An AsCas12f-based compact genome-editing tool derived by deep mutational scanning and structural analysis.

SpCas9 and AsCas12a are widely utilized as genome-editing tools in human cells. However, their relatively large size poses a limitation for delivery by cargo-size-limited adeno-associated virus (AAV) vectors. The type V-F Cas12f from Acidibacillus sulfuroxidans is exceptionally compact (422 amino acids) and has been harnessed as a compact genome-editing tool. Here, we developed an approach, combining deep mutational scanning and structure-informed design, to successfully generate two AsCas12f activity-enhanced (enAsCas12f) variants. Remarkably, the enAsCas12f variants exhibited genome-editing activities in human cells comparable with those of SpCas9 and AsCas12a. The cryoelectron microscopy (cryo-EM) structures revealed that the mutations stabilize the dimer formation and reinforce interactions with nucleic acids to enhance their DNA cleavage activities. Moreover, enAsCas12f packaged with partner genes in an all-in-one AAV vector exhibited efficient knock-in/knock-out activities and transcriptional activation in mice. Taken together, enAsCas12f variants could offer a minimal genome-editing platform for in vivo gene therapy.

A Proteome-wide Fission Yeast Interactome Reveals Network Evolution Principles from Yeasts to Human.

Here, we present FissionNet, a proteome-wide binary protein interactome for S. pombe, comprising 2,278 high-quality interactions, of which ∼ 50% were previously not reported in any species. FissionNet unravels previously unreported interactions implicated in processes such as gene silencing and pre-mRNA splicing. We developed a rigorous network comparison framework that accounts for assay sensitivity and specificity, revealing extensive species-specific network rewiring between fission yeast, budding yeast, and human. Surprisingly, although genes are better conserved between the yeasts, S. pombe interactions are significantly better conserved in human than in S. cerevisiae. Our framework also reveals that different modes of gene duplication influence the extent to which paralogous proteins are functionally repurposed. Finally, cross-species interactome mapping demonstrates that coevolution of interacting proteins is remarkably prevalent, a result with important implications for studying human disease in model organisms. Overall, FissionNet is a valuable resource for understanding protein functions and their evolution.

Dynamic regulation of mitotic ubiquitin ligase APC/C by coordinated Plx1 kinase and PP2A phosphatase action on a flexible Apc1 loop.

The anaphase-promoting complex/cyclosome (APC/C), a multi-subunit ubiquitin ligase essential for cell cycle control, is regulated by reversible phosphorylation. APC/C phosphorylation by cyclin-dependent kinase 1 (Cdk1) promotes Cdc20 co-activator loading in mitosis to form active APC/C-Cdc20. However, detailed phospho-regulation of APC/C dynamics through other kinases and phosphatases is still poorly understood. Here, we show that an interplay between polo-like kinase (Plx1) and PP2A-B56 phosphatase on a flexible loop domain of the subunit Apc1 (Apc1-loop500 ) controls APC/C activity and mitotic progression. Plx1 directly binds to the Apc1-loop500 in a phosphorylation-dependent manner and promotes the formation of APC/C-Cdc20 via Apc3 phosphorylation. Upon phosphorylation of loop residue T532, PP2A-B56 is recruited to the Apc1-loop500 and differentially promotes dissociation of Plx1 and PP2A-B56 through dephosphorylation of Plx1-binding sites. Stable Plx1 binding, which prevents PP2A-B56 recruitment, prematurely activates the APC/C and delays APC/C dephosphorylation during mitotic exit. Furthermore, the phosphorylation status of the Apc1-loop500 is controlled by distant Apc3-loop phosphorylation. Our study suggests that phosphorylation-dependent feedback regulation through flexible loop domains within a macromolecular complex coordinates the activity and dynamics of the APC/C during the cell cycle.

APC/C-dependent degradation of Spd2 regulates centrosome asymmetry in Drosophila neural stem cells.

A functional centrosome is vital for the development and physiology of animals. Among numerous regulatory mechanisms of the centrosome, ubiquitin-mediated proteolysis is known to be critical for the precise regulation of centriole duplication. However, its significance beyond centrosome copy number control remains unclear. Using an in vitro screen for centrosomal substrates of the APC/C ubiquitin ligase in Drosophila, we identify several conserved pericentriolar material (PCM) components, including the inner PCM protein Spd2. We show that Spd2 levels are controlled by the interphase-specific form of APC/C, APC/CFzr , in cultured cells and developing brains. Increased Spd2 levels compromise neural stem cell-specific asymmetric PCM recruitment and microtubule nucleation at interphase centrosomes, resulting in partial randomisation of the division axis and segregation patterns of the daughter centrosome in the following mitosis. We further provide evidence that APC/CFzr -dependent Spd2 degradation restricts the amount and mobility of Spd2 at the daughter centrosome, thereby facilitating the accumulation of Polo-dependent Spd2 phosphorylation for PCM recruitment. Our study underpins the critical role of cell cycle-dependent proteolytic regulation of the PCM in stem cells.

PP2A-B56 binds to Apc1 and promotes Cdc20 association with the APC/C ubiquitin ligase in mitosis.

Cell cycle progression and genome stability are regulated by a ubiquitin ligase, the anaphase-promoting complex/cyclosome (APC/C). Cyclin-dependent kinase 1 (Cdk1) has long been implicated in APC/C activation; however, the molecular mechanisms of governing this process in vivo are largely unknown. Recently, a Cdk1-dependent phosphorylation relay within Apc3-Apc1 subunits has been shown to alleviate Apc1-mediated auto-inhibition by which a mitotic APC/C co-activator Cdc20 binds to and activates the APC/C. However, the underlying mechanism for dephosphorylation of Cdc20 and APC/C remains elusive. Here, we show that a disordered loop domain of Apc1 (Apc1-loop500 ) directly binds the B56 regulatory subunit of protein phosphatase 2A (PP2A) and stimulates Cdc20 loading to the APC/C. Using the APC/C reconstitution system in Xenopus egg extracts, we demonstrate that mutations in Apc1-loop500 that abolish B56 binding decrease Cdc20 loading and APC/C-dependent ubiquitylation. Conversely, a non-phosphorylatable mutant Cdc20 can efficiently bind the APC/C even when PP2A-B56 binding is impeded. Furthermore, PP2A-B56 preferentially dephosphorylates Cdc20 over the Apc1 inhibitory domain. These results indicate that Apc1-loop500 plays a role in dephosphorylating Cdc20, promoting APC/C-Cdc20 complex formation in mitosis.

Plasmodium P-Type Cyclin CYC3 Modulates Endomitotic Growth during Oocyst Development in Mosquitoes.

Cell-cycle progression and cell division in eukaryotes are governed in part by the cyclin family and their regulation of cyclin-dependent kinases (CDKs). Cyclins are very well characterised in model systems such as yeast and human cells, but surprisingly little is known about their number and role in Plasmodium, the unicellular protozoan parasite that causes malaria. Malaria parasite cell division and proliferation differs from that of many eukaryotes. During its life cycle it undergoes two types of mitosis: endomitosis in asexual stages and an extremely rapid mitotic process during male gametogenesis. Both schizogony (producing merozoites) in host liver and red blood cells, and sporogony (producing sporozoites) in the mosquito vector, are endomitotic with repeated nuclear replication, without chromosome condensation, before cell division. The role of specific cyclins during Plasmodium cell proliferation was unknown. We show here that the Plasmodium genome contains only three cyclin genes, representing an unusual repertoire of cyclin classes. Expression and reverse genetic analyses of the single Plant (P)-type cyclin, CYC3, in the rodent malaria parasite, Plasmodium berghei, revealed a cytoplasmic and nuclear location of the GFP-tagged protein throughout the lifecycle. Deletion of cyc3 resulted in defects in size, number and growth of oocysts, with abnormalities in budding and sporozoite formation. Furthermore, global transcript analysis of the cyc3-deleted and wild type parasites at gametocyte and ookinete stages identified differentially expressed genes required for signalling, invasion and oocyst development. Collectively these data suggest that cyc3 modulates oocyst endomitotic development in Plasmodium berghei.

Dephosphorylation of Cdc20 is required for its C-box-dependent activation of the APC/C.

The anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligase is tightly regulated to ensure programmed proteolysis in cells. The activity of the APC/C is positively controlled by cyclin-dependent kinase (CDK), but a second level of control must also exist because phosphorylation inactivates Cdc20, a mitotic APC/C co-activator. How Cdc20 is dephosphorylated specifically, when CDK is high, has remained unexplained. Here, we show that phosphatases are crucial to activate the APC/C. Cdc20 is phosphorylated at six conserved residues (S50/T64/T68/T79/S114/S165) by CDK in Xenopus egg extracts. When all the threonine residues are phosphorylated, Cdc20 binding to and activation of the APC/C are inhibited. Their dephosphorylation is regulated depending on the sites and protein phosphatase 2A, active in mitosis, is essential to dephosphorylate the threonine residues and activate the APC/C. Consistently, most of the Cdc20 bound to the APC/C in anaphase evades phosphorylation at T79. Furthermore, we show that the 'activation domain' of Cdc20 associates with the Apc6 and Apc8 core subunits. Our data suggest that dephosphorylation of Cdc20 is required for its loading and activation of the APC/C ubiquitin ligase.

A role for the Fizzy/Cdc20 family of proteins in activation of the APC/C distinct from substrate recruitment.

The Fizzy/Cdc20 family of proteins are essential activators of the anaphase-promoting complex/cyclosome (APC/C), a multisubunit E3 ubiquitin ligase. However, apart from the well-established role of the C-terminal WD40 domain in substrate recognition, the precise roles of the activators remain elusive. Here we show that Nek2A, which directly binds the APC/C, can be ubiquitylated and destroyed in Fizzy/Cdc20-depleted Xenopus egg extracts when only the N-terminal domain of Fizzy/Cdc20 (N-Cdc20) is added. This activity is dependent upon the C box and is conserved in the alternative activator, Fizzy-related/Cdh1. In contrast, canonical substrates such as cyclin B and securin require both the N-terminal and WD40 domains, unless N-Cdc20 is fused to substrates when the WD40 domain becomes dispensable. Furthermore, in Cdc20-depleted cells, N-Cdc20 can facilitate Nek2A destruction in a C box-dependent manner. Our results reveal a role for the N-terminal domain of the Fizzy/Cdc20 family of activators in triggering substrate ubiquitylation by the APC/C.

Anaphase-promoting complex/cyclosome-mediated proteolysis of Ams2 in the G1 phase ensures the coupling of histone gene expression to DNA replication in fission yeast.

Histone transcription and deposition are tightly regulated with the DNA replication cycle to maintain genetic integrity. Ams2 is a GATA-containing transcription factor responsible for core histone gene expression and for CENP-A loading at centromeres in fission yeast. Ams2 levels are cell cycle-regulated, and after the S phase Ams2 is degraded by the SCF(pof3) ubiquitin ligase; however, the regulation of Ams2 in G(1) or meiosis is poorly understood. Here we show that another ubiquitin ligase, the anaphase-promoting complex/cyclosome (APC/C) targets Ams2 for destruction in G(1). Ubiquitylation and destruction of Ams2 is dependent upon a coactivator Cdh1/Ste9 and the KEN box in the C terminus of Ams2. We also find that stabilization of Ams2 sensitizes cells to the anti-microtubule drug thiabendazole and the histone deacetylase inhibitor tricostatin A when a histone deacetylase gene hst4 is deleted, suggesting that histone acetylation together with Ams2 stability ensures the coupling of mitosis to DNA replication. Furthermore, in meiosis, the failure of the APC/C-mediated destruction of Ams2 is deleterious, and pre-meiotic DNA replication is barely completed. These data suggest that Ams2 destruction via both the APC/C and the SCF ubiquitin ligases underlies the coordination of histone expression and DNA replication.

The C-terminal disordered loop domain of Apc8 unlocks APC/C mitotic activation.

The anaphase-promoting complex/cyclosome (APC/C) is a critical and tightly regulated E3 ligase that orchestrates the cellular life cycle by controlling the degradation of cell cycle regulators. An intriguing feature of this complex is an autoinhibition mechanism: an intrinsically disordered loop domain, Apc1-300L, blocks Cdc20 coactivator binding, yet phosphorylation of Apc1-300L counteracts this autoinhibition. Many such disordered loops within APC/C remain unexplored. Our systematic analysis of loop-deficient APC/C mutants uncovered a pivotal role for Apc8's C-terminal loop (Apc8-L) in mitotic activation. Apc8-L directly recruits the CDK adaptor protein, Xe-p9/Cks2, positioning the Xe-p9-CDK-CycB complex near Apc1-300L. This stimulates the phosphorylation and removal of Apc1-300L, prompting the formation of active APC/CCdc20. Strikingly, without both Apc8-L and Apc3-L, the APC/C is rendered inactive during mitosis, highlighting Apc8-L's synergistic role with other loops and kinases. This study broadens our understanding of the intricate dynamics in APC/C regulation and provides insights on the regulation of macromolecular complexes.

CDK1-PP2A-B55 interplay ensures cell cycle oscillation via Apc1-loop300.

Cell cycle control relies on a delicate balance of phosphorylation with CDK1 and phosphatases like PP1 and PP2A-B55. Yet, identifying the primary substrate responsible for cell cycle oscillations remains a challenge. We uncover the pivotal role of phospho-regulation in the anaphase-promoting complex/cyclosome (APC/C), particularly through the Apc1-loop300 domain (Apc1-300L), orchestrated by CDK1 and PP2A-B55. Premature activation of PP2A-B55 during mitosis, induced by Greatwall kinase depletion, leads to Apc1-300L dephosphorylation, stalling APC/C activity and delaying Cyclin B degradation. This effect can be counteracted using the B55-specific inhibitor pEnsa or by removing Apc1-300L. We also show Cdc20's dynamic APC/C interaction across cell cycle stages, but dephosphorylation of Apc1-300L specifically inhibits further Cdc20 recruitment. Our study underscores APC/C's central role in cell cycle oscillation, identifying it as a primary substrate regulated by the CDK-PP2A partnership.

Early mitotic degradation of Nek2A depends on Cdc20-independent interaction with the APC/C.

The temporal control of mitotic protein degradation remains incompletely understood. In particular, it is unclear why the mitotic checkpoint prevents the anaphase-promoting complex/cyclosome (APC/C)-mediated degradation of cyclin B and securin in early mitosis, but not cyclin A. Here, we show that another APC/C substrate, NIMA-related kinase 2A (Nek2A), is also destroyed in pro-metaphase in a checkpoint-independent manner and that this depends on an exposed carboxy-terminal methionine-arginine (MR) dipeptide tail. Truncation of the Nek2A C terminus delays its degradation until late mitosis, whereas Nek2A C-terminal peptides interfere with APC/C activity in an MR-dependent manner. Most importantly, we show that Nek2A binds directly to the APC/C, also in an MR-dependent manner, even in the absence of the adaptor protein Cdc20. As similar C-terminal dipeptide tails promote direct association of Cdc20, Cdh1 and Apc10-Doc1 with core APC/C subunits, we propose that this sequence also allows a substrate, Nek2A, to directly bind the APC/C. Thus, although Cdc20 is required for the degradation of Nek2A, it is not required for its recruitment and this renders its degradation insensitive to the mitotic checkpoint.

Zfp296 knockout enhances chromatin accessibility and induces a unique state of pluripotency in embryonic stem cells.

The Zfp296 gene encodes a zinc finger-type protein. Its expression is high in mouse embryonic stem cells (ESCs) but rapidly decreases following differentiation. Zfp296-knockout (KO) ESCs grew as flat colonies, which were reverted to rounded colonies by exogenous expression of Zfp296. KO ESCs could not form teratomas when transplanted into mice but could efficiently contribute to germline-competent chimeric mice following blastocyst injection. Transcriptome analysis revealed that Zfp296 deficiency up- and down-regulates a distinct group of genes, among which Dppa3, Otx2, and Pou3f1 were markedly downregulated. Chromatin immunoprecipitation sequencing demonstrated that ZFP296 binding is predominantly seen in the vicinity of the transcription start sites (TSSs) of a number of genes, and ZFP296 was suggested to negatively regulate transcription. Consistently, chromatin accessibility assay clearly showed that ZFP296 binding reduces the accessibility of the TSS regions of target genes. Zfp296-KO ESCs showed increased histone H3K9 di- and trimethylation. Co-immunoprecipitation analyses revealed interaction of ZFP296 with G9a and GLP. These results show that ZFP296 plays essential roles in maintaining the global epigenetic state of ESCs through multiple mechanisms including activation of Dppa3, attenuation of chromatin accessibility, and repression of H3K9 methylation, but that Zfp296-KO ESCs retain a unique state of pluripotency while lacking the teratoma-forming ability.

Structural analysis sheds light on APC/C-mediated ubiquitylation.

In the December 22nd issue of Molecular Cell, two groups report refined cryo-electron microscopic structures of the APC/C at approximately 20 A resolution. They also reveal important new features including multiple copies of subunits, dimerization and structural flexibility of the APC/C, which give a hint to solve the mechanisms of the APC/C-dependent ubiquitylation.

Fission yeast Mes1p ensures the onset of meiosis II by blocking degradation of cyclin Cdc13p.

Meiosis is a special form of nuclear division to generate eggs, sperm and spores in eukaryotes. Meiosis consists of the first (MI) and the second (MII) meiotic divisions, which occur consecutively. MI is reductional, in which homologous chromosomes derived from parents segregate. MI is supported by an elaborate mechanism involving meiosis-specific cohesin and its protector. MII is equational, in which replicated sister-chromatids separate as in mitosis. MII is generally considered to mimic mitosis in mechanism. However, fission yeast Mes1p is essential for MII but dispensable for mitosis. The mes1-B44 mutant arrests before MII. Transcription of mes1 is low in vegetative cells and boosted in a narrow window between late MI and late MII. The mes1 mRNA undergoes meiosis-specific splicing. Here we show that Mes1p is a factor that suppresses the degradation of cyclin Cdc13p at anaphase I. Mes1p binds to Slp1p, an activator of APC/C (anaphase promoting complex/cyclosome), and counteracts its function to engage Cdc13p in proteolysis. Inhibition of APC/C-dependent degradation of Cdc13p by Mes1p was reproduced in a Xenopus egg extract. We therefore propose that Mes1p has a key function in saving a sufficient level of MPF (M-phase-promoting factor) activity required for the execution of MII.

The APC/C and CBP/p300 cooperate to regulate transcription and cell-cycle progression.

The anaphase-promoting complex/cyclosome (APC/C) is a multicomponent E3 ubiquitin ligase that, by targeting protein substrates for 26S proteasome-mediated degradation through ubiquitination, coordinates the temporal progression of eukaryotic cells through mitosis and the subsequent G1 phase of the cell cycle. Other functions of the APC/C are, however, less well defined. Here we show that two APC/C components, APC5 and APC7, interact directly with the coactivators CBP and p300 through protein-protein interaction domains that are evolutionarily conserved in adenovirus E1A. This interaction stimulates intrinsic CBP/p300 acetyltransferase activity and potentiates CBP/p300-dependent transcription. We also show that APC5 and APC7 suppress E1A-mediated transformation in a CBP/p300-dependent manner, indicating that these components of the APC/C may be targeted during cellular transformation. Furthermore, we establish that CBP is required in APC/C function; specifically, gene ablation of CBP by RNA-mediated interference markedly reduces the E3 ubiquitin ligase activity of the APC/C and the progression of cells through mitosis. Taken together, our results define discrete roles for the APC/C-CBP/p300 complexes in growth regulation.

A role for Cdc2- and PP2A-mediated regulation of Emi2 in the maintenance of CSF arrest.

BACKGROUND: Vertebrate oocytes are arrested in metaphase II of meiosis prior to fertilization by cytostatic factor (CSF). CSF enforces a cell-cycle arrest by inhibiting the anaphase-promoting complex (APC), an E3 ubiquitin ligase that targets Cyclin B for degradation. Although Cyclin B synthesis is ongoing during CSF arrest, constant Cyclin B levels are maintained. To achieve this, oocytes allow continuous slow Cyclin B degradation, without eliminating the bulk of Cyclin B, which would induce release from CSF arrest. However, the mechanism that controls this continuous degradation is not understood. RESULTS: We report here the molecular details of a negative feedback loop wherein Cyclin B promotes its own destruction through Cdc2/Cyclin B-mediated phosphorylation and inhibition of the APC inhibitor Emi2. Emi2 bound to the core APC, and this binding was disrupted by Cdc2/Cyclin B, without affecting Emi2 protein stability. Cdc2-mediated phosphorylation of Emi2 was antagonized by PP2A, which could bind to Emi2 and promote Emi2-APC interactions. CONCLUSIONS: Constant Cyclin B levels are maintained during a CSF arrest through the regulation of Emi2 activity. A balance between Cdc2 and PP2A controls Emi2 phosphorylation, which in turn controls the ability of Emi2 to bind to and inhibit the APC. This balance allows proper maintenance of Cyclin B levels and Cdc2 kinase activity during CSF arrest.

In vitro assays for the anaphase-promoting complex/cyclosome (APC/C) in Xenopus egg extracts.

The anaphase-promoting complex/cyclosome (APC/C), a large (20S) multisubunit E3 ligase, has an essential role to ubiquitylate numerous substrates at specific times during mitosis and G1 phase as well as in meiosis. The deregulation of the APC/C causes cell death or genomic instability, which is a hallmark of cancers. Although 13 years have passed since its discovery, the molecular mechanisms of the APC/C-dependent ubiquitylation and proteolysis are still poorly understood. The development of in vitro systems enables the identification of new substrates and investigation of the molecular mechanisms by which the APC/C recognizes its substrates. This chapter describes in vitro assays reconstituted in Xenopus egg extracts.

APC/C: current understanding and future perspectives.

The separation of sister chromatids at anaphase, which is regulated by an E3 ubiquitin ligase called the anaphase-promoting complex/cyclosome (APC/C), is arguably the most important irrevocable event during the cell cycle. The APC/C and cyclin-dependent kinase 1 (Cdk1) are just two of the many significant cell cycle regulators and exert control through ubiquitylation and phosphorylation, respectively. The temporal and spatial regulation of the APC/C is achieved by multiple mechanisms, including phosphorylation, interaction with the structurally related co-activators Cdc20 and Cdh1, loading of distinct E2 ubiquitin-conjugating enzymes, binding with inhibitors and differential affinities for various substrates. Since the discovery of APC/C 25 years ago, intensive studies have uncovered many aspects of APC/C regulation, but we are still far from a full understanding of this important cellular machinery. Recent high-resolution cryogenic electron microscopy analysis and reconstitution of the APC/C have greatly advanced our understanding of molecular mechanisms underpinning the enzymatic properties of APC/C. In this review, we will examine the historical background and current understanding of APC/C regulation.