Meitinger et al. perform a genome-wide CRISPR/Cas9 screen for centrinone resistance and identify a 53BP1-USP28 module as critical for communicating mitotic challenges to the p53 circuit and TRIM37 as an enforcer of the singularity of centrosome assembly.

Spatial and timely coordination of cytokinesis is crucial for the maintenance of organelle inheritance and genome integrity. The mitotic exit network (MEN) pathway controls both the timely initiation of mitotic exit and cytokinesis in budding yeast. Here we identified the conserved F-BAR protein Hof1 as a substrate of the MEN kinase complex Dbf2-Mob1 during cytokinesis. We show that polo-like kinase Cdc5 first phosphorylates Hof1 to allow subsequent phosphorylation by Dbf2-Mob1. This releases Hof1 from the septin ring and facilitates Hof1 binding to the medial actomyosin ring (AMR), where Hof1 promotes AMR contraction and membrane ingression. Domain structure analysis established that the central, unstructured, region of Hof1, named the ring localization sequence (RLS), is sufficient to mediate Hof1's binding to the medial ring in a cell cycle-dependent manner. Genetic and functional data support a model in which Dbf2-Mob1 regulates Hof1 by inducing domain rearrangements, leading to the exposure of the Hof1 RLS domain during telophase.

The CCCTC-binding factor (CTCF) works together with the cohesin complex to drives the formation of chromatin loops and topologically associating domains, but its role in gene regulation has not been fully defined. Here, we investigated the effects of acute CTCF loss on chromatin architecture and transcriptional programs in mouse embryonic stem cells undergoing differentiation to neural precursor cells. We identified CTCF-dependent enhancer-promoter contacts genome-wide and found that they disproportionally affect genes that are bound by CTCF at promoter and dependent on long-distance enhancers. Disruption of promoter-proximal CTCF binding reduced both long-range enhancer-promoter contacts and transcription, which are restored by artificial tethering of CTCF to the promoter. Promoter-proximal CTCF binding is correlated with transcription of over 2,000 genes, across a diverse set of adult tissues. Taken together, our study shows that CTCF binding to promoters may promote long-distance-enhancer dependent transcription at specific genes in diverse cell types.

SummaryThe mitotic-exit network (MEN) is a signaling pathway that is essential for the coordination of mitotic exit and cytokinesis. Whereas the role of the MEN in mitotic exit is well established, the molecular mechanisms by which MEN components regulate cytokinesis remain poorly understood. Here, we show that the MEN controls components involved in septum formation, including Inn1, Cyk3 and Chs2. MEN-deficient mutants, forced to exit mitosis as a result of Cdk1 inactivation, show defects in targeting Cyk3 and Inn1 to the bud-neck region. In addition, we found that the chitin synthase Chs2 did not efficiently localize at the bud neck in the absence of MEN activity. Ultrastructural analysis of the bud neck revealed that low MEN activity led to unilateral, uncoordinated extension of the primary and secondary septa. This defect was partially suppressed by increased levels of Cyk3. We therefore propose that the MEN directly controls cytokinesis via targeting of Inn1, Cyk3 and Chs2 to the bud neck.

Centrosomes catalyze microtubule formation for mitotic spindle assembly 1 . Centrosomes duplicate once per cell cycle in a process controlled the kinase PLK4 2 , 3 . Following chemical PLK4 inhibition, cell division in the absence of centrosome duplication generates centrosome-less cells that exhibit delayed, acentrosomal spindle assembly 4 . Whether PLK4 inhibitors can be leveraged for cancer treatment is not yet clear. Here, we show that acentrosomal spindle assembly following PLK4 inhibition depends on levels of the centrosomal ubiquitin ligase TRIM37. Low TRIM37 accelerates acentrosomal spindle assembly and improves proliferation following PLK4 inhibition, whereas high TRIM37 inhibits acentrosomal spindle assembly, leading to mitotic failure and cessation of proliferation. The Chr17q region containing the TRIM37 gene is frequently amplified in neuroblastoma and in breast cancer 5 – 8 , which renders these cancer types highly sensitive to PLK4 inhibition. TRIM37 inactivation improves acentrosomal mitosis because TRIM37 prevents PLK4 self-assembly into centrosome-independent condensates that serve as ectopic microtubule-organizing centers. By contrast, elevated TRIM37 expression inhibits acentrosomal spindle assembly via a distinct mechanism that involves degradation of the centrosomal component CEP192. Thus, TRIM37 is a critical determinant of mitotic vulnerability to PLK4 inhibition. Linkage of TRIM37 to prevalent cancer-associated genomic changes, including 17q gain in neuroblastoma and 17q23 amplification in breast cancer, may offer an opportunity to use PLK4 inhibition to trigger selective mitotic failure and provide new avenues to treatments for these cancers.

Mitotic duration is determined by activation of the anaphase-promoting complex/cyclosome (APC/C) bound to its coactivator, Cdc20. Kinetochores, the microtubule-interacting machines on chromosomes, restrain mitotic exit when not attached to spindle microtubules by generating a Cdc20-containing complex that inhibits the APC/C. Here, we show that flux of Cdc20 through kinetochores also accelerates mitotic exit by promoting its dephosphorylation by kinetochore-localized protein phosphatase 1, which allows Cdc20 to activate the APC/C. Both APC/C activation and inhibition depend on Cdc20 fluxing through the same binding site at kinetochores. The microtubule attachment status of kinetochores therefore optimizes mitotic duration by controlling the balance between opposing Cdc20 fates.

In budding yeast, the mitotic exit network (MEN) is a signaling pathway best known for its role in driving cells out of mitosis through activation of the conserved phosphatase Cdc14. However, work over the past few years show that MEN components and Cdc14 also have a direct role in promoting cytokinesis by acting upon components of the contractile actomyosin ring and cell separation machineries. In this review, we discuss the current view on the role of MEN kinases and Cdc14 in cytokinesis and comment on the cytokinesis-related function of MEN and Cdc14 orthologs in higher eukaryotes.

Cdc42 is a highly conserved master regulator of cell polarity. Here, we investigated the mechanism by which yeast cells never re-establish polarity at cortical sites (cytokinesis remnants [CRMs]) that have previously supported Cdc42-mediated growth as a paradigm to mechanistically understand how Cdc42-inhibitory polarity cues are established. We revealed a two-step mechanism of loading the Cdc42 antagonist Nba1 into CRMs to mark these compartments as refractory for a second round of Cdc42 activation. Our data indicate that Nba1 together with a cortically tethered adaptor protein confers memory of previous polarization events to translate this spatial legacy into a biochemical signal that ensures the local singularity of Cdc42 activation. "Memory loss" mutants that repeatedly use the same polarity site over multiple generations display nuclear segregation defects and a shorter lifespan. Our work thus established CRMs as negative polarity cues that prevent Cdc42 reactivation to sustain the fitness of replicating cells.