RESUMO
Transcription initiation by RNA polymerase II (RNA Pol II) requires preinitiation complex (PIC) assembly at gene promoters. In the dynamic nucleus, where thousands of promoters are broadly distributed in chromatin, it is unclear how multiple individual components converge on any target to establish the PIC. Here we use live-cell, single-molecule tracking in S. cerevisiae to visualize constrained exploration of the nucleoplasm by PIC components and Mediator's key role in guiding this process. On chromatin, TFIID/TATA-binding protein (TBP), Mediator, and RNA Pol II instruct assembly of a short-lived PIC, which occurs infrequently but efficiently within a few seconds on average. Moreover, PIC exclusion by nucleosome encroachment underscores regulated promoter accessibility by chromatin remodeling. Thus, coordinated nuclear exploration and recruitment to accessible targets underlies dynamic PIC establishment in yeast. Our study provides a global spatiotemporal model for transcription initiation in live cells.
Assuntos
Complexo Mediador/metabolismo , RNA Polimerase II/metabolismo , Iniciação da Transcrição Genética/fisiologia , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina/fisiologia , Complexo Mediador/genética , Nucleossomos/metabolismo , Regiões Promotoras Genéticas/genética , Ligação Proteica/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Análise Espaço-Temporal , Proteína de Ligação a TATA-Box/genética , Fator de Transcrição TFIID/genética , Transcrição Gênica/genéticaRESUMO
In budding yeast, which possesses simple point centromeres, we discovered that all of its centromeres express long noncoding RNAs (cenRNAs), especially in S phase. Induction of cenRNAs coincides with CENP-ACse4 loading time and is dependent on DNA replication. Centromeric transcription is repressed by centromere-binding factor Cbf1 and histone H2A variant H2A.ZHtz1 Deletion of CBF1 and H2A.ZHTZ1 results in an up-regulation of cenRNAs; an increased loss of a minichromosome; elevated aneuploidy; a down-regulation of the protein levels of centromeric proteins CENP-ACse4, CENP-A chaperone HJURPScm3, CENP-CMif2, SurvivinBir1, and INCENPSli15; and a reduced chromatin localization of CENP-ACse4, CENP-CMif2, and Aurora BIpl1 When the RNA interference system was introduced to knock down all cenRNAs from the endogenous chromosomes, but not the cenRNA from the circular minichromosome, an increase in minichromosome loss was still observed, suggesting that cenRNA functions in trans to regulate centromere activity. CenRNA knockdown partially alleviates minichromosome loss in cbf1Δ, htz1Δ, and cbf1Δ htz1Δ in a dose-dependent manner, demonstrating that cenRNA level is tightly regulated to epigenetically control point centromere function.
Assuntos
Centrômero/metabolismo , Segregação de Cromossomos/fisiologia , RNA não Traduzido/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Saccharomycetales/metabolismo , Aurora Quinases/metabolismo , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Proteínas de Transporte/metabolismo , Centrômero/genética , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Cromossomos Fúngicos , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Deleção de Genes , Histonas/genética , Histonas/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Interferência de RNA/fisiologia , Fase S , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Regulação para CimaRESUMO
Endogenous chromosomes contain centromeres to direct equal chromosomal segregation in mitosis and meiosis. The location and function of existing centromeres is usually maintained through cell cycles and generations. Recent studies have investigated how the centromere-specific histone H3 variant CENP-A is assembled and replenished after DNA replication to epigenetically propagate the centromere identity. However, existing centromeres occasionally become inactivated, with or without change in underlying DNA sequences, or lost after chromosomal rearrangements, resulting in acentric chromosomes. New centromeres, known as neocentromeres, may form on ectopic, non-centromeric chromosomal regions to rescue acentric chromosomes from being lost, or form dicentric chromosomes if the original centromere is still active. In addition, de novo centromeres can form after chromatinization of purified DNA that is exogenously introduced into cells. Here, we review the phenomena of naturally occurring and experimentally induced new centromeres and summarize the genetic (DNA sequence) and epigenetic features of these new centromeres. We compare the characteristics of new and native centromeres to understand whether there are different requirements for centromere establishment and propagation. Based on our understanding of the mechanisms of new centromere formation, we discuss the perspectives of developing more stably segregating human artificial chromosomes to facilitate gene delivery in therapeutics and research.
Assuntos
Centrômero/genética , Epigênese Genética , Epigenômica , Genômica , Animais , Centrômero/metabolismo , Cromatina/genética , Cromatina/metabolismo , Instabilidade Cromossômica , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Aberrações Cromossômicas , Cromossomos Artificiais Humanos , Cromossomos de Plantas , Suscetibilidade a Doenças , Epigenômica/métodos , Evolução Molecular , Regulação da Expressão Gênica , Genômica/métodos , Humanos , Meiose/genética , Mitose/genética , Plantas/genética , Deleção de SequênciaRESUMO
The formation of de novo centromeres on artificial chromosomes in humans (HACs) and fission yeast (SpYACs) has provided much insights to the epigenetic and genetic control on regional centromere establishment and maintenance. Similarly, the use of artificial chromosomes in point centromeric budding yeast Saccharomyces cerevisiae (ScYACs) and holocentric Caenorhabditis elegans (WACs) has revealed epigenetic regulation in the originally thought purely genetically-determined point centromeres and some centromeric DNA sequence features in holocentromeres, respectively. These relatively extreme and less characterized centromere organizations, on the endogenous chromosomes and artificial chromosomes, will be discussed and compared to the more well-studied regional centromere systems. This review will highlight some of the common epigenetic and genetic features in different centromere architectures, including the presence of the centromeric histone H3 variant, CENP-A or CenH3, centromeric and pericentric transcription, AT-richness and repetitiveness of centromeric DNA sequences.
Assuntos
Proteína Centromérica A/genética , Centrômero/metabolismo , Cromatina/química , Proteínas Cromossômicas não Histona/genética , Epigênese Genética , Histonas/genética , Animais , Bombyx/genética , Bombyx/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Candida albicans/genética , Candida albicans/metabolismo , Centrômero/ultraestrutura , Proteína Centromérica A/metabolismo , Cromatina/genética , Proteínas Cromossômicas não Histona/metabolismo , Cromossomos Artificiais/química , Cromossomos Artificiais/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Histonas/metabolismo , Humanos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Zea mays/genética , Zea mays/metabolismoRESUMO
To ensure proper chromosome segregation during cell division, the centromere in many organisms is transcribed to produce a low level of long non-coding RNA to regulate the activity of the kinetochore. In the budding yeast point centromere, our recent work has shown that the level of centromeric RNAs (cenRNAs) is tightly regulated and repressed by the kinetochore protein Cbf1 and histone H2A variant H2A.ZHtz1, and de-repressed during S phase of the cell cycle. Too little or too much cenRNAs will disrupt centromere activity. Here, we discuss the current advance in the understanding of the action and regulation of cenRNAs at the point centromere of Saccharomyces cerevisiae. We further show that budding yeast cenRNAs are cryptic unstable transcripts (CUTs) that can be degraded by the nuclear RNA decay pathway. CenRNA provides an example that even CUTs, when present at the right time with the right level, can serve important cellular functions.
Assuntos
Centrômero/genética , Epigênese Genética , RNA não Traduzido/genética , Centrômero/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Regulação da Expressão Gênica , Instabilidade Genômica , Histonas/metabolismo , Transcrição GênicaRESUMO
Efficient gene expression requires RNA polymerase II (RNAPII) to find chromatin targets precisely in space and time. How RNAPII manages this complex diffusive search in three-dimensional nuclear space remains largely unknown. The disordered carboxy-terminal domain (CTD) of RNAPII, which is essential for recruiting transcription-associated proteins, forms phase-separated droplets in vitro, hinting at a potential role in modulating RNAPII dynamics. In the present study, we use single-molecule tracking and spatiotemporal mapping in living yeast to show that the CTD is required for confining RNAPII diffusion within a subnuclear region enriched for active genes, but without apparent phase separation into condensates. Both Mediator and global chromatin organization are required for sustaining RNAPII confinement. Remarkably, truncating the CTD disrupts RNAPII spatial confinement, prolongs target search, diminishes chromatin binding, impairs pre-initiation complex formation and reduces transcription bursting. The present study illuminates the pivotal role of the CTD in driving spatiotemporal confinement of RNAPII for efficient gene expression.
Assuntos
RNA Polimerase II , Proteínas de Saccharomyces cerevisiae , RNA Polimerase II/metabolismo , Transcrição Gênica , Cromatina/genética , Cromatina/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , FosforilaçãoRESUMO
Efficient gene expression requires RNA Polymerase II (RNAPII) to find chromatin targets precisely in space and time. How RNAPII manages this complex diffusive search in 3D nuclear space remains largely unknown. The disordered carboxy-terminal domain (CTD) of RNAPII, which is essential for recruiting transcription-associated proteins, forms phase-separated droplets in vitro, hinting at a potential role in modulating RNAPII dynamics. Here, we use single-molecule tracking and spatiotemporal mapping in living yeast to show that the CTD is required for confining RNAPII diffusion within a subnuclear region enriched for active genes, but without apparent phase separation into condensates. Both Mediator and global chromatin organization are required for sustaining RNAPII confinement. Remarkably, truncating the CTD disrupts RNAPII spatial confinement, prolongs target search, diminishes chromatin binding, impairs pre-initiation complex formation, and reduces transcription bursting. This study illuminates the pivotal role of the CTD in driving spatiotemporal confinement of RNAPII for efficient gene expression.
RESUMO
The coiled-coil alpha-helical rod protein 1 (CCHCR1) was first identified as a candidate gene in psoriasis and has lately been found to be associated with a wide range of clinical conditions including COVID-19. CCHCR1 is located within P-bodies and centrosomes, but its exact role in these two subcellular structures and its transcriptional control remain largely unknown. Here, we showed that CCHCR1 shares a bidirectional promoter with its neighboring gene, TCF19. This bidirectional promoter is activated by the G1/S-regulatory transcription factor E2F1, and both genes are co-induced during the G1/S transition of the cell cycle. A luciferase reporter assay suggests that the short intergenic sequence, only 287 bp in length, is sufficient for the G1/S induction of both genes, but the expression of CCHCR1 is further enhanced by the presence of exon 1 from both TCF19 and CCHCR1. This research uncovers the transcriptional regulation of the CCHCR1 gene, offering new perspectives on its function. These findings contribute to the broader understanding of diseases associated with CCHCR1 and may serve as a foundational benchmark for future research in these vital medical fields.
Assuntos
Fator de Transcrição E2F1 , Psoríase , Humanos , Fator de Transcrição E2F1/genética , Fator de Transcrição E2F1/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Psoríase/genética , Psoríase/metabolismo , Regulação da Expressão Gênica , Ciclo Celular , Fatores de Transcrição E2F/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Isomalabaricanes are a small class of rearranged triterpenoids obtained from marine sponges. Most of these are cytotoxic to tumor cells, but the underlying mechanism is not clear. In this study, it was demonstrated that stellettin A (1), obtained from Geodia japonica, inhibited the growth of B16F10 murine melanoma cells by the induction of endoplasmic reticulum stress and accumulation of unfolded proteins. Immunoblotting analysis revealed abnormal glycosylation patterns of two melanoma marker proteins, tyrosinase and tyrosinase-related protein 1, and the retention of these proteins in the endoplasmic reticulum. Compound 1 induced the upregulation of the unfolded protein chaperone, glucose-regulated protein 78, in a dose-dependent manner. Increase of autophagosome-associated protein light chain 3 (LC3) in a membrane-bound form (LC3II) and its immunofluorescence co-localization with tyrosinase suggest the possible removal of deglycosylated and unfolded proteins by autophagy of the cells. There was no change in either the expression of the apoptosis marker protein Bcl-2 or the appearance of apoptotic nuclei in 1-treated cells. Taken together, 1 is an endoplasmic reticulum stressor that inhibits the growth of B16 melanoma cells by induction of abnormal protein glycosylation and autophagy.
Assuntos
Antineoplásicos/isolamento & purificação , Antineoplásicos/farmacologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Geodia/química , Triterpenos/isolamento & purificação , Triterpenos/farmacologia , Animais , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Glicosilação/efeitos dos fármacos , Humanos , Biologia Marinha , Melanoma Experimental/metabolismo , Camundongos , Estrutura Molecular , Triterpenos/químicaRESUMO
Geoditin A, an isomalabaricane triterpene isolated from marine sponge Geodia japonica, has been demonstrated to induce apoptosis in leukemia HL60 cells and human colon HT29 cancer cells through an oxidative stress, a process also interfering with normal melanogenesis in pigment cells. Treatment of murine melanoma B16 cells with geoditin A decreased expression of melanogenic proteins and cell melanogenesis which was aggravated with adenylate cyclase inhibitor SQ22536, indicating melanogenic inhibition was mediated through a cAMP-dependent signaling pathway. Immunofluorescence microscopy and glycosylation studies revealed abnormal glycosylation patterns of melanogenic proteins (tyrosinase and tyrosinase-related protein 1), and a co-localization of tyrosinase with calnexin (CNX) and lysosome-associated membrane protein 1 (LAMP-1), implicating a post-translational modification in the ER and a degradation of tyrosinase in the lysosome. Taken together, potent anti-melanogenic property and the relatively low cytotoxicity of geoditin A have demonstrated its therapeutic potential as a skin lightening agent.
Assuntos
Antineoplásicos/farmacologia , Melanoma/tratamento farmacológico , Resorcinóis/farmacologia , Triterpenos/farmacologia , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/patologia , Geodia/metabolismo , Glicosilação/efeitos dos fármacos , Concentração Inibidora 50 , Levodopa/metabolismo , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Lisossomos/patologia , Melaninas/metabolismo , Melanoma/metabolismo , Melanoma/patologia , Camundongos , Monofenol Mono-Oxigenase/metabolismo , Proteínas de Neoplasias/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismoRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Cordyceps sinensis is a fungus used in traditional Chinese medicine as a tonic to soothe the lung for the treatment of fatigue and respiratory diseases. Idiopathic pulmonary fibrosis is a chronic, irreversible and debilitating lung disease showing fibroblast/myofibroblast expansion and excessive deposition of extracellular matrix in the interstitium leading to breathing difficulty. Our previous observation revealed a partial relief of lung fibrosis in patients suffering from severe acute respiratory syndrome (SARS). We hypothesize that Cordyceps has beneficial effects on lung fibrosis and the objective of this study is to explore the target(s) of Cordyceps in the relief of lung fibrosis in animal and cell models and to gain insight into its underlying mechanisms. MATERIAL AND METHODS: A rat model of bleomycin (BLM)-induced lung fibrosis and a fibrotic cell model with transforming growth factor beta-1 induction were employed in the studies. RESULTS: Reduction of infiltration of inflammatory cells, deposition of fibroblastic loci and collagen, formation of reactive oxygen species, and production of cytokines, as well as recovery from imbalance of MMP-9/TIMP-1, were observed in fibrotic rats after treatment with Cordyceps in preventive (from the day of BLM administration) and therapeutic (from 14 days after BLM) regimens. In a fibrotic cell model with transforming growth factor beta-1 induction, the human lung epithelial A549 acquired a mesenchymal phenotype and an increase of vimentin expression with a concomitant decrease of E-cadherin. This epithelial-mesenchymal transition could be partially reverted by cordycepin, a major component of Cordyceps. CONCLUSION: The findings provide an insight into the preventive and therapeutic potentials of Cordyceps for the treatment of lung fibrosis.