RESUMO
Young sex chromosomes possess unique and ongoing dynamics that allow us to understand processes that have an impact on their evolution and divergence. The genus Silene includes species with evolutionarily young sex chromosomes, and two species of section Melandrium, namely Silene latifolia (24, XY) and Silene dioica (24, XY), are well-established models of sex chromosome evolution, Y chromosome degeneration, and sex determination. In both species, the X and Y chromosomes are strongly heteromorphic and differ in the genomic composition compared to the autosomes. It is generally accepted that for proper cell division, the longest chromosomal arm must not exceed half of the average length of the spindle axis at telophase. Yet, it is not clear what are the dynamics between males and females during mitosis and how the cell compensates for the presence of the large Y chromosome in one sex. Using hydroxyurea cell synchronization and 2D/3D microscopy, we determined the position of the sex chromosomes during the mitotic cell cycle and determined the upper limit for the expansion of sex chromosome non-recombining region. Using 3D specimen preparations, we found that the velocity of the large chromosomes is compensated by the distant positioning from the central interpolar axis, confirming previous mathematical modulations.
Assuntos
Cromátides/fisiologia , Cromossomos Sexuais/fisiologia , Silene/fisiologia , Cromossomos de Plantas/fisiologia , Evolução Molecular , Hidroxiureia/farmacologia , Hibridização in Situ Fluorescente , Microscopia Confocal , Mitose , Silene/genéticaRESUMO
OBJECTIVE: Poly(ADP-ribose) polymerase1 (PARP1) interacts and poly(ADP-ribosyl)ates telomere repeat binding factor 2 (TRF2), which acts as a platform to recruit a large number of proteins at the telomere. Since the discovery of TRF2-SLX4 interaction, SLX4 is becoming the key player in telomere length (TL) maintenance and repair by telomere sister chromatid exchange (T-SCE). Defective TL maintenance pathway results in a spectrum of diseases called telomeropathies like dyskeratosis congenita, aplastic anemia, fanconi anemia, cancer. We aimed to study the role of SLX4 and PARP1 on each other's telomere localization, T-SCE, and TL maintenance in human telomerase-negative osteosarcoma U2OS cells to understand some of the molecular mechanisms of telomere homeostasis. MATERIALS AND METHODS: We checked the role of SLX4 and PARP1 on each other's telomere localization by telomere immunofluorescence. We have cloned full-length wild-type and catalytically inactive mutant PARP1 to understand the role of poly(ADP-ribosyl)ation reaction by PARP1 in telomere length homeostasis. TL of U2OS cells was measured by Q-FISH. T-SCE was measured by Telomere-FISH. KEY FINDINGS: We observed that SLX4 has no role in the telomere localization of PARP1. However, reduced localization of SLX4 at undamaged and damaged telomere upon PARP1 depletion was reversed by overexpression of exogenous wild-type PARP1 but not by overexpression of catalytically inactive mutant PARP1. PARP1 depletion synergized SLX4 depletion-mediated reduction of T-SCE. Furthermore, SLX4 depletion elongated TL, and combined insufficiency of SLX4 with PARP1 further elongated TL. CONCLUSION: So, PARP1 controls SLX4 recruitment at telomere by poly(ADP-ribosyl)ation reaction, thereby regulating SLX4-mediated T-SCE and TL homeostasis.
Assuntos
Poli(ADP-Ribose) Polimerase-1/metabolismo , Recombinases/metabolismo , Troca de Cromátide Irmã/fisiologia , Linhagem Celular Tumoral , Cromátides/metabolismo , Cromátides/fisiologia , Reparo do DNA , Homeostase , Humanos , Poli(ADP-Ribose) Polimerase-1/fisiologia , Poli(ADP-Ribose) Polimerases/genética , Recombinases/genética , Recombinases/fisiologia , Telomerase/metabolismo , Telômero/fisiologia , Homeostase do Telômero/fisiologia , Proteína 2 de Ligação a Repetições Teloméricas/metabolismoRESUMO
Cohesin helps mediate sister chromatid cohesion, chromosome condensation, DNA repair, and transcription regulation. We exploited proximity-dependent labeling to define the in vivo interactions of cohesin domains with DNA or with other cohesin domains that lie within the same or in different cohesin complexes. Our results suggest that both cohesin's head and hinge domains are proximal to DNA, and cohesin structure is dynamic with differential folding of its coiled coil regions to generate butterfly confirmations. This method also reveals that cohesins form ordered clusters on and off DNA. The levels of cohesin clusters and their distribution on chromosomes are cell cycle-regulated. Cohesin clustering is likely necessary for cohesion maintenance because clustering and maintenance uniquely require the same subset of cohesin domains and the auxiliary cohesin factor Pds5p. These conclusions provide important new mechanistic and biological insights into the architecture of the cohesin complex, cohesin-cohesin interactions, and cohesin's tethering and loop-extruding activities.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/genética , Cromátides/fisiologia , Proteínas Cromossômicas não Histona/genética , Cromossomos Fúngicos , Reparo do DNA , Domínios Proteicos , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , CoesinasRESUMO
Nonrandom DNA segregation (NDS) is a mitotic event in which sister chromatids carrying the old (parent) DNA strands are distributed exclusively to one of the two daughter cells. Although this phenomenon occurs in multiple organisms, the low frequency poses an obstacle to observation. Here, we present an improved protocol to induce NDS under replication stress. This protocol can be modified to accommodate various cell lines. For complete details on the use and execution of this protocol, please refer to Xing et al. (2020).
Assuntos
Segregação de Cromossomos/efeitos dos fármacos , Replicação do DNA/fisiologia , Microscopia de Fluorescência/métodos , Linhagem Celular , Cromátides/metabolismo , Cromátides/fisiologia , Segregação de Cromossomos/genética , Segregação de Cromossomos/fisiologia , DNA/genética , Replicação do DNA/genética , Imunofluorescência/métodos , Humanos , Mitose/genética , Coloração e Rotulagem/métodosRESUMO
Chromosome fusion is a frequent intermediate in oncogenic chromosome rearrangements and has been proposed to cause multiple tumor-driving abnormalities. In conventional experimental systems, however, these abnormalities were often induced by randomly induced chromosome fusions involving multiple different chromosomes. It was therefore not well understood whether a single defined type of chromosome fusion, which is reminiscent of a sporadic fusion in tumor cells, has the potential to cause chromosome instabilities. Here, we developed a human cell-based sister chromatid fusion visualization system (FuVis), in which a single defined sister chromatid fusion is induced by CRISPR/Cas9 concomitantly with mCitrine expression. The fused chromosome subsequently developed extra-acentric chromosomes, including chromosome scattering, indicative of chromothripsis. Live-cell imaging and statistical modeling indicated that sister chromatid fusion generated micronuclei (MN) in the first few cell cycles and that cells with MN tend to display cell cycle abnormalities. The powerful FuVis system thus demonstrates that even a single sporadic sister chromatid fusion can induce chromosome instability and destabilize the cell cycle through MN formation.
Assuntos
Instabilidade Cromossômica/genética , Análise de Célula Única/métodos , Troca de Cromátide Irmã/fisiologia , Sistemas CRISPR-Cas/genética , Ciclo Celular/genética , Divisão Celular/genética , Cromátides/genética , Cromátides/patologia , Cromátides/fisiologia , Instabilidade Cromossômica/fisiologia , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Engenharia Genética/métodos , Células HCT116 , Humanos , Microscopia de Fluorescência/métodos , Neoplasias/genética , Troca de Cromátide Irmã/genéticaRESUMO
Chromosome segregation requires both compaction and disentanglement of sister chromatids. We describe SisterC, a chromosome conformation capture assay that distinguishes interactions between and along identical sister chromatids. SisterC employs 5-bromo-2'-deoxyuridine (BrdU) incorporation during S-phase to label newly replicated strands, followed by Hi-C and then the destruction of 5-bromodeoxyuridine-containing strands via Hoechst/ultraviolet treatment. After sequencing of the remaining intact strands, this allows assignment of Hi-C products as inter- and intra-sister interactions based on the strands that reads are mapped to. We performed SisterC on mitotic Saccharomyces cerevisiae cells. We find precise alignment of sister chromatids at centromeres. Along arms, sister chromatids are less precisely aligned, with inter-sister connections every ~35 kilobase (kb). Inter-sister interactions occur between cohesin binding sites that are often offset by 5 to 25 kb. Along sister chromatids, cohesin results in the formation of loops of up to 50 kb. SisterC allows study of the complex interplay between sister chromatid compaction and their segregation during mitosis.
Assuntos
Cromátides/fisiologia , Cromatina/fisiologia , Segregação de Cromossomos/fisiologia , Animais , Reparo do DNA , Replicação do DNA , Regulação da Expressão Gênica , Mitose/fisiologia , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologiaRESUMO
Sister-chromatid cohesion describes the orderly association of newly replicated DNA molecules behind replication forks. It plays an essential role in the maintenance and faithful transmission of genetic information. Cohesion is created by DNA topological links and proteinaceous bridges, whose formation and deposition could be potentially affected by many processes. Current knowledge on cohesion has been mainly gained by fluorescence microscopy observation. However, the resolution limit of microscopy and the restricted number of genomic positions that can be simultaneously visualized considerably hampered progress. Here, we present a high-throughput methodology to monitor sister-chromatid contacts (Hi-SC2). Using the multi-chromosomal Vibrio cholerae bacterium as a model, we show that Hi-SC2 permits to monitor local variations in sister-chromatid cohesion at a high resolution over a whole genome.
Assuntos
Cromátides/fisiologia , Técnicas Genéticas , Vibrio cholerae/genética , Cromossomos Bacterianos/fisiologia , Replicação do DNA , DNA Bacteriano , Sequenciamento de Nucleotídeos em Larga Escala , Integrases/metabolismo , Conformação de Ácido NucleicoRESUMO
During cell division, mitotic chromosomes assemble and are equally distributed into two new daughter cells. The chromosome organisation of the two chromatids is essential for even distribution of genetic materials. Although the 11-nm fibre or nucleosome structure is well-understood as a fundamental fibrous structure of chromosomes, the reports on organisation of 30-nm basic chromatin fibres have been controversial, with debates on the contribution of 30-nm or thicker fibres to the higher order inner structure of chromosomes. Here, we used focused ion beam/scanning electron microscopy (FIB/SEM) to show that both 11-nm and 30-nm fibres are present in the human metaphase chromosome, although the higher-order periodical structure could not be detected under the conditions employed. We directly dissected the chromosome every 10-nm and observed 224 cross-section SEM images. We demonstrated that the chromosome consisted of chromatin fibres of an average diameter of 16.9-nm. The majority of the chromatin fibres had diameters between 5 and 25-nm, while those with 30-nm were in the minority. The reduced packaging ratio of the chromatin fibres was detected at axial regions of each chromatid. Our results provide a strong basis for further discussions on the chromosome higher-order structure.
Assuntos
Cromatina/fisiologia , Cromossomos/metabolismo , Metáfase/fisiologia , Cromátides/metabolismo , Cromátides/fisiologia , Cromatina/metabolismo , Cromossomos/genética , Cromossomos Humanos , Células HeLa , Humanos , Microscopia Eletrônica de Varredura , Nucleossomos/fisiologiaRESUMO
Meiotic pairing between parental chromosomes (homologs) is required for formation of haploid gametes. Homolog pairing depends on recombination initiation via programmed double-strand breaks (DSBs). Although DSBs appear prior to pairing, the homolog, rather than the sister chromatid, is used as repair partner for crossing over. Here, we show that Mph1, the budding yeast ortholog of Fanconi anemia helicase FANCM, prevents precocious DSB strand exchange between sister chromatids before homologs have completed pairing. By dissociating precocious DNA displacement loops (D-loops) between sister chromatids, Mph1FANCM ensures high levels of crossovers and non-crossovers between homologs. Later-occurring recombination events are protected from Mph1-mediated dissociation by synapsis protein Zip1. Increased intersister repair in absence of Mph1 triggers a shift among remaining interhomolog events from non-crossovers to crossover-specific strand exchange, explaining Mph1's apparent anti-crossover function. Our findings identify temporal coordination between DSB strand exchange and homolog pairing as a critical determinant for recombination outcome.
Assuntos
Cromossomos Fúngicos/genética , RNA Helicases DEAD-box/metabolismo , Recombinação Homóloga , Meiose , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Cromátides/fisiologia , Segregação de Cromossomos , RNA Helicases DEAD-box/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Proteínas Nucleares/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Cohesin is a conserved, ring-shaped protein complex that topologically entraps DNA. This ability makes this member of the structural maintenance of chromosomes (SMC) complex family a central hub of chromosome dynamics regulation. Besides its essential role in sister chromatid cohesion, cohesin shapes the interphase chromatin domain architecture and plays important roles in transcriptional regulation and DNA repair. Cohesin is loaded onto chromosomes at centromeres, at the promoters of highly expressed genes, as well as at DNA replication forks and sites of DNA damage. However, the features that determine these binding sites are still incompletely understood. We recently described a role of the budding yeast RSC chromatin remodeler in cohesin loading onto chromosomes. RSC has a dual function, both as a physical chromatin receptor of the Scc2/Scc4 cohesin loader complex, as well as by providing a nucleosome-free template for cohesin loading. Here, we show that the role of RSC in sister chromatid cohesion is conserved in fission yeast. We discuss what is known about the broader conservation of the contribution of chromatin remodelers to cohesin loading onto chromatin.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Cromátides/fisiologia , Montagem e Desmontagem da Cromatina , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/genética , Adenosina Trifosfatases/metabolismo , Cromatina/genética , Cromossomos Fúngicos/genética , Cromossomos Fúngicos/metabolismo , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Fatores de Transcrição/metabolismo , CoesinasRESUMO
Concomitant with DNA replication, the chromosomal cohesin complex establishes cohesion between newly replicated sister chromatids. Several replication-fork-associated "cohesion establishment factors," including the multifunctional Ctf18-RFC complex, aid this process in as yet unknown ways. Here, we show that Ctf18-RFC's role in sister chromatid cohesion correlates with PCNA loading but is separable from its role in the replication checkpoint. Ctf18-RFC loads PCNA with a slight preference for the leading strand, which is dispensable for DNA replication. Conversely, the canonical Rfc1-RFC complex preferentially loads PCNA onto the lagging strand, which is crucial for DNA replication but dispensable for sister chromatid cohesion. The downstream effector of Ctf18-RFC is cohesin acetylation, which we place toward a late step during replication maturation. Our results suggest that Ctf18-RFC enriches and balances PCNA levels at the replication fork, beyond the needs of DNA replication, to promote establishment of sister chromatid cohesion and possibly other post-replicative processes.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Cromátides/fisiologia , Proteínas Cromossômicas não Histona/metabolismo , Cromossomos Fúngicos/fisiologia , Replicação do DNA , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Acetiltransferases/genética , Acetiltransferases/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/genética , Segregação de Cromossomos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Antígeno Nuclear de Célula em Proliferação/genética , Proteína de Replicação C/genética , Proteína de Replicação C/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , CoesinasRESUMO
Meiosis produces gametes through a specialized, two-step cell division, which is highly error prone in humans. Reductional meiosis I, where maternal and paternal chromosomes (homologs) segregate, is followed by equational meiosis II, where sister chromatids separate. Uniquely during meiosis I, sister kinetochores are monooriented and pericentromeric cohesin is protected. Here, we demonstrate that these key adaptations for reductional chromosome segregation are achieved through separable control of multiple kinases by the meiosis-I-specific budding yeast Spo13 protein. Recruitment of Polo kinase to kinetochores directs monoorientation, while independently, cohesin protection is achieved by containing the effects of cohesin kinases. Therefore, reductional chromosome segregation, the defining feature of meiosis, is established by multifaceted kinase control by a master regulator. The recent identification of Spo13 orthologs, fission yeast Moa1 and mouse MEIKIN, suggests that kinase coordination by a meiosis I regulator may be a general feature in the establishment of reductional chromosome segregation.
Assuntos
Segregação de Cromossomos/fisiologia , Cinetocoros/fisiologia , Meiose/fisiologia , Proteínas de Ciclo Celular/metabolismo , Centrômero/metabolismo , Cromátides/fisiologia , Proteínas Cromossômicas não Histona/metabolismo , Segregação de Cromossomos/genética , Cinetocoros/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/fisiologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas de Schizosaccharomyces pombe/fisiologia , CoesinasRESUMO
Cohesin is a fundamental protein complex that holds sister chromatids together. Separase protease cleaves a cohesin subunit Rad21/SCC1, causing the release of cohesin from DNA to allow chromosome segregation. To understand the functional organization of cohesin, we employed next-generation whole-genome sequencing and identified numerous extragenic suppressors that overcome either inactive separase/Cut1 or defective cohesin in the fission yeast Schizosaccharomyces pombe Unexpectedly, Cut1 is dispensable if suppressor mutations cause disorders of interfaces among essential cohesin subunits Psm1/SMC1, Psm3/SMC3, Rad21/SCC1, and Mis4/SCC2, the crystal structures of which suggest physical and functional impairment at the interfaces of Psm1/3 hinge, Psm1 head-Rad21, or Psm3 coiled coil-Rad21. Molecular-dynamics analysis indicates that the intermolecular ß-sheets in the cohesin hinge of cut1 suppressor mutants remain intact, but a large mobility change occurs at the coiled coil bound to the hinge. In contrast, suppressors of rad21-K1 occur in either the head ATPase domains or the Psm3 coiled coil that interacts with Rad21. Suppressors of mis4-G1326E reside in the head of Psm3/1 or the intragenic domain of Mis4. These may restore the binding of cohesin to DNA. Evidence is provided that the head and hinge of SMC subunits are proximal, and that they coordinate to form arched coils that can hold or release DNA by altering the angles made by the arched coiled coils. By combining molecular modeling with suppressor sequence analysis, we propose a cohesin structure designated the "hold-and-release" model, which may be considered as an alternative to the prevailing "ring" model.
Assuntos
Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/metabolismo , DNA Fúngico/metabolismo , Mutação , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/genética , Proteínas de Ciclo Celular/genética , Cromátides/fisiologia , Proteínas Cromossômicas não Histona/genética , Segregação de Cromossomos , DNA Fúngico/genética , Modelos Moleculares , Proteínas Nucleares/genética , Fosfoproteínas/genética , Fosforilação , Conformação Proteica , Subunidades Proteicas , Schizosaccharomyces/crescimento & desenvolvimento , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Supressão Genética , CoesinasRESUMO
Cohesin is a conserved protein complex required for sister chromatid cohesion, chromosome condensation, DNA damage repair, and regulation of transcription. Although cohesin functions to tether DNA duplexes, the contribution of its individual domains to this activity remains poorly understood. We interrogated the Smc3p subunit of cohesin by random insertion mutagenesis. Analysis of a mutant in the Smc3p hinge revealed an unexpected role for this domain in cohesion maintenance and condensation. Further investigation revealed that the Smc3p hinge functions at a step following cohesin's stable binding to chromosomes and independently of Smc3p's regulation by the Eco1p acetyltransferase. Hinge mutant phenotypes resemble loss of Pds5p, which binds opposite the hinge near Smc3p's head domain. We propose that a specific conformation of the Smc3p hinge and Pds5p cooperate to promote cohesion maintenance and condensation.
Assuntos
Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/fisiologia , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/fisiologia , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/fisiologia , Proteínas de Ciclo Celular/metabolismo , Cromátides/genética , Cromátides/fisiologia , Proteínas Cromossômicas não Histona/metabolismo , Segregação de Cromossomos/genética , Cromossomos Fúngicos/metabolismo , Reparo do DNA , Mutação , Proteínas Nucleares/metabolismo , Domínios Proteicos/genética , Domínios Proteicos/fisiologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , CoesinasRESUMO
Three key steps in meiosis allow diploid organisms to produce haploid gametes: (1) homologous chromosomes (homologs) pair and undergo crossovers; (2) homologs segregate to opposite poles; and (3) sister chromatids segregate to opposite poles. The XX/XO sex determination system found in many nematodes [1] facilitates the study of meiosis because variation is easily recognized [2-4]. Here we show that meiotic segregation of X chromosomes in the trioecious nematode Auanema rhodensis [5] varies according to sex (hermaphrodite, female, or male) and type of gametogenesis (oogenesis or spermatogenesis). In this species, XO males exclusively produce X-bearing sperm [6, 7]. The unpaired X precociously separates into sister chromatids, which co-segregate with the autosome set to generate a functional haplo-X sperm. The other set of autosomes is discarded into a residual body. Here we explore the X chromosome behavior in female and hermaphrodite meioses. Whereas X chromosomes segregate following the canonical pattern during XX female oogenesis to yield haplo-X oocytes, during XX hermaphrodite oogenesis they segregate to the first polar body to yield nullo-X oocytes. Thus, crosses between XX hermaphrodites and males yield exclusively male progeny. During hermaphrodite spermatogenesis, the sister chromatids of the X chromosomes separate during meiosis I, and homologous X chromatids segregate to the functional sperm to create diplo-X sperm. Given these intra-species, intra-individual, and intra-gametogenesis variations in the meiotic program, A. rhodensis is an ideal model for studying the plasticity of meiosis and how it can be modulated.
Assuntos
Cromátides/fisiologia , Segregação de Cromossomos/fisiologia , Rhabditoidea/fisiologia , Cromossomo X/fisiologia , Animais , Feminino , Organismos Hermafroditas/genética , Organismos Hermafroditas/fisiologia , Masculino , Meiose , Oogênese/fisiologia , Rhabditoidea/genética , Espermatogênese/fisiologiaRESUMO
Condensins I and II are multisubunit complexes that play a central role in mitotic chromosome assembly. Although both complexes become concentrated along the axial region of each chromatid by metaphase, it remains unclear exactly how such axes might assemble and contribute to chromosome shaping. To address these questions from a physico-chemical point of view, we have established a set of two-step protocols for inducing reversible assembly of chromosome structure in situ, namely within a whole cell. In this assay, mitotic chromosomes are first expanded in a hypotonic buffer containing a Mg2+-chelating agent and then converted into different shapes in a NaCl concentration-dependent manner. Both chromatin and condensin-positive chromosome axes are converted into near-original shapes at 100 mM NaCl. This assay combined with small interfering RNA depletion demonstrates that the recovery of chromatin shapes and the reorganization of axes are highly sensitive to depletion of condensin II but less sensitive to depletion of condensin I or topoisomerase IIα. Furthermore, quantitative morphological analyses using the machine-learning algorithm wndchrm support the notion that chromosome shaping is tightly coupled to the reorganization of condensin II-based axes. We propose that condensin II makes a primary contribution to mitotic chromosome architecture and maintenance in human cells.
Assuntos
Adenosina Trifosfatases/fisiologia , Cromatina/fisiologia , Cromossomos Humanos/fisiologia , Proteínas de Ligação a DNA/fisiologia , Complexos Multiproteicos/fisiologia , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Proteínas de Ciclo Celular/genética , Cromátides/química , Cromátides/fisiologia , Cromatina/química , Proteínas Cromossômicas não Histona/genética , Cromossomos Humanos/genética , Cromossomos Humanos/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células HeLa , Humanos , Mitose/fisiologia , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Proteínas Nucleares/genética , RNA Interferente PequenoRESUMO
Sister chromatids are tethered together by the cohesin complex from the time they are made until their separation at anaphase. The ability of cohesin to tether sister chromatids together depends on acetylation of its Smc3 subunit by members of the Eco1 family of cohesin acetyltransferases. Vertebrates express two orthologs of Eco1, called Esco1 and Esco2, both of which are capable of modifying Smc3, but their relative contributions to sister chromatid cohesion are unknown. We therefore set out to determine the precise contributions of Esco1 and Esco2 to cohesion in vertebrate cells. Here we show that cohesion establishment is critically dependent upon Esco2. Although most Smc3 acetylation is Esco1 dependent, inactivation of the ESCO1 gene has little effect on mitotic cohesion. The unique ability of Esco2 to promote cohesion is mediated by sequences in the N terminus of the protein. We propose that Esco1-dependent modification of Smc3 regulates almost exclusively the noncohesive activities of cohesin, such as DNA repair, transcriptional control, chromosome loop formation, and/or stabilization. Collectively, our data indicate that Esco1 and Esco2 contribute to distinct and separable activities of cohesin in vertebrate cells.
Assuntos
Acetiltransferases/metabolismo , Cromátides/fisiologia , Proteínas Cromossômicas não Histona/metabolismo , Acetilação , Acetiltransferases/fisiologia , Sequência de Bases , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/fisiologia , Divisão Celular/fisiologia , Cromátides/metabolismo , Proteínas Cromossômicas não Histona/fisiologia , Segregação de Cromossomos/fisiologia , Replicação do DNA/fisiologia , Regulação da Expressão Gênica/genética , Humanos , Proteínas Nucleares/metabolismo , CoesinasRESUMO
Two sister chromatids must be held together by a cohesion process from their synthesis during S phase to segregation in anaphase. Despite its pivotal role in accurate chromosome segregation, how cohesion is established remains elusive. Here, we demonstrate that yeast Rtt101-Mms1, Cul4 family E3 ubiquitin ligases are stronger dosage suppressors of loss-of-function eco1 mutants than PCNA The essential cohesion reaction, Eco1-catalyzed Smc3 acetylation is reduced in the absence of Rtt101-Mms1. One of the adaptor subunits, Mms22, associates directly with Eco1. Point mutations (L61D/G63D) in Eco1 that abolish the interaction with Mms22 impair Smc3 acetylation. Importantly, an eco1LGpol30A251V double mutant displays additive Smc3ac reduction. Moreover, Smc3 acetylation and cohesion defects also occur in the mutants of other replication-coupled nucleosome assembly (RCNA) factors upstream or downstream of Rtt101-Mms1, indicating unanticipated cross talk between histone modifications and cohesin acetylation. These data suggest that fork-associated Cul4-Ddb1 E3s, together with PCNA, coordinate chromatin reassembly and cohesion establishment on the newly replicated sister chromatids, which are crucial for maintaining genome and chromosome stability.
Assuntos
Cromátides/fisiologia , Proteínas Culina/metabolismo , Nucleossomos/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Acetilação , Acetiltransferases/genética , Acetiltransferases/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cromátides/genética , Montagem e Desmontagem da Cromatina , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Culina/genética , Replicação do DNA , Período de Replicação do DNA , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Nucleossomos/genética , Mutação Puntual , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Orderly execution of two critical events during the cell cycle--DNA replication and chromosome segregation--ensures the stable transmission of genetic materials. The cohesin complex physically connects sister chromatids during DNA replication in a process termed sister chromatid cohesion. Timely establishment and dissolution of sister chromatid cohesion is a prerequisite for accurate chromosome segregation, and is tight regulated by the cell cycle machinery and cohesin-associated proteins. In this review, we discuss recent progress in the molecular understanding of sister chromatid cohesion during the mitotic cell cycle.
Assuntos
Cromátides/fisiologia , Segregação de Cromossomos/fisiologia , Cinetocoros/fisiologia , Mitose/fisiologia , Animais , Proteínas de Ciclo Celular/metabolismo , Replicação do DNA/genética , Humanos , Microtúbulos/metabolismo , Mitose/genética , Modelos BiológicosRESUMO
Several genetic and epigenetic events that take place in the nucleus (i.e. meiotic recombination, meiotic silencing, chromatin reorganization and histone replacement) are crucial for the spermatogenesis process, as well as, is the assembling of cytoplasmic bodies (or chromatoid bodies). In this minireview, we give special attention to the most recent research approaches involved in the molecular structure and physiology of the chromatoid body (CB). Though it was described several decades ago, the CB is still a very intriguing cytoplasmic structure of male germ cells. It plays roles in the most important steps of the spermatozoon formation, such as mRNA regulation, smallRNA-mediated gene control, and cell communication among round spermatids. Studies that have been done on the CB largely focus on two main topics: (1) CB proteome, in this minireview focused on 'Evidences linking the nucleolar cycle and the CB assembling; and Circadian proteins found in the CB'; and (2) CB transcriptome, in this minireview focused on 'miRNAs and piRNAs pathways; and X but not Y chromosome transcripts enriching the CB'. Herein, we described the most relevant results produced in each of these subjects in order to clarify the main physiological role played by this intriguing cytoplasmic structure in the germ cells of male mammals, which though long since described, still fascinates researchers in the field.