RESUMEN
Eukaryotic genomes are organized by condensin into 3D chromosomal architectures suitable for chromosomal segregation during mitosis. However, molecular mechanisms underlying the condensin-mediated chromosomal organization remain largely unclear. Here, we investigate the role of newly identified interaction between the Cnd1 condensin and Pmc4 mediator subunits in fission yeast, Schizosaccharomyces pombe. We develop a condensin mutation, cnd1-K658E, that impairs the condensin-mediator interaction and find that this mutation diminishes condensinmediated chromatin domains during mitosis and causes chromosomal segregation defects. The condensin-mediator interaction is involved in recruiting condensin to highly transcribed genes and mitotically activated genes, the latter of which demarcate condensin-mediated domains. Furthermore, this study predicts that mediator-driven transcription of mitotically activated genes contributes to forming domain boundaries via phase separation. This study provides a novel insight into how genome-wide gene expression during mitosis is transformed into the functional chromosomal architecture suitable for chromosomal segregation.
RESUMEN
During meiotic prophase, cohesin-dependent axial structures are formed in the synaptonemal complex (SC). However, the functional correlation between these structures and cohesion remains elusive. Here, we examined the formation of cohesin-dependent axial structures in the fission yeast Schizosaccharomyces pombe. This organism forms atypical SCs composed of linear elements (LinEs) resembling the lateral elements of SC but lacking the transverse filaments. Hi-C analysis using a highly synchronous population of meiotic S. pombe cells revealed that the axis-loop chromatin structure formed in meiotic prophase was dependent on the Rec8 cohesin complex. In contrast, the Rec8-mediated formation of the axis-loop structure occurred in cells lacking components of LinEs. To dissect the functions of Rec8, we identified a rec8-F204S mutant that lost the ability to assemble the axis-loop structure without losing cohesion of sister chromatids. This mutant showed defects in the formation of the axis-loop structure and LinE assembly and thus exhibited reduced meiotic recombination. Collectively, our results demonstrate that the Rec8-dependent axis-loop structure provides a structural platform essential for LinE assembly, facilitating meiotic recombination of homologous chromosomes, independently of its role in sister chromatid cohesion.
Asunto(s)
Meiosis , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Proteínas de Ciclo Celular , Cromatina , Proteínas Cromosómicas no Histona , Fosfoproteínas/genética , Schizosaccharomyces/citología , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Complejo Sinaptonémico , CohesinasRESUMEN
Telomere binding protein Stn1 forms the CST (Cdc13/CTC1-STN1-TEN1) complex in budding yeast and mammals. Likewise, fission yeast Stn1 and Ten1 form a complex indispensable for telomere protection. We have previously reported that stn1-1, a high-temperature sensitive mutant, rapidly loses telomere DNA at the restrictive temperature due to frequent failure of replication fork progression at telomeres and subtelomeres, both containing repetitive sequences. It is unclear, however, whether Stn1 is required for maintaining other repetitive DNAs such as ribosomal DNA. In this study, we have demonstrated that stn1-1 cells, even when grown at the permissive temperature, exhibited dynamic rearrangements in the telomere-proximal regions of subtelomere and ribosomal DNA repeats. Furthermore, Rad52 and γH2A accumulation was observed at ribosomal DNA repeats in the stn1-1 mutant. The phenotypes exhibited by the stn1-1 allele were largely suppressed in the absence of Reb1, a replication fork barrier-forming protein, suggesting that Stn1 is involved in the maintenance of the arrested replication forks. Collectively, we propose that Stn1 maintains the stability of repetitive DNAs at subtelomeres and rDNA regions.
Asunto(s)
ADN de Hongos/química , ADN Ribosómico/química , Secuencias Repetitivas de Ácidos Nucleicos , Proteínas de Schizosaccharomyces pombe/fisiología , Schizosaccharomyces/genética , Proteínas de Unión a Telómeros/fisiología , Proteínas de Unión al ADN/genética , Viabilidad Microbiana , Mutación , Recombinación Genética , Reparación del ADN por Recombinación , Proteínas de Schizosaccharomyces pombe/genética , Telómero , Proteínas de Unión a Telómeros/genética , Factores de Transcripción/genéticaRESUMEN
Genome sequences have been determined for many model organisms; however, repetitive regions such as centromeres, telomeres, and subtelomeres have not yet been sequenced completely. Here, we report the complete sequences of subtelomeric homologous (SH) regions of the fission yeast Schizosaccharomyces pombe. We overcame technical difficulties to obtain subtelomeric repetitive sequences by constructing strains that possess single SH regions of a standard laboratory strain. In addition, some natural isolates of S. pombe were analyzed using previous sequencing data. Whole sequences of SH regions revealed that each SH region consists of two distinct parts with mosaics of multiple common segments or blocks showing high variation among subtelomeres and strains. Subtelomere regions show relatively high frequency of nucleotide variations among strains compared with the other chromosomal regions. Furthermore, we identified subtelomeric RecQ-type helicase genes, tlh3 and tlh4, which add to the already known tlh1 and tlh2, and found that the tlh1-4 genes show high sequence variation with missense mutations, insertions, and deletions but no severe effects on their RNA expression. Our results indicate that SH sequences are highly polymorphic and hot spots for genome variation. These features of subtelomeres may have contributed to genome diversity and, conversely, various diseases.
Asunto(s)
Variación Genética , Genoma Fúngico , Schizosaccharomyces/genética , Telómero/genética , Secuencia de Bases , Mutación INDEL/genética , Mosaicismo , Familia de Multigenes , Nucleótidos/genética , Filogenia , ARN de Hongos/genética , RecQ Helicasas/genética , Schizosaccharomyces/aislamiento & purificaciónRESUMEN
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.
Asunto(s)
Inestabilidad Cromosómica/genética , Análisis de la Célula Individual/métodos , Intercambio de Cromátides Hermanas/fisiología , Sistemas CRISPR-Cas/genética , Ciclo Celular/genética , División Celular/genética , Cromátides/genética , Cromátides/patología , Cromátides/fisiología , Inestabilidad Cromosómica/fisiología , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Ingeniería Genética/métodos , Células HCT116 , Humanos , Microscopía Fluorescente/métodos , Neoplasias/genética , Intercambio de Cromátides Hermanas/genéticaRESUMEN
Senescence is induced by various stimuli such as oncogene expression and telomere shortening, referred to as oncogene-induced senescence (OIS) and replicative senescence (RS), respectively, and accompanied by global transcriptional alterations and 3D genome reorganization. Here, we demonstrate that the human condensin II complex participates in senescence via gene regulation and reorganization of euchromatic A and heterochromatic B compartments. Both OIS and RS are accompanied by A-to-B and B-to-A compartmental transitions, the latter of which occur more frequently and are undergone by 14% (430 Mb) of the human genome. Mechanistically, condensin is enriched in A compartments and implicated in B-to-A transitions. The full activation of senescence genes (SASP genes and p53 targets) requires condensin; its depletion impairs senescence markers. This study describes that condensin reinforces euchromatic A compartments and promotes B-to-A transitions, both of which are coupled to optimal expression of senescence genes, thereby allowing condensin to contribute to senescent processes.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfatasas/farmacología , Senescencia Celular/genética , Senescencia Celular/fisiología , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Complejos Multiproteicos/metabolismo , Complejos Multiproteicos/farmacología , Proteínas de Ciclo Celular/genética , Línea Celular , Cromatina , Perfilación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Genómica , Humanos , Proteínas Nucleares/genética , Oncogenes , Regiones Promotoras Genéticas , Acortamiento del Telómero , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Genomic rearrangements (gross chromosomal rearrangements, GCRs) threatens genome integrity and cause cell death or tumor formation. At the terminus of linear chromosomes, a telomere-binding protein complex, called shelterin, ensures chromosome stability by preventing chromosome end-to-end fusions and regulating telomere length homeostasis. As such, shelterin-mediated telomere functions play a pivotal role in suppressing GCR formation. However, it remains unclear whether the shelterin proteins play any direct role in inhibiting GCR at non-telomeric regions. Here, we have established a GCR assay for the first time in fission yeast and measured GCR rates in various mutants. We found that fission yeast cells lacking shelterin components Taz1 or Rap1 (mammalian TRF1/2 or RAP1 homologues, respectively) showed higher GCR rates compared to wild-type, accumulating large chromosome deletions. Genetic dissection of Rap1 revealed that Rap1 contributes to inhibiting GCRs via two independent pathways. The N-terminal BRCT-domain promotes faithful DSB repair, as determined by I-SceI-mediated DSB-induction experiments; moreover, association with Poz1 mediated by the central Poz1-binding domain regulates telomerase accessibility to DSBs, leading to suppression of de novo telomere additions. Our data highlight unappreciated functions of the shelterin components Taz1 and Rap1 in maintaining genome stability, specifically by preventing non-telomeric GCRs.
Asunto(s)
Reparación del ADN , Reordenamiento Génico , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/genética , Proteínas de Unión a Telómeros/metabolismo , Roturas del ADN de Doble Cadena , Inestabilidad Genómica , Mutación , Proteínas de Schizosaccharomyces pombe/genética , Complejo Shelterina , Homeostasis del Telómero , Proteínas de Unión a Telómeros/genéticaRESUMEN
The subtelomere, a telomere-adjacent chromosomal domain, contains species-specific homologous DNA sequences, in addition to various genes. However, the functions of subtelomeres, particularly subtelomeric homologous (SH) sequences, remain elusive. Here, we report the first comprehensive analyses of the cellular functions of SH sequences in the fission yeast, Schizosaccharomyces pombe. Complete removal of SH sequences from the genome revealed that they are dispensable for mitosis, meiosis and telomere length control. However, when telomeres are lost, SH sequences prevent deleterious inter-chromosomal end fusion by facilitating intra-chromosomal circularization. Surprisingly, SH-deleted cells sometimes survive telomere loss through inter-chromosomal end fusions via homologous loci such as LTRs, accompanied by centromere inactivation of either chromosome. Moreover, SH sequences function as a buffer region against the spreading of subtelomeric heterochromatin into the neighboring gene-rich regions. Furthermore, we found a nucleosome-free region at the subtelomeric border, which may be a second barrier that blocks heterochromatin spreading into the subtelomere-adjacent euchromatin. Thus, our results demonstrate multiple defense functions of subtelomeres in chromosome homeostasis and gene expression.
Asunto(s)
Cromosomas Fúngicos/fisiología , Expresión Génica , Homeostasis/genética , Proteínas de Schizosaccharomyces pombe/genética , Schizosaccharomyces/genética , Telómero/fisiología , Centrómero/metabolismo , Inestabilidad Cromosómica/genética , Regulación Fúngica de la Expresión Génica , Heterocromatina/metabolismo , Organismos Modificados Genéticamente , Eliminación de Secuencia , Proteínas de Unión a Telómeros/metabolismoRESUMEN
A chromosome is composed of structurally and functionally distinct domains. However, the molecular mechanisms underlying the formation of chromatin structure and the function of subtelomeres, the telomere-adjacent regions, remain obscure. Here we report the roles of the conserved centromeric protein Shugoshin 2 (Sgo2) in defining chromatin structure and functions of the subtelomeres in the fission yeast Schizosaccharomyces pombe. We show that Sgo2 localizes at the subtelomeres preferentially during G2 phase and is essential for the formation of a highly condensed subtelomeric chromatin body 'knob'. Furthermore, the absence of Sgo2 leads to the derepression of the subtelomeric genes and premature DNA replication at the subtelomeric late origins. Thus, the subtelomeric specialized chromatin domain organized by Sgo2 represses both transcription and replication to ensure proper gene expression and replication timing.
Asunto(s)
Cromatina/metabolismo , Proteínas Cromosómicas no Histona/química , Proteínas Cromosómicas no Histona/metabolismo , Momento de Replicación del ADN , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Telómero/metabolismo , Cromatina/genética , Proteínas Cromosómicas no Histona/genética , Fase G2 , Estructura Terciaria de Proteína , Transporte de Proteínas , Schizosaccharomyces/química , Schizosaccharomyces/citología , Proteínas de Schizosaccharomyces pombe/genética , Telómero/genética , Transcripción GenéticaRESUMEN
Facultative heterochromatin is reversibly established and disrupted during differentiation, but its regulation remains mechanistically unclear. Here, we show that two meiotic gene loci in fission yeast, mei4 and ssm4, comprise facultative heterochromatin that is regulated in a developmental stage-dependent manner. This heterochromatin coordinates expression levels by associating with a chromodomain protein Chp1 and an antisilencing factor Epe1. It has been recently shown that an RNA surveillance machinery for eliminating meiotic gene transcripts, which involves a cis-element called the determinant of selective removal (DSR) and transacting factors, Mmi1 and Red1, also participates in heterochromatin formation at the meiotic genes, but the molecular mechanism underlying the process is largely unknown. By dissecting the mei4 gene, we identified a region that promotes DSR-dependent methylation of histone H3 lysine 9 (H3K9). Integration of this mei4 region together with DSR into an unrelated gene results in ectopic H3K9 methylation. Moreover, our results suggest that transcription of these elements induces chromatin association of Mmi1, which, in turn, recruits Red1 interacting with Clr4/Suv39h H3K9 methyltransferase. Mmi1 remains associated in cells lacking Red1, suggesting that the recruitment of Red1 follows the chromatin association of Mmi1. Overall, we provide detailed insights into the facultative heterochromatin regulation in fission yeast.