RESUMO
Heat-shocked cells prioritize the translation of heat shock (HS) mRNAs, but the underlying mechanism is unclear. We report that HS in budding yeast induces the disassembly of the eIF4F complex, where eIF4G and eIF4E assemble into translationally arrested mRNA ribonucleoprotein particles (mRNPs) and HS granules (HSGs), whereas eIF4A promotes HS translation. Using in vitro reconstitution biochemistry, we show that a conformational rearrangement of the thermo-sensing eIF4A-binding domain of eIF4G dissociates eIF4A and promotes the assembly with mRNA into HS-mRNPs, which recruit additional translation factors, including Pab1p and eIF4E, to form multi-component condensates. Using extracts and cellular experiments, we demonstrate that HS-mRNPs and condensates repress the translation of associated mRNA and deplete translation factors that are required for housekeeping translation, whereas HS mRNAs can be efficiently translated by eIF4A. We conclude that the eIF4F complex is a thermo-sensing node that regulates translation during HS.
Assuntos
Fator de Iniciação 4F em Eucariotos , Fator de Iniciação Eucariótico 4G , Resposta ao Choque Térmico , Proteínas de Ligação a Poli(A) , Biossíntese de Proteínas , RNA Mensageiro , Ribonucleoproteínas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Resposta ao Choque Térmico/genética , Fator de Iniciação 4F em Eucariotos/metabolismo , Fator de Iniciação 4F em Eucariotos/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fator de Iniciação Eucariótico 4G/metabolismo , Fator de Iniciação Eucariótico 4G/genética , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas/genética , Fator de Iniciação 4E em Eucariotos/metabolismo , Fator de Iniciação 4E em Eucariotos/genética , Fator de Iniciação 4A em Eucariotos/metabolismo , Fator de Iniciação 4A em Eucariotos/genética , Regulação Fúngica da Expressão Gênica , Ligação Proteica , RNA Fúngico/metabolismo , RNA Fúngico/genéticaRESUMO
CTCF is crucial to the organization of mammalian genomes into loop structures. According to recent studies, the transcription apparatus is compartmentalized and concentrated at super-enhancers to form phase-separated condensates and drive the expression of cell-identity genes. However, it remains unclear whether and how transcriptional condensates are coupled to higher-order chromatin organization. Here, we show that CTCF is essential for RNA polymerase II (Pol II)-mediated chromatin interactions, which occur as hyperconnected spatial clusters at super-enhancers. We also demonstrate that CTCF clustering, unlike Pol II clustering, is independent of liquid-liquid phase-separation and resistant to perturbation of transcription. Interestingly, clusters of Pol II, BRD4, and MED1 were found to dissolve upon CTCF depletion, but were reinstated upon restoration of CTCF, suggesting a potent instructive function for CTCF in the formation of transcriptional condensates. Overall, we provide evidence suggesting that CTCF-mediated chromatin looping acts as an architectural prerequisite for the assembly of phase-separated transcriptional condensates.
Assuntos
Fator de Ligação a CCCTC/metabolismo , Montagem e Desmontagem da Cromatina , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cromatina/química , Cromatina/genética , Cromatina/metabolismo , Epigênese Genética , Células HCT116 , Humanos , Subunidade 1 do Complexo Mediador/genética , Subunidade 1 do Complexo Mediador/metabolismo , RNA Polimerase II/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
One of the most commonly used drugs in chemotherapy, 5-fluorouracil (5-FU) has been shown to be effective in only 10-15% of patients with colon cancer. Thus, studies of the mechanisms affecting 5-FU sensitivity in these patients are necessary. The tumor suppressor protein p53 is a transcription factor that serves important roles in cell apoptosis by regulating the cell cycle. It has also been characterized as a key factor influencing drug sensitivity. Furthermore, accessible chromatin is a hallmark of active DNA regulatory elements and functions as a crucial epigenetic factor regulating cancer mechanisms. The present study assessed the genetic regulatory landscape in colon cancer by performing RNA sequencing and Assay for Transposase-Accessible Chromatin sequencing, and investigated the effects of 5-FU on chromatin accessibility and gene expression. Notably, while treatment with 5-FU mediated global increases in chromatin accessibility, chromatin organization in several genomic regions differed depending on the expression status of p53. Since the occupancy of p53 does not overlap with accessible chromatin regions, the 5-FU-mediated changes in chromatin accessibility were not regulated by direct binding of p53. In the p53-expressing condition, the 5-FU-mediated accessible chromatin region was primarily associated with genes encoding cell death pathways. Additionally, 5-FU was revealed to induce open chromatin conformation at regions containing binding motifs for AP-1 family transcription factors, which may drive expression of apoptosis pathway genes. In conclusion, expression of p53 may confer 5-FU sensitivity by regulating chromatin accessibility of distinct genes associated with cell apoptosis in a transcription-independent manner.