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.

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.

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.

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.

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.

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 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.

Cyclin-Specific Docking Mechanisms Reveal the Complexity of M-CDK Function in the Cell Cycle.

Cyclin-dependent kinases (CDKs) coordinate hundreds of molecular events during the cell cycle. Multiple cyclins are involved, but the global role of cyclin-specific phosphorylation has remained unsolved. We uncovered a cyclin docking motif, LxF, that mediates binding of replication factor Cdc6 to mitotic cyclin. This interaction leads to phospho-adaptor Cks1-mediated inhibition of M-CDK to facilitate Cdc6 accumulation and sequestration in mitosis. The LxF motif and Cks1 also mediate the mutual inhibition between M-CDK and the tyrosine kinase Swe1. Additionally, the LxF motif is critical for targeting M-CDK to phosphorylate several mitotic regulators; for example, Spo12 is targeted via LxF to release the phosphatase Cdc14. The results complete the full set of G1, S, and M-CDK docking mechanisms and outline the unified role of cyclin specificity and CDK activity thresholds. Cooperation of cyclin and Cks1 docking creates a variety of CDK thresholds and switching orders, including combinations of last in, first out (LIFO) and first in, first out (FIFO) ordering.

CDK12 phosphorylates 4E-BP1 to enable mTORC1-dependent translation and mitotic genome stability.

The RNA polymerase II (RNAPII) C-terminal domain kinase, CDK12, regulates genome stability, expression of DNA repair genes, and cancer cell resistance to chemotherapy and immunotherapy. In addition to its role in mRNA biosynthesis of DNA repair genes, we show here that CDK12 phosphorylates the mRNA 5' cap-binding repressor, 4E-BP1, to promote translation of mTORC1-dependent mRNAs. In particular, we found that phosphorylation of 4E-BP1 by mTORC1 (T37 and T46) facilitates subsequent CDK12 phosphorylation at two Ser-Pro sites (S65 and T70) that control the exchange of 4E-BP1 with eIF4G at the 5' cap of CHK1 and other target mRNAs. RNA immunoprecipitation coupled with deep sequencing (RIP-seq) revealed that CDK12 regulates release of 4E-BP1, and binding of eIF4G, to many mTORC1 target mRNAs, including those needed for MYC transformation. Genome-wide ribosome profiling (Ribo-seq) further identified specific CDK12 "translation-only" target mRNAs, including many mTORC1 target mRNAs as well as many subunits of mitotic and centromere/centrosome complexes. Accordingly, confocal imaging analyses revealed severe chromosome misalignment, bridging, and segregation defects in cells deprived of CDK12 or CCNK. We conclude that the nuclear RNAPII-CTD kinase CDK12 cooperates with mTORC1, and controls a specialized translation network that is essential for mitotic chromosome stability.

Sall1 and Sall4 cooperatively interact with Myocd and SRF to promote cardiomyocyte proliferation by regulating CDK and cyclin genes.

Sall1 and Sall4 (Sall1/4), zinc-finger transcription factors, are expressed in the progenitors of the second heart field (SHF) and in cardiomyocytes during the early stages of mouse development. To understand the function of Sall1/4 in heart development, we generated heart-specific Sall1/4 functionally inhibited mice by forced expression of the truncated form of Sall4 (ΔSall4) in the heart. The ΔSall4-overexpression mice exhibited a hypoplastic right ventricle and outflow tract, both of which were derived from the SHF, and a thinner ventricular wall. We found that the numbers of proliferative SHF progenitors and cardiomyocytes were reduced in ΔSall4-overexpression mice. RNA-sequencing data showed that Sall1/4 act upstream of the cyclin-dependent kinase (CDK) and cyclin genes, and of key transcription factor genes for the development of compact cardiomyocytes, including myocardin (Myocd) and serum response factor (Srf). In addition, ChIP-sequencing and co-immunoprecipitation analyses revealed that Sall4 and Myocd form a transcriptional complex with SRF, and directly bind to the upstream regulatory regions of the CDK and cyclin genes (Cdk1 and Ccnb1). These results suggest that Sall1/4 are critical for the proliferation of cardiac cells via regulation of CDK and cyclin genes that interact with Myocd and SRF.