RESUMO
The structural maintenance of chromosomes (SMC) protein complexes-cohesin, condensin, and the Smc5/6 complex (Smc5/6)-are essential for chromosome function. At the molecular level, these complexes fold DNA by loop extrusion. Accordingly, cohesin creates chromosome loops in interphase, and condensin compacts mitotic chromosomes. However, the role of Smc5/6's recently discovered DNA loop extrusion activity is unknown. Here, we uncover that Smc5/6 associates with transcription-induced positively supercoiled DNA at cohesin-dependent loop boundaries on budding yeast (Saccharomyces cerevisiae) chromosomes. Mechanistically, single-molecule imaging reveals that dimers of Smc5/6 specifically recognize the tip of positively supercoiled DNA plectonemes and efficiently initiate loop extrusion to gather the supercoiled DNA into a large plectonemic loop. Finally, Hi-C analysis shows that Smc5/6 links chromosomal regions containing transcription-induced positive supercoiling in cis. Altogether, our findings indicate that Smc5/6 controls the three-dimensional organization of chromosomes by recognizing and initiating loop extrusion on positively supercoiled DNA.
Assuntos
Proteínas de Ciclo Celular , Proteínas de Saccharomyces cerevisiae , Proteínas de Ciclo Celular/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , DNA Super-Helicoidal/genética , Coesinas , DNA/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Cromossomos/metabolismoRESUMO
Structural maintenance of chromosomes (SMC) protein complexes are essential for the spatial organization of chromosomes1. Whereas cohesin and condensin organize chromosomes by extrusion of DNA loops, the molecular functions of the third eukaryotic SMC complex, Smc5/6, remain largely unknown2. Using single-molecule imaging, we show that Smc5/6 forms DNA loops by extrusion. Upon ATP hydrolysis, Smc5/6 reels DNA symmetrically into loops at a force-dependent rate of one kilobase pair per second. Smc5/6 extrudes loops in the form of dimers, whereas monomeric Smc5/6 unidirectionally translocates along DNA. We also find that the subunits Nse5 and Nse6 (Nse5/6) act as negative regulators of loop extrusion. Nse5/6 inhibits loop-extrusion initiation by hindering Smc5/6 dimerization but has no influence on ongoing loop extrusion. Our findings reveal functions of Smc5/6 at the molecular level and establish DNA loop extrusion as a conserved mechanism among eukaryotic SMC complexes.
Assuntos
Proteínas de Ciclo Celular , Cromossomos Fúngicos , DNA Fúngico , Saccharomyces cerevisiae , Trifosfato de Adenosina/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona , Cromossomos Fúngicos/química , Cromossomos Fúngicos/metabolismo , DNA Fúngico/química , DNA Fúngico/metabolismo , Hidrólise , Complexos Multiproteicos , Imagem Individual de Molécula , CoesinasRESUMO
Although our knowledge of chromatin organization has advanced significantly in recent years, much about the relationships between different features of genome architecture is still unknown. Folding of mammalian genomes into spatial domains is thought to depend on architectural proteins, other DNA-binding proteins, and different forms of RNA. In addition, emerging evidence points towards the possibility that the three-dimensional organisation of the genome is controlled by DNA topology. In this scenario, cohesin, CCCTC-binding factor (CTCF), transcription, DNA supercoiling, and topoisomerases are integrated to dictate different layers of genome organization, and the contribution of all four to gene control is an important direction of future studies. In this perspective, we review recent studies that give new insight on how DNA supercoiling shape chromatin structure.
Assuntos
Proteínas de Ciclo Celular/química , Cromatina/química , Proteínas Cromossômicas não Histona/química , DNA Topoisomerases/química , DNA Super-Helicoidal/química , Conformação de Ácido Nucleico , Animais , Fator de Ligação a CCCTC/química , Fator de Ligação a CCCTC/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , DNA Topoisomerases/metabolismo , DNA Super-Helicoidal/metabolismo , Genoma , Humanos , RNA/química , RNA/metabolismo , Transcrição Gênica , CoesinasRESUMO
Targeted protein degradation systems developed for eukaryotes employ cytoplasmic machineries to perform proteolysis. This has prevented mitochondria-specific analysis of proteins that localize to multiple locations, for example, the mitochondria and the nucleus. Here, we present an inducible mitochondria-specific protein degradation system in Saccharomyces cerevisiae based on the Mesoplasma florum Lon (mf-Lon) protease and its corresponding ssrA tag (called PDT). We show that mitochondrially targeted mf-Lon protease efficiently and selectively degrades a PDT-tagged reporter protein localized to the mitochondrial matrix. The degradation can be induced by depleting adenine from the medium, and tuned by altering the promoter strength of the MF-LON gene. We furthermore demonstrate that mf-Lon specifically degrades endogenous, PDT-tagged mitochondrial proteins. Finally, we show that mf-Lon-dependent PDT degradation can also be achieved in human mitochondria. In summary, this system provides an efficient tool to selectively analyze the mitochondrial function of dually localized proteins.
Assuntos
Mitocôndrias , Protease La , Humanos , Proteólise , Mitocôndrias/metabolismo , Protease La/genética , Protease La/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
Genome function depends on regulated chromosome folding, and loop extrusion by the protein complex cohesin is essential for this multilayered organization. The chromosomal positioning of cohesin is controlled by transcription, and the complex also localizes to stalled replication forks. However, the role of transcription and replication in chromosome looping remains unclear. Here, we show that reduction of chromosome-bound RNA polymerase weakens normal cohesin loop extrusion boundaries, allowing cohesin to form new long-range chromosome cis interactions. Stress response genes induced by transcription inhibition are also shown to act as new loop extrusion boundaries. Furthermore, cohesin loop extrusion during early S phase is jointly controlled by transcription and replication units. Together, the results reveal that replication and transcription machineries are chromosome-folding regulators that block the progression of loop-extruding cohesin, opening for new perspectives on cohesin's roles in genome function and stability.
RESUMO
In C. elegans, the conserved transcription factor DAF-16/FOXO is a powerful aging regulator, relaying dire conditions into expression of stress resistance and longevity promoting genes. For some of these functions, including low insulin/IGF signaling (IIS), DAF-16 depends on the protein SMK-1/SMEK, but how SMK-1 exerts this role has remained unknown. We show that SMK-1 functions as part of a specific Protein Phosphatase 4 complex (PP4SMK-1). Loss of PP4SMK-1 hinders transcriptional initiation at several DAF-16-activated genes, predominantly by impairing RNA polymerase II recruitment to their promoters. Search for the relevant substrate of PP4SMK-1 by phosphoproteomics identified the conserved transcriptional regulator SPT-5/SUPT5H, whose knockdown phenocopies the loss of PP4SMK-1. Phosphoregulation of SPT-5 is known to control transcriptional events such as elongation and termination. Here we also show that transcription initiating events are influenced by the phosphorylation status of SPT-5, particularly at DAF-16 target genes where transcriptional initiation appears rate limiting, rendering PP4SMK-1 crucial for many of DAF-16's physiological roles.
Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Fatores de Transcrição Forkhead/genética , Regulação da Expressão Gênica/genética , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , Fatores de Elongação da Transcrição/metabolismo , Envelhecimento/genética , Animais , Caenorhabditis elegans/genética , Proteínas Cromossômicas não Histona/genética , Longevidade/genética , Complexos Multiproteicos/metabolismo , Interferência de RNA , RNA Polimerase II/metabolismo , Estresse Fisiológico/genética , Transcrição Gênica/genética , Fatores de Elongação da Transcrição/genéticaRESUMO
Pseudomonas are a common cause of hospital-acquired infections that may be lethal. ADP-ribosyltransferase activities of Pseudomonas exotoxin-S and -T depend on 14-3-3 proteins inside the host cell. By binding in the 14-3-3 phosphopeptide binding groove, an amphipathic C-terminal helix of ExoS and ExoT has been thought to be crucial for their activation. However, crystal structures of the 14-3-3ß:ExoS and -ExoT complexes presented here reveal an extensive hydrophobic interface that is sufficient for complex formation and toxin activation. We show that C-terminally truncated ExoS ADP-ribosyltransferase domain lacking the amphipathic binding motif is active when co-expressed with 14-3-3. Moreover, swapping the amphipathic C-terminus with a fragment from Vibrio Vis toxin creates a 14-3-3 independent toxin that ADP-ribosylates known ExoS targets. Finally, we show that 14-3-3 stabilizes ExoS against thermal aggregation. Together, this indicates that 14-3-3 proteins activate exotoxin ADP-ribosyltransferase domains by chaperoning their hydrophobic surfaces independently of the amphipathic C-terminal segment.