Spindle checkpoint silencing at kinetochores with submaximal microtubule occupancy.

The spindle assembly checkpoint (SAC) ensures proper chromosome segregation by monitoring kinetochore-microtubule interactions. SAC proteins are shed from kinetochores once stable attachments are achieved. Human kinetochores consist of hundreds of SAC protein recruitment modules and bind up to 20 microtubules, raising the question of how the SAC responds to intermediate attachment states. We show that one protein module ('RZZS-MAD1-MAD2') of the SAC is removed from kinetochores at low microtubule occupancy and remains absent at higher occupancies, while another module ('BUB1-BUBR1') is retained at substantial levels irrespective of attachment states. These behaviours reflect different silencing mechanisms: while BUB1 displacement is almost fully dependent on MPS1 inactivation, MAD1 (also known as MAD1L1) displacement is not. Artificially tuning the affinity of kinetochores for microtubules further shows that ∼50% occupancy is sufficient to shed MAD2 and silence the SAC. Kinetochores thus respond as a single unit to shut down SAC signalling at submaximal occupancy states, but retain one SAC module. This may ensure continued SAC silencing on kinetochores with fluctuating occupancy states while maintaining the ability for fast SAC re-activation.

Integrated cistromic and expression analysis of amplified NKX2-1 in lung adenocarcinoma identifies LMO3 as a functional transcriptional target.

The NKX2-1 transcription factor, a regulator of normal lung development, is the most significantly amplified gene in human lung adenocarcinoma. To study the transcriptional impact of NKX2-1 amplification, we generated an expression signature associated with NKX2-1 amplification in human lung adenocarcinoma and analyzed DNA-binding sites of NKX2-1 by genome-wide chromatin immunoprecipitation. Integration of these expression and cistromic analyses identified LMO3, itself encoding a transcription regulator, as a candidate direct transcriptional target of NKX2-1. Further cistromic and overexpression analyses indicated that NKX2-1 can cooperate with the forkhead box transcription factor FOXA1 to regulate LMO3 gene expression. RNAi analysis of NKX2-1-amplified cells compared with nonamplified cells demonstrated that LMO3 mediates cell survival downstream from NKX2-1. Our findings provide new insight into the transcriptional regulatory network of NKX2-1 and suggest that LMO3 is a transcriptional signal transducer in NKX2-1-amplified lung adenocarcinomas.

Biallelic TRIP13 mutations predispose to Wilms tumor and chromosome missegregation.

Through exome sequencing, we identified six individuals with biallelic loss-of-function mutations in TRIP13. All six developed Wilms tumor. Constitutional mosaic aneuploidies, microcephaly, developmental delay and seizures, which are features of mosaic variegated aneuploidy (MVA) syndrome, were more variably present. Through functional studies, we show that TRIP13-mutant patient cells have no detectable TRIP13 and have substantial impairment of the spindle assembly checkpoint (SAC), leading to a high rate of chromosome missegregation. Accurate segregation, as well as SAC proficiency, is rescued by restoring TRIP13 function. Individuals with biallelic TRIP13 or BUB1B mutations have a high risk of embryonal tumors, and here we show that their cells display severe SAC impairment. MVA due to biallelic CEP57 mutations, or of unknown cause, is not associated with embryonal tumors and cells from these individuals show minimal SAC deficiency. These data provide insights into the complex relationships between aneuploidy and carcinogenesis.

Attachment issues: kinetochore transformations and spindle checkpoint silencing.

Cell division culminates in the segregation of duplicated chromosomes in opposite directions prior to cellular fission. This process is guarded by the spindle assembly checkpoint (SAC), which prevents the anaphase of cell division until stable connections between spindle microtubules and the kinetochores of all chromosomes are established. The anaphase inhibitor is generated at unattached kinetochores and inhibitor production is prevented when microtubules are captured. Understanding the molecular changes in the kinetochore that are evoked by microtubule attachments is crucial for understanding the mechanisms of SAC signaling and silencing. Here, we highlight the most recent findings on these events, pinpoint some remaining mysteries, and argue for incorporating holistic views of kinetochore dynamics in order to understand SAC silencing.

Kinetochore-microtubule attachment is sufficient to satisfy the human spindle assembly checkpoint.

The spindle assembly checkpoint (SAC) is a genome surveillance mechanism that protects against aneuploidization. Despite profound progress on understanding mechanisms of its activation, it remains unknown what aspect of chromosome-spindle interactions is monitored by the SAC: kinetochore-microtubule attachment or the force generated by dynamic microtubules that signals stable biorientation of chromosomes? To answer this, we uncoupled these two processes by expressing a non-phosphorylatable version of the main microtubule-binding protein at kinetochores (HEC1-9A), causing stabilization of incorrect kinetochore-microtubule attachments despite persistent activity of the error-correction machinery. The SAC is fully functional in HEC1-9A-expressing cells, yet cells in which chromosomes cannot biorient but are stably attached to microtubules satisfy the SAC and exit mitosis. SAC satisfaction requires neither intra-kinetochore stretching nor dynamic microtubules. Our findings support the hypothesis that in human cells the end-on interactions of microtubules with kinetochores are sufficient to satisfy the SAC without the need for microtubule-based pulling forces.