RESUMEN
Accurate gene model annotation of reference genomes is critical for making them useful. The modENCODE project has improved the D. melanogaster genome annotation by using deep and diverse high-throughput data. Since transcriptional activity that has been evolutionarily conserved is likely to have an advantageous function, we have performed large-scale interspecific comparisons to increase confidence in predicted annotations. To support comparative genomics, we filled in divergence gaps in the Drosophila phylogeny by generating draft genomes for eight new species. For comparative transcriptome analysis, we generated mRNA expression profiles on 81 samples from multiple tissues and developmental stages of 15 Drosophila species, and we performed cap analysis of gene expression in D. melanogaster and D. pseudoobscura. We also describe conservation of four distinct core promoter structures composed of combinations of elements at three positions. Overall, each type of genomic feature shows a characteristic divergence rate relative to neutral models, highlighting the value of multispecies alignment in annotating a target genome that should prove useful in the annotation of other high priority genomes, especially human and other mammalian genomes that are rich in noncoding sequences. We report that the vast majority of elements in the annotation are evolutionarily conserved, indicating that the annotation will be an important springboard for functional genetic testing by the Drosophila community.
Asunto(s)
Biología Computacional/métodos , Drosophila melanogaster/genética , Perfilación de la Expresión Génica , Anotación de Secuencia Molecular , Transcriptoma , Animales , Análisis por Conglomerados , Drosophila melanogaster/clasificación , Evolución Molecular , Exones , Femenino , Genoma de los Insectos , Humanos , Masculino , Motivos de Nucleótidos , Filogenia , Posición Específica de Matrices de Puntuación , Regiones Promotoras Genéticas , Edición de ARN , Sitios de Empalme de ARN , Empalme del ARN , Reproducibilidad de los Resultados , Sitio de Iniciación de la TranscripciónRESUMEN
BACKGROUND: Gene dosage change is a mild perturbation that is a valuable tool for pathway reconstruction in Drosophila. While it is often assumed that reducing gene dose by half leads to two-fold less expression, there is partial autosomal dosage compensation in Drosophila, which may be mediated by feedback or buffering in expression networks. RESULTS: We profiled expression in engineered flies where gene dose was reduced from two to one. While expression of most one-dose genes was reduced, the gene-specific dose responses were heterogeneous. Expression of two-dose genes that are first-degree neighbors of one-dose genes in novel network models also changed, and the directionality of change depended on the response of one-dose genes. CONCLUSIONS: Our data indicate that expression perturbation propagates in network space. Autosomal compensation, or the lack thereof, is a gene-specific response, largely mediated by interactions with the rest of the transcriptome.
Asunto(s)
Compensación de Dosificación (Genética) , Drosophila/genética , Redes Reguladoras de Genes , Genes de Insecto , Animales , Animales Modificados Genéticamente/genética , Cromosomas de Insectos/genética , Femenino , Dosificación de Gen , Heterogeneidad Genética , Masculino , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Transcriptoma , Cromosoma X/genéticaRESUMEN
At the latest stage of terminal differentiation in the epidermis, granular keratinocytes (GKs) undergo cornification, a programmed cell death required for the establishment of a functional skin barrier. A complex genetic regulatory network orchestrates the underlying biochemical modifications, but very few transcription factors specific to this programme have been identified to date. Here, we describe a large-scale, multi-technique approach performed on cells purified from normal human epidermis, primarily focusing on the identification of regulators. We combined data from microarray analysis of cell fractions enriched in GKs or basal keratinocytes, from an expressed sequence tag (EST) library built from GKs and from an in silico promoter analysis of 52 differentiation-associated genes. Among 3576 genes potentially expressed in GK, 298 candidates were selected, and half were directly profiled for the first time in the different layers of the epidermis by quantitative real-time PCR. Forty-nine genes upregulated during terminal differentiation, associated with numerous function of GK including lipid synthesis and secretion, were identified. Of 94 transcription factors detected, 37 were found to be either positively or negatively regulated, suggesting their involvement as regulators of gene expression in the GKs. These results largely extend the number of genes known as involved in the latest step of the terminal differentiation of human epidermis as well as the number of transcription factors known to control the expression of these genes.
Asunto(s)
Diferenciación Celular/fisiología , Células Epidérmicas , Queratinocitos/citología , Análisis por Matrices de Proteínas/métodos , Diferenciación Celular/genética , Permeabilidad de la Membrana Celular/genética , Permeabilidad de la Membrana Celular/fisiología , Polaridad Celular/genética , Polaridad Celular/fisiología , Citoesqueleto/genética , Citoesqueleto/fisiología , Epidermis/fisiología , Humanos , Queratinocitos/fisiología , Transducción de Señal/genética , Transducción de Señal/fisiologíaRESUMEN
Drosophila melanogaster is one of the most well studied genetic model organisms; nonetheless, its genome still contains unannotated coding and non-coding genes, transcripts, exons and RNA editing sites. Full discovery and annotation are pre-requisites for understanding how the regulation of transcription, splicing and RNA editing directs the development of this complex organism. Here we used RNA-Seq, tiling microarrays and cDNA sequencing to explore the transcriptome in 30 distinct developmental stages. We identified 111,195 new elements, including thousands of genes, coding and non-coding transcripts, exons, splicing and editing events, and inferred protein isoforms that previously eluded discovery using established experimental, prediction and conservation-based approaches. These data substantially expand the number of known transcribed elements in the Drosophila genome and provide a high-resolution view of transcriptome dynamics throughout development.
Asunto(s)
Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/genética , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica/genética , Transcripción Genética/genética , Empalme Alternativo/genética , Animales , Secuencia de Bases , Proteínas de Drosophila/genética , Drosophila melanogaster/embriología , Exones/genética , Femenino , Genes de Insecto/genética , Genoma de los Insectos/genética , Masculino , MicroARNs/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Isoformas de Proteínas/genética , Edición de ARN/genética , ARN Mensajero/análisis , ARN Mensajero/genética , ARN Pequeño no Traducido/análisis , ARN Pequeño no Traducido/genética , Análisis de Secuencia , Caracteres SexualesRESUMEN
Corneodesmosin (CDSN) is specific to desmosomes of epithelia undergoing cornification, mainly the epidermis and the inner root sheath of the hair follicles. CDSN nonsense mutations are associated with hypotrichosis simplex of the scalp, a rare disease that leads to complete baldness in young adults. CDSN displays adhesive properties, mostly attributable to its N-terminal glycine-rich domain, and is sequentially proteolyzed as corneocytes migrate towards the skin surface. K14-promoter driven Cre-mediated deletion of Cdsn in mice resulted in neonatal death as a result of epidermal tearing upon minor mechanical stress. Ultrastructural analyses revealed a desmosomal break at the interface between the living and cornified layers. After grafting onto nude mice, knockout skin showed a chronic defect in the epidermal permeability barrier. The epidermis was first hyperproliferative with a thick cornified layer, then, both the epidermis and the hair follicles degenerated. In adults, Cdsn deletion resulted in similar histological abnormalities and in a lethal barrier defect. We demonstrate that Cdsn is not essential for skin-barrier formation in utero, but is vital throughout life to preserve this barrier by maintaining desmosome integrity. The strong adhesive function that the protein confers on corneodesmosomes also seems necessary for maintaining the architecture of the hair follicle.
Asunto(s)
Desmosomas/ultraestructura , Epidermis/ultraestructura , Eliminación de Gen , Glicoproteínas/fisiología , Folículo Piloso/anomalías , Hipotricosis/patología , Animales , Western Blotting , Desmosomas/metabolismo , Epidermis/metabolismo , Femenino , Hipotricosis/genética , Integrasas/metabolismo , Masculino , Ratones , Ratones Noqueados , Ratones Desnudos , Ratones Transgénicos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Trasplante de PielRESUMEN
BACKGROUND: During epidermal differentiation, keratinocytes progressing through the suprabasal layers undergo complex and tightly regulated biochemical modifications leading to cornification and desquamation. The last living cells, the granular keratinocytes (GKs), produce almost all of the proteins and lipids required for the protective barrier function before their programmed cell death gives rise to corneocytes. We present here the first analysis of the transcriptome of human GKs, purified from healthy epidermis by an original approach. RESULTS: Using the ORESTES method, 22,585 expressed sequence tags (ESTs) were produced that matched 3,387 genes. Despite normalization provided by this method (mean 4.6 ORESTES per gene), some highly transcribed genes, including that encoding dermokine, were overrepresented. About 330 expressed genes displayed less than 100 ESTs in UniGene clusters and are most likely to be specific for GKs and potentially involved in barrier function. This hypothesis was tested by comparing the relative expression of 73 genes in the basal and granular layers of epidermis by quantitative RT-PCR. Among these, 33 were identified as new, highly specific markers of GKs, including those encoding a protease, protease inhibitors and proteins involved in lipid metabolism and transport. We identified filaggrin 2 (also called ifapsoriasin), a poorly characterized member of the epidermal differentiation complex, as well as three new lipase genes clustered with paralogous genes on chromosome 10q23.31. A new gene of unknown function, C1orf81, is specifically disrupted in the human genome by a frameshift mutation. CONCLUSION: These data increase the present knowledge of genes responsible for the formation of the skin barrier and suggest new candidates for genodermatoses of unknown origin.