RESUMEN
The mechanisms and timescales controlling de novo establishment of chromatin-mediated transcriptional silencing by Polycomb repressive complex 2 (PRC2) are unclear. Here, we investigate PRC2 silencing at Arabidopsis FLOWERING LOCUS C (FLC), known to involve co-transcriptional RNA processing, histone demethylation activity, and PRC2 function, but so far not mechanistically connected. We develop and test a computational model describing proximal polyadenylation/termination mediated by the RNA-binding protein FCA that induces H3K4me1 removal by the histone demethylase FLD. H3K4me1 removal feeds back to reduce RNA polymerase II (RNA Pol II) processivity and thus enhance early termination, thereby repressing productive transcription. The model predicts that this transcription-coupled repression controls the level of transcriptional antagonism to PRC2 action. Thus, the effectiveness of this repression dictates the timescale for establishment of PRC2/H3K27me3 silencing. We experimentally validate these mechanistic model predictions, revealing that co-transcriptional processing sets the level of productive transcription at the locus, which then determines the rate of the ON-to-OFF switch to PRC2 silencing.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Regulación de la Expresión Génica de las Plantas , Silenciador del Gen , Histonas , Proteínas de Dominio MADS , Complejo Represivo Polycomb 2 , ARN Polimerasa II , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Histonas/metabolismo , Histonas/genética , ARN Polimerasa II/metabolismo , ARN Polimerasa II/genética , Complejo Represivo Polycomb 2/metabolismo , Complejo Represivo Polycomb 2/genética , Proteínas de Dominio MADS/genética , Proteínas de Dominio MADS/metabolismo , Transcripción Genética , Poliadenilación , Histona Demetilasas/metabolismo , Histona Demetilasas/genética , Terminación de la Transcripción Genética , Cromatina/metabolismo , Cromatina/genética , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genéticaRESUMEN
Oxidative phosphorylation (OXPHOS) complexes, encoded by both mitochondrial and nuclear DNA, are essential producers of cellular ATP, but how nuclear and mitochondrial gene expression steps are coordinated to achieve balanced OXPHOS subunit biogenesis remains unresolved. Here, we present a parallel quantitative analysis of the human nuclear and mitochondrial messenger RNA (mt-mRNA) life cycles, including transcript production, processing, ribosome association, and degradation. The kinetic rates of nearly every stage of gene expression differed starkly across compartments. Compared with nuclear mRNAs, mt-mRNAs were produced 1,100-fold more, degraded 7-fold faster, and accumulated to 160-fold higher levels. Quantitative modeling and depletion of mitochondrial factors LRPPRC and FASTKD5 identified critical points of mitochondrial regulatory control, revealing that the mitonuclear expression disparities intrinsically arise from the highly polycistronic nature of human mitochondrial pre-mRNA. We propose that resolving these differences requires a 100-fold slower mitochondrial translation rate, illuminating the mitoribosome as a nexus of mitonuclear co-regulation.
Asunto(s)
Mitocondrias , Ribosomas Mitocondriales , Humanos , Mitocondrias/genética , Mitocondrias/metabolismo , Ribosomas Mitocondriales/metabolismo , Biosíntesis de Proteínas , Fosforilación Oxidativa , Proteínas Mitocondriales/metabolismo , ADN Mitocondrial/genética , ADN Mitocondrial/metabolismoRESUMEN
Dissecting the regulatory mechanisms controlling mammalian transcripts from production to degradation requires quantitative measurements of mRNA flow across the cell. We developed subcellular TimeLapse-seq to measure the rates at which RNAs are released from chromatin, exported from the nucleus, loaded onto polysomes, and degraded within the nucleus and cytoplasm in human and mouse cells. These rates varied substantially, yet transcripts from genes with related functions or targeted by the same transcription factors and RNA-binding proteins flowed across subcellular compartments with similar kinetics. Verifying these associations uncovered a link between DDX3X and nuclear export. For hundreds of RNA metabolism genes, most transcripts with retained introns were degraded by the nuclear exosome, while the remaining molecules were exported with stable cytoplasmic lifespans. Transcripts residing on chromatin for longer had extended poly(A) tails, whereas the reverse was observed for cytoplasmic mRNAs. Finally, machine learning identified molecular features that predicted the diverse life cycles of mRNAs.
Asunto(s)
Núcleo Celular , Cromatina , ARN Helicasas DEAD-box , ARN Mensajero , Animales , Humanos , Ratones , ARN Mensajero/metabolismo , ARN Mensajero/genética , Núcleo Celular/metabolismo , Núcleo Celular/genética , ARN Helicasas DEAD-box/metabolismo , ARN Helicasas DEAD-box/genética , Cromatina/metabolismo , Cromatina/genética , Citoplasma/metabolismo , Citoplasma/genética , Estabilidad del ARN , Transporte Activo de Núcleo Celular , Polirribosomas/metabolismo , Polirribosomas/genética , Aprendizaje Automático , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Exosomas/metabolismo , Exosomas/genéticaRESUMEN
Glycine-12 mutations in the GTPase KRAS (KRASG12) are an initiating event for development of lung adenocarcinoma (LUAD). KRASG12 mutations promote cell-intrinsic rewiring of alveolar type-II progenitor (AT2) cells, but to what extent such changes interplay with lung homeostasis and cell fate pathways is unclear. Here, we generated single-cell RNA-seq (scRNA-seq) profiles from AT2-mesenchyme organoid co-cultures, mice, and stage-IA LUAD patients, identifying conserved regulators of AT2 transcriptional dynamics and defining the impact of KRASG12D mutation with temporal resolution. In AT2WT organoids, we found a transient injury/plasticity state preceding AT2 self-renewal and AT1 differentiation. Early-stage AT2KRAS cells exhibited perturbed gene expression dynamics, most notably retention of the injury/plasticity state. The injury state in AT2KRAS cells of patients, mice, and organoids was distinguishable from AT2WT states via altered receptor expression, including co-expression of ITGA3 and SRC. The combination of clinically relevant KRASG12D and SRC inhibitors impaired AT2KRAS organoid growth. Together, our data show that an injury/plasticity state essential for lung repair is co-opted during AT2 self-renewal and LUAD initiation, suggesting that early-stage LUAD may be susceptible to interventions that target specifically the oncogenic nature of this cell state.
Asunto(s)
Neoplasias Pulmonares , Organoides , Proteínas Proto-Oncogénicas p21(ras) , Animales , Humanos , Ratones , Adenocarcinoma del Pulmón/genética , Adenocarcinoma del Pulmón/patología , Adenocarcinoma del Pulmón/metabolismo , Diferenciación Celular , Regulación Neoplásica de la Expresión Génica , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/metabolismo , Mutación , Organoides/metabolismo , Organoides/patología , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Familia-src Quinasas/metabolismo , Familia-src Quinasas/genéticaRESUMEN
The basis of quantitative regulation of gene expression is still poorly understood. In Arabidopsis thaliana, quantitative variation in expression of FLOWERING LOCUS C (FLC) influences the timing of flowering. In ambient temperatures, FLC expression is quantitatively modulated by a chromatin silencing mechanism involving alternative polyadenylation of antisense transcripts. Investigation of this mechanism unexpectedly showed that RNA polymerase II (Pol II) occupancy changes at FLC did not reflect RNA fold changes. Mathematical modeling of these transcriptional dynamics predicted a tight coordination of transcriptional initiation and elongation. This prediction was validated by detailed measurements of total and chromatin-bound FLC intronic RNA, a methodology appropriate for analyzing elongation rate changes in a range of organisms. Transcription initiation was found to vary â¼ 25-fold with elongation rate varying â¼ 8- to 12-fold. Premature sense transcript termination contributed very little to expression differences. This quantitative variation in transcription was coincident with variation in H3K36me3 and H3K4me2 over the FLC gene body. We propose different chromatin states coordinately influence transcriptional initiation and elongation rates and that this coordination is likely to be a general feature of quantitative gene regulation in a chromatin context.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas , Proteínas de Dominio MADS/genética , ARN Polimerasa II/metabolismo , Elongación de la Transcripción Genética , Iniciación de la Transcripción Genética , Proteínas de Arabidopsis/metabolismo , Cromatina/metabolismo , Flores/genética , Silenciador del Gen , Variación Genética , Histona Desacetilasas/metabolismo , Histonas/metabolismo , Proteínas de Dominio MADS/metabolismo , Modelos Genéticos , Poliadenilación , Pliegue del ARN , Empalme del ARN , Proteínas de Unión al ARN/metabolismoRESUMEN
A high proportion of all eukaryotic genes express antisense RNA (asRNA), which accumulates to varying degrees at different loci. Whether there is a general function for asRNA is unknown, but its widespread occurrence and frequent regulation by stress suggest an important role. The best-characterized plant gene exhibiting a complex antisense transcript pattern is the Arabidopsis floral regulator FLOWERING LOCUS C (FLC). Changes occur in the accumulation, splicing, and polyadenylation of this antisense transcript, termed COOLAIR, in different environments and genotypes. These changes are associated with altered chromatin regulation and differential FLC expression, provoking mechanistic comparisons with many well-studied loci in yeast and mammals. Detailed analysis of these specific examples may shed light on the complex interplay between asRNA and chromatin modifications in different genomes.
Asunto(s)
Arabidopsis/crecimiento & desarrollo , Arabidopsis/genética , Cromatina , Flores/crecimiento & desarrollo , Flores/genética , ARN sin Sentido/genética , Animales , Proteínas de Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas , HumanosRESUMEN
Low copy number plasmids in bacteria require segregation for stable inheritance through cell division. This is often achieved by a parABC locus, comprising an ATPase ParA, DNA-binding protein ParB and a parC region, encoding ParB-binding sites. These minimal components space plasmids equally over the nucleoid, yet the underlying mechanism is not understood. Here we investigate a model where ParA-ATP can dynamically associate to the nucleoid and is hydrolyzed by plasmid-associated ParB, thereby creating nucleoid-bound, self-organizing ParA concentration gradients. We show mathematically that differences between competing ParA concentrations on either side of a plasmid can specify regular plasmid positioning. Such positioning can be achieved regardless of the exact mechanism of plasmid movement, including plasmid diffusion with ParA-mediated immobilization or directed plasmid motion induced by ParB/parC-stimulated ParA structure disassembly. However, we find experimentally that parABC from Escherichia coli plasmid pB171 increases plasmid mobility, inconsistent with diffusion/immobilization. Instead our observations favor directed plasmid motion. Our model predicts less oscillatory ParA dynamics than previously believed, a prediction we verify experimentally. We also show that ParA localization and plasmid positioning depend on the underlying nucleoid morphology, indicating that the chromosomal architecture constrains ParA structure formation. Our directed motion model unifies previously contradictory models for plasmid segregation and provides a robust mechanistic basis for self-organized plasmid spacing that may be widely applicable.
Asunto(s)
ADN Bacteriano/química , ADN Bacteriano/genética , Espacio Intracelular/química , Espacio Intracelular/genética , Plásmidos/química , Plásmidos/genética , Biología Computacional , ADN Bacteriano/metabolismo , Difusión , Escherichia coli/citología , Escherichia coli/genética , Espacio Intracelular/metabolismo , Modelos Biológicos , Plásmidos/metabolismoRESUMEN
Oxidative phosphorylation (OXPHOS) complexes, encoded by both mitochondrial and nuclear DNA, are essential producers of cellular ATP, but how nuclear and mitochondrial gene expression steps are coordinated to achieve balanced OXPHOS biogenesis remains unresolved. Here, we present a parallel quantitative analysis of the human nuclear and mitochondrial messenger RNA (mt-mRNA) life cycles, including transcript production, processing, ribosome association, and degradation. The kinetic rates of nearly every stage of gene expression differed starkly across compartments. Compared to nuclear mRNAs, mt-mRNAs were produced 700-fold higher, degraded 5-fold faster, and accumulated to 170-fold higher levels. Quantitative modeling and depletion of mitochondrial factors, LRPPRC and FASTKD5, identified critical points of mitochondrial regulatory control, revealing that the mitonuclear expression disparities intrinsically arise from the highly polycistronic nature of human mitochondrial pre-mRNA. We propose that resolving these differences requires a 100-fold slower mitochondrial translation rate, illuminating the mitoribosome as a nexus of mitonuclear co-regulation.
RESUMEN
The controlled release of promoter-proximal paused RNA polymerase II (Pol II) into productive elongation is a major step in gene regulation. However, functional analysis of Pol II pausing is difficult because factors that regulate pause release are almost all essential. In this study, we identified heterozygous loss-of-function mutations in SUPT5H , which encodes SPT5, in individuals with ß-thalassemia unlinked to HBB mutations. During erythropoiesis in healthy human cells, cell cycle genes were highly paused at the transition from progenitors to precursors. When the pathogenic mutations were recapitulated by SUPT5H editing, Pol II pause release was globally disrupted, and the transition from progenitors to precursors was delayed, marked by a transient lag in erythroid-specific gene expression and cell cycle kinetics. Despite this delay, cells terminally differentiate, and cell cycle phase distributions normalize. Therefore, hindering pause release perturbs proliferation and differentiation dynamics at a key transition during erythropoiesis, revealing a role for Pol II pausing in the temporal coordination between the cell cycle and differentiation.
RESUMEN
Controlled release of promoter-proximal paused RNA polymerase II (RNA Pol II) is crucial for gene regulation. However, studying RNA Pol II pausing is challenging, as pause-release factors are almost all essential. In this study, we identified heterozygous loss-of-function mutations in SUPT5H, which encodes SPT5, in individuals with ß-thalassemia. During erythropoiesis in healthy human cells, cell cycle genes were highly paused as cells transition from progenitors to precursors. When the pathogenic mutations were recapitulated by SUPT5H editing, RNA Pol II pause release was globally disrupted, and as cells began transitioning from progenitors to precursors, differentiation was delayed, accompanied by a transient lag in erythroid-specific gene expression and cell cycle kinetics. Despite this delay, cells terminally differentiate, and cell cycle phase distributions normalize. Therefore, hindering pause release perturbs proliferation and differentiation dynamics at a key transition during erythropoiesis, identifying a role for RNA Pol II pausing in temporally coordinating the cell cycle and erythroid differentiation.
Asunto(s)
Regulación de la Expresión Génica , ARN Polimerasa II , Humanos , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , Diferenciación Celular , Ciclo Celular , Transcripción Genética , Proteínas Nucleares/metabolismo , Factores de Elongación Transcripcional/genéticaRESUMEN
A bottleneck in high-throughput functional genomics experiments is identifying the most important genes and their relevant functions from a list of gene hits. Gene Ontology (GO) enrichment methods provide insight at the gene set level. Here, we introduce GeneWalk ( github.com/churchmanlab/genewalk ) that identifies individual genes and their relevant functions critical for the experimental setting under examination. After the automatic assembly of an experiment-specific gene regulatory network, GeneWalk uses representation learning to quantify the similarity between vector representations of each gene and its GO annotations, yielding annotation significance scores that reflect the experimental context. By performing gene- and condition-specific functional analysis, GeneWalk converts a list of genes into data-driven hypotheses.
Asunto(s)
Bases de Datos Genéticas , Redes Reguladoras de Genes , Animales , Biflavonoides , Encéfalo , Ontología de Genes , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Ratones , RNA-Seq , TranscriptomaRESUMEN
BACKGROUND: Adverse drug reactions (ADRs) are one of the leading causes of morbidity and mortality in health care. Understanding which drug targets are linked to ADRs can lead to the development of safer medicines. METHODS: Here, we analyse in vitro secondary pharmacology of common (off) targets for 2134 marketed drugs. To associate these drugs with human ADRs, we utilized FDA Adverse Event Reports and developed random forest models that predict ADR occurrences from in vitro pharmacological profiles. FINDINGS: By evaluating Gini importance scores of model features, we identify 221 target-ADR associations, which co-occur in PubMed abstracts to a greater extent than expected by chance. Amongst these are established relations, such as the association of in vitro hERG binding with cardiac arrhythmias, which further validate our machine learning approach. Evidence on bile acid metabolism supports our identification of associations between the Bile Salt Export Pump and renal, thyroid, lipid metabolism, respiratory tract and central nervous system disorders. Unexpectedly, our model suggests PDE3 is associated with 40 ADRs. INTERPRETATION: These associations provide a comprehensive resource to support drug development and human biology studies. FUNDING: This study was not supported by any formal funding bodies.
Asunto(s)
Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/genética , Aprendizaje Automático , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/epidemiología , Humanos , PubMedRESUMEN
Single-cell quantification of transcription kinetics and variability promotes a mechanistic understanding of gene regulation. Here, using single-molecule RNA fluorescence in situ hybridization and mathematical modeling, we dissect cellular RNA dynamics for Arabidopsis FLOWERING LOCUS C (FLC). FLC expression quantitatively determines flowering time and is regulated by antisense (COOLAIR) transcription. In cells without observable COOLAIR expression, we quantify FLC transcription initiation, elongation, intron processing, and lariat degradation, as well as mRNA release from the locus and degradation. In these heterogeneously sized cells, FLC mRNA number increases linearly with cell size, resulting in a large cell-to-cell variability in transcript level. This variation is accounted for by cell-size-dependent, Poissonian FLC mRNA production, but not by large transcriptional bursts. In COOLAIR-expressing cells, however, antisense transcription increases with cell size and contributes to FLC transcription decreasing with cell size. Our analysis therefore reveals an unexpected role for antisense transcription in modulating the scaling of transcription with cell size.