed by an additional 8-10 nt to enable self-dimerization into a partially complementary duplex. Analysis of numerous diverse structures demonstrates that molecules composed of two short helices separated by a loop can efficiently enter and activate the RNA-induced silencing complex (RISC). This finding enables the design of highly effective single-oligo compounds for any mRNA target.


Asunto(s)
Oligonucleótidos/química , Interferencia de ARN , ARN Interferente Pequeño/química , Complejo Silenciador Inducido por ARN/metabolismo , Células Cultivadas , Silenciador del Gen , Células HeLa , Humanos , Conformación de Ácido Nucleico , ARN/química , ARN/metabolismo , ARN Mensajero/química , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Complejo Silenciador Inducido por ARN/química
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