PIASgamma is required for faithful chromosome segregation in human cells.

BACKGROUND: The precision of the metaphase-anaphase transition ensures stable genetic inheritance. The spindle checkpoint blocks anaphase onset until the last chromosome biorients at metaphase plate, then the bonds between sister chromatids are removed and disjoined chromatids segregate to the spindle poles. But, how sister separation is triggered is not fully understood. PRINCIPAL FINDINGS: We identify PIASgamma as a human E3 sumo ligase required for timely and efficient sister chromatid separation. In cells lacking PIASgamma, normal metaphase plates form, but the spindle checkpoint is activated, leading to a prolonged metaphase block. Sister chromatids remain cohered even if cohesin is removed by depletion of hSgo1, because DNA catenations persist at centromeres. PIASgamma-depleted cells cannot properly localize Topoisomerase II at centromeres or in the cores of mitotic chromosomes, providing a functional link between PIASgamma and Topoisomerase II. CONCLUSIONS: PIASgamma directs Topoisomerase II to specific chromosome regions that require efficient removal of DNA catenations prior to anaphase. The lack of this activity activates the spindle checkpoint, protecting cells from non-disjunction. Because DNA catenations persist without PIASgamma in the absence of cohesin, removal of catenations and cohesin rings must be regulated in parallel.

Regulated separation of sister centromeres depends on the spindle assembly checkpoint but not on the anaphase promoting complex/cyclosome.

Key to faithful genetic inheritance is the cohesion between sister centromeres that physically links replicated sister chromatids and is then abruptly lost at the onset of anaphase. Misregulated cohesion causes aneuploidy, birth defects and perhaps initiates cancers. Loss of centromere cohesion is controlled by the spindle checkpoint and is thought to depend on a ubiquitin ligase, the Anaphase Promoting Complex/Cyclosome (APC). But here we present evidence that the APC pathway is dispensable for centromere separation at anaphase in mammals, and that anaphase proceeds in the presence of cyclin B and securin. Arm separation is perturbed in the absence of APC, compromising the fidelity of segregation, but full sister chromatid separation is achieved after a delayed anaphase. Thereafter, cells arrest terminally in telophase with high levels of cyclin B. Extending these findings we provide evidence that the spindle checkpoint regulates centromere cohesion through an APC-independent pathway. We propose that this Centromere Linkage Pathway (CLiP) is a second branch that stems from the spindle checkpoint to regulate cohesion preferentially at the centromeres and that Sgo1 is one of its components.

Separase is required at multiple pre-anaphase cell cycle stages in human cells.

The yeast separase proteins Esp1 and Cut1 are required for loss of sister chromatid cohesion that occurs at the moment of anaphase onset. Circumstantial evidence has linked human separase to centromere separation at anaphase, but a direct test that the role of this enzyme is functionally conserved with the yeast proteins is lacking. Here we describe the effects of separase depletion from human cells using RNA interference. Surprisingly, HeLa cells lacking separase are delayed or arrest at the G2-M phase transition. This arrest is not likely due to the activation of a known checkpoint control, but may be a result of a failure to construct a mitotic chromosome. Without separase, cells also have a prolonged prometaphase, perhaps resulting from defects in spindle assembly or dynamics. In cells that reach mitosis, sister arm resolution and separation are perturbed, whereas in anaphase cells sister centromeres do appear to separate. These data indicate that separase function is not restricted to anaphase initiation and that its role in promoting loss of sister chromatid cohesion might be preferentially at arms but not centromeres.

DNA catenations that link sister chromatids until the onset of anaphase are maintained by a checkpoint mechanism.

Treatment of Allium cepa meristematic cells in metaphase with the topoisomerase II inhibitor ICRF-193, results in bridging of the sister chromatids at anaphase. Separation of the sisters in experimentally generated acentric chromosomal fragments was also inhibited by ICRF-193, indicating that some non-centromeric catenations also persist in metaphase chromosomes. Thus, catenations must be resolved by DNA topoisomerase II at the metaphase-to-anaphase transition to allow segregation of sisters. A passive mechanism could maintain catenations holding sisters until the onset of anaphase. At this point the opposite tension exerted on sister chromatids could render the decatenation reaction physically more favorable than catenation. But this possibility was dismissed as acentric chromosome fragments were able to separate their sister chromatids at anaphase. A timing mechanism (a common trigger for two processes taking different times to be completed) could passively couple the resolution of the last remaining catenations to the moment of anaphase onset. This possibility was also discarded as cells arrested in metaphase with microtubule-destabilising drugs still displayed anaphase bridges when released in the presence of ICRF-193. It is possible that a checkpoint mechanism prevents the release of the last catenations linking sisters until the onset of anaphase. To test whether cells are competent to fully resolve catenations before anaphase onset, we generated multinucleate plant cells. In this system, the nuclei within a single multinucleate cell displayed differences in chromosome condensation at metaphase, but initiated anaphase synchronously. When multinucleates were treated with ICRF-193 at the metaphase-toanaphase transition, tangled and untangled anaphases were observed within the same cell. This can only occur if cells are competent to disentangle sister chromatids before the onset of anaphase, but are prevented from doing so by a checkpoint mechanism.

Reiniciación de la transcripción en el ciclo celular: Nucleologénesis / Reinitiation of the Transcription in the celular cycle. Nucleologenesis

La transición de telofase al periodo G1 interfásico incluye el proceso de nucleologénesis o reorganización nucelolar en la que específicas regiones (NOR) de determinados cromosomas en cada cariotipo se encuentran involucradas en la formación de los nuevos nucleolos.Los estudios realizados en células que efectúan esta transición muestran que tanto la síntesis de proteínas como la de RNA, que son inexistentes en las etapas medias de la mitosis, aumentan su intensidad al final de la telofase, precisamente durante el periodo de reorganización nucleolar, justamente cuando son observados los llamados cuerpos cuerpos prenucleolares en la superficie de los cromosomas. Para abordar de qué manera podrían influir la biosíntesis de proteínas y del RNA en la nucleologénesis se partió de varios hechos: a) El marcado citocinesis por inhibición mediante cafeína; b) el conocimiento de que la nucleologénesis abarca a un 7,4 por ciento de la duración del ciclo celular y se sitúa entre telofase y G1 temprano; c) la aplicación de inhibidores de síntesis de proteínas o de RNA antes, durante y posteriormente al marcado binucleado y su análisis sobre la reorganización celular.Experimentalmente se demostró que los cuerpos prenucleolares no eran productos inmediatos de la actividad transcripcional de los cromosomas post-metafásicos, apareciendo independientemente de una simultánea síntesis de proteínas. La correlación cronológica perfecta entre la desaparición de los cuerpos prenucleolares y la nucleologénesis completa, así como su adelantamiento mediante inhibición de síntesis de proteínas o su bloqueo por inhibición de la síntesis de RNA mostraron concluyentemente qué factores se encontraban en el proceso de nucleologénesis. La existencia del NOR no siempre asegura la reorganización de un nucleolo. Puede existir dominancia de unos NORs sobre otros, tanto inter- como intra-específicos, formándose un número de nucleolos inferior al número de NORs, debiéndose a proteínas de unión al DNA condicionadas por el nivel de metilación de sus citosinas. Esta dominancia en la nucleologénesis es detallada en sus diferentes fases (AU)