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.

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.

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.

Interplay between Phosphatases and the Anaphase-Promoting Complex/Cyclosome in Mitosis.

Accurate division of cells into two daughters is a process that is vital to propagation of life. Protein phosphorylation and selective degradation have emerged as two important mechanisms safeguarding the delicate choreography of mitosis. Protein phosphatases catalyze dephosphorylation of thousands of sites on proteins, steering the cells through establishment of the mitotic phase and exit from it. A large E3 ubiquitin ligase, the anaphase-promoting complex/cyclosome (APC/C) becomes active during latter stages of mitosis through G1 and marks hundreds of proteins for destruction. Recent studies have revealed the complex interregulation between these two classes of enzymes. In this review, we highlight the direct and indirect mechanisms by which phosphatases and the APC/C mutually influence each other to ensure accurate spatiotemporal and orderly progression through mitosis, with a particular focus on recent insights and conceptual advances.

Targeting of Fzr/Cdh1 for timely activation of the APC/C at the centrosome during mitotic exit.

A multi-subunit ubiquitin ligase, the anaphase-promoting complex/cyclosome (APC/C), regulates critical cellular processes including the cell cycle. To accomplish its diverse functions, APC/C activity must be precisely regulated in time and space. The interphase APC/C activator Fizzy-related (Fzr or Cdh1) is localized at centrosomes in animal cells. However, neither the mechanism of its localization nor its importance is clear. Here we identify the centrosome component Spd2 as a major partner of Fzr in Drosophila. The localization of Fzr to the centriole during interphase depends on direct interaction with Spd2. By generating Spd2 mutants unable to bind Fzr, we show that centrosomal localization of Fzr is essential for optimal APC/C activation towards its centrosomal substrate Aurora A. Finally, we show that Spd2 is also a novel APC/C(Fzr) substrate. Our study is the first to demonstrate the critical importance of distinct subcellular pools of APC/C activators in the spatiotemporal control of APC/C activity.

A synthetic lethal interaction between APC/C and topoisomerase poisons uncovered by proteomic screens.

The Anaphase-promoting complex/cyclosome (APC/C) cofactor Cdh1 modulates cell proliferation by targeting multiple cell-cycle regulators for ubiquitin-dependent degradation. Lack of Cdh1 results in structural and numerical chromosome aberrations, a hallmark of genomic instability. By using a proteomic approach in Cdh1-null cells and mouse tissues, we have identified kinesin Eg5 and topoisomerase 2α as Cdh1 targets involved in the maintenance of genomic stability. These proteins are ubiquitinated and degraded through specific KEN and D boxes in a Cdh1-dependent manner. Whereas Cdh1-null cells display partial resistance to Eg5 inhibitors such as monastrol, lack of Cdh1 results in a dramatic sensitivity to Top2α poisons as a consequence of increased levels of trapped Top2α-DNA complexes. Chemical inhibition of the APC/C in cancer cells results in increased sensitivity to Top2α poisons. This work identifies in vivo targets of the mammalian APC/C-Cdh1 complex and reveals synthetic lethal interactions of relevance in anticancer treatments.

Targeting mitotic exit leads to tumor regression in vivo: Modulation by Cdk1, Mastl, and the PP2A/B55α,δ phosphatase.

Targeting mitotic exit has been recently proposed as a relevant therapeutic approach against cancer. By using genetically engineered mice, we show that the APC/C cofactor Cdc20 is essential for anaphase onset in vivo in embryonic or adult cells, including progenitor/stem cells. Ablation of Cdc20 results in efficient regression of aggressive tumors, whereas current mitotic drugs display limited effects. Yet, Cdc20 null cells can exit from mitosis upon inactivation of Cdk1 and the kinase Mastl (Greatwall). This mitotic exit depends on the activity of PP2A phosphatase complexes containing B55α or B55δ regulatory subunits. These data illustrate the relevance of critical players of mitotic exit in mammals and their implications in the balance between cell death and mitotic exit in tumor cells.

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.

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.

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.

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.

Requirement of the SCFPop1/Pop2 Ubiquitin Ligase for Degradation of the Fission Yeast S Phase Cyclin Cig2.

Two multiprotein E3 (ubiquitin-protein ligase) ubiquitin ligases, the SCF (Skp1-Cullin-1-F-box) and the APC/C (anaphase promoting complex/cyclosome), are vital in ensuring the temporal order of the cell cycle. Particularly, timely destruction of cyclins via these two E3s is essential for down-regulation of cyclin-dependent kinase. In general, G(1) and S phase cyclins are ubiquitylated by the SCF, whereas ubiquitylation of mitotic cyclins is catalyzed by the APC/C. Here we show that fission yeast S phase cyclin Cig2 is ubiquitylated and degraded via both the SCF and the APC/C. Cig2 instability during G(2) and M phase is dependent upon the SCF complex, whereas the APC/C is responsible for Cig2 destruction during anaphase and G(1), thereby ensuring a spike pattern of Cig2 levels, peaking only at S phase. Two F-box/WD proteins Pop1 and Pop2, homologues of budding yeast Cdc4 and human Fbw7, are responsible for Cig2 instability. Pop1 binds Cig2 in vivo. An in vitro binding assay shows that an internal 93 amino acid residues comprising a part of the cyclin box are necessary and sufficient for this binding. Cig2 phosphorylation is also required for interaction with Pop1. We previously showed that transcriptional oscillation of cig2(+) requires Pop1 and Pop2 function. SCF(Pop1/Pop2) therefore regulates Cig2 levels in a dual manner, transcriptionally and post-translationally. Our results also highlight a collaborative action of the APC/C and the SCF toward the common substrate Cig2. This type of composite degradation control may be more general as the regulatory mechanism in other complex systems.