RESUMEN
Microscopy-based spatially resolved omic methods are transforming the life sciences. However, these methods rely on high numerical aperture objectives and cannot resolve crowded molecular targets, limiting the amount of extractable biological information. To overcome these limitations, here we develop Deconwolf, an open-source, user-friendly software for high-performance deconvolution of widefield fluorescence microscopy images, which efficiently runs on laptop computers. Deconwolf enables accurate quantification of crowded diffraction limited fluorescence dots in DNA and RNA fluorescence in situ hybridization images and allows robust detection of individual transcripts in tissue sections imaged with ×20 air objectives. Deconvolution of in situ spatial transcriptomics images with Deconwolf increased the number of transcripts identified more than threefold, while the application of Deconwolf to images obtained by fluorescence in situ sequencing of barcoded Oligopaint probes drastically improved chromosome tracing. Deconwolf greatly facilitates the use of deconvolution in many bioimaging applications.
Asunto(s)
Procesamiento de Imagen Asistido por Computador , Hibridación Fluorescente in Situ , Microscopía Fluorescente , Programas Informáticos , Microscopía Fluorescente/métodos , Hibridación Fluorescente in Situ/métodos , Procesamiento de Imagen Asistido por Computador/métodos , Animales , Ratones , HumanosRESUMEN
The linear sequence of DNA provides invaluable information about genes and their regulatory elements along chromosomes. However, to fully understand gene function and regulation, we need to dissect how genes physically fold in the three-dimensional nuclear space. Here we describe immuno-OligoSTORM, an imaging strategy that reveals the distribution of nucleosomes within specific genes in super-resolution, through the simultaneous visualization of DNA and histones. We combine immuno-OligoSTORM with restraint-based and coarse-grained modeling approaches to integrate super-resolution imaging data with Hi-C contact frequencies and deconvoluted micrococcal nuclease-sequencing information. The resulting method, called Modeling immuno-OligoSTORM, allows quantitative modeling of genes with nucleosome resolution and provides information about chromatin accessibility for regulatory factors, such as RNA polymerase II. With Modeling immuno-OligoSTORM, we explore intercellular variability, transcriptional-dependent gene conformation, and folding of housekeeping and pluripotency-related genes in human pluripotent and differentiated cells, thereby obtaining the highest degree of data integration achieved so far to our knowledge.
Asunto(s)
Nucleasa Microcócica , Nucleosomas , Cromatina/genética , ADN/genética , Histonas/genética , Humanos , Nucleasa Microcócica/metabolismo , Nucleosomas/genética , ARN Polimerasa II/genéticaRESUMEN
The chromatin fiber folds into loops, but the mechanisms controlling loop extrusion are still poorly understood. Using super-resolution microscopy, we visualize that loops in intact nuclei are formed by a scaffold of cohesin complexes from which the DNA protrudes. RNA polymerase II decorates the top of the loops and is physically segregated from cohesin. Augmented looping upon increased loading of cohesin on chromosomes causes disruption of Lamin at the nuclear rim and chromatin blending, a homogeneous distribution of chromatin within the nucleus. Altering supercoiling via either transcription or topoisomerase inhibition counteracts chromatin blending, increases chromatin condensation, disrupts loop formation, and leads to altered cohesin distribution and mobility on chromatin. Overall, negative supercoiling generated by transcription is an important regulator of loop formation in vivo.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Cromatina/química , Cromatina/genética , Proteínas Cromosómicas no Histona/metabolismo , Transcripción Genética/fisiología , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Línea Celular , Núcleo Celular/genética , Proteoglicanos Tipo Condroitín Sulfato/genética , Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/química , Proteínas Cromosómicas no Histona/genética , ADN-Topoisomerasas de Tipo I/genética , ADN-Topoisomerasas de Tipo I/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Femenino , Humanos , Laminas/genética , Laminas/metabolismo , ARN Polimerasa II/metabolismo , Imagen Individual de Molécula/métodos , CohesinasRESUMEN
Centromeres are essential chromosomal regions that mediate kinetochore assembly and spindle attachments during cell division. Despite their functional conservation, centromeres are among the most rapidly evolving genomic regions and can shape karyotype evolution and speciation across taxa. Although significant progress has been made in identifying centromere-associated proteins, the highly repetitive centromeres of metazoans have been refractory to DNA sequencing and assembly, leaving large gaps in our understanding of their functional organization and evolution. Here, we identify the sequence composition and organization of the centromeres of Drosophila melanogaster by combining long-read sequencing, chromatin immunoprecipitation for the centromeric histone CENP-A, and high-resolution chromatin fiber imaging. Contrary to previous models that heralded satellite repeats as the major functional components, we demonstrate that functional centromeres form on islands of complex DNA sequences enriched in retroelements that are flanked by large arrays of satellite repeats. Each centromere displays distinct size and arrangement of its DNA elements but is similar in composition overall. We discover that a specific retroelement, G2/Jockey-3, is the most highly enriched sequence in CENP-A chromatin and is the only element shared among all centromeres. G2/Jockey-3 is also associated with CENP-A in the sister species D. simulans, revealing an unexpected conservation despite the reported turnover of centromeric satellite DNA. Our work reveals the DNA sequence identity of the active centromeres of a premier model organism and implicates retroelements as conserved features of centromeric DNA.
Asunto(s)
Centrómero/genética , Drosophila/genética , Retroelementos/genética , Animales , Proteína A Centromérica/genética , Cromatina/metabolismo , Elementos Transponibles de ADN/genética , ADN Satélite/genética , Drosophila/embriología , Proteínas de Drosophila/genética , Embrión no Mamífero/metabolismo , Genoma de los Insectos , Secuencias Repetidas Terminales/genéticaRESUMEN
This study explores the relationship between three-dimensional genome organization and ultraconserved elements (UCEs), an enigmatic set of DNA elements that are perfectly conserved between the reference genomes of distantly related species. Examining both human and mouse genomes, we interrogate the relationship of UCEs to three features of chromosome organization derived from Hi-C studies. We find that UCEs are enriched within contact domains and, further, that the subset of UCEs within domains shared across diverse cell types are linked to kidney-related and neuronal processes. In boundaries, UCEs are generally depleted, with those that do overlap boundaries being overrepresented in exonic UCEs. Regarding loop anchors, UCEs are neither overrepresented nor underrepresented, but those present in loop anchors are enriched for splice sites. Finally, as the relationships between UCEs and human Hi-C features are conserved in mouse, our findings suggest that UCEs contribute to interspecies conservation of genome organization and, thus, genome stability.
Asunto(s)
Secuencia Conservada/genética , Genoma , Mamíferos/genética , Animales , Cromosomas de los Mamíferos/genética , ADN Intergénico/genética , Exones/genética , Humanos , Intrones/genética , Riñón/metabolismo , Ratones , Procesamiento Postranscripcional del ARN/genética , Sitio de Iniciación de la TranscripciónRESUMEN
Oligonucleotide (oligo)-based FISH has emerged as an important tool for the study of chromosome organization and gene expression and has been empowered by the commercial availability of highly complex pools of oligos. However, a dedicated bioinformatic design utility has yet to be created specifically for the purpose of identifying optimal oligo FISH probe sequences on the genome-wide scale. Here, we introduce OligoMiner, a rapid and robust computational pipeline for the genome-scale design of oligo FISH probes that affords the scientist exact control over the parameters of each probe. Our streamlined method uses standard bioinformatic file formats, allowing users to seamlessly integrate new and existing utilities into the pipeline as desired, and introduces a method for evaluating the specificity of each probe molecule that connects simulated hybridization energetics to rapidly generated sequence alignments using supervised machine learning. We demonstrate the scalability of our approach by performing genome-scale probe discovery in numerous model organism genomes and showcase the performance of the resulting probes with diffraction-limited and single-molecule superresolution imaging of chromosomal and RNA targets. We anticipate that this pipeline will make the FISH probe design process much more accessible and will more broadly facilitate the design of pools of hybridization probes for a variety of applications.
Asunto(s)
Genómica/métodos , Hibridación Fluorescente in Situ/métodos , Sondas de Oligonucleótidos/química , Sondas de Oligonucleótidos/genética , Animales , Arabidopsis , ADN/genética , ADN/metabolismo , Minería de Datos , Humanos , Ratones , Modelos Genéticos , Sondas de Oligonucleótidos/metabolismoRESUMEN
Polycomb group (PcG) proteins are major chromatin-bound factors that can read and modify chromatin states to maintain gene silencing throughout development. Here we focus on a close homolog of the PcG protein Posterior sex combs to better understand how these proteins affect regulation. This homolog, called Suppressor 2 of zeste [Su(z)2] is composed of two regions: the N-terminal homology region (HR), which serves as a hub for protein interactions, and the C-terminal region (CTR), which is believed to harbor the core activity of compacting chromatin. Here, we describe our classical genetic studies to dissect the structure of Su(z)2 Surprisingly, we found that the CTR is dispensable for viability. Furthermore, the core activity of Su(z)2 seems to reside in the HR instead of the CTR. Remarkably, our data also suggest a regulatory cascade between CTR and HR of Su(z)2, which, in turn, may help prioritize the myriad of PcG interactions that occur with the HR.
Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas de Drosophila/genética , Proteínas del Grupo Polycomb/genética , Animales , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Proteínas del Grupo Polycomb/metabolismo , Dominios ProteicosRESUMEN
The impact of genetic technologies is being felt in many aspects of society, including medicine and the legal system, as well as the personal lives of individuals. How do we make sure that all segments of the population are equally aware of these technologies and have ample opportunity to voice opinions and shape the future? One ongoing effort, which began ten years ago and in which we are directly involved, is the Personal Genetics Education Project, a nonprofit initiative housed within and largely supported by the Department of Genetics at Harvard Medical School. The goal of pgEd is to raise public awareness and promote conversations about the benefits and implications of the genetics revolution in ways that are inclusive of all voices regardless of socioeconomic, educational, ethnic, cultural, or religious background. This essay describes our approach and experience in hopes that they will be of help to others engaged in similar efforts.
Asunto(s)
Concienciación , Técnicas Genéticas , Pruebas Genéticas , Genoma Humano , Mapeo Cromosómico , Conocimientos, Actitudes y Práctica en Salud , Humanos , Estados UnidosRESUMEN
The spatial organization of chromatin critically affects genome function. Recent chromosome-conformation-capture studies have revealed topologically associating domains (TADs) as a conserved feature of chromatin organization, but how TADs are spatially organized in individual chromosomes remains unknown. Here, we developed an imaging method for mapping the spatial positions of numerous genomic regions along individual chromosomes and traced the positions of TADs in human interphase autosomes and X chromosomes. We observed that chromosome folding deviates from the ideal fractal-globule model at large length scales and that TADs are largely organized into two compartments spatially arranged in a polarized manner in individual chromosomes. Active and inactive X chromosomes adopt different folding and compartmentalization configurations. These results suggest that the spatial organization of chromatin domains can change in response to regulation.
Asunto(s)
Cromatina/química , Cromosomas Humanos X/química , Genoma Humano , Interfase , Línea Celular , Cromosomas Humanos Par 20/química , Cromosomas Humanos Par 22/química , Regulación de la Expresión Génica , Humanos , Imagen Molecular/métodosRESUMEN
Metazoan genomes are spatially organized at multiple scales, from packaging of DNA around individual nucleosomes to segregation of whole chromosomes into distinct territories. At the intermediate scale of kilobases to megabases, which encompasses the sizes of genes, gene clusters and regulatory domains, the three-dimensional (3D) organization of DNA is implicated in multiple gene regulatory mechanisms, but understanding this organization remains a challenge. At this scale, the genome is partitioned into domains of different epigenetic states that are essential for regulating gene expression. Here we investigate the 3D organization of chromatin in different epigenetic states using super-resolution imaging. We classified genomic domains in Drosophila cells into transcriptionally active, inactive or Polycomb-repressed states, and observed distinct chromatin organizations for each state. All three types of chromatin domains exhibit power-law scaling between their physical sizes in 3D and their domain lengths, but each type has a distinct scaling exponent. Polycomb-repressed domains show the densest packing and most intriguing chromatin folding behaviour, in which chromatin packing density increases with domain length. Distinct from the self-similar organization displayed by transcriptionally active and inactive chromatin, the Polycomb-repressed domains are characterized by a high degree of chromatin intermixing within the domain. Moreover, compared to inactive domains, Polycomb-repressed domains spatially exclude neighbouring active chromatin to a much stronger degree. Computational modelling and knockdown experiments suggest that reversible chromatin interactions mediated by Polycomb-group proteins play an important role in these unique packaging properties of the repressed chromatin. Taken together, our super-resolution images reveal distinct chromatin packaging for different epigenetic states at the kilobase-to-megabase scale, a length scale that is directly relevant to genome regulation.
Asunto(s)
Ensamble y Desensamble de Cromatina , Cromatina/genética , Cromatina/metabolismo , Drosophila melanogaster/genética , Epigénesis Genética , Animales , Línea Celular , Posicionamiento de Cromosoma , Drosophila melanogaster/citología , Represión Epigenética , Fractales , Genoma/genética , Proteínas del Grupo Polycomb/metabolismo , Transcripción GenéticaRESUMEN
Synthetic oligonucleotides are the main cost factor for studies in DNA nanotechnology, genetics and synthetic biology, which all require thousands of these at high quality. Inexpensive chip-synthesized oligonucleotide libraries can contain hundreds of thousands of distinct sequences, however only at sub-femtomole quantities per strand. Here we present a selective oligonucleotide amplification method, based on three rounds of rolling-circle amplification, that produces nanomole amounts of single-stranded oligonucleotides per millilitre reaction. In a multistep one-pot procedure, subsets of hundreds or thousands of single-stranded DNAs with different lengths can selectively be amplified and purified together. These oligonucleotides are used to fold several DNA nanostructures and as primary fluorescence in situ hybridization probes. The amplification cost is lower than other reported methods (typically around US$ 20 per nanomole total oligonucleotides produced) and is dominated by the use of commercial enzymes.
Asunto(s)
ADN de Cadena Simple , Biblioteca de Genes , Técnicas de Amplificación de Ácido Nucleico/métodos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Oligonucleótidos , Cromatografía Líquida de Alta Presión , Hibridación Fluorescente in Situ , Nanoestructuras , Nanotecnología , Técnicas de Amplificación de Ácido Nucleico/economía , Análisis de Secuencia por Matrices de Oligonucleótidos/economía , Biología SintéticaRESUMEN
Oligonucleotide microarrays allow the production of complex custom oligonucleotide libraries for nucleic acid detection-based applications such as fluorescence in situ hybridization (FISH). We have developed a PCR-free method to make single-stranded DNA (ssDNA) fluorescent probes through an intermediate RNA library. A double-stranded oligonucleotide library is amplified by transcription to create an RNA library. Next, dye- or hapten-conjugate primers are used to reverse transcribe the RNA to produce a dye-labeled cDNA library. Finally the RNA is hydrolyzed under alkaline conditions to obtain the single-stranded fluorescent probes library. Starting from unique oligonucleotide library constructs, we present two methods to produce single-stranded probe libraries. The two methods differ in the type of reverse transcription (RT) primer, the incorporation of fluorescent dye, and the purification of fluorescent probes. The first method employs dye-labeled reverse transcription primers to produce multiple differentially single-labeled probe subsets from one microarray library. The fluorescent probes are purified from excess primers by oligonucleotide-bead capture. The second method uses an RNA:DNA chimeric primer and amino-modified nucleotides to produce amino-allyl probes. The excess primers and RNA are hydrolyzed under alkaline conditions, followed by probe purification and labeling with amino-reactive dyes. The fluorescent probes created by the combination of transcription and reverse transcription can be used for FISH and to detect any RNA and DNA targets via hybridization.
Asunto(s)
ADN de Cadena Simple/genética , Colorantes Fluorescentes/metabolismo , Biblioteca de Genes , Sondas de Oligonucleótidos/genética , ARN/genética , Transcripción Reversa , Secuencia de Bases , Línea Celular , Cartilla de ADN/química , Cartilla de ADN/genética , ADN de Cadena Simple/química , Colorantes Fluorescentes/química , Humanos , Hibridación Fluorescente in Situ/métodos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Sondas de Oligonucleótidos/química , ARN/química , Transcripción GenéticaRESUMEN
Fluorescence in situ hybridization (FISH) is a powerful single-cell technique for studying nuclear structure and organization. Here we report two advances in FISH-based imaging. We first describe the in situ visualization of single-copy regions of the genome using two single-molecule super-resolution methodologies. We then introduce a robust and reliable system that harnesses single-nucleotide polymorphisms (SNPs) to visually distinguish the maternal and paternal homologous chromosomes in mammalian and insect systems. Both of these new technologies are enabled by renewable, bioinformatically designed, oligonucleotide-based Oligopaint probes, which we augment with a strategy that uses secondary oligonucleotides (oligos) to produce and enhance fluorescent signals. These advances should substantially expand the capability to query parent-of-origin-specific chromosome positioning and gene expression on a cell-by-cell basis.
Asunto(s)
Pintura Cromosómica/métodos , Cromosomas/genética , Haplotipos , Hibridación Fluorescente in Situ/métodos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Animales , Línea Celular , Drosophila , Biblioteca de Genes , Sondas de Oligonucleótidos/metabolismo , Coloración y EtiquetadoRESUMEN
In mammals, several classes of monoallelic genes have been identified, including those subject to X-chromosome inactivation (XCI), genomic imprinting, and random monoallelic expression (RMAE). However, the extent to which these epigenetic phenomena are influenced by underlying genetic variation is unknown. Here we perform a systematic classification of allelic imbalance in mouse hybrids derived from reciprocal crosses of divergent strains. We observe that deviation from balanced biallelic expression is common, occurring in â¼20% of the mouse transcriptome in a given tissue. Allelic imbalance attributed to genotypic variation is by far the most prevalent class and typically is tissue-specific. However, some genotype-based imbalance is maintained across tissues and is associated with greater genetic variation, especially in 5' and 3' termini of transcripts. We further identify novel random monoallelic and imprinted genes and find that genotype can modify penetrance of parental origin even in the setting of large imprinted regions. Examination of nascent transcripts in single cells from inbred parental strains reveals that genes showing genotype-based imbalance in hybrids can also exhibit monoallelic expression in isogenic backgrounds. This surprising observation may suggest a competition between alleles and/or reflect the combined impact of cis- and trans-acting variation on expression of a given gene. Our findings provide novel insights into gene regulation and may be relevant to human genetic variation and disease.
Asunto(s)
Desequilibrio Alélico , Transcriptoma , Alelos , Animales , Análisis por Conglomerados , Cruzamientos Genéticos , Perfilación de la Expresión Génica , Variación Genética , Impresión Genómica , Genotipo , Ratones , Especificidad de Órganos/genéticaRESUMEN
Oligopaint probes are fluorescently labeled, single-stranded DNA oligonucleotides that can be used to visualize genomic regions ranging in size from tens of kilobases to many megabases. This unit details how Oligopaint probes can be synthesized using basic molecular biological techniques, and provides protocols for FISH, 3D-FISH, and sample preparation.
Asunto(s)
ADN de Cadena Simple/genética , Genoma/genética , Hibridación Fluorescente in Situ/métodos , Sondas de Oligonucleótidos/genética , Oligonucleótidos/genética , HumanosRESUMEN
A host of observations demonstrating the relationship between nuclear architecture and processes such as gene expression have led to a number of new technologies for interrogating chromosome positioning. Whereas some of these technologies reconstruct intermolecular interactions, others have enhanced our ability to visualize chromosomes in situ. Here, we describe an oligonucleotide- and PCR-based strategy for fluorescence in situ hybridization (FISH) and a bioinformatic platform that enables this technology to be extended to any organism whose genome has been sequenced. The oligonucleotide probes are renewable, highly efficient, and able to robustly label chromosomes in cell culture, fixed tissues, and metaphase spreads. Our method gives researchers precise control over the sequences they target and allows for single and multicolor imaging of regions ranging from tens of kilobases to megabases with the same basic protocol. We anticipate this technology will lead to an enhanced ability to visualize interphase and metaphase chromosomes.
Asunto(s)
Pintura Cromosómica/métodos , Genoma/genética , Hibridación Fluorescente in Situ/métodos , Sondas de Oligonucleótidos/metabolismo , Animales , Caenorhabditis elegans/genética , Núcleo Celular/metabolismo , Cromosomas/genética , Drosophila melanogaster/citología , Drosophila melanogaster/genética , Femenino , Biblioteca de Genes , Humanos , Interfase/genética , Metafase/genética , Ratones , Ovario/citología , Ovario/metabolismo , Coloración y EtiquetadoRESUMEN
BACKGROUND: Solid lipid nanoparticle (SLN) systems have been applied to various drugs and delivery routes. Vitamin K1 is an important cofactor for maintaining hemostasis and preventing hemorrhage. METHOD: Vitamin K1-loaded SLNs are systematically being developed by optimizing triglycerides and lipophilic and hydrophilic surfactants based on the size and stability of the resulting SLNs. Concentrations of the surfactants, Myverol and Pluronic, were optimized by a central composite design and response surface methodology. Vitamin K1 (phylloquinone) was used as a lipophilic drug in the SLN system to evaluate the potential for oral delivery. RESULTS: Vitamin K1-loaded SLNs had a mean size of 125 nm and a zeta potential of -23 mV as measured by photon correlation spectroscopy. The prepared SLNs were examined by differential scanning calorimetry and transmission electron microscopy and found to have an imperfect crystalline lattice and a spherical morphology. Effects of ultrasonication duration and drug load on the particle size and entrapment efficiency of the SLNs were also evaluated. CONCLUSION: More than 85% of the vitamin K1 was entrapped in SLNs when the payload was <5%. The vitamin K1 in SLNs was stable for a 54-h duration in simulated gastric and intestinal fluids. The particle size and vitamin K1 entrapped in the SLN were stable after 4 months of storage at 25 degrees C. The results demonstrated that SLNs prepared herein can potentially be exploited as carriers for the oral delivery of vitamin K1.
Asunto(s)
Antifibrinolíticos/administración & dosificación , Portadores de Fármacos/química , Lípidos/química , Nanopartículas/química , Vitamina K 1/administración & dosificación , Animales , Antifibrinolíticos/química , Rastreo Diferencial de Calorimetría , Química Farmacéutica , Composición de Medicamentos , Estabilidad de Medicamentos , Emulsionantes/química , Microscopía Electrónica de Rastreo , Tamaño de la Partícula , Ratas , Tensoactivos/química , Tecnología Farmacéutica , Vitamina K 1/químicaRESUMEN
The Su(z)2 complex contains Posterior sex combs (Psc) and Suppressor 2 of zeste [Su(z)2], two paralogous genes that likely arose by gene duplication. Psc encodes a Polycomb group protein that functions as a central component of the PRC1 complex, which maintains transcriptional repression of a wide array of genes. Although much is known about Psc, very little is known about Su(z)2, the analysis of which has been hampered by a dearth of alleles. We have generated new alleles of Su(z)2 and analyzed them at the genetic and molecular levels. Some of these alleles display negative complementation in that they cause lethality when heterozygous with the gain-of-function Su(z)2(1) allele but are hemizygous and, in some cases, homozygous viable. Interestingly, alleles of this class identify protein domains within Su(z)2 that are highly conserved in Psc and the mammalian Bmi-1 and Mel-18 proteins. We also find several domains of intrinsic disorder in the C-terminal regions of both Psc and Su(z)2 and suggest that these domains may contribute to the essential functions of both proteins.
Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas de Drosophila/genética , Alelos , Secuencia de Bases , Proteínas de Unión al ADN/química , Proteínas de Drosophila/química , Genes de Insecto , Genes Letales , Prueba de Complementación Genética , Datos de Secuencia Molecular , Complejo Represivo Polycomb 1 , Estructura Terciaria de ProteínaRESUMEN
The piRNA class of small RNAs are distinct from other small RNAs by their approximately 26-31 nucleotide size, single-strandedness and strand-specificity as well as by the clustered arrangement of their origins. Here, we highlight how these features are reminiscent of the mechanisms of DNA replication, and then present three models suggesting that the origin of piRNAs may be mechanistically similar to key processes in DNA replication.
Asunto(s)
Replicación del ADN/genética , Modelos Genéticos , ARN/biosíntesis , ARN/genética , Animales , HumanosRESUMEN
Polycomb group (PcG) genes propagate patterns of transcriptional repression throughout development. The products of several such genes are part of Polycomb repressive complex 1 (PRC1), which inhibits chromatin remodeling and transcription in vitro. Genetic and biochemical studies suggest the product of the Posterior sex combs (Psc) gene plays a central role in both PcG-mediated gene repression in vivo and PRC1 activity in vitro. To dissect the relationship between the in vivo and in vitro activities of Psc, we identified the lesions associated with 11 genetically characterized Psc mutations and asked how the corresponding mutant proteins affect Psc activity on nucleosomal templates in vitro. Analysis of both single-mutant Psc proteins and recombinant complexes containing mutant protein revealed that Psc encodes at least two functions, complex formation and the inhibition of remodeling and transcription, which require different regions of the protein. There is an excellent correlation between the in vivo phenotypes of mutant Psc alleles and the structure and in vitro activities of the corresponding proteins, suggesting that the in vitro activities of PRC1 reflect essential functions of Psc in vivo.