The cell cycle is the fundamental process of cell duplication, essential for growth, development, and tissue homeostasis in multicellular organisms. Precise control of the cell cycle is crucial for maintaining genomic stability and preventing aberrant cell division, which can lead to diseases such as cancer. To ensure this fidelity, cells have evolved sophisticated mitotic signalling mechanisms mediated by cell cycle regulators (CCRs). CCRs consist of several key enzyme classes: cyclin-dependent kinases (Cdks) including Cdk1, Cdk2, and Cdk4; mitotic kinases such as Plk1, Aurora A, and Aurora B; and ubiquitin ligase complexes like Anaphase-Promoting Complex/Cyclosome (APC/C) and the Skp1, Cul1, F-box protein (SCF) complex (reviewed in Refs. [1,2]). These CCRs primarily use post-translational modifications (PTMs) to orchestrate the sequential events of cell division and ensure faithful genetic inheritance.

While the basic principles of the cell cycle are conserved across species, multicellularity has added new layers of complexity. The primary challenge lies in adapting the processes originally designed for single-cell organisms to the development of specialised organs with heterogenous cellular compositions in precise proportions while maintaining homeostasis throughout the life span. This adaptation requires intricate coordination between cell proliferation and differentiation, which must be precisely balanced to maintain tissue architecture and function.

CCRs play a central role in this coordination. Unlike classical developmental mechanisms, which rely largely on transcriptional control and chromatin reorganisation, CCRs operate with greater speed and flexibility through PTMs like phosphorylation and ubiquitination. These PTMs enable rapid and dynamic control of key cell cycle transitions, such as the decision window at mitotic exit when cells must choose between continued proliferation and cell cycle exit or entry into quiescence. In addition, CCR-mediated mechanisms ensure the unidirectional progression of the cell cycle, primarily through ubiquitin-dependent proteolysis, which irreversibly degrades specific proteins to prevent backward progression. Together, these features equip CCRs with the temporal precision and unidirectional control needed to integrate cell reproduction processes with cell fate decisions in multicellular organisms (reviewed in Refs. [1,3,4]).

Recent studies exploiting advanced methodologies in diverse progenitor cell models, such as neural progenitors, early embryonic precursors, and embryonic stem cells (ESCs), have provided deeper mechanical insights into noncanonical functions of CCRs. In the following sections, we highlight key studies focusing on three major processes: the regulation of asymmetric cell division, the maintenance of epigenetic identity, and cell cycle alterations (Figure 1).

Asymmetric cell division is a specialised mode of cell division fundamental to developmental patterning and tissue homeostasis, enabling the generation of distinct daughter cells from a single progenitor. Progenitor cells and stem cells may divide symmetrically to expand cell numbers or divide asymmetrically either by segregating internal fate determinants or by positioning daughter cells into distinct microenvironments that differentially influence their fate. CCRs play active roles in these processes, exemplifying how they integrate cell cycle progression with cell fate decisions. Various progenitors orient the mitotic spindle in response to polarity cues, ensuring that differentiation signals are segregated into one daughter cell while the other retains factors necessary for maintaining proliferative capacity or that the daughter cells are positioned into distinct microenvironments that influence their subsequent differentiation and function [5, 6, 7, 8]. The ability of CCRs to induce rapid PTMs makes them well-suited to coordinate spindle orientation and other dynamic events in the asymmetric division process.

Past work has illuminated specific roles for CCRs, particularly cell cycle kinases, in asymmetric divisions in well-characterised invertebrate models. The Cdk2-Cyclin E complex is essential for asymmetric division in Drosophila neuroblasts [9] and Caenorhabditis elegans embryos [10], while Plk1 and Aurora mitotic kinases phosphorylate fate determinants and adaptor proteins to regulate spindle orientation and polarity establishment [5,11,12]. In C. elegans, Plk1 and Aurora A also play significant roles during the oocyte-to-embryo transition and early embryonic patterning, establishing polarity and symmetry breaking through interactions with the Par complex and other developmental regulators [13, 14, 15, 16, 17, 18, 19, 20]. Asymmetric distribution of cell fate determinants has also been observed in several mammalian systems, including neural progenitors and epidermal stem cells [21, 22, 23]. Given that Plk1 phosphorylates the Notch signalling inhibitor Numb [24,25] and that Aurora A phosphorylates NuMA to align the mitotic spindle in human cell lines [26], functions of these CCRs are likely conserved in vertebrates.

While these studies have revealed their noncanonical roles using classic mutations or RNA interference (RNAi)-mediated depletion of CCRs, prolonged knockdown presents challenges in achieving a high temporal resolution of these events and disentangling cell cycle roles from noncanonical functions particularly in higher eukaryotic cells and tissues. Recent Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9-based approaches [27] allow acute CCR inactivation and PTM target identification in vivo, facilitating precise investigation of CCR functions. A previous study uncovered a critical role of CDK1 in asymmetric division of Drosophila neuroblasts [28]. However, its specific role in asymmetric division remained unclear. To address this, Loyer et al. created a novel, analogue-sensitive (AS) allele of Cdk1 using CRISPR/Cas9 [29], enabling acute and graded Cdk1 inhibition with the addition of an ATP analogue [30]. Applying this mutant to 4D culture of Drosophila larval brains [31], they showed that partial inhibition of Cdk1, which still allows mitotic entry, weakens apical polarity in neuroblasts, leading to defects in the polarisation of fate determinants [29]. Furthermore, Cdk1 was shown to phosphorylate a specific residue (serine 180) on Bazooka, a phosphorylation event conserved in its human orthologue PAR-3, which is crucial for proper fate-determinant polarisation [29].

Beyond phosphorylation-mediated regulation, CCRs also regulate asymmetric division through ubiquitination, particularly via APC/C activity. Activation of APC/C by Cdc20 (Fzy) is required for establishing polarity in C. elegans embryos [32], while mutations in APC/C subunits impair localisation of fate determinants in Drosophila neuroblasts [33]. However, it remained unclear whether this is mediated by ubiquitin-dependent proteolysis and what the key target of APC/C was.

Recent work by Meghini et al. [34], conducting quantitative analysis of mitotic spindle orientation in long-term live imaging of Drosophila neuroblasts, demonstrated that APC/C-dependent ubiquitination of the conserved centrosome component Spd2 [35,36] is critical for maintaining spindle orientation across multiple rounds of mitosis. APC/CCDH1-mediated degradation of Spd2 restricts its levels and mobility at the daughter centrosome, facilitating the accumulation of phosphorylated Spd2 for PCM recruitment [34]. Disrupting the Spd2–Cdh1 interaction compromised the ability of neuroblasts to retain the memory of the apical–basal division axis and led to an irregular neuroblast number [34,36], highlighting the critical role of cell cycle–dependent proteolysis in preserving spindle orientation through successive asymmetric divisions.

Together, these studies illustrate how CCRs coordinate polarity establishment and spindle orientation with cell cycle progression via PTMs to ensure a faithful asymmetric division.

Cell cycle events, such as DNA replication and mitosis, pose significant challenges to maintaining cell identity, which is defined by a cell’s specific transcriptional program. During DNA replication, epigenetic marks must be preserved as histones are temporarily displaced and then reassembled. During mitosis, more dramatic changes occur, including chromosome condensation, nuclear envelope breakdown, and an almost global halt in transcription. Despite these disruptions, cells typically re-establish their transcriptional programs with remarkable accuracy after mitosis, indicating the presence of robust mechanisms that preserve gene expression information across cell divisions [37].

Earlier work established that APC/C targets various chromatin regulatory proteins for degradation [38]. However, the biological relevance of this regulation remained elusive. A recent study by Franks et al. used in silico analysis to identify additional putative APC/C substrates, revealing a significant enrichment for chromatin regulatory proteins, including the DNA methylation factor UHRF1 [39]. UHRF1 is essential for maintaining DNA methylation patterns during DNA replication, and its dysregulation is associated with epigenetic instability [40]. Stabilisation of UHRF1 at mitotic exit not only accelerates G1 progression but also disrupts genome-wide methylation patterns [39]. Furthermore, UHRF1 was also shown to be regulated by the mitotic kinase Plk1, which phosphorylates UHRF1 at serine 265 to enhance its stability via USP7-mediated deubiquitylation [41]. Inhibition of Plk1 leads to accelerated degradation of UHRF1, genome-wide DNA hypomethylation, and reduced cell viability [41]. These findings suggest that CCR-dependent control of epigenetic regulators may safeguard transcriptional integrity during cell cycle transitions, a process likely to be relevant for stem and progenitor cells.

Indeed, recent studies using ESCs have demonstrated direct links to stem cell maintenance. A genome-wide short hairpin RNA screen targeting ubiquitylation regulators in human ESCs identified the APC/C as critical for maintaining pluripotency, whereas the counteracting deubiquitylase USP44 promoted differentiation [42]. Mechanistically, the chromatin-associated proteins WDR5 and TBP recruit APC/C to the transcription start sites of pluripotency-associated genes during interphase [42]. During the following mitosis, the APC/C adds Lys11- and Lys48-linked ubiquitin chains to histones at these loci, targeting them for degradation and opening the chromatin for rapid reactivation of pluripotency genes upon mitotic exit [42]. This mitotic bookmarking helps ESCs re-establish their transcriptional landscape post-mitosis, preserving stem cell identity across divisions.

While this mechanism has been primarily observed in ESC self-renewal, it is plausible that similar mechanisms might also function during differentiation, where prodifferentiation signals could direct APC/C to specific genomic locations to influence gene expression and cell fate decisions in the following G1 phase.

Building upon this idea, a recent study using 16-plex tandem mass tag quantitative mass spectrometry in APC/C mutant mouse cerebellum identified the chromosome passenger complex, topoisomerase 2α, and Ki-67 as major chromatin factors targeted by the APC/C during neuronal differentiation [43]. The clearance of these factors from distinct nuclear subdomains is crucial for epigenetic changes that occur during cell cycle exit and terminal differentiation [43]. Together, these findings underscore the broad role of APC/C in modulating the epigenetic landscape during both stem cell maintenance and the exit from the cell cycle. Importantly, the length of the G1 phase has been suggested as a critical factor in cell fate commitment in various progenitor cells [44, 45, 46]. The ability of APC/C to modulate chromatin accessibility at key gene loci may contribute to such timing-dependent fate decisions.

In addition to ubiquitination by APC/C, phosphorylation by cell cycle kinases also plays a pivotal role in epigenetic regulation [47]. A significant advance has recently been made through the generation of a knock-in mouse model harbouring an AS version of Cdk1, allowing for acute inhibition and thiol-phosphor-labelling of direct CDK1 substrates in vivo [48]. Utilising this labelling technique in ESCs, Michowski et al. discovered that Cdk1 regulates several chromatin modifiers, including Dot1l, which catalyses the formation of H3K79me2, a histone mark tightly associated with active transcription and ESC differentiation [49,50]. It was shown that Cdk1 phosphorylation prevents Dot1l from entering the nucleus, thereby inhibiting the formation of H3K79me2 marks on differentiation-associated genes and maintaining ESCs in a pluripotent state [48]. When cells are exposed to prodifferentiation signals such as retinoic acid or Wnt3a/activin A, this inhibition is relieved, allowing Dot1l to enter the nucleus and activate the prodifferentiation genes [48]. Notably, the identified phosphorylation sites on chromatin regulators are unique to metazoans [48], suggesting that CCRs have evolved specialised functions to meet the intricate regulatory demands of multicellular organisms. In a developmentally distinct context, during the oocyte-to-embryo transition in Drosophila, the mitotic kinase Greatwall (Gwl/Mastl) was shown to regulate maternal messenger RNA stability and translation, further highlighting the capacity of CCRs to modulate gene expression through post-transcriptional mechanisms [51]. These findings reveal a mechanistic link between mitotic chromatin regulation and the preservation of progenitor cell identity.

Beyond their roles in asymmetric cell division and epigenetic regulation, CCRs uniquely adapt their activity to alter cell cycle modes, facilitating differentiation in specialised cell types such as multiciliated cells, which arise from basal progenitor populations, such as those lining the airways, ependymal cells in the brain, and cells in the female reproductive system. In most somatic cells, centriole duplication is tightly coupled with cell cycle progression to maintain proper centrosome numbers and prevent multipolar spindle formation. However, multiciliated cells form supernumerary centrioles while maintaining their ploidy. This raises the question: how do they decouple centriole duplication from cell cycle progression?

Advances in single-cell genomics have shed new light on this unusual cellular behaviour. A recent study by Choksi et al. applied single-cell RNA sequencing to differentiating airway epithelial progenitors in the mouse tracheal epithelium to uncover an alternative cell cycle regulation mechanism that enables centriole amplification without re-entering the cell cycle [52]. Specifically, the study identified E2F7, a member of the E2F family, as a key regulator that suppresses the initiation of the S phase while permitting continuous centriole amplification by CCRs [52].

Further studies have revealed that this cell cycle variant in multiciliated cells involves the temporal replacement of the canonical cyclins, cyclin E2 and cyclin A2, with noncanonical cyclin O and cyclin A1, alongside the repression of the APC/C inhibitor Emi1 [53]. Remarkably, re-introducing the canonical cyclins or Emi1 can partially revert these cells to a normal somatic cell cycle. Furthermore, mutations in cyclin O have been linked to a subtype of primary ciliary dyskinesia that results in a reduced generation of motile cilia, underscoring the clinical relevance of these findings [54].

These examples underscore the adaptability of CCRs in facilitating alternative cell cycle modes necessary for the differentiation of specialised cell types, further emphasising their crucial role in modulating progenitor behaviour and enabling the cellular diversity of multicellular organisms.