RESUMO
Coordinated regulation of gene expression that involves activation of lineage specific genes and repression of pluripotency genes drives differentiation of embryonic stem cells (ESC). For complete repression of pluripotency genes during ESC differentiation, chromatin at their enhancers is silenced by the activity of the Lsd1-Mi2/NuRD complex. The mechanism/s that regulate DNA methylation at these enhancers are largely unknown. Here, we investigated the affect of the Lsd1-Mi2/NuRD complex on the dynamic regulatory switch that induces the local interaction of histone tails with the Dnmt3 ATRX-DNMT3-DNMT3L (ADD) domain, thus promoting DNA methylation at the enhancers of a subset of pluripotency genes. This is supported by previous structural studies showing a specific interaction between Dnmt3-ADD domain with H3K4 unmethylated histone tails that is disrupted by histone H3K4 methylation and histone acetylation. Our data suggest that Dnmt3a activity is triggered by Lsd1-Mi2/NuRD-mediated histone deacetylation and demethylation at these pluripotency gene enhancers when they are inactivated during mouse ESC differentiation. Using Dnmt3 knockout ESCs and the inhibitors of Lsd1 and p300 histone modifying enzymes during differentiation of E14Tg2A and ZHBTc4 ESCs, our study systematically reveals this mechanism and establishes that Dnmt3a is both reader and effector of the epigenetic state at these target sites.
Assuntos
Diferenciação Celular/genética , Metilação de DNA/genética , Elementos Facilitadores Genéticos/genética , Epigênese Genética , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Células-Tronco Pluripotentes/metabolismo , Acetilação , Animais , DNA (Citosina-5-)-Metiltransferases/química , DNA (Citosina-5-)-Metiltransferases/metabolismo , DNA Metiltransferase 3A , Histona Desmetilases/metabolismo , Histonas/metabolismo , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/metabolismo , Camundongos , Modelos Biológicos , Fator 3 de Transcrição de Octâmero/metabolismo , Ligação Proteica/genética , Domínios ProteicosRESUMO
PREMISE OF THE STUDY: Gravity regulates the magnitude and direction of a trans-cell calcium current in germinating spores of Ceratopteris richardii. Blocking this current with nifedipine blocks the spore's downward polarity alignment, a polarization that is fixed by gravity â¼10 h after light induces the spores to germinate. RNA-seq analysis at 10 h was used to identify genes potentially important for the gravity response. The data set will be valuable for other developmental and phylogenetic studies. METHODS: De novo Newbler assembly of 958 527 reads from Roche 454 sequencing was executed. The sequences were identified and analyzed using in silico methods. The roles of endomembrane Ca(2+)-ATPase pumps and apyrases in the gravity response were further tested using pharmacological agents. KEY RESULTS: Transcripts related to calcium signaling and ethylene biosynthesis were identified as notable constituents of the transcriptome. Inhibiting the activity of endomembrane Ca(2+)-ATPase pumps with 2,5-di-(t-butyl)-1,4-hydroquinone diminished the trans-cell current, but increased the orientation of the polar axis to gravity. The effects of applied nucleotides and purinoceptor antagonists gave novel evidence implicating extracellular nucleotides as regulators of the gravity response in these fern spores. CONCLUSIONS: In addition to revealing general features of the transcriptome of germinating spores, the results highlight a number of calcium-responsive and light-receptive transcripts. Pharmacologic assays indicate endomembrane Ca(2+)-ATPases and extracellular nucleotides may play regulatory roles in the gravity response of Ceratopteris spores.
Assuntos
Apirase/metabolismo , ATPases Transportadoras de Cálcio/metabolismo , Cálcio/metabolismo , Gravitação , Pteridaceae/fisiologia , Análise de Sequência de RNA/métodos , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Apirase/genética , ATPases Transportadoras de Cálcio/antagonistas & inibidores , ATPases Transportadoras de Cálcio/química , Polaridade Celular/efeitos dos fármacos , Bases de Dados Genéticas , Inibidores Enzimáticos/farmacologia , Espaço Extracelular/efeitos dos fármacos , Espaço Extracelular/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Genes de Plantas/genética , Dados de Sequência Molecular , Fotorreceptores de Plantas/metabolismo , Pteridaceae/citologia , Pteridaceae/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Esporos/efeitos dos fármacosRESUMO
Heading date is one of the most important quantitative traits responsible for the domestication of rice. We compared a 155-kb reference segment of the Oryza sativa ssp. japonica cv. Nipponbare genome surrounding Hd1, a major heading date gene in rice, with orthologous regions from nine diploid Oryza species that diverged over a relatively short time frame (â¼16 My) to study sequence evolution around a domestication locus. The orthologous Hd1 region from Sorghum bicolor was included to compare and contrast the evolution in a more distant relative of rice. Consistent with other observations at the adh1/adh2, monoculm1, and sh2/a1 loci in grass species, we found high gene colinearity in the Hd1 region amidst size differences that were lineage specific and long terminal repeat retrotransposon driven. Unexpectedly, the Hd1 gene was deleted in O. glaberrima, whereas the O. rufipogon and O. punctata copies had degenerative mutations, suggesting that other heading date loci might compensate for the loss or nonfunctionality of Hd1 in these species. Compared with the japonica Hd1 region, the orthologous region in sorghum exhibited micro-rearrangements including gene translocations, seven additional genes, and a gene triplication and truncation event predating the divergence from Oryza.
Assuntos
Diploide , Genes de Plantas/genética , Oryza/genética , Homologia de Sequência do Ácido Nucleico , Sorghum/genética , Sintenia/genética , Sequência de Bases , Cromossomos Artificiais Bacterianos/genética , Cromossomos Artificiais de Bacteriófago P1/genética , Sequência Consenso/genética , Sequência Conservada/genética , Elementos de DNA Transponíveis/genética , Bases de Dados de Ácidos Nucleicos , Loci Gênicos/genética , Especiação Genética , Dados de Sequência Molecular , Filogenia , Especificidade da Espécie , Sequências Repetidas Terminais/genética , Fatores de TempoRESUMO
The role the mammary epithelial circadian clock plays in gland development and lactation is unknown. We hypothesized that mammary epithelial clocks function to regulate mammogenesis and lactogenesis, and propose the core clock transcription factor BMAL1:CLOCK regulates genes that control mammary epithelial development and milk synthesis. Our objective was to identify transcriptional targets of BMAL1 in undifferentiated (UNDIFF) and lactogen differentiated (DIFF) mammary epithelial cells (HC11) using ChIP-seq. Ensembl gene IDs with the nearest transcriptional start site to ChIP-seq peaks were explored as potential targets, and represented 846 protein coding genes common to UNDIFF and DIFF cells and 2773 unique to DIFF samples. Genes with overlapping peaks between samples (1343) enriched cell-cell adhesion, membrane transporters and lipid metabolism categories. To functionally verify targets, an HC11 line with Bmal1 gene knocked out (BMAL1-KO) using CRISPR-CAS was created. BMAL1-KO cultures had lower cell densities over an eight-day growth curve, which was associated with increased (p<0.05) levels of reactive oxygen species and lower expression of superoxide dismutase 3 (Sod3). RT-qPCR analysis also found lower expression of the putative targets, prolactin receptor (Prlr), Ppara, and beta-casein (Csn2). Findings support our hypothesis and highlight potential importance of clock in mammary development and substrate transport.
Assuntos
Fatores de Transcrição ARNTL/fisiologia , Células Epiteliais/metabolismo , Glândulas Mamárias Animais/metabolismo , Proteínas do Leite/metabolismo , Fatores de Transcrição ARNTL/metabolismo , Anemia Falciforme , Animais , Proteína 9 Associada à CRISPR , Sistemas CRISPR-Cas , Feminino , Edição de Genes , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Glândulas Mamárias Animais/fisiologia , CamundongosRESUMO
A comparative physical map of the AA genome (Oryza sativa) and the BB genome (O. punctata) was constructed by aligning a physical map of O. punctata, deduced from 63,942 BAC end sequences (BESs) and 34,224 fingerprints, onto the O. sativa genome sequence. The level of conservation of each chromosome between the two species was determined by calculating a ratio of BES alignments. The alignment result suggests more divergence of intergenic and repeat regions in comparison to gene-rich regions. Further, this characteristic enabled localization of heterochromatic and euchromatic regions for each chromosome of both species. The alignment identified 16 locations containing expansions, contractions, inversions, and transpositions. By aligning 40% of the punctata BES on the map, 87% of the punctata FPC map covered 98% of the O. sativa genome sequence. The genome size of O. punctata was estimated to be 8% larger than that of O. sativa with individual chromosome differences of 1.5-16.5%. The sum of expansions and contractions observed in regions >500 kb were similar, suggesting that most of the contractions/expansions contributing to the genome size difference between the two species are small, thus preserving the macro-collinearity between these species, which diverged approximately 2 million years ago.
Assuntos
Genoma de Planta/genética , Oryza/classificação , Oryza/genética , Mapeamento Físico do Cromossomo , Inversão Cromossômica/genética , Cromossomos Artificiais Bacterianos/genética , Cromossomos de Plantas/genética , Células Clonais , Dados de Sequência Molecular , Translocação GenéticaRESUMO
Genetic and genomic approaches have much to offer in terms of ecology, evolution, and conservation. To better understand the biology of the gray whale Eschrichtius robustus (Lilljeborg, 1861), we sequenced the genome and produced an assembly that contains â¼95% of the genes known to be highly conserved among eukaryotes. From this assembly, we annotated 22,711 genes and identified 2,057,254 single-nucleotide polymorphisms (SNPs). Using this assembly, we generated a curated list of candidate genes potentially subject to strong natural selection, including genes associated with osmoregulation, oxygen binding and delivery, and other aspects of marine life. From these candidate genes, we queried 92 autosomal protein-coding markers with a panel of 96 SNPs that also included 2 sexing and 2 mitochondrial markers. Genotyping error rates, calculated across loci and across 69 intentional replicate samples, were low (0.021%), and observed heterozygosity was 0.33 averaged over all autosomal markers. This level of variability provides substantial discriminatory power across loci (mean probability of identity of 1.6 × 10-25 and mean probability of exclusion >0.999 with neither parent known), indicating that these markers provide a powerful means to assess parentage and relatedness in gray whales. We found 29 unique multilocus genotypes represented among our 36 biopsies (indicating that we inadvertently sampled 7 whales twice). In total, we compiled an individual data set of 28 western gray whales (WGSs) and 1 presumptive eastern gray whale (EGW). The lone EGW we sampled was no more or less related to the WGWs than expected by chance alone. The gray whale genomes reported here will enable comparative studies of natural selection in cetaceans, and the SNP markers should be highly informative for future studies of gray whale evolution, population structure, demography, and relatedness.
Assuntos
Genoma/genética , Baleias/genética , Animais , Variação Genética , Genética Populacional , Genótipo , Polimorfismo de Nucleotídeo Único/genética , Especificidade da EspécieRESUMO
BACKGROUND: With the completion of the genome sequence for rice (Oryza sativa L.), the focus of rice genomics research has shifted to the comparison of the rice genome with genomes of other species for gene cloning, breeding, and evolutionary studies. The genus Oryza includes 23 species that shared a common ancestor 8-10 million years ago making this an ideal model for investigations into the processes underlying domestication, as many of the Oryza species are still undergoing domestication. This study integrates high-throughput, hybridization-based markers with BAC end sequence and fingerprint data to construct physical maps of rice chromosome 1 orthologues in two wild Oryza species. Similar studies were undertaken in Sorghum bicolor, a species which diverged from cultivated rice 40-50 million years ago. RESULTS: Overgo markers, in conjunction with fingerprint and BAC end sequence data, were used to build sequence-ready BAC contigs for two wild Oryza species. The markers drove contig merges to construct physical maps syntenic to rice chromosome 1 in the wild species and provided evidence for at least one rearrangement on chromosome 1 of the O. sativa versus Oryza officinalis comparative map. When rice overgos were aligned to available S. bicolor sequence, 29% of the overgos aligned with three or fewer mismatches; of these, 41% gave positive hybridization signals. Overgo hybridization patterns supported colinearity of loci in regions of sorghum chromosome 3 and rice chromosome 1 and suggested that a possible genomic inversion occurred in this syntenic region in one of the two genomes after the divergence of S. bicolor and O. sativa. CONCLUSION: The results of this study emphasize the importance of identifying conserved sequences in the reference sequence when designing overgo probes in order for those probes to hybridize successfully in distantly related species. As interspecific markers, overgos can be used successfully to construct physical maps in species which diverged less than 8 million years ago, and can be used in a more limited fashion to examine colinearity among species which diverged as much as 40 million years ago. Additionally, overgos are able to provide evidence of genomic rearrangements in comparative physical mapping studies.
Assuntos
Mapeamento Cromossômico/métodos , Cromossomos de Plantas/genética , Sondas de DNA , Marcadores Genéticos , Genoma de Planta , Hibridização de Ácido Nucleico , Oryza/genética , Sorghum/genética , Cromossomos Artificiais Bacterianos/genética , Impressões Digitais de DNA , Evolução Molecular , Biblioteca Gênica , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico , Especificidade da EspécieRESUMO
The finding in animal species of complexes homologous to the products of six Saccharomyces cerevisiae genes, origin of replication recognition complex (ORC), has suggested that ORC-related mechanisms have been conserved in all eukaryotes. In plants, however, the only cloned putative homologs of ORC subunits are the Arabidopsis ORC2 and the rice ORC1. Homologs of other subunits of plant origin have not been cloned and characterized. A striking observation was the absence from the Arabidopsis genome of an obvious candidate gene-homolog of ORC4. This fact raised compelling questions of whether plants, in general, and Arabidopsis, in particular, may have lost the ORC4 gene, whether ORC-homologous subunits function within a complex in plants, whether an ORC complex may form and function without an ORC4 subunit, whether a functional (but not sequence) protein homolog may have taken up the role of ORC4 in Arabidopsis, and whether lack of ORC4 is a plant feature, in general. Here, we report the first cloned and molecularly characterized five genes coding for the maize putative homologs of ORC subunits ZmORC1, ZmORC2, ZmORC3, ZmORC4 and ZmORC5. Their expression profiles in tissues with different cell-dividing activities are compatible with a role in DNA replication. Based on the potential of ORC-homologous maize proteins to bind each other in yeast, we propose a model for their possible assembly within a maize ORC. The isolation and molecular characterization of an ORC4-homologous gene from maize argues that, in its evolution, Arabidopsis may have lost the homologous ORC4 gene.
Assuntos
Proteínas de Ligação a DNA/genética , Zea mays/genética , Replicação do DNA/genética , DNA Complementar/química , DNA Complementar/genética , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica de Plantas , Dados de Sequência Molecular , Complexo de Reconhecimento de Origem , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Ligação Proteica , Saccharomyces cerevisiae/genética , Análise de Sequência de DNA , Técnicas do Sistema de Duplo-HíbridoRESUMO
Parent-specific gene expression (PSGE) is little known outside of mammals and plants. PSGE occurs when the expression level of a gene depends on whether an allele was inherited from the mother or the father. Kin selection theory predicts that there should be extensive PSGE in social insects because social insect parents can gain inclusive fitness benefits by silencing parental alleles in female offspring. We searched for evidence of PSGE in honey bees using transcriptomes from reciprocal crosses between European and Africanized strains. We found 46 transcripts with significant parent-of-origin effects on gene expression, many of which overexpressed the maternal allele. Interestingly, we also found a large proportion of genes showing a bias toward maternal alleles in only one of the reciprocal crosses. These results indicate that PSGE may occur in social insects. The nonreciprocal effects could be largely driven by hybrid incompatibility between these strains. Future work will help to determine if these are indeed parent-of-origin effects that can modulate inclusive fitness benefits.
Assuntos
Abelhas/genética , Expressão Gênica , Alelos , Animais , Abelhas/crescimento & desenvolvimento , Abelhas/metabolismo , Encéfalo/metabolismo , Troca Genética , Feminino , Ligação Genética , Genótipo , Larva/metabolismo , Masculino , Polimorfismo de Nucleotídeo ÚnicoRESUMO
We analyzed the transcriptome of Escherichia coli K-12 by strand-specific RNA sequencing at single-nucleotide resolution during steady-state (logarithmic-phase) growth and upon entry into stationary phase in glucose minimal medium. To generate high-resolution transcriptome maps, we developed an organizational schema which showed that in practice only three features are required to define operon architecture: the promoter, terminator, and deep RNA sequence read coverage. We precisely annotated 2,122 promoters and 1,774 terminators, defining 1,510 operons with an average of 1.98 genes per operon. Our analyses revealed an unprecedented view of E. coli operon architecture. A large proportion (36%) of operons are complex with internal promoters or terminators that generate multiple transcription units. For 43% of operons, we observed differential expression of polycistronic genes, despite being in the same operons, indicating that E. coli operon architecture allows fine-tuning of gene expression. We found that 276 of 370 convergent operons terminate inefficiently, generating complementary 3' transcript ends which overlap on average by 286 nucleotides, and 136 of 388 divergent operons have promoters arranged such that their 5' ends overlap on average by 168 nucleotides. We found 89 antisense transcripts of 397-nucleotide average length, 7 unannotated transcripts within intergenic regions, and 18 sense transcripts that completely overlap operons on the opposite strand. Of 519 overlapping transcripts, 75% correspond to sequences that are highly conserved in E. coli (>50 genomes). Our data extend recent studies showing unexpected transcriptome complexity in several bacteria and suggest that antisense RNA regulation is widespread. Importance: We precisely mapped the 5' and 3' ends of RNA transcripts across the E. coli K-12 genome by using a single-nucleotide analytical approach. Our resulting high-resolution transcriptome maps show that ca. one-third of E. coli operons are complex, with internal promoters and terminators generating multiple transcription units and allowing differential gene expression within these operons. We discovered extensive antisense transcription that results from more than 500 operons, which fully overlap or extensively overlap adjacent divergent or convergent operons. The genomic regions corresponding to these antisense transcripts are highly conserved in E. coli (including Shigella species), although it remains to be proven whether or not they are functional. Our observations of features unearthed by single-nucleotide transcriptome mapping suggest that deeper layers of transcriptional regulation in bacteria are likely to be revealed in the future.
Assuntos
Escherichia coli/genética , Óperon/genética , Análise de Sequência de RNA/métodos , Regulação Bacteriana da Expressão Gênica , Transcrição Gênica/genética , Transcriptoma/genéticaRESUMO
Research is uncovering the genetic and biochemical effects of consuming large quantities of alcohol. One prime example is the J- or U-shaped relationship between the levels of alcohol consumption and the risk of atherosclerotic cardiovascular disease. Moderate alcohol consumption in humans (about 30 g ethanol/d) is associated with reduced risk of coronary heart disease, while abstinence and heavier alcohol intake is linked to increased risk. However, the hepatic consequences of moderate alcohol drinking are largely unknown. Previous data from alcohol-preferring (P) rats showed that chronic consumption does not produce significant hepatic steatosis in this well-established model. Therefore, free-choice alcohol drinking in P rats may mimic low risk or nonhazardous drinking in humans, and chronic exposure in P animals can illuminate the molecular underpinnings of free-choice drinking in the liver. To address this gap, we captured the global, steady-state liver transcriptome following a 23 week free-choice, moderate alcohol consumption regimen (â¼ 7.43 g ethanol/kg/day) in inbred alcohol-preferring (iP10a) rats. Chronic consumption led to down-regulation of nine genes in the cholesterol biosynthesis pathway, including HMG-CoA reductase, the rate-limiting step for cholesterol synthesis. These findings corroborate our phenotypic analyses, which indicate that this paradigm produced animals whose hepatic triglyceride levels, cholesterol levels and liver histology were indistinguishable from controls. These findings explain, at least in part, the J- or U-shaped relationship between cardiovascular risk and alcohol intake, and provide outstanding candidates for future studies aimed at understanding the mechanisms that underlie the salutary cardiovascular benefits of chronic low risk and nonhazardous alcohol intake.
Assuntos
Acil Coenzima A/genética , Consumo de Bebidas Alcoólicas/genética , Colesterol/genética , Etanol/administração & dosagem , Perfilação da Expressão Gênica/métodos , Fígado/metabolismo , Fígado/patologia , Consumo de Bebidas Alcoólicas/patologia , Animais , Comportamento Animal , Comportamento de Escolha , Colesterol/metabolismo , Modelos Animais de Doenças , Regulação para Baixo , Feminino , Humanos , Dados de Sequência Molecular , Ratos , Análise de Sequência de RNA/métodosRESUMO
Evolutionary and reproductive success of angiosperms, the most diverse group of land plants, relies on visual and olfactory cues for pollinator attraction. Previous work has focused on elucidating the developmental regulation of pathways leading to the formation of pollinator-attracting secondary metabolites such as scent compounds and flower pigments. However, to date little is known about how flowers control their entire metabolic network to achieve the highly regulated production of metabolites attracting pollinators. Integrative analysis of transcripts and metabolites in snapdragon sepals and petals over flower development performed in this study revealed a profound developmental remodeling of gene expression and metabolite profiles in petals, but not in sepals. Genes up-regulated during petal development were enriched in functions related to secondary metabolism, fatty acid catabolism, and amino acid transport, whereas down-regulated genes were enriched in processes involved in cell growth, cell wall formation, and fatty acid biosynthesis. The levels of transcripts and metabolites in pathways leading to scent formation were coordinately up-regulated during petal development, implying transcriptional induction of metabolic pathways preceding scent formation. Developmental gene expression patterns in the pathways involved in scent production were different from those of glycolysis and the pentose phosphate pathway, highlighting distinct developmental regulation of secondary metabolism and primary metabolic pathways feeding into it.
Assuntos
Antirrhinum/crescimento & desenvolvimento , Antirrhinum/metabolismo , Flores/crescimento & desenvolvimento , Flores/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Redes e Vias Metabólicas/genética , Antirrhinum/genética , Biomassa , Regulação para Baixo/genética , Flores/genética , Perfilação da Expressão Gênica , Genes de Plantas/genética , Glicólise/genética , Metaboloma/genética , Metabolômica , Anotação de Sequência Molecular , Via de Pentose Fosfato/genética , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sacarose/metabolismo , Regulação para Cima/genética , VolatilizaçãoRESUMO
Gibberella zeae is a broad host range pathogen that infects many crop plants, including wheat and barley, and causes head blight and rot diseases throughout the world. To better understand fungal development and pathogenicity, we have generated 7996 ESTs from three cDNA libraries. Two libraries were generated from carbon-(C-) and nitrogen- (N-) starved mycelia and one library was generated from cultures of maturing perithecia (P). In other fungal pathogens, starvation conditions have been shown to act as cues to induce infection-related gene expression. To assign putative function to cDNAs, sequences were initially assembled using StackPack. The estimated total number of genes identified from the three EST databases was 2110: 1088 contigs and 1022 singleton sequences. These 2110 sequences were compared to a yeast protein sequence reference set and to the GenBank nonredundant database using BLASTX. Based on presumptive gene function identified by this process, we found that the two starved cultures had similar, but not identical, patterns of gene expression, whereas the developmental cultures were distinct in their pattern of expression. Of the three libraries, the perithecium library had the greatest percentage (46%) of ESTS falling into the "unclassified" category. Homologues of some known fungal virulence or pathogenicity factors were found primarily in the N- and C-libraries. Comparisons also were made with ESTs from the related fungi, Neurospora crassa and Magnaporthe grisea and the genomic sequence of N. crassa.