Normal levels of p27Xic1 are necessary for somite segmentation and determining pronephric organ size

The Xenopus laevis cyclin dependent kinase inhibitor p27^Xic1 has been shown to be involved in exit from the cell cycle and differentiation of cells into a quiescent state in the nervous system, muscle tissue, heart and retina. We show that p27^Xic1 is expressed in the developing kidney in the nephrostomal regions. Using overexpression and morpholino oligonucleotide (MO) knock-down approaches we show normal levels of p27^Xic1 regulate pronephros organ size by regulating cell cycle exit. Knock-down of p27^Xic1 expression using a MO prevented myogenesis, as previously reported; an effect that subsequently inhibits pronephrogenesis. Furthermore, we show that normal levels of p27^Xic1 are required for somite segmentation also through its cell cycle control function. Finally, we provide evidence to suggest correct paraxial mesoderm segmentation is not necessary for pronephric induction in the intermediate mesoderm. These results indicate novel developmental roles for p27^Xic1, and reveal its differentiation function is not universally utilised in all developing tissues.     

The cdk inhibitor p27Xic1 is required for differentiation of primary neurones in Xenopus

We have investigated the role of the cyclin-dependent kinase inhibitor, p27(Xic1), in the coordination of cell cycle exit and differentiation during early neurogenesis. We demonstrate that p27(Xic1) is highly expressed in cells destined to become primary neurones and is essential for an early stage of neurogenesis. Ablation of p27(Xic1) protein prevents differentiation of primary neurones, while overexpressing p27(Xic1) promotes their formation. p27(Xic1) may enhance neurogenesis by stabilising the bHLH protein, neurogenin. Moreover, the ability of p27(Xic1) to stabilise neurogenin and enhance neurogenesis localises to an N-terminal domain of the molecule and is separable from its ability to inhibit the cell cycle.     

p27Kip1 is required to maintain proliferative quiescence in the adult cochlea and pituitary

Cell cycle inhibitors, such as the cyclin-dependent kinase (Cdk) inhibitor proteins and retinoblastoma (Rb) family members, control exit from the cell cycle during the development of a variety of terminally differentiated tissues. It is unclear whether sustained expression of these proteins is required to prevent cell cycle re-entry in quiescent and terminally differentiated cells. The organ of Corti (cochlear sensory epithelium) and pars intermedia (intermediate lobe of the pituitary) are two tissues that share the characteristic of ongoing cell division in mice lacking either the p27^Kip1 Cdk inhibitor, Ink4 proteins, or Rb. Here, we use tamoxifen-inducible mouse models to delete p27^Kip1 in postnatal animals and show this is sufficient to induce proliferation in both the organ of Corti and pars intermedia. Thus, these tissues remain sensitive to the presence of p27^Kip1 even after their developmental exit from the cell cycle. The neonatal cochlea displayed heightened sensitivity to changes in p27^Kip1 expression, with a proliferative response higher than that of constitutive null mice. In adults, the proliferative response was reduced but was accompanied by increased cell survival. In contrast, re-establishment of normal p27^Kip1 expression in animals with established pituitary tumors, in an inducible “knock-on” model, led to cessation of pituitary tumor growth, indicating the cells had maintained their susceptibility to p27-mediated growth suppression. Although restoration of p27^Kip1 did not induce apoptosis, it did lead to resolution of pathological features and normalization of gene expression. Our data underscore the importance of p27^Kip1 expression in the maintenance of cellular quiescence and terminal differentiation.     

CDK inhibitors,p21 and p27, participate in cell cycle exit of mammalian cardiomyocytes

Mammalian cardiomyocytes actively proliferate during embryonic stages, following which cardiomyocytes exit their cell cycle after birth. The irreversible cell cycle exit inhibits cardiac regeneration by the proliferation of pre-existing cardiomyocytes. Exactly how the cell cycle exit occurs remains largely unknown. Previously, we showed that cyclin E- and cyclin A-CDK activities are inhibited before the CDKs levels decrease in postnatal stages. This result suggests that factors such as CDK inhibitors (CKIs) inhibit CDK activities, and contribute to the cell cycle exit. In the present study, we focused on a Cip/Kip family, which can inhibit cyclin E- and cyclin A-CDK activities. Expression of p21^Cip1 and p27^Kip1 but not p57^Kip2 showed a peak around postnatal day 5, when cyclin E- and cyclin A-CDK activities start to decrease. p21^Cip1 and p27^Kip1 bound to cyclin E, cyclin A and CDK2 at postnatal stages. Cell cycle distribution patterns of postnatal cardiomyocytes in p21^Cip1 and p27^Kip1 knockout mice showed failure in the cell cycle exit at G1-phase, and endoreplication. These results indicate that p21^Cip1 and p27^Kip play important roles in the cell cycle exit of postnatal cardiomyocytes.     

Identification of Xenopus cyclin-dependent kinase inhibitors, p16Xic2 and p17Xic3

The Cip/Kip family of mammalian cyclin-dependent kinase (cdk) inhibitors plays important roles in development, particularly in cell fate determination and differentiation, in addition to their function of blocking cell cycle progression. We have identified two novel members of the Kip/Cip cdk inhibitor family, p16Xic2 and p17Xic3, from Xenopus laevis. Sequence analysis revealed that p16Xic2 and p17Xic3 are orthologues of mammalian p21Cip1 and p27Kip1, respectively. Overexpression of these inhibitors results in cell cycle arrest by inhibition of cdk2 activity. Interestingly, the expression of these inhibitors is highly developmentally regulated. p16Xic2 is highly expressed in differentiating somite, tail bud, lens, and cement gland, while p17Xic3 is expressed in the central nervous system. In a retinal cell fate determination assay, both p16Xic2 and p17Xic3 have an activity that influences cell fate determination. These observations suggest that p16Xic2 and p17Xic3 might be involved in cell fate determination in a tissue-specific manner by coordinating proliferation and differentiation as observed with p27Xic1.     

p27Kip1 is required to maintain proliferative quiescence in the adult cochlea and pituitary

Cell cycle inhibitors, such as the cyclin-dependent kinase (Cdk) inhibitor proteins and retinoblastoma (Rb) family members, control exit from the cell cycle during the development of a variety of terminally differentiated tissues. It is unclear whether sustained expression of these proteins is required to prevent cell cycle re-entry in quiescent and terminally differentiated cells. The organ of Corti (cochlear sensory epithelium) and pars intermedia (intermediate lobe of the pituitary) are two tissues that share the characteristic of ongoing cell division in mice lacking either the p27^Kip1 Cdk inhibitor, Ink4 proteins or Rb. Here, we use tamoxifen-inducible mouse models to delete p27^Kip1 in postnatal animals and show this is sufficient to induce proliferation in both the organ of Corti and pars intermedia. Thus, these tissues remain sensitive to the presence of p27^Kip1 even after their developmental exit from the cell cycle. The neonatal cochlea displayed heightened sensitivity to changes in p27^Kip1 expression, with a proliferative response higher than that of constitutive null mice. In adults, the proliferative response was reduced but was accompanied by increased cell survival. In contrast, re-establishment of normal p27^Kip1 expression in animals with established pituitary tumors, in an inducible “knock-on” model, led to cessation of pituitary tumor growth, indicating the cells had maintained their susceptibility to p27-mediated growth suppression. Although restoration of p27^Kip1 did not induce apoptosis, it did lead to resolution of pathological features and normalization of gene expression. Our data underscore the importance of p27^Kip1 expression in the maintenance of cellular quiescence and terminal differentiation.Key words: proliferation, cell cycle, p27, Cdk inhibitor, auditory, cochlea, pituitary     

p21cip1 is required for the differentiation of oligodendrocytes independently of cell cycle withdrawal

Differentiation of most cell types requires both establishment of G₁ arrest and the induction of a program related to achieving quiescence. We have chosen to study the differentiation of oligodendrocyte cells to determine the role of p27 and p21 in this process. Here we report that both p27 and p21 are required for the appropriate differentiation of these cells. p27 is required for proper withdrawal from the cell cycle, p21 is not. Instead, p21 is required for the establishment of the differentiation program following growth arrest. Similar observations were made in vivo. We show that p21^–/– cells withdraw from the cell cycle similar to wild-type cells; however, early in animal life, the brain is hypomyelinated, inferring that the loss of p21 delayed myelination in the cerebellum. We found that we could complement or bypass the differentiation failure in p21^–/– cells with either PD98059, an inhibitor of Mek1, or by transducing them with a tat–p16^Ink4a protein. We concluded that the two cdk inhibitors serve non-redundant roles in this program of differentiation, with p27 being responsible for arrest and p21 having a function in differentiation independent of its ability to control exit from the cell cycle.     

The Down syndrome-related protein kinase DYRK1A phosphorylates p27Kip1 and Cyclin D1 and induces cell cycle exit and neuronal differentiation

A fundamental question in neurobiology is how the balance between proliferation and differentiation of neuronal precursors is maintained to ensure that the proper number of brain neurons is generated. Substantial evidence implicates DYRK1A (dual specificity tyrosine-phosphorylation-regulated kinase 1A) as a candidate gene responsible for altered neuronal development and brain abnormalities in Down syndrome. Recent findings support the hypothesis that DYRK1A is involved in cell cycle control. Nonetheless, how DYRK1A contributes to neuronal cell cycle regulation and thereby affects neurogenesis remains poorly understood. In the present study we have investigated the mechanisms by which DYRK1A affects cell cycle regulation and neuronal differentiation in a human cell model, mouse neurons, and mouse brain. Dependent on its kinase activity and correlated with the dosage of overexpression, DYRK1A blocked proliferation of SH-SY5Y neuroblastoma cells within 24 h and arrested the cells in G₁ phase. Sustained overexpression of DYRK1A induced G₀ cell cycle exit and neuronal differentiation. Furthermore, we provide evidence that DYRK1A modulated protein stability of cell cycle-regulatory proteins. DYRK1A reduced cellular Cyclin D1 levels by phosphorylation on Thr286, which is known to induce proteasomal degradation. In addition, DYRK1A phosphorylated p27^Kip1 on Ser10, resulting in protein stabilization. Inhibition of DYRK1A kinase activity reduced p27^Kip1 Ser10 phosphorylation in cultured hippocampal neurons and in embryonic mouse brain. In aggregate, these results suggest a novel mechanism by which overexpression of DYRK1A may promote premature neuronal differentiation and contribute to altered brain development in Down syndrome.     

Retinoblastoma tumor suppressor protein-dependent methylation of histone H3 lysine 27 is associated with irreversible cell cycle exit

The retinoblastoma tumor suppressor protein (pRb) is involved in mitotic exit, promoting the arrest of myoblasts, and myogenic differentiation. However, it is unclear how permanent cell cycle exit is maintained in differentiated muscle. Using RNA interference, expression profiling, and chromatin immunoprecipitations, we show that pRb is essential for cell cycle exit and the differentiation of myoblasts and is also uniquely required to maintain this arrest in myotubes. Remarkably, we also uncover a function for the pRb-related proteins p107 and p130 as enforcers of a G2/M phase checkpoint that prevents progression into mitosis in cells that have lost pRb. We further demonstrate that pRb effects permanent cell cycle exit in part by maintaining trimethylation of histone H3 lysine 27 (H3K27) on cell cycle genes. H3K27 trimethylation silences other genes, including Cyclin D1, in a pRb-independent but polycomb-dependent manner. Thus, our data distinguish two distinct chromatin-based regulatory mechanisms that lead to terminal differentiation.     

The cell cycle inhibitor p27Kip1 controls self-renewal and pluripotency of human embryonic stem cells by regulating the cell cycle,Brachyury and Twist

The continued turn over of human embryonic stem cells (hESC) while maintaining an undifferentiated state is dependent on the regulation of the cell cycle. Here we asked the question if a single cell cycle gene could regulate the self-renewal or pluripotency properties of hESC. We identified that the protein expression of the p27^Kip1 cell cycle inhibitor is low in hESC cells and increased with differentiation. By adopting a gain and loss of function strategy we forced or reduced its expression in undifferentiating conditions to define its functional role in self-renewal and pluripotency. Using undifferentiation conditions, overexpression of p27^Kip1 in hESC lead to a G₁ phase arrest with an enlarged and flattened hESC morphology and consequent loss of self-renewal ability. Loss of p27^Kip1 caused an elongated/scatter cell-like phenotype involving upregulation of Brachyury and Twist gene expression. We demonstrate the novel finding that p27^Kip1 protein occupies the Twist1 gene promoter and manipulation of p27^Kip1 by gain and loss of function is associated with Twist gene expression changes. These results define p27^Kip1 expression levels as critical for self-renewal and pluripotency in hESC and suggest a role for p27^Kip1 in controlling an epithelial to mesenchymal transition (EMT) in hESC.     

A cell-autonomous requirement for Cip/Kip cyclin-kinase inhibitors in regulating neuronal cell cycle exit but not differentiation in the developing spinal cord

Control over cell cycle exit is fundamental to the normal generation of the wide array of distinct cell types that comprise the mature vertebrate CNS. Here, we demonstrate a critical role for Cip/Kip class cyclin-kinase inhibitory (CKI) proteins in regulating this process during neurogenesis in the embryonic spinal cord. Using immunohistochemistry, we show that all three identified Cip/Kip CKI proteins are expressed in both distinct and overlapping populations of nascent and post-mitotic neurons during early neurogenesis, with p27(Kip1) having the broadest expression, and both p57(Kip2) and p21(Cip1) showing transient expression in restricted populations. Loss- and gain-of-function approaches were used to establish the unique and redundant functions of these proteins in spinal cord neurogenesis. Using genetic lineage tracing, we provide evidence that, in the absence of p57, nascent neurons re-enter the cell cycle inappropriately but later exit to begin differentiation. Analysis of p57(Kip2);p27(Kip1) double mutants, where p21 expression is confined to only a small population of interneurons, demonstrates that Cip/Kip CKI-independent factors initiate progenitor cell cycle exit for the majority of interneurons generated in the developing spinal cord. Our studies indicate that p57 plays a critical cell-autonomous role in timing cell cycle exit at G1/S by opposing the activity of Cyclin D1, which promotes cell cycle progression. These studies support a multi-step model for neuronal progenitor cell cycle withdrawal that involves p57(Kip2) in a central role opposing latent Cyclin D1 and other residual cell cycle promoting activities in progenitors targeted for differentiation.     

The CRL4Cdt2 Ubiquitin Ligase Mediates the Proteolysis of Cyclin-Dependent Kinase Inhibitor Xic1 through a Direct Association with PCNA

During DNA polymerase switching, the Xenopus laevis Cip/Kip-type cyclin-dependent kinase inhibitor Xic1 associates with trimeric proliferating cell nuclear antigen (PCNA) and is recruited to chromatin, where it is ubiquitinated and degraded. In this study, we show that the predominant E3 for Xic1 in the egg is the Cul4-DDB1-XCdt2 (Xenopus Cdt2) (CRL4^Cdt2) ubiquitin ligase. The addition of full-length XCdt2 to the Xenopus extract promotes Xic1 turnover, while the N-terminal domain of XCdt2 (residues 1 to 400) cannot promote Xic1 turnover, despite its ability to bind both Xic1 and DDB1. Further analysis demonstrated that XCdt2 binds directly to PCNA through its C-terminal domain (residues 401 to 710), indicating that this interaction is important for promoting Xic1 turnover. We also identify the cis-acting sequences required for Xic1 binding to Cdt2. Xic1 binds to Cdt2 through two domains (residues 161 to 170 and 179 to 190) directly flanking the Xic1 PCNA binding domain (PIP box) but does not require PIP box sequences (residues 171 to 178). Similarly, human p21 binds to human Cdt2 through residues 156 to 161, adjacent to the p21 PIP box. In addition, we identify five lysine residues (K180, K182, K183, K188, and K193) immediately downstream of the Xic1 PIP box and within the second Cdt2 binding domain as critical sites for Xic1 ubiquitination. Our studies suggest a model in which both the CRL4^Cdt2 E3- and PIP box-containing substrates, like Xic1, are recruited to chromatin through independent direct associations with PCNA.The eukaryotic cell cycle is positively regulated by cyclin-dependent kinases (CDKs) and negatively regulated by CDK inhibitors (CKIs) (22, 25, 27, 28). A complete knockout of all CDK inhibitor function, although as yet not attained in mammalian cells, has been accomplished in Saccharomyces cerevisiae and is shown to result in genomic instability due to premature entry into S phase (19). Conversely, the overexpression of cyclin E in mammalian cells has also been observed to induce chromosome instability (31). These studies suggest that CDK inhibitor function can play a critical role in maintaining genomic stability through the proper regulation of DNA replication initiation. Mammalian Cip/Kip-type CDK inhibitors p27 and p21 are stoichiometric inhibitors of CDK2-cyclins that regulate the entry into S phase and are targeted by ubiquitin- and proteasome-dependent proteolysis during the G₁-to-S-phase transition (4, 5, 33, 35). In the frog, Xenopus laevis, three types of CDK inhibitors have been identified that share sequence and functional similarities with mammalian p27 and p21. The first type of CDK inhibitor includes the Xenopus inhibitor of CDK (p27^Xic1 or Xic1) and kinase inhibitor from Xenopus (p28^Kix1 or Kix1), which share ∼90% amino acid sequence identity with each other, preferentially inhibit the activity of CDK2-cyclin E or A and bind all CDK-cyclins and proliferating cell nuclear antigen (PCNAs) (30, 32). The second and third types of Xenopus CDK inhibitors are p16^Xic2 and p17^Xic3, which share sequence homology with p21 and p27, respectively, and exhibit restricted developmental expression but have not been extensively characterized biochemically (9).In an effort to study the molecular mechanism of Cip/Kip-type CDK inhibitor proteolysis in the context of the temporal events of DNA replication initiation, we utilize the biochemically tractable Xenopus egg extract system. This extract can recapitulate all of the events of semiconservative DNA replication and fully support protein ubiquitination and degradation in the context of DNA replication initiation (3, 36). Using this system, we have shown that during DNA polymerase switching, Xic1 is recruited to sites of DNA replication initiation through its association with proliferating cell nuclear antigen (PCNA) and is targeted for ubiquitination and degradation (6). Using a strategy of PCNA reconstitution to PCNA-depleted extracts, our studies showed that Xic1 ubiquitination and turnover required not only PCNA binding but also the ability of PCNA to be loaded at a site of DNA replication initiation by replication factor C (RFC) (6). Our previous study indicated that like mammalian p27 and p21, Xic1 could be ubiquitinated in vitro by SCF^XSkp2 (21), but our subsequent studies suggested that Xenopus Skp2 (XSkp2) levels were very low in the early embryo, and XSkp2 immunodepletion did not stabilize Xic1 in the Xenopus egg extract (our unpublished observations). Therefore, we postulated that in the interphase egg extract, Xic1 was targeted for ubiquitination by an alternate ubiquitin ligase.In this study, we identify Cul4-DDB1-XCdt2 (CRL4^Cdt2) as the ubiquitin ligase for Xic1 in the egg. We also identify both the critical residues of Xic1 required for association to Cdt2 and the critical lysine residues of Xic1 ubiquitinated by CRL4^Cdt2. Importantly, we report a direct interaction between the C-terminal domain of Cdt2 and PCNA and show that the C-terminal domain of Cdt2 is required to promote the proteolysis of Xic1. Our studies suggest a model for Xic1 ubiquitination and proteolysis which requires the Xic1 PIP box for association with PCNA and Xic1 chromatin recruitment, the Xic1 sequences flanking the PIP box for association with Cdt2, specific lysine residues within the Cdt2 binding domain of Xic1 for efficient Xic1 ubiquitination, and a direct association between the Cdt2 C terminus and PCNA.     

The Notch ligand JAG1 is required for sensory progenitor development in the mammalian inner ear

In mammals, six separate sensory regions in the inner ear are essential for hearing and balance function. Each sensory region is made up of hair cells, which are the sensory cells, and their associated supporting cells, both arising from a common progenitor. Little is known about the molecular mechanisms that govern the development of these sensory organs. Notch signaling plays a pivotal role in the differentiation of hair cells and supporting cells by mediating lateral inhibition via the ligands Delta-like 1 and Jagged (JAG) 2. However, another Notch ligand, JAG1, is expressed early in the sensory patches prior to cell differentiation, indicating that there may be an earlier role for Notch signaling in sensory development in the ear. Here, using conditional gene targeting, we show that the Jag1 gene is required for the normal development of all six sensory organs within the inner ear. Cristae are completely lacking in Jag1-conditional knockout (cko) mutant inner ears, whereas the cochlea and utricle show partial sensory development. The saccular macula is present but malformed. Using SOX2 and p27^kip1 as molecular markers of the prosensory domain, we show that JAG1 is initially expressed in all the prosensory regions of the ear, but becomes down-regulated in the nascent organ of Corti by embryonic day 14.5, when the cells exit the cell cycle and differentiate. We also show that both SOX2 and p27^kip1 are down-regulated in Jag1-cko inner ears. Taken together, these data demonstrate that JAG1 is expressed early in the prosensory domains of both the cochlear and vestibular regions, and is required to maintain the normal expression levels of both SOX2 and p27^kip1. These data demonstrate that JAG1-mediated Notch signaling is essential during early development for establishing the prosensory regions of the inner ear.     

Proteolysis of <Emphasis Type="Italic">Xenopus</Emphasis> Cip-type CDK inhibitor,p16<Superscript>Xic2</Superscript>, is regulated by PCNA binding and CDK2 phosphorylation

Background

Cell division is positively regulated by cyclin-dependent kinases (CDKs) partnered with cyclins and negatively regulated by CDK inhibitors. In the frog, Xenopus laevis, three types of CDK inhibitors have been described: p27^Xic1 (Xic1) which shares sequence homology with both p21^Cip1 and p27^Kip1 from mammals, p16^Xic2 (Xic2) which shares sequence homology with p21^Cip1, and p17^Xic3 (Xic3) which shares sequence homology with p27^Kip1. While past studies have demonstrated that during DNA polymerase switching, Xic1 is targeted for protein turnover dependent upon DNA, Proliferating Cell Nuclear Antigen (PCNA), and the ubiquitin ligase CRL4^Cdt2, little is known about the processes that regulate Xic2 or Xic3.

Methods

We used the Xenopus interphase egg extract as a model system to examine the regulation of Xic2 by proteolysis and phosphorylation.

Results

Our studies indicated that following primer synthesis during the initiation of DNA replication, Xic2 is targeted for DNA- and PCNA-dependent ubiquitin-mediated proteolysis and that Cdt2 can promote Xic2 turnover. Additionally, during interphase, Xic2 is phosphorylated by CDK2 at Ser-98 and Ser-131 in a DNA-independent manner, inhibiting Xic2 turnover. In the presence of double-stranded DNA ends, Xic2 is also phosphorylated at Ser-78 and Ser-81 by a caffeine-sensitive kinase, but this phosphorylation does not alter Xic2 turnover. Conversely, in the presence or absence of DNA, Xic3 was stable in the Xenopus interphase egg extract and did not exhibit a shift indicative of phosphorylation.

Conclusions

During interphase, Xic2 is targeted for DNA- and PCNA-dependent proteolysis that is negatively regulated by CDK2 phosphorylation. During a response to DNA damage, Xic2 may be alternatively regulated by phosphorylation by a caffeine-sensitive kinase. Our studies suggest that the three types of Xenopus CDK inhibitors, Xic1, Xic2, and Xic3 appear to be uniquely regulated which may reflect their specialized roles during cell division or early development in the frog.