RESUMO
Fertilization in Arabidopsis (Arabidopsis thaliana) is a highly coordinated process that begins with a pollen tube delivering the 2 sperm cells into the embryo sac. Each sperm cell can then fertilize either the egg or the central cell to initiate embryo or endosperm development, respectively. The success of this double fertilization process requires a tight cell cycle synchrony between the male and female gametes to allow karyogamy (nuclei fusion). However, the cell cycle status of the male and female gametes during fertilization remains elusive as DNA quantification and DNA replication assays have given conflicting results. Here, to reconcile these results, we quantified the DNA replication state by DNA sequencing and performed microscopic analyses of fluorescent markers covering all phases of the cell cycle. We show that male and female Arabidopsis gametes are both arrested prior to DNA replication at maturity and initiate their DNA replication only during fertilization.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Sementes/genética , Sementes/metabolismo , Reprodução , Fertilização , Proteínas de Arabidopsis/metabolismo , Divisão Celular , Células Germinativas/metabolismoRESUMO
Repeated herbicide applications in agricultural fields exert strong selection on weeds such as blackgrass (Alopecurus myosuroides), which is a major threat for temperate climate cereal crops. This inadvertent selection pressure provides an opportunity for investigating the underlying genetic mechanisms and evolutionary processes of rapid adaptation, which can occur both through mutations in the direct targets of herbicides and through changes in other, often metabolic, pathways, known as non-target-site resistance. How much target-site resistance (TSR) relies on de novo mutations vs. standing variation is important for developing strategies to manage herbicide resistance. We first generated a chromosome-level reference genome for A. myosuroides for population genomic studies of herbicide resistance and genome-wide diversity across Europe in this species. Next, through empirical data in the form of highly accurate long-read amplicons of alleles encoding acetyl-CoA carboxylase (ACCase) and acetolactate synthase (ALS) variants, we showed that most populations with resistance due to TSR mutations-23 out of 27 and six out of nine populations for ACCase and ALS, respectively-contained at least two TSR haplotypes, indicating that soft sweeps are the norm. Finally, through forward-in-time simulations, we inferred that TSR is likely to mainly result from standing genetic variation, with only a minor role for de novo mutations.
Assuntos
Resistência a Herbicidas , Herbicidas , Resistência a Herbicidas/genética , Poaceae/genética , Poaceae/metabolismo , Mutação , Haplótipos , Europa (Continente) , Herbicidas/farmacologia , Acetil-CoA Carboxilase/genética , Acetil-CoA Carboxilase/metabolismoRESUMO
Structural variants and presence/absence polymorphisms are common in plant genomes, yet they are routinely overlooked in genome-wide association studies (GWAS). Here, we expand the type of genetic variants detected in GWAS to include major deletions, insertions and rearrangements. We first use raw sequencing data directly to derive short sequences, k-mers, that mark a broad range of polymorphisms independently of a reference genome. We then link k-mers associated with phenotypes to specific genomic regions. Using this approach, we reanalyzed 2,000 traits in Arabidopsis thaliana, tomato and maize populations. Associations identified with k-mers recapitulate those found with SNPs, but with stronger statistical support. Importantly, we discovered new associations with structural variants and with regions missing from reference genomes. Our results demonstrate the power of performing GWAS before linking sequence reads to specific genomic regions, which allows the detection of a wider range of genetic variants responsible for phenotypic variation.
Assuntos
Genoma de Planta/genética , Polimorfismo de Nucleotídeo Único/genética , Variação Biológica da População , Estudo de Associação Genômica Ampla/métodos , Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Solanum lycopersicum/genética , Fenótipo , Análise de Sequência de DNA/métodos , Zea mays/genéticaRESUMO
Splicing expands, reshapes, and regulates the transcriptome of eukaryotic organisms. Despite its importance, key questions remain unanswered, including the following: Can splicing evolve when organisms adapt to new challenges? How does evolution optimize inefficiency of introns' splicing and of the splicing machinery? To explore these questions, we evolved yeast cells that were engineered to contain an inefficiently spliced intron inside a gene whose protein product was under selection for an increased expression level. We identified a combination of mutations in Cis (within the gene of interest) and in Trans (in mRNA-maturation machinery). Surprisingly, the mutations in Cis resided outside of known intronic functional sites and improved the intron's splicing efficiency potentially by easing tight mRNA structures. One of these mutations hampered a protein's domain that was not under selection, demonstrating the evolutionary flexibility of multi-domain proteins as one domain functionality was improved at the expense of the other domain. The Trans adaptations resided in two proteins, Npl3 and Gbp2, that bind pre-mRNAs and are central to their maturation. Interestingly, these mutations either increased or decreased the affinity of these proteins to mRNA, presumably allowing faster spliceosome recruitment or increased time before degradation of the pre-mRNAs, respectively. Altogether, our work reveals various mechanistic pathways toward optimizations of intron splicing to ultimately adapt gene expression patterns to novel demands.
Assuntos
Adaptação Biológica/genética , Splicing de RNA/genética , Trans-Splicing/genética , Adaptação Biológica/fisiologia , Evolução Molecular , Expressão Gênica/genética , Regulação Fúngica da Expressão Gênica/genética , Íntrons/genética , Mutação , Precursores de RNA/metabolismo , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Spliceossomos/metabolismoRESUMO
DNA replication introduces a dosage imbalance between early and late replicating genes. In budding yeast, buffering gene expression against this imbalance depends on marking replicated DNA by H3K56 acetylation (H3K56ac). Whether additional processes are required for suppressing transcription from H3K56ac-labeled DNA remains unknown. Here, using a database-guided candidate screen, we find that COMPASS, the H3K4 methyltransferase, and its upstream effector, PAF1C, act downstream of H3K56ac to buffer expression. Replicated genes show reduced abundance of the transcription activating mark H3K4me3 and accumulate the transcription inhibitory mark H3K4me2 near transcription start sites. Notably, in hydroxyurea-exposed cells, the S phase checkpoint stabilizes H3K56ac and becomes essential for buffering. We suggest that H3K56ac suppresses transcription of replicated genes by interfering with post-replication recovery of epigenetic marks and assign a new function for the S phase checkpoint in stabilizing this mechanism during persistent dosage imbalance.
Assuntos
Replicação do DNA/fisiologia , Histonas/metabolismo , Acetilação , Pontos de Checagem do Ciclo Celular/genética , Replicação do DNA/genética , Epigênese Genética/fisiologia , Epigenômica/métodos , Regulação Fúngica da Expressão Gênica/genética , Histona Acetiltransferases/metabolismo , Histona Metiltransferases/metabolismo , Histonas/fisiologia , Homeostase/genética , Lisina/metabolismo , Proteínas Nucleares/metabolismo , Processamento de Proteína Pós-Traducional/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Sphingolipids (SLs) are essential components of cell membranes and are broad-range bioactive signaling molecules. SL levels must be tightly regulated as imbalances affect cellular function and contribute to pathologies ranging from neurodegenerative and metabolic disorders to cancer and aging. Deciphering how SL homeostasis is maintained and uncovering new regulators is required for understanding lipid biology and for identifying new targets for therapeutic interventions. Here we combine omics technologies to identify the changes of the transcriptome, proteome, and phosphoproteome in the yeast Saccharomyces cerevisiae upon SL depletion induced by myriocin. Surprisingly, while SL depletion triggers important changes in the expression of regulatory proteins involved in SL homeostasis, the most dramatic regulation occurs at the level of the phosphoproteome, suggesting that maintaining SL homeostasis demands rapid responses. To discover which of the phosphoproteomic changes are required for the cell's first-line response to SL depletion, we overlaid our omics results with systematic growth screens for genes required during growth in myriocin. By following the rate of SL biosynthesis in those candidates that are both affecting growth and are phosphorylated in response to the drug, we uncovered Atg9, Stp4, and Gvp36 as putative new regulators of SL homeostasis.
Assuntos
Ácido Aspártico Endopeptidases/genética , Proteínas Relacionadas à Autofagia/genética , Regulação Fúngica da Expressão Gênica , Proteínas de Membrana/genética , Proteínas de Transporte de Monossacarídeos/genética , Fosfoproteínas/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Antifúngicos/farmacologia , Ácido Aspártico Endopeptidases/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , Ácidos Graxos Monoinsaturados/farmacologia , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Homeostase/efeitos dos fármacos , Homeostase/genética , Proteínas de Membrana/metabolismo , Proteínas de Transporte de Monossacarídeos/metabolismo , Fosfoproteínas/metabolismo , Fosforilação/efeitos dos fármacos , Proteômica/métodos , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Esfingolipídeos/antagonistas & inibidores , Esfingolipídeos/biossínteseRESUMO
Chromatin can function as an integrator of DNA-related processes, allowing communication, for example, between DNA replication and gene transcription. Such communication is needed to overcome the gene-dosage imbalance introduced during DNA replication, when certain genes are replicated prior to others. Increased transcription of early replicating genes could alter regulatory balances. This does not occur, suggesting a mechanism that suppresses expression from newly replicated DNA. Critical to this buffering is Rtt109, which acetylates the internal K56 residue of newly synthesized histone H3 prior to incorporation onto DNA. H3K56ac distinguishes replicated from non-replicated DNA, communicating this information to the transcription machinery to ensure expression homeostasis during S phase.
Assuntos
Replicação do DNA/genética , Dosagem de Genes , Histona Acetiltransferases/metabolismo , Cromatina/genética , Regulação da Expressão Gênica , Homeostase , HumanosRESUMO
DNA replication perturbs the dosage balance between genes that replicate early during S phase and those that replicate late. If propagated to influence protein content, this dosage imbalance could influence cellular functions. In bacteria, mechanisms have evolved to use this imbalance to tune certain processes with the rate of cell growth. By contrast, eukaryotes buffer this dosage imbalance to ensure gene expression homeostasis also during S phase. Here, we outline classical and more recent studies describing how different organisms deal with this replication-dependent dosage imbalance, and describe recent results linking the eukaryotic buffering mechanism to replication-dependent histone acetylation. Finally, we discuss the possible implications of this buffering mechanism and speculate why it is specific to eukaryote cells.
Assuntos
Replicação do DNA/genética , Dosagem de Genes/genética , Fase S/genética , Transcrição Gênica , Acetilação , Bactérias/genética , Ciclo Celular/genética , Eucariotos/genética , Histonas/genéticaRESUMO
Chromatin is composed of DNA and histones, which provide a unified platform for regulating DNA-related processes, mostly through their post-translational modification. During DNA replication, histone arrangement is perturbed, first to allow progression of DNA polymerase and then during repackaging of the replicated DNA. To study how DNA replication influences the pattern of histone modification, we followed the cell-cycle dynamics of 10 histone marks in budding yeast. We find that histones deposited on newly replicated DNA are modified at different rates: While some marks appear immediately upon replication (e.g., H4K16ac, H3K4me1), others increase with transcription-dependent delays (e.g., H3K4me3, H3K36me3). Notably, H3K9ac was deposited as a wave preceding the replication fork by â¼5-6 kb. This replication-guided H3K9ac was fully dependent on the acetyltransferase Rtt109, while expression-guided H3K9ac was deposited by Gcn5. Further, topoisomerase depletion intensified H3K9ac in front of the replication fork and in sites where RNA polymerase II was trapped, suggesting supercoiling stresses trigger H3K9 acetylation. Our results assign complementary roles for DNA replication and gene expression in defining the pattern of histone modification.
Assuntos
Replicação do DNA/genética , Histona Acetiltransferases/genética , Código das Histonas/genética , Proteínas de Saccharomyces cerevisiae/genética , Acetilação , Cromatina , DNA Polimerase Dirigida por DNA/genética , Histona-Lisina N-Metiltransferase/genética , Histonas , RNA Polimerase II/genética , Saccharomycetales/genéticaRESUMO
Genome replication introduces a stepwise increase in the DNA template available for transcription. Genes replicated early in S phase experience this increase before late-replicating genes, raising the question of how expression levels are affected by DNA replication. We show that in budding yeast, messenger RNA (mRNA) synthesis rate is buffered against changes in gene dosage during S phase. This expression homeostasis depends on acetylation of H3 on its internal K56 site by Rtt109/Asf1. Deleting these factors, mutating H3K56 or up-regulating its deacetylation, increases gene expression in S phase in proportion to gene replication timing. Therefore, H3K56 acetylation on newly deposited histones reduces transcription efficiency from replicated DNA, complementing its role in guarding genome stability. Our study provides molecular insight into the mechanism maintaining expression homeostasis during DNA replication.
Assuntos
Replicação do DNA/fisiologia , Regulação Fúngica da Expressão Gênica , Instabilidade Genômica , Homeostase/genética , Saccharomyces cerevisiae/fisiologia , Acetilação , Proteínas de Ciclo Celular/metabolismo , Replicação do DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Dosagem de Genes , Genoma Fúngico , Histona Acetiltransferases/metabolismo , Histonas/metabolismo , Chaperonas Moleculares/metabolismo , RNA Fúngico/biossíntese , RNA Mensageiro/biossíntese , Fase S , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Moldes GenéticosRESUMO
4sUDRB-seq separately measures, on a genomic scale, the distinct contributions of transcription elongation speed and rate of RNA polymerase II (Pol II) transition into active elongation (TAE) to the overall mRNA production rate. It uses reversible inhibition of transcription elongation with 5,6-dichloro-1-ß-D-ribofuranosylbenzimidazole (DRB), combined with a pulse of 4-thiouridine (4sU), to tag newly transcribed RNA. After DRB removal, cells are collected at several time points, and tagged RNA is biotinylated, captured on streptavidin beads and sequenced. 4sUDRB-seq enables the comparison of elongation speeds between different developmental stages or different cell types, and it allows the impact of specific transcription factors on transcription elongation speed versus TAE to be studied. RNA preparation takes â¼4 d to complete, with deep sequencing requiring an additional â¼4-11 d plus 1-3 d for bioinformatics analysis. The experimental protocol requires basic molecular biology skills, whereas data analysis requires knowledge in bioinformatics, particularly MATLAB and the Linux environment.
Assuntos
Diclororribofuranosilbenzimidazol/química , RNA Polimerase II/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Análise de Sequência de RNA/métodos , Tiouridina/metabolismo , Elongação da Transcrição Genética , Biotina/química , Genoma , Células HeLa , Humanos , RNA/isolamento & purificação , RNA Polimerase II/química , RNA Polimerase II/genética , Reprodutibilidade dos Testes , Análise de Sequência de RNA/instrumentação , Estreptavidina/química , Tiouridina/químicaRESUMO
Various histone modifications decorate nucleosomes within transcribed genes. Among these, monoubiquitylation of histone H2B (H2Bub1) and methylation of histone H3 on lysines 36 (H3K36me2/3) and 79 (H3K79me2/3) correlate positively with gene expression. By measuring the progression of the transcriptional machinery along genes within live cells, we now report that H2B monoubiquitylation occurs cotranscriptionally and accurately reflects the advance of RNA polymerase II (Pol II). In contrast, H3K36me3 and H3K79me2 are less dynamic and represent Pol II movement less faithfully. High-resolution ChIP-seq reveals that H2Bub1 levels are selectively reduced at exons and decrease in an exon-dependent stepwise manner toward the 3' end of genes. Exonic depletion of H2Bub1 in gene bodies is highly correlated with Pol II pausing at exons, suggesting elongation rate changes associated with intron-exon structure. In support of this notion, H2Bub1 levels were found to be significantly correlated with transcription elongation rates measured in various cell lines. Overall, our data shed light on the organization of H2Bub1 within transcribed genes and single out H2Bub1 as a reliable marker for ongoing transcription elongation.
Assuntos
Histonas/genética , Histonas/metabolismo , RNA Polimerase II/metabolismo , Elongação da Transcrição Genética , Linhagem Celular Tumoral , Éxons , Células HeLa , Humanos , Dados de Sequência Molecular , RNA Mensageiro/metabolismo , Análise de Sequência de DNA , UbiquitinaçãoRESUMO
Various histone modifications are widely associated with gene expression, but their functional selectivity at individual genes remains to be characterized. Here, we identify widespread differences between genome-wide patterns of two prominent marks, H3K9ac and H3K4me3, in budding yeasts. As well as characteristic gene profiles, relative modification levels vary significantly amongst genes, irrespective of expression. Interestingly, we show that these differences couple to contrasting features: higher methylation to essential, periodically expressed, 'DPN' (Depleted Proximal Nucleosome) genes, and higher acetylation to non-essential, responsive, 'OPN' (Occupied Proximal Nucleosome) genes. Thus, H3K4me3 may generally associate with expression stability, and H3K9ac, with variability. To evaluate this notion, we examine their association with expression divergence between the closely related species, S. cerevisiae and S. paradoxus. Although individually well conserved at orthologous genes, changes between modifications are mostly uncorrelated, indicating largely non-overlapping regulatory mechanisms. Notably, we find that inter-species differences in methylation, but not acetylation, are well correlated with expression changes, thereby proposing H3K4me3 as a candidate regulator of expression divergence. Taken together, our results suggest distinct evolutionary roles for expression-linked modifications, wherein H3K4me3 may contribute to stabilize average expression, whilst H3K9ac associates with more indirect aspects such as responsiveness.
Assuntos
Histonas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Imunoprecipitação da Cromatina , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Processamento de Proteína Pós-Traducional , Saccharomyces cerevisiae/genética , Análise de Sequência de RNARESUMO
Although transcriptional elongation by RNA polymerase II is coupled with many RNA-related processes, genomewide elongation rates remain unknown. We describe a method, called 4sUDRB-seq, based on reversible inhibition of transcription elongation coupled with tagging newly transcribed RNA with 4-thiouridine and high throughput sequencing to measure simultaneously with high confidence genome-wide transcription elongation rates in cells. We find that most genes are transcribed at about 3.5 Kb/min, with elongation rates varying between 2 Kb/min and 6 Kb/min. 4sUDRB-seq can facilitate genomewide exploration of the involvement of specific elongation factors in transcription and the contribution of deregulated transcription elongation to various pathologies.
Assuntos
Diclororribofuranosilbenzimidazol/farmacologia , RNA Polimerase II/metabolismo , Tiouridina/metabolismo , Elongação da Transcrição Genética , Genoma Humano , Células HeLa , Humanos , Análise de Sequência de RNA/métodosRESUMO
Cells adapt to environmental changes through genetic mutations that stabilize novel phenotypes. Often, this adaptation involves regulatory changes which modulate gene expression. In the budding yeast, ribosomal-related gene expression correlates with cell growth rate across different environments. To examine whether the same relationship between gene expression and growth rate is observed also across natural populations, we measured gene expression, growth rate and ethanol production of twenty-four wild type yeast strains originating from diverse habitats, grown on the pentose sugar xylulose. We found that expression of ribosome-related genes did not correlate with growth rate. Rather, growth rate was correlated with the expression of amino acid biosynthesis genes. Searching other databases, we observed a similar correlation between growth rate and amino-acid biosyntehsis genes in a library of gene deletions. We discuss the implications of our results for understanding how cells coordinate their translation capacity with available nutrient resources.
Assuntos
Regulação Fúngica da Expressão Gênica , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Aminoácidos/biossíntese , Antifúngicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Evolução Molecular , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Mutação , Fenótipo , Ribossomos/genética , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Especificidade da Espécie , Xilulose/farmacologiaRESUMO
Gene expression shows a significant variation (noise) between genetically identical cells. Noise depends on the gene expression process regulated by the chromatin environment. We screened for chromatin factors that modulate noise in S. cerevisiae and analyzed the results using a theoretical model that infers regulatory mechanisms from the noise versus mean relationship. Distinct activities of the Rpd3(L) and Set3 histone deacetylase complexes were predicted. Both HDACs repressed expression. Yet, Rpd3(L)C decreased the frequency of transcriptional bursts, while Set3C decreased the burst size, as did H2B monoubiquitination (ubH2B). We mapped the acetylation of H3 lysine 9 (H3K9ac) upon deletion of multiple subunits of Set3C and Rpd3(L)C and of ubH2B effectors. ubH2B and Set3C appear to function in the same pathway to reduce the probability that an elongating PolII produces a functional transcript (PolII processivity), while Rpd3(L)C likely represses gene expression at a step preceding elongation.