Essential roles of the ANKRD31-REC114 interaction in meiotic recombination and mouse spermatogenesis.

Meiotic DNA double-strand breaks (DSBs) initiate homologous recombination and are crucial for ensuring proper chromosome segregation. In mice, ANKRD31 recently emerged as a regulator of DSB timing, number, and location, with a particularly important role in targeting DSBs to the pseudoautosomal regions (PARs) of sex chromosomes. ANKRD31 interacts with multiple proteins, including the conserved and essential DSB-promoting factor REC114, so it was hypothesized to be a modular scaffold that "anchors" other proteins together and to meiotic chromosomes. To determine whether and why the REC114 interaction is important for ANKRD31 function, we generated mice with Ankrd31 mutations that either reduced (missense mutation) or eliminated (C-terminal truncation) the ANKRD31-REC114 interaction without diminishing contacts with other known partners. A complete lack of the ANKRD31-REC114 interaction mimicked an Ankrd31 null, with delayed DSB formation and recombination, defects in DSB repair, and altered DSB locations including failure to target DSBs to the PARs. In contrast, when the ANKRD31-REC114 interaction was substantially but not completely disrupted, spermatocytes again showed delayed DSB formation globally, but recombination and repair were hardly affected and DSB locations were similar to control mice. The missense Ankrd31 allele showed a dosage effect, wherein combining it with the null or C-terminal truncation allele resulted in intermediate phenotypes for DSB formation, recombination, and DSB locations. Our results show that ANKRD31 function is critically dependent on its interaction with REC114 and that defects in ANKRD31 activity correlate with the severity of the disruption of the interaction.

Multiple assembly mechanisms anchor the KMN spindle checkpoint platform at human mitotic kinetochores.

During mitosis, the spindle checkpoint senses kinetochores not properly attached to spindle microtubules and prevents precocious sister-chromatid separation and aneuploidy. The constitutive centromere-associated network (CCAN) at inner kinetochores anchors the KMN network consisting of Knl1, the Mis12 complex (Mis12C), and the Ndc80 complex (Ndc80C) at outer kinetochores. KMN is a critical kinetochore receptor for both microtubules and checkpoint proteins. Here, we show that nearly complete inactivation of KMN in human cells through multiple strategies produced strong checkpoint defects even when all kinetochores lacked microtubule attachment. These KMN-inactivating strategies reveal multiple KMN assembly mechanisms at human mitotic kinetochores. In one mechanism, the centromeric kinase Aurora B phosphorylates Mis12C and strengthens its binding to the CCAN subunit CENP-C. In another, CENP-T contributes to KMN attachment in a CENP-H-I-K-dependent manner. Our study provides insights into the mechanisms of mitosis-specific assembly of the checkpoint platform KMN at human kinetochores.

Tracking spindle checkpoint signals from kinetochores to APC/C.

Accurate chromosome segregation during mitosis is critical for maintaining genomic stability. The kinetochore--a large protein assembly on centromeric chromatin--functions as the docking site for spindle microtubules and a signaling hub for the spindle checkpoint. At metaphase, spindle microtubules from opposing spindle poles capture each pair of sister kinetochores, exert pulling forces, and create tension across sister kinetochores. The spindle checkpoint detects improper kinetochore-microtubule attachments and translates these defects into biochemical activities that inhibit the anaphase-promoting complex or cyclosome (APC/C) throughout the cell to delay anaphase onset. A deficient spindle checkpoint leads to premature sister-chromatid separation and aneuploidy. Here, we review recent progress on the generation, propagation, transmission, and silencing of the spindle checkpoint signals from kinetochores to APC/C.

Structure of human Mad1 C-terminal domain reveals its involvement in kinetochore targeting.

The spindle checkpoint prevents aneuploidy by delaying anaphase onset until all sister chromatids achieve proper microtubule attachment. The kinetochore-bound checkpoint protein complex Mad1-Mad2 promotes the conformational activation of Mad2 and serves as a catalytic engine of checkpoint signaling. How Mad1 is targeted to kinetochores is not understood. Here, we report the crystal structure of the conserved C-terminal domain (CTD) of human Mad1. Mad1 CTD forms a homodimer and, unexpectedly, has a fold similar to those of the kinetochore-binding domains of Spc25 and Csm1. Nonoverlapping Mad1 fragments retain detectable kinetochore targeting. Deletion of the CTD diminishes, does not abolish, Mad1 kinetochore localization. Mutagenesis studies further map the functional interface of Mad1 CTD in kinetochore targeting and implicate Bub1 as its receptor. Our results indicate that CTD is a part of an extensive kinetochore-binding interface of Mad1, and rationalize graded kinetochore targeting of Mad1 during checkpoint signaling.

Mutual regulation between the spindle checkpoint and APC/C.

Accurate chromosome segregation during mitosis is critical for maintaining genomic stability. The spindle checkpoint is a cellular surveillance system that ensures the fidelity of chromosome segregation. In response to sister chromatids not properly captured by spindle microtubules, the spindle checkpoint interferes with the functions of Cdc20, the mitotic activator of the anaphase-promoting complex or cyclosome (APC/C), thereby blocking APC/C-mediated degradation of securin and cyclin B to delay anaphase onset. This review summarizes the recent progress on the mechanisms by which checkpoint proteins inhibit APC/C, the conformational and enzymatic activation of checkpoint proteins, and the emerging roles of APC/C-dependent ubiquitination in checkpoint inactivation.

Mitotic centromeric targeting of HP1 and its binding to Sgo1 are dispensable for sister-chromatid cohesion in human cells.

Human Shugoshin 1 (Sgo1) protects centromeric sister-chromatid cohesion during prophase and prevents premature sister-chromatid separation. Heterochromatin protein 1 (HP1) has been proposed to protect centromeric sister-chromatid cohesion by directly targeting Sgo1 to centromeres in mitosis. Here we show that HP1α is targeted to mitotic centromeres by INCENP, a subunit of the chromosome passenger complex (CPC). Biochemical and structural studies show that both HP1-INCENP and HP1-Sgo1 interactions require the binding of the HP1 chromo shadow domain to PXVXL/I motifs in INCENP or Sgo1, suggesting that the INCENP-bound, centromeric HP1α is incapable of recruiting Sgo1. Consistently, a Sgo1 mutant deficient in HP1 binding is functional in centromeric cohesion protection and localizes normally to centromeres in mitosis. By contrast, INCENP or Sgo1 mutants deficient in HP1 binding fail to localize to centromeres in interphase. Therefore, our results suggest that HP1 binding by INCENP or Sgo1 is dispensable for centromeric cohesion protection during mitosis of human cells, but might regulate yet uncharacterized interphase functions of CPC or Sgo1 at the centromeres.

Phosphorylation of the spindle checkpoint protein Mad2 regulates its conformational transition.

Regulated conformational changes of proteins are critical for cellular signal transduction. The spindle checkpoint protein Mad2 is an unusual protein with two native folds: the latent open conformer (O-Mad2) and the activated closed conformer (C-Mad2). During mitosis, cytosolic O-Mad2 binds to the Mad1-Mad2 core complex at unattached kinetochores and undergoes conformational activation to become C-Mad2. C-Mad2 binds to and inhibits Cdc20, an activator of APC/C, to prevent precocious anaphase onset. Here, we show that the conformational transition of Mad2 is regulated by phosphorylation of S195 in its C-terminal region. The phospho-mimicking Mad2(S195D) mutant and the phospho-S195 Mad2 protein obtained using intein-mediated semisynthesis do not form C-Mad2 on their own. Mad2(S195D) fails to bind to Cdc20, a low-affinity ligand, but still binds to high-affinity ligands, such as Mad1 and MBP1, forming ligand-bound C-Mad2. Overexpression of Mad2(S195D) in human cells causes checkpoint defects. Our results indicate that Mad2 phosphorylation inhibits its function through differentially regulating its binding to Mad1 and Cdc20 and establish that the conformational change of Mad2 is regulated by posttranslational mechanisms.