Duane A. Compton

Accurate chromosome segregation relies on proper centromere and kinetochore formation and phospho-regulation. We previously demonstrated that a pluripotent state confers a low fidelity of chromosome segregation, however it is unknown how a pluripotent state impacts centromere and kinetochore function. Here, we demonstrate that both centromere and kinetochore structural organization and phosphorylation in mitosis are developmentally regulated. CENP-A, CENP-C, and HEC1 protein abundance is reduced at mitotic centromeres and kinetochores of human pluripotent stem cells (hPSCs) compared to isogenic somatic cells; however, elevating their levels does not improve chromosome segregation fidelity. Rather, we find that reduced phosphorylation of kinetochores is responsible for their low fidelity. HEC1 is hypophosphorylated at kinetochores of hPSCs compared to isogenic somatic cells at Cyclin B/Cdk1 and Aurora kinase phospho-sites. Inhibiting PP2A phosphatase activity or differentiation increases HEC1 phosphorylation at hPSC kinetochores decreasing chromosome segregation errors. Thus, mitotic fidelity in non-transformed human cells depends on the developmental regulation of the kinase and phosphatase networks controlling kinetochore phosphorylation.

Faithful chromosome segregation requires the spatial and temporal remodeling of cell structures driven largely by cyclin-dependent kinase (Cdk) activity. In some experimental systems the timely elevation of one cyclin isoform is sufficient to support mitotic entry and progression. In human somatic cells, however, three cyclins – Cyclin A2, Cyclin B1, and Cyclin B2 – are present at mitotic entry, and their distinct contributions during mitosis remain largely unclear. We demonstrate that Cyclin A2 promotes the kinetochore localization of Cyclin B1, Cyclin B2 and the fibrous corona component CENPF, while suppressing recruitment of the kinetochore-microtubule (k-MT) stabilizer Astrin. Extending Cyclin A2 into metaphase by expressing a non-degradable mutant causes persistent kinetochore localization of Cyclin B1, Cyclin B2, and CENPF. Conversely, Cyclin B1 limits kinetochore localization of Cyclin B2 and CENPF, promotes Astrin recruitment, and stabilizes k-MT attachments and Cyclin B1 overexpression further reduces kinetochore localization of CENPF during prometaphase. These findings reveal an interdependence among mitotic cyclins in early mitosis and the countervailing activities of Cyclin A2 versus Cyclin B1/B2 in regulating key mitotic events. We propose that this circuitry enforces a switch-like transition from prometaphase – marked by corona assembly and high k-MT turnover – to metaphase – marked by corona disassembly, stabilization of end-on k-MT attachments, and eventual spindle assembly checkpoint satisfaction – to choreograph the structural changes required to ensure faithful chromosome segregation.

The biorientation-defective (BOD) protein family, comprising BOD1, BOD1L1 (BOD1-like 1), and BOD1L2, plays critical and diverse roles in fundamental cellular processes, including mitosis, DNA repair, neurological function, and metabolism. BOD1 and BOD1L2 are small proteins of less than 200 amino acids, whereas BOD1L1 contains a long C-terminal extension, totaling 3042 amino acids. BOD1 was originally identified in Xenopus laevis oocyte chromatin extracts. Subsequent work in mitotic human cells demonstrated that BOD1 is an outer kinetochore protein that regulates PP2A-B56 phosphatase function and consequently is vital for chromosome biorientation and segregation fidelity, hence the name. BOD1 also has important roles in neurological function and lipid metabolism as a component of the COMPASS (complex of proteins associated with SET1)–SETD1B complex. In contrast, BOD1L1 was first identified as a phosphorylated target of the ATM kinase and then highlighted in a screen for DNA replication fork components. Further work demonstrated a role for BOD1L1 in DNA double-stranded break repair, where BOD1L1 is required to recruit the protein RIF1 to damaged chromatin to enable efficient DNA repair and control sensitivity to radio/chemotherapeutics. Consistently, BOD1L1 binds known DNA damage/repair/replication proteins, including FANCD2, RIF1, and BRCA2. Loss of BOD1L1 causes catastrophic genome instability through misrepair and/or overprocessing of damaged DNA. Recently, BOD1L1 has also been shown to regulate the PP2A-B56 phosphatase at kinetochores in mitotic human cells. In contrast, little is known about BOD1L2, which is only expressed in sperm cells and precursors. In this review, we describe recent progress in understanding the functions of this protein family and discuss future avenues of research.

To ensure genomic fidelity, a series of spatially and temporally coordinated events is executed during prometaphase of mitosis, including bipolar spindle formation, chromosome attachment to spindle microtubules at kinetochores, the correction of erroneous kinetochore-microtubule (k-MT) attachments, and chromosome congression to the spindle equator. Cyclin A/Cdk1 kinase plays a key role in destabilizing k-MT attachments during prometaphase to promote correction of erroneous k-MT attachments. However, it is unknown whether Cyclin A/Cdk1 kinase regulates other events during prometaphase. Here, we investigate additional roles of Cyclin A/Cdk1 in prometaphase by using an siRNA knockdown strategy to deplete endogenous Cyclin A from human cells. We find that depleting Cyclin A significantly extends mitotic duration, specifically prometaphase, because chromosome alignment is delayed. Unaligned chromosomes display erroneous monotelic, syntelic, or lateral k-MT attachments suggesting that bioriented k-MT attachment formation is delayed in the absence of Cyclin A. Mechanistically, chromosome alignment is likely impaired because the localization of the kinetochore proteins BUB1 kinase, KNL1, and MPS1 kinase are reduced in Cyclin A-depleted cells. Moreover, we find that Cyclin A promotes BUB1 kinetochore localization independently of its role in destabilizing k-MT attachments. Thus, Cyclin A/Cdk1 facilitates chromosome alignment during prometaphase to support timely mitotic progression.

To ensure genomic fidelity a series of spatially and temporally coordinated events are executed during prometaphase of mitosis, including bipolar spindle formation, chromosome attachment to spindle microtubules at kinetochores, the correction of erroneous kinetochore-microtubule (k-MT) attachments, and chromosome congression to the spindle equator. Cyclin A/Cdk1 kinase plays a key role in destabilizing k-MT attachments during prometaphase to promote correction of erroneous k-MT attachments. However, it is unknown if Cyclin A/Cdk1 kinase regulates other events during prometaphase. Here, we investigate additional roles of Cyclin A/Cdk1 in prometaphase by using an siRNA knockdown strategy to deplete endogenous Cyclin A from human cells. We find that depleting Cyclin A significantly extends mitotic duration, specifically prometaphase, because chromosome alignment is delayed. Unaligned chromosomes display erroneous monotelic, syntelic, or lateral k-MT attachments suggesting that bioriented k-MT attachment formation is delayed in the absence of Cyclin A. Mechanistically, chromosome alignment is likely impaired because the localization of the kinetochore proteins BUB1 kinase, KNL1, and MPS1 kinase are reduced in Cyclin A-depleted cells. Moreover, we find that Cyclin A promotes BUB1 kinetochore localization independently of its role in destabilizing k-MT attachments. Thus, Cyclin A/Cdk1 facilitates chromosome alignment during prometaphase to support timely mitotic progression.

Summary: Cancer cells are often aneuploid and frequently display elevated rates of chromosome mis-segregation in a phenomenon called chromosomal instability (CIN). CIN is commonly caused by hyperstable kinetochore-microtubule (K-MT) attachments that reduces the efficiency of correction of erroneous K-MT attachments. We recently showed that UMK57, a chemical agonist of MCAK (alias KIF2C) improves chromosome segregation fidelity in CIN cancer cells although cells rapidly develop adaptive resistance. To determine the mechanism of resistance we performed unbiased proteomic screens which revealed increased phosphorylation in cells adapted to UMK57 at two Aurora kinase A phosphoacceptor sites on BOD1L1 (alias FAM44A). BOD1L1 depletion or Aurora kinase A inhibition eliminated resistance to UMK57 in CIN cancer cells. BOD1L1 localizes to spindles/kinetochores during mitosis, interacts with the PP2A phosphatase, and regulates phosphorylation levels of kinetochore proteins, chromosome alignment, mitotic progression and fidelity. Moreover, the BOD1L1 gene is mutated in a subset of human cancers, and BOD1L1 depletion reduces cell growth in combination with clinically relevant doses of taxol or Aurora kinase A inhibitor. Thus, an Aurora kinase A-BOD1L1-PP2A axis promotes faithful chromosome segregation during mitosis. (175 words)

Effects of USP33 knock-down on growth inhibition induced by CDK2 antagonism in human lung cancer cells. (A) A549 and (B) Hop62 cells were independently transfected with each of two different USP33 targeting siRNAs or control siRNA. Twenty-four hours after USP33 knock-down, cells were treated with seliciclib (10 μM) for 72 hours and proliferation was measured. The symbols indicate * P < 0.05 and ** P < 0.01.

Effects of CP110 phosphorylation sites on CP110 protein stability. KRAS wild-type ED-1 cells were transfected with CP110-WT or CP110-MUT 24 hours before treatment with or without cycloheximide (CHX, 40μg/ml) for indicated time periods. Expression of CP110 in each time point was shown by immunoblot analyses and ImageJ quantifications.

Ser 551 and Ser 906 of CP110 are not critical for protecting cells from undergoing anaphase catastrophe. (A) ED-1 cells overexpressing wild-type CP110 (CP110-WT), 8 CDK phosphorylation sites mutated CP110 (CP110-MUT), 10 CDK phosphorylation sites mutated CP110 (CP110-ALL MUT) or an empty vector were treated with the indicated dosage of seliciclib. After 24 hours treatment, ED-1 cells were fixed and scored for multipolar anaphases (NS, not significant). (B) Schematic of CP110-WT, CP110-MUT (8 sites) and CP110-ALL MUT (10 sites) sequences.