Cytoplasmic division cycles without the nucleus and mitotic CDK/cyclin complexes.

Cytoplasmic divisions are thought to rely on nuclear divisions and mitotic signals. We demonstrate in Drosophila embryos that cytoplasm can divide repeatedly without nuclei and mitotic CDK/cyclin complexes. Cdk1 normally slows an otherwise faster cytoplasmic division cycle, coupling it with nuclear divisions, and when uncoupled, cytoplasm starts dividing before mitosis. In developing embryos where CDK/cyclin activity can license mitotic microtubule (MT) organizers like the spindle, cytoplasmic divisions can occur without the centrosome, a principal organizer of interphase MTs. However, centrosomes become essential in the absence of CDK/cyclin activity, implying that the cytoplasm can employ either the centrosome-based interphase or CDK/cyclin-dependent mitotic MTs to facilitate its divisions. Finally, we present evidence that autonomous cytoplasmic divisions occur during unperturbed fly embryogenesis and that they may help extrude mitotically stalled nuclei during blastoderm formation. We postulate that cytoplasmic divisions occur in cycles governed by a yet-to-be-uncovered clock mechanism autonomous from CDK/cyclin complexes.

A Jasmonate Signaling Network Activates Root Stem Cells and Promotes Regeneration.

Plants are sessile and have to cope with environmentally induced damage through modification of growth and defense pathways. How tissue regeneration is triggered in such responses and whether this involves stem cell activation is an open question. The stress hormone jasmonate (JA) plays well-established roles in wounding and defense responses. JA also affects growth, which is hitherto interpreted as a trade-off between growth and defense. Here, we describe a molecular network triggered by wound-induced JA that promotes stem cell activation and regeneration. JA regulates organizer cell activity in the root stem cell niche through the RBR-SCR network and stress response protein ERF115. Moreover, JA-induced ERF109 transcription stimulates CYCD6;1 expression, functions upstream of ERF115, and promotes regeneration. Soil penetration and response to nematode herbivory induce and require this JA-mediated regeneration response. Therefore, the JA tissue damage response pathway induces stem cell activation and regeneration and activates growth after environmental stress.

A Non-catalytic Function of SETD1A Regulates Cyclin K and the DNA Damage Response.

MLL/SET methyltransferases catalyze methylation of histone 3 lysine 4 and play critical roles in development and cancer. We assessed MLL/SET proteins and found that SETD1A is required for survival of acute myeloid leukemia (AML) cells. Mutagenesis studies and CRISPR-Cas9 domain screening show the enzymatic SET domain is not necessary for AML cell survival but that a newly identified region termed the "FLOS" (functional location on SETD1A) domain is indispensable. FLOS disruption suppresses DNA damage response genes and induces p53-dependent apoptosis. The FLOS domain acts as a cyclin-K-binding site that is required for chromosomal recruitment of cyclin K and for DNA-repair-associated gene expression in S phase. These data identify a connection between the chromatin regulator SETD1A and the DNA damage response that is independent of histone methylation and suggests that targeting SETD1A and cyclin K complexes may represent a therapeutic opportunity for AML and, potentially, for other cancers.

CDK-independent role of D-type cyclins in regulating DNA mismatch repair.

Although mismatch repair (MMR) is essential for correcting DNA replication errors, it can also recognize other lesions, such as oxidized bases. In G0 and G1, MMR is kept in check through unknown mechanisms as it is error-prone during these cell cycle phases. We show that in mammalian cells, D-type cyclins are recruited to sites of oxidative DNA damage in a PCNA- and p21-dependent manner. D-type cyclins inhibit the proteasomal degradation of p21, which competes with MMR proteins for binding to PCNA, thereby inhibiting MMR. The ability of D-type cyclins to limit MMR is CDK4- and CDK6-independent and is conserved in G0 and G1. At the G1/S transition, the timely, cullin-RING ubiquitin ligase (CRL)-dependent degradation of D-type cyclins and p21 enables MMR activity to efficiently repair DNA replication errors. Persistent expression of D-type cyclins during S-phase inhibits the binding of MMR proteins to PCNA, increases the mutational burden, and promotes microsatellite instability.

Depletion of cap-binding protein eIF4E dysregulates amino acid metabolic gene expression.

Protein synthesis is metabolically costly and must be tightly coordinated with changing cellular needs and nutrient availability. The cap-binding protein eIF4E makes the earliest contact between mRNAs and the translation machinery, offering a key regulatory nexus. We acutely depleted this essential protein and found surprisingly modest effects on cell growth and recovery of protein synthesis. Paradoxically, impaired protein biosynthesis upregulated genes involved in the catabolism of aromatic amino acids simultaneously with the induction of the amino acid biosynthetic regulon driven by the integrated stress response factor GCN4. We further identified the translational control of Pho85 cyclin 5 (PCL5), a negative regulator of Gcn4, that provides a consistent protein-to-mRNA ratio under varied translation environments. This regulation depended in part on a uniquely long poly(A) tract in the PCL5 5' UTR and poly(A) binding protein. Collectively, these results highlight how eIF4E connects protein synthesis to metabolic gene regulation, uncovering mechanisms controlling translation during environmental challenges.

The CDK-APC/C Oscillator Predominantly Entrains Periodic Cell-Cycle Transcription.

Throughout cell-cycle progression, the expression of multiple transcripts oscillate, and whether these are under the centralized control of the CDK-APC/C proteins or can be driven by a de-centralized transcription factor (TF) cascade is a fundamental question for understanding cell-cycle regulation. In budding yeast, we find that the transcription of nearly all genes, as assessed by RNA-seq or fluorescence microscopy in single cells, is dictated by CDK-APC/C. Three exceptional genes are transcribed in a pulsatile pattern in a variety of CDK-APC/C arrests. Pursuing one of these transcripts, the SIC1 inhibitor of B-type cyclins, we use a combination of mathematical modeling and experimentation to provide evidence that, counter-intuitively, Sic1 provides a failsafe mechanism promoting nuclear division when levels of mitotic cyclins are low.

CDK Substrate Phosphorylation and Ordering the Cell Cycle.

S phase and mitotic onset are brought about by the action of multiple different cyclin-CDK complexes. However, it has been suggested that changes in the total level of CDK kinase activity, rather than substrate specificity, drive the temporal ordering of S phase and mitosis. Here, we present a phosphoproteomics-based systems analysis of CDK substrates in fission yeast and demonstrate that the phosphorylation of different CDK substrates can be temporally ordered during the cell cycle by a single cyclin-CDK. This is achieved by rising CDK activity and the differential sensitivity of substrates to CDK activity over a wide dynamic range. This is combined with rapid phosphorylation turnover to generate clearly resolved substrate-specific activity thresholds, which in turn ensures the appropriate ordering of downstream cell-cycle events. Comparative analysis with wild-type cells expressing multiple cyclin-CDK complexes reveals how cyclin-substrate specificity works alongside activity thresholds to fine-tune the patterns of substrate phosphorylation.

Post-transcriptional Wnt Signaling Governs Epididymal Sperm Maturation.

The canonical Wnt signaling pathway is of paramount importance in development and disease. An emergent question is whether the upstream cascade of the canonical Wnt pathway has physiologically relevant roles beyond ß-catenin-mediated transcription, which is difficult to study due to the pervasive role of this protein. Here, we show that transcriptionally silent spermatozoa respond to Wnt signals released from the epididymis and that mice mutant for the Wnt regulator Cyclin Y-like 1 are male sterile due to immotile and malformed spermatozoa. Post-transcriptional Wnt signaling impacts spermatozoa through GSK3 by (1) reducing global protein poly-ubiquitination to maintain protein homeostasis; (2) inhibiting septin 4 phosphorylation to establish a membrane diffusion barrier in the sperm tail; and (3) inhibiting protein phosphatase 1 to initiate sperm motility. The results indicate that Wnt signaling orchestrates a rich post-transcriptional sperm maturation program and invite revisiting transcription-independent Wnt signaling in somatic cells as well.

Compartmentalization of a bistable switch enables memory to cross a feedback-driven transition.

Cells make accurate decisions in the face of molecular noise and environmental fluctuations by relying not only on present pathway activity, but also on their memory of past signaling dynamics. Once a decision is made, cellular transitions are often rapid and switch-like due to positive feedback loops in the regulatory network. While positive feedback loops are good at promoting switch-like transitions, they are not expected to retain information to inform subsequent decisions. However, this expectation is based on our current understanding of network motifs that accounts for temporal, but not spatial, dynamics. Here, we show how spatial organization of the feedback-driven yeast G1/S switch enables the transmission of memory of past pheromone exposure across this transition. We expect this to be one of many examples where the exquisite spatial organization of the eukaryotic cell enables previously well-characterized network motifs to perform new and unexpected signal processing functions.

An alternative cell cycle coordinates multiciliated cell differentiation.

The canonical mitotic cell cycle coordinates DNA replication, centriole duplication and cytokinesis to generate two cells from one1. Some cells, such as mammalian trophoblast giant cells, use cell cycle variants like the endocycle to bypass mitosis2. Differentiating multiciliated cells, found in the mammalian airway, brain ventricles and reproductive tract, are post-mitotic but generate hundreds of centrioles, each of which matures into a basal body and nucleates a motile cilium3,4. Several cell cycle regulators have previously been implicated in specific steps of multiciliated cell differentiation5,6. Here we show that differentiating multiciliated cells integrate cell cycle regulators into a new alternative cell cycle, which we refer to as the multiciliation cycle. The multiciliation cycle redeploys many canonical cell cycle regulators, including cyclin-dependent kinases (CDKs) and their cognate cyclins. For example, cyclin D1, CDK4 and CDK6, which are regulators of mitotic G1-to-S progression, are required to initiate multiciliated cell differentiation. The multiciliation cycle amplifies some aspects of the canonical cell cycle, such as centriole synthesis, and blocks others, such as DNA replication. E2F7, a transcriptional regulator of canonical S-to-G2 progression, is expressed at high levels during the multiciliation cycle. In the multiciliation cycle, E2F7 directly dampens the expression of genes encoding DNA replication machinery and terminates the S phase-like gene expression program. Loss of E2F7 causes aberrant acquisition of DNA synthesis in multiciliated cells and dysregulation of multiciliation cycle progression, which disrupts centriole maturation and ciliogenesis. We conclude that multiciliated cells use an alternative cell cycle that orchestrates differentiation instead of controlling proliferation.

The Crk4-Cyc4 complex regulates G2/M transition in Toxoplasma gondii.

A versatile division of apicomplexan parasites and a dearth of conserved regulators have hindered the progress of apicomplexan cell cycle studies. While most apicomplexans divide in a multinuclear fashion, Toxoplasma gondii tachyzoites divide in the traditional binary mode. We previously identified five Toxoplasma CDK-related kinases (Crk). Here, we investigated TgCrk4 and its cyclin partner TgCyc4. We demonstrated that TgCrk4 regulates conventional G2 phase processes, such as repression of chromosome rereplication and centrosome reduplication, and acts upstream of the spindle assembly checkpoint. The spatial TgCyc4 dynamics supported the TgCrk4-TgCyc4 complex role in the coordination of chromosome and centrosome cycles. We also identified a dominant TgCrk4-TgCyc4 complex interactor, TgiRD1 protein, related to DNA replication licensing factor CDT1 but played no role in licensing DNA replication in the G1 phase. Our results showed that TgiRD1 also plays a role in controlling chromosome and centrosome reduplication. Global phosphoproteome analyses identified TgCrk4 substrates, including TgORC4, TgCdc20, TgGCP2, and TgPP2ACA. Importantly, the phylogenetic and structural studies suggest the Crk4-Cyc4 complex is limited to a minor group of the binary dividing apicomplexans.

Non-canonical functions of cell cycle cyclins and cyclin-dependent kinases.

The roles of cyclins and their catalytic partners, the cyclin-dependent kinases (CDKs), as core components of the machinery that drives cell cycle progression are well established. Increasing evidence indicates that mammalian cyclins and CDKs also carry out important functions in other cellular processes, such as transcription, DNA damage repair, control of cell death, differentiation, the immune response and metabolism. Some of these non-canonical functions are performed by cyclins or CDKs, independently of their respective cell cycle partners, suggesting that there was a substantial divergence in the functions of these proteins during evolution.

A super-assembly of Whi3 encodes memory of deceptive encounters by single cells during yeast courtship.

Cellular behavior is frequently influenced by the cell's history, indicating that single cells may memorize past events. We report that budding yeast permanently escape pheromone-induced cell-cycle arrest when experiencing a deceptive mating attempt, i.e., not reaching their putative partner within reasonable time. This acquired behavior depends on super-assembly and inactivation of the G1/S inhibitor Whi3, which liberates the G1 cyclin Cln3 from translational inhibition. Super-assembly of Whi3 is a slow response to pheromone, driven by polyQ and polyN domains, counteracted by Hsp70, and stable over generations. Unlike prion aggregates, Whi3 super-assemblies are not inherited mitotically but segregate to the mother cell. We propose that such polyQ- and polyN-based elements, termed here mnemons, act as cellular memory devices to encode previous environmental conditions.

Core control principles of the eukaryotic cell cycle.

Cyclin-dependent kinases (CDKs) lie at the heart of eukaryotic cell cycle control, with different cyclin-CDK complexes initiating DNA replication (S-CDKs) and mitosis (M-CDKs)1,2. However, the principles on which cyclin-CDK complexes organize the temporal order of cell cycle events are contentious3. One model proposes that S-CDKs and M-CDKs are functionally specialized, with substantially different substrate specificities to execute different cell cycle events4-6. A second model proposes that S-CDKs and M-CDKs are redundant with each other, with both acting as sources of overall CDK activity7,8. In this model, increasing CDK activity, rather than CDK substrate specificity, orders cell cycle events9,10. Here we reconcile these two views of core cell cycle control. Using phosphoproteomic assays of in vivo CDK activity in fission yeast, we find that S-CDK and M-CDK substrate specificities are remarkably similar, showing that S-CDKs and M-CDKs are not completely specialized for S phase and mitosis alone. Normally, S-CDK cannot drive mitosis but can do so when protein phosphatase 1 is removed from the centrosome. Thus, increasing S-CDK activity in vivo is sufficient to overcome substrate specificity differences between S-CDK and M-CDK, and allows S-CDK to carry out M-CDK function. Therefore, we unite the two opposing views of cell cycle control, showing that the core cell cycle engine is largely based on a quantitative increase in CDK activity through the cell cycle, combined with minor and surmountable qualitative differences in catalytic specialization of S-CDKs and M-CDKs.

Differential Scaling of Gene Expression with Cell Size May Explain Size Control in Budding Yeast.

Yeast cells must grow to a critical size before committing to division. It is unknown how size is measured. We find that as cells grow, mRNAs for some cell-cycle activators scale faster than size, increasing in concentration, while mRNAs for some inhibitors scale slower than size, decreasing in concentration. Size-scaled gene expression could cause an increasing ratio of activators to inhibitors with size, triggering cell-cycle entry. Consistent with this, expression of the CLN2 activator from the promoter of the WHI5 inhibitor, or vice versa, interfered with cell size homeostasis, yielding a broader distribution of cell sizes. We suggest that size homeostasis comes from differential scaling of gene expression with size. Differential regulation of gene expression as a function of cell size could affect many cellular processes.

CDK11 requires a critical activator SAP30BP to regulate pre-mRNA splicing.

CDK11 is an emerging druggable target for cancer therapy due to its prevalent roles in phosphorylating critical transcription and splicing factors and in facilitating cell cycle progression in cancer cells. Like other cyclin-dependent kinases, CDK11 requires its cognate cyclin, cyclin L1 or cyclin L2, for activation. However, little is known about how CDK11 activities might be modulated by other regulators. In this study, we show that CDK11 forms a tight complex with cyclins L1/L2 and SAP30BP, the latter of which is a poorly characterized factor. Acute degradation of SAP30BP mirrors that of CDK11 in causing widespread and strong defects in pre-mRNA splicing. Furthermore, we demonstrate that SAP30BP facilitates CDK11 kinase activities in vitro and in vivo, through ensuring the stabilities and the assembly of cyclins L1/L2 with CDK11. Together, these findings uncover SAP30BP as a critical CDK11 activator that regulates global pre-mRNA splicing.

COSA-1 reveals robust homeostasis and separable licensing and reinforcement steps governing meiotic crossovers.

Crossovers (COs) between homologous chromosomes ensure their faithful segregation during meiosis. We identify C. elegans COSA-1, a cyclin-related protein conserved in metazoa, as a key component required to convert meiotic double-strand breaks (DSBs) into COs. During late meiotic prophase, COSA-1 localizes to foci that correspond to the single CO site on each homolog pair and indicate sites of eventual concentration of other conserved CO proteins. Chromosomes gain and lose competence to load CO proteins during meiotic progression, with competence to load COSA-1 requiring prior licensing. Our data further suggest a self-reinforcing mechanism maintaining CO designation. Modeling of a nonlinear dose-response relationship between IR-induced DSBs and COSA-1 foci reveals efficient conversion of DSBs into COs when DSBs are limiting and a robust capacity to limit cytologically differentiated CO sites when DSBs are in excess. COSA-1 foci serve as a unique live cell readout for investigating CO formation and CO interference.

Cyclin F-mediated degradation of ribonucleotide reductase M2 controls genome integrity and DNA repair.

F-box proteins are the substrate binding subunits of SCF (Skp1-Cul1-F-box protein) ubiquitin ligase complexes. Using affinity purifications and mass spectrometry, we identified RRM2 (the ribonucleotide reductase family member 2) as an interactor of the F-box protein cyclin F. Ribonucleotide reductase (RNR) catalyzes the conversion of ribonucleotides to deoxyribonucleotides (dNTPs), which are necessary for both replicative and repair DNA synthesis. We found that, during G2, following CDK-mediated phosphorylation of Thr33, RRM2 is degraded via SCF(cyclin F) to maintain balanced dNTP pools and genome stability. After DNA damage, cyclin F is downregulated in an ATR-dependent manner to allow accumulation of RRM2. Defective elimination of cyclin F delays DNA repair and sensitizes cells to DNA damage, a phenotype that is reverted by expressing a nondegradable RRM2 mutant. In summary, we have identified a biochemical pathway that controls the abundance of dNTPs and ensures efficient DNA repair in response to genotoxic stress.

CDK-dependent Hsp70 Phosphorylation controls G1 cyclin abundance and cell-cycle progression.

In budding yeast, the essential functions of Hsp70 chaperones Ssa1-4 are regulated through expression level, isoform specificity, and cochaperone activity. Suggesting a novel regulatory paradigm, we find that phosphorylation of Ssa1 T36 within a cyclin-dependent kinase (CDK) consensus site conserved among Hsp70 proteins alters cochaperone and client interactions. T36 phosphorylation triggers displacement of Ydj1, allowing Ssa1 to bind the G1 cyclin Cln3 and promote its degradation. The stress CDK Pho85 phosphorylates T36 upon nitrogen starvation or pheromone stimulation, destabilizing Cln3 to delay onset of S phase. In turn, the mitotic CDK Cdk1 phosphorylates T36 to block Cln3 accumulation in G2/M. Suggesting broad conservation from yeast to human, CDK-dependent phosphorylation of Hsc70 T38 similarly regulates Cyclin D1 binding and stability. These results establish an active role for Hsp70 chaperones as signal transducers mediating growth control of G1 cyclin abundance and activity.

Cyclin F Controls Cell-Cycle Transcriptional Outputs by Directing the Degradation of the Three Activator E2Fs.

E2F1, E2F2, and E2F3A, the three activators of the E2F family of transcription factors, are key regulators of the G1/S transition, promoting transcription of hundreds of genes critical for cell-cycle progression. We found that during late S and in G2, the degradation of all three activator E2Fs is controlled by cyclin F, the substrate receptor of 1 of 69 human SCF ubiquitin ligase complexes. E2F1, E2F2, and E2F3A interact with the cyclin box of cyclin F via their conserved N-terminal cyclin binding motifs. In the short term, E2F mutants unable to bind cyclin F remain stable throughout the cell cycle, induce unscheduled transcription in G2 and mitosis, and promote faster entry into the next S phase. However, in the long term, they impair cell fitness. We propose that by restricting E2F activity to the S phase, cyclin F controls one of the main and most critical transcriptional engines of the cell cycle.