cdc2


EVOLUTIONARY HOMOLOGS


Table of contents

Targets of cdc2 involved directly in mitosis

In fission yeast, Cdc2 kinase has both positive and negative roles in regulating DNA replication, being first necessary for the transition from G1 to S phase and later required to prevent the re-initiation of DNA replication during G2. Cdc2 interacts with Orp2, a protein similar to the Orc2 replication factor subunit of Saccharomyces cerevisiae origin recognition complex (ORC). ORC binds chromosomal origins and is essential for chromosomal replication initiation. Fission yeast Orp2 is required for DNA replication; it interacts with the rate-limiting replication activator Cdc18. Cells lacking Orp2 undergo aberrant mitosis, indicating that Orp2 is involved in generating a checkpoint signal. These findings suggest that ORC functions are conserved among eukaryotes and provide evidence that Cdc2 controls DNA replication initiation by acting directly at chromosomal origins (Leatherwood, 1996).

Two specific components are required for the ubiquitination of mitotic cyclins: E2-C, a cyclin-selective ubiquitin carrier protein that is constitutively active during the cell cycle, and E3-C, a cyclin-selective ubiquitin ligase that purifies as part of an approximately 1500-kDa complex, termed the cyclosome, which is active only near the end of mitosis. The cyclosome has been separated from its ultimate upstream activator, cdc2. The mitotic, active form of the cyclosome can be inactivated by incubation with a partially purified, endogenous okadaic acid-sensitive phosphatase; addition of cdc2 restores activity to the cyclosome after a lag that reproduces one seen previously in intact cells and in crude extracts. These results demonstrate that activity of cyclin-ubiquitin ligase is controlled by reversible phosphorylation of the cyclosome complex (Lahav-Baratz, 1995).

A human homolog of Xenopus Eg5, a kinesin-related motor protein is implicated in the assembly and dynamics of the mitotic spindle. Microinjection of antibodies against human Eg5 (HsEg5) blocks centrosome migration and causes HeLa cells to arrest in mitosis with monoastral microtubule arrays. An evolutionarily conserved cdc2 phosphorylation site (Thr-927) in HsEg5 is phosphorylated specifically during mitosis in HeLa cells and by p34cdc2/cyclin B in vitro. Mutation of Thr-927 to nonphosphorylatable residues prevents HsEg5 from binding to centrosomes, indicating that phosphorylation controls the association of this motor with the spindle apparatus. These results indicate that HsEg5 is required for establishing a bipolar spindle and that p34cdc2 protein kinase directly regulates its localization (Blangy, 1995).

Cdc2 is localized to the centrosome region and is tightly bound to the nuclear matrix-intermediate filament scaffold. Antibodies to Cdc2 and to the centrosome-specific protein, pericentrin, label the centrosome in an apparently cell cycle independent manner. Isolated centrosomes also label similarly with both antibodies. Essentially, all cells show Cdc2 labeling of the centrosomes, implying their independence of the cell cycle phase, a conclusion supported by studies of synchronized cells. In contrast to the labeling of every cell with the Cdc2 monoclonal antibody, fewer centrosomes are labeled with an antibody to the PSTAIRE domain of Cdc2. Both Cdc2 and pericentrin antibodies decorate the amorphous pericentriolar material, while the Cdc2 antibodies also decorate the centrioles themselves. The constitutive presence of Cdc2 at the centrosome suggests a continuing role in the dynamics of centrosome function throughout the cell cycle (Pockwinse, 1997).

Cdc2 kinase triggers the entry of mammalian cells into mitosis, the only cell cycle phase in which transcription is globally repressed. Cdc2 kinase phosphorylates components of the RNA polymerase II transcription machinery, including the RNA polymerase II carboxy-terminal repeat domain (CTD). To test the specific effect of CTD phosphorylation by Cdc2 kinase, a yeast in vitro transcription extract was used that is dependent on exogenous RNA polymerase II, which contains a CTD. Phosphorylation was carried out using immobilized Cdc2, allowing the kinase to be removed from the phosphorylated polymerase. ATP gamma S and Cdc2 kinase were used to produce an RNA polymerase IIO that is not detectably dephosphorylated in the transcription extract. RNA polymerase IIO produced in this way is defective in promoter-dependent transcription, suggesting that phosphorylation of the CTD by Cdc2 kinase can mediate transcription repression during mitosis. In addition, phosphorylation of pol II with the human TFIIH-associated kinase Cdk7 also decreases transcription activity despite a different pattern of CTD phosphorylation by this kinase. These results extend previous findings that RNA polymerase IIO is defective in preinitiation complex formation. This study shows that phosphorylation of the CTD by cyclin-dependent kinases with different phosphoryl acceptor specificities can inhibit transcription in a CTD-dependent transcription system (Gebara, 1997).

Protein phosphatase 1 (PP-1) is known to be a critical component of eukaryotic cell cycle progression. In vitro, cdc2 kinase phosphorylates Thr-320 (T320) in PP-1, and this leads to inhibition of enzyme activity. To examine directly the phosphorylation of PP-1 in intact mammalian cells, an antibody has been prepared that specifically recognizes PP-1C alpha phosphorylated at T320. Cell synchronization studies reveal in a variety of cell types that T320 of PP-1 is phosphorylated to high levels only during early to mid-mitosis. The phosphorylation of T320 of PP-1 is reduced by the cyclin-dependent protein kinase inhibitor, olomoucine, and increased by the PP-1/PP-2A inhibitor, calyculin A. In NIH 3T3 cells the phosphorylation of PP-1 begins to increase from basal levels in prophase and to peak at metaphase. Phospho-PP-1 is localized exclusively to nonchromosomal regions. In cell fractionation studies of mitotic cells, phospho-PP-1 is detectable only in the soluble fraction. These observations suggest that phosphorylation by cdc2 kinase in early to mid-mitosis and inhibition of PP-1 activity is likely to contribute to the increased state of phosphorylation of proteins that is critical to the initiation of normal cell division (Kwon, 1997).

Sam68 (Src-associated in mitosis) is an SH3 (Src-homology 3), SH2 (Src-homology 2), and RNA binding protein that both associates with and is tyrosine phosphorylated by wild-type and activated forms of c-Src in a mitosis-specific manner. Sam68 immunoprecipitated from either HeLa S3 or NIH3T3 cells is phosphorylated exclusively on threonine residues during mitosis, as well as on serine residues during both interphase and mitosis. After incubation with mitotic lysates, recombinant Sam68, expressed as a glutathione S-transferase (GST) fusion protein, is phosphorylated on threonine and serine residues several-fold more extensively than after incubation with unsynchronized lysates. Cdc2 was identified as the kinase responsible for the mitotic threonine phosphorylation by (1) immunodepletion of the mitotic Sam68 kinase from cell lysates with anti-Cdc2 antibodies, (2) inhibition of Sam68 phosphorylation in vitro and in vivo by the cyclin-dependent kinase inhibitor olomoucine and (3) phosphorylation of Sam68 by purified Cdc2. These data demonstrate that Sam68 is a direct target of Cdc2 and may therefore mediate some of its biological effects during mitosis (Resnick, 1997).

Fanconi anemia (FA) is an autosomal recessive disorder characterized by developmental defects, bone marrow failure, and cancer susceptibility. Cells derived from FA patients are sensitive to crosslinking agents and have a prolonged G2 phase, suggesting a cell cycle abnormality. Although transfection of type-C FA cells with the FAC cDNA corrects these cellular abnormalities, the molecular function of the FAC polypeptide remains unknown. Expression of the FAC polypeptide is regulated during cell cycle progression. In synchronized HeLa cells, FAC protein expression increases during S phase, is maximal at the G2/M transition, and declines during M phase. In addition, the FAC protein coimmunoprecipitates with the cyclin-dependent kinase, cdc2. Various mutant forms of the FAC polypeptide were tested for binding to cdc2. A patient-derived mutant FAC polypeptide, containing a point mutation at L554P, fails to bind to cdc2. The FAC/cdc2 binding interaction therefore correlates with the functional activity of the FAC protein. Binding of FAC to cdc2 is mediated by the carboxyl-terminal 50 amino acids of FAC in a region of the protein required for FAC function. Taken together, these results suggest that the binding of FAC and cdc2 is required for normal G2/M progression in mammalian cells. Absence of a functional interaction between FAC and cdc2 in FA cells may underlie the cell cycle abnormality and clinical abnormalities of FA (Kupfer, 1997).

A novel gene, psp1(+), which functionally complements a temperature-sensitive mutant defective in cell cycle progression both in G1/S and G2/M has been isolated from the genomic and cDNA libraries of Schizosaccharomyces pombe. Disruption of this gene is lethal for cell growth at 30 degrees C indicating that it is an essential gene for vegetative cell growth. The Psp1 protein exists in two different molecular weight forms depending on the growth state of the cell. In vitro experiments with a phosphatase show that this difference is due to phosphorylation. The dephosphorylated form of the protein is dominant in actively growing cells whereas the phosphorylated form becomes the major species when cells enter the stationary phase. The Cdc2-Cdc13 complex is shown to phosphorylate the protein in vitro; the serine residue at position 333 in the carboxyl-terminal region is required for phosphorylation. This protein tends to be localized to both ends of the cell upon entry into the stationary phase of cell growth. However, overexpression of the novel protein Psp1 in actively growing cells inhibits cell growth, causing accumulation of DNA (4n or 8n). Thus it is speculated that Psp1 can function at both G1/S and G2/M phases, complementing the defect of the new mutant that has been isolated. It is likely that Psp1 is required both for proper DNA replication and for the process of mitosis (Jang, 1997).

The kinesin-related motor HsEg5 is essential for centrosome separation; its association with centrosomes appears to be regulated by phosphorylation of tail residue threonine 927 by the p34(cdc2) protein kinase. To identify proteins able to interact with the tail of HsEg5, a yeast two-hybrid screen was performed with a HsEg5 stalk-tail construct as bait. A cDNA was isolated coding for the central, alpha-helical region of human p150(Glued), a prominent component of the dynactin complex. The interaction between HsEg5 and p150(Glued) is enhanced upon activation of p34(CDC28), the budding yeast homolog of p34(cdc2), provided that HsEg5 has a phosphorylatable residue at position 927. Phosphorylation also enhances the specific binding of p150(Glued) to the tail domain of HsEg5 in vitro, indicating that the two proteins are able to interact directly. Immunofluorescence microscopy reveals co-localization of HsEg5 and p150(Glued) during mitosis but not during interphase, consistent with a cell cycle-dependent association between the two proteins. Taken together, these results suggest that HsEg5 and p150(Glued) may interact in mammalian cells in vivo and that p34(cdc2) may regulate this interaction. Furthermore, they imply that the dynactin complex may functionally interact not only with dynein but also with kinesin-related motors (Blangy, 1997).

Degradation of mitotic cyclins on exit from M phase occurs by ubiquitin-mediated proteolysis. The ubiquitination of mitotic cyclins is regulated by the anaphase-promoting complex (APC) or cyclosome. Xe-p9, the Xenopus homolog of the Suc1/Cks protein, is required for some step in the destruction of mitotic cyclin in Xenopus egg extracts. In addition to cyclin and Cdk, a third protein component of Cdk/cyclin complexes belongs to the Suc1/Cks family of proteins. This protein has been implicated in Cdk regulation through both genetic and biochemical methods. Suc1 (in fission yeast) and Cks1 (in budding yeast) were both identified on the basis of their ability to suppress certain temperature-sensitive mutations of Cdks. In human cells, two homologs of the Cks1 protein have been isolated. If Xenopus p9 is removed from interphase egg extracts, these p9-depleted extracts are unable to carry out the proteolysis of cyclin B after entry into mitosis and thus remain arrested in M phase. To explore the molecular basis of this defect, p9 was depleted from extracts that had already entered M phase and thus contained an active APC. Ubiquitin-mediated proteolysis of cyclin B is not compromised under these circumstances, suggesting that p9 is not directly required for ubiquitination or proteolysis. Further analysis of extracts from which p9 had been removed during interphase shows that, at the beginning of mitosis, these extracts are unable to carry out the hyperphosphorylation of the Cdc27 component of the APC, which coincides with the initial activation of the APC. p9 can be found in a complex with a small fraction of the Cdc27 protein during M phase but not interphase. The phosphorylation of the Cdc27 protein (either associated with the APC or in an isolated, bacterially expressed form) by recombinant Cdc2/cyclin B is strongly enhanced by p9. These results indicate that p9 directly regulates the phosphorylation of the APC by Cdc2/cyclin B. These studies indicate that the Suc1/Cks protein modulates substrate recognition by a cyclin-dependent kinase (Patra, 1998).

Molecular markers of the zebrafish inner nuclear membrane (NEP55) and nuclear lamina (L68) were identified, partially characterized and used to demonstrate that disassembly of the zebrafish nuclear envelope requires sequential phosphorylation events by first PKC, then Cdc2 kinase. NEP55 and L68 are immunologically and functionally related to human LAP2beta and lamin B, respectively. Exposure of zebrafish nuclei to meiotic cytosol elicits rapid phosphorylation of NEP55 and L68, and disassembly of both proteins. L68 phosphorylation is completely inhibited by simultaneous inhibition of Cdc2 and PKC and only partially blocked by inhibition of either kinase. NEP55 phosphorylation is completely prevented by inhibition or immunodepletion of cytosolic Cdc2. Inhibition of cAMP-dependent kinase, MEK or CaM kinase II does not affect NEP55 or L68 phosphorylation. In vitro, nuclear envelope disassembly requires phosphorylation of NEP55 and L68 by both mammalian PKC and Cdc2. Inhibition of either kinase is sufficient to abolish NE disassembly. Furthermore, novel two-step phosphorylation assays in cytosol and in vitro indicate that PKC-mediated phosphorylation of L68 prior to Cdc2-mediated phosphorylation of L68 and NEP55 is essential to elicit nuclear envelope breakdown. Phosphorylation elicited by Cdc2 prior to PKC prevents nuclear envelope disassembly even though NEP55 is phosphorylated. The results indicate that sequential phosphorylation events elicited by PKC, followed by Cdc2, are required for zebrafish nuclear disassembly. They also argue that phosphorylation of inner nuclear membrane integral proteins is not sufficient to promote nuclear envelope breakdown, and suggest a multiple-level regulation of disassembly of nuclear envelope components during meiosis and at mitosis (Collas, 1999).

The cyclosome/anaphase-promoting complex is a multisubunit ubiquitin ligase that targets for degradation mitotic cyclins and some other cell cycle regulators in exit from mitosis. It becomes enzymatically active at the end of mitosis. The activation of the cyclosome is initiated by its phosphorylation, a process necessary for its conversion to an active form by the ancillary protein Cdc20/Fizzy. Previous reports have implicated either cyclin-dependent kinase 1-cyclin B or polo-like kinase as the major protein kinase that directly phosphorylates and activates the cyclosome. These conflicting results could be due to the use of partially purified cyclosome preparations or of immunoprecipitated cyclosome, whose interactions with protein kinases or ancillary factors may be hampered by binding to immobilized antibody. To examine this problem, cyclosome has been purified from HeLa cells by a combination of affinity chromatography and ion exchange procedures. With the use of purified preparations, it was found that both cyclin-dependent kinase 1-cyclin B and polo-like kinase directly phosphorylate the cyclosome, but the pattern of the phosphorylation of the different cyclosome subunits by the two protein kinases is not similar. Plk1 and Cdk1/cyclin-B have additive effects in phosphorylating and activating the APC/C; the former preferentially phosphorylates Cdc16 and Cdc23, and the latter preferentially phosphorylates Cdc27. Each protein kinase can restore only partially the cyclin-ubiquitin ligase activity of dephosphorylated cyclosome. However, following phosphorylation by both protein kinases, an additive and nearly complete restoration of cyclin-ubiquitin ligase activity is observed. It is suggested that this joint activation may be due to the complementary phosphorylation of different cyclosome subunits by the two protein kinases (Golan, 2002).

Spindle assembly is subject to the regulatory controls of both the cell-cycle machinery and the Ran-signaling pathway. An important question is how the two regulatory pathways communicate with each other to achieve coordinated regulation in mitosis. Cdc2 kinase phosphorylates the serines located in or near the nuclear localization signal (NLS) of human RCC1, the nucleotide exchange factor for Ran. This phosphorylation is necessary for RCC1 to generate RanGTP on mitotic chromosomes in mammalian cells, which in turn is required for spindle assembly and chromosome segregation. Moreover, phosphorylation of the NLS of RCC1 is required to prevent the binding of importin alpha and beta to RCC1, thereby allowing RCC1 to couple RanGTP production to chromosome binding. These findings reveal that the cell-cycle machinery directly regulates the Ran-signaling pathway by placing a high RanGTP concentration on the mitotic chromosome in mammalian cells (Li, 2004).

The survival of eukaryotes depends on the accurate coordination of mitosis with cytokinesis. Key for the coordination of both processes is the chromosomal passenger complex (CPC) comprising Aurora-B, INCENP, survivin, and borealin. The translocation of the CPC from centromeres to the spindle midzone, a structure composed of antiparallel microtubules, at anaphase onset is critical for the completion of cytokinesis. In mammalian cells, the mitotic kinesin Mklp2 is essential for recruitment of the CPC to the spindle midzone. However, the mechanism regulating the binding of Mklp2 to microtubules has remained unknown. This study demonstrates that Mklp2 and the CPC mutually depend on each other for midzone localization; i.e., Mklp2 is mislocalized in INCENP-RNAi cells and vice versa. Remarkably, INCENP is required for localization of Mklp2 to the ends of stable microtubules in cells with low Cdk1 activity. In vitro assays revealed that the association between the CPC and Mklp2 is negatively regulated by Cdk1. Collectively, these data suggest that anaphase onset triggers the association between the CPC and Mklp2 and that this association targets the CPC-Mklp2 complex to the ends of stable microtubules in the spindle midzone (Hümmer, 2009).

Entry into M phase is governed by cyclin B-Cdk1, which undergoes both an initial activation and subsequent autoregulatory activation. A key part of the autoregulatory activation is the cyclin B-Cdk1-dependent inhibition of the protein phosphatase 2A (PP2A)-B55, which antagonizes cyclin B-Cdk1. Greatwall kinase (Gwl) is believed to be essential for the autoregulatory activation because Gwl is activated downstream of cyclin B-Cdk1 to phosphorylate and activate alpha-endosulfine (Ensa)/Arpp19, an inhibitor of PP2A-B55. However, cyclin B-Cdk1 becomes fully activated in some conditions lacking Gwl, yet how this is accomplished remains unclear. This study shows that cyclin B-Cdk1 can directly phosphorylate Arpp19 on a different conserved site, resulting in inhibition of PP2A-B55. Importantly, this novel bypass is sufficient for cyclin B-Cdk1 autoregulatory activation. Gwl-dependent phosphorylation of Arpp19 is nonetheless necessary for downstream mitotic progression because chromosomes fail to segregate properly in the absence of Gwl. Such a biphasic regulation of Arpp19 results in different levels of PP2A-B55 inhibition and hence might govern its different cellular roles (Okumura, 2014).

Cdc2 and the mitotic spindle and mitotic spindle checkpoint

The spindle checkpoint prevents anaphase onset until completion of mitotic spindle assembly by restraining activation of the ubiquitin ligase anaphase-promoting complex/cyclosome-Cdc20 (APC/CCdc20: Fizzy is the Drosophila homolog of Cdc20). The spindle checkpoint requires mitotic cyclin-dependent kinase (cdk) activity. Inhibiting cdk activity overrides checkpoint-dependent arrest in Xenopus egg extracts and human cells. Following inhibition, the interaction between APC/C and Cdc20 transiently increases while the inhibitory checkpoint protein Mad2 dissociates from Cdc20. Cdk inhibition also overcomes Mad2-induced mitotic arrest. In addition, in vitro cdk1-phosphorylated Cdc20 interacts with Mad2 rather than APC/C. Thus, cdk activity is required to restrain APC/CCdc20 activation until completion of spindle assembly (D'Angiolella, 2003).

A possible target for cdk action in regulating Cdc20 interaction with APC/C and Mad2 could be Cdc20 itself. Cdc20 contains multiple cdk phosphorylation sites and is phosphorylated during mitosis and under checkpoint conditions. Mutation of several of these sites substantially reduces Cdc20 phosphorylation by mitotic egg extracts and by cdk1. In addition, it has been shown by in vitro studies that phosphorylated Cdc20 is a poorer stimulator of APC/C activity than non- or hypo-phosphorylated Cdc20 and that cdk-dependent phosphorylation of Cdc20 inhibits binding to APC componenet Cdc27 (see Drosophila Cdc27/Makos). Thus, under checkpoint conditions, cdk1-dependent phosphorylation of Cdc20 may affect the ability of Cdc20 to bind APC/C and Mad2. Phosphorylated Cdc20 has retarded mobility on SDS-PAGE. To analyze the phosphorylation status of Cdc20 in checkpoint-arrested cells after cdk inhibition, HeLa proteins were separated in longer SDS-PAGE runs to better visualize changes in Cdc20 mobility. Cdc20 mobility increases after addition of cdk inhibitor Roscovitine to checkpoint-arrested cells, suggesting that Cdc20 becomes dephosphorylated. Cdc20 isolated from checkpoint-arrested cells also shows increased mobility after treatment with lambda protein phosphatase, indicating that the increased mobility can be ascribed to dephosphorylation. The timing of Cdc20 dephosphorylation is compatible with the hypothesis that dephosphorylation of Cdc20 increases binding to APC/C while it decreases it to Mad2 (D'Angiolella, 2003).

Egg extracts were used to study in more detail the relevance of Cdc20 phosphorylation for the interaction with APC/C and Mad2. Does Cdc20 phosphorylation depend on cdk1 or cdk2 activity in egg extracts? The results indicate that cdk1 rather than cdk2 is responsible for Cdc20 phosphorylation (D'Angiolella, 2003).

To know whether cdk1-dependent phosphorylation affects the ability of Cdc20 to interact with Cdc27 and Mad2 in egg extracts, [35S]-labeled Cdc20 wild type and a mutant Cdc20 version nonphosphorylatable at seven cdk phosphorylation sites (7A) were used. Using this mutant, it has been shown that cdk1-phosphorylated Cdc20 binds poorly to Cdc27 in vitro. Cdc20 wild type and 7A were independently pretreated with active cdk1 and then incubated in CSF-arrested extracts with recombinant Mad2. Cdc27 or Mad2 were isolated 30 min later and the amount of bound Cdc20 wild type or Cdc20 7A was determined. Cdc27 binds more highly to Cdc20 7A than to Cdc20 wild type. Conversely, Mad2 preferentially binds to Cdc20 wild type than to Cdc20 7A. Although cdk-dependent phosphorylation of Cdc20 in vivo may not be so extensive as in vitro, these data show that cdk-phosphorylated Cdc20 interacts more efficiently with Mad2 than Cdc27. Mad2-dependent arrest is maintained in the presence of the cdk1-phosphorylated wild-type Cdc20 but not in the presence of the phosphorylation-resistant mutant Cdc20 version. These findings are consistent with the in vivo findings that cdk inhibition induces dissociation of Cdc20 from Mad2 and a transient increase of Cdc20 binding to Cdc27 (D'Angiolella, 2003).

These observations, from Xenopus egg extracts and human somatic cells, indicate a crucial role for cdk1 activity in the regulation of the spindle checkpoint. Cdk1 activity appears to be required to inhibit APC/CCdc20 activation by stabilizing Cdc20-Mad2 interaction and reducing Cdc20 binding to APC/C via direct phosphorylation of Cdc20 (D'Angiolella, 2003).

In Xenopus egg extracts and embryos, cyclin E-cdk2 activity is relevant for many aspects of the M phase; it is also involved in checkpoint control. It is possible that, under checkpoint conditions, cyclin E-cdk2 is required to sustain cdk1 activity, and that this is ultimately responsible to inhibit APC/CCdc20 activation. It is presently unclear why cyclin E-cdk2 plays a role in mitotic events in this system, while it does not appear to be so relevant for mitosis in somatic cells. Many reports also indicate that MAPK participates in checkpoint regulation in egg extracts. MAPK may participate directly in checkpoint regulation, and also indirectly, like cdk2, by sustaining cdk1 activity (D'Angiolella, 2003).

In mammalian cells, cdk2 may not have a crucial role in spindle checkpoint-dependent arrest because the abundance of its partners, cyclin A and E, is very low under checkpoint conditions, and the relevance of the MAPK pathway in mitosis and spindle checkpoint is not yet completely understood . Some human cancers, however, show deregulated cyclin E expression and cyclin E-cdk2 activity is present at high levels also in mitosis. Observations in the embryonic system may provide a framework to explain some aspects of the oncogenic potential of deregulated cyclin E expression and why this causes chromosomal instability in somatic mammalian cells (D'Angiolella, 2003).

During mitosis, cdk1 may perform positive phosphorylations of APC/C, a prerequisite for its activity, but at the same time, by phosphorylating Cdc20, it lessens interaction between APC/C and Cdc20 and provides a precondition for inhibitory checkpoint proteins, activated at unattached kinetochores like Mad2, to interact with Cdc20. Thus, upon completion of mitotic spindle assembly, activated checkpoint proteins are no longer produced and active APC/Cdc20 can begin to form. An alternative hypothesis could be that the checkpoint mechanism acts also by sustaining the pathways that control and maintain cdk activation. Thus, upon completion of mitotic spindle assembly, an early and transient proteolysis-independent drop in cdk1 activity may help rapid APC/Cdc20 reactivation by increasing the Cdc20-APC/C interaction and inducing the dissociation of activated checkpoint proteins from Cdc20 (D'Angiolella, 2003).

The bipolar mitotic spindle is responsible for segregating sister chromatids at anaphase. Microtubule motor proteins generate spindle bipolarity and enable the spindle to perform mechanical work. A major change in spindle architecture occurs at anaphase onset when central spindle assembly begins. This structure regulates the initiation of cytokinesis and is essential for its completion. Central spindle assembly requires the centralspindlin complex composed of the Caenorhabditis elegans ZEN-4 (mammalian orthologue MKLP1) kinesin-like protein and the Rho family GAP CYK-4 (MgcRacGAP). This study describes a regulatory mechanism that controls the timing of central spindle assembly. The mitotic kinase Cdk1/cyclin B phosphorylates the motor domain of ZEN-4 on a conserved site within a basic amino-terminal extension characteristic of the MKLP1 subfamily. Phosphorylation by Cdk1 diminishes the motor activity of ZEN-4 by reducing its affinity for microtubules. Preventing Cdk1 phosphorylation of ZEN-4/MKLP1 causes enhanced metaphase spindle localization and defects in chromosome segregation. Thus, phosphoregulation of the motor domain of MKLP1 kinesin ensures that central spindle assembly occurs at the appropriate time in the cell cycle and maintains genomic stability (Mishima, 2004).

GRASP65, a structural protein of the Golgi apparatus, has been linked to the sensing of Golgi structure and the integration of this information with the control of mitotic entry in the form of a Golgi checkpoint. Cdk1-cyclin B is the major kinase phosphorylating GRASP65 in mitosis, and phosphorylated GRASP65 interacts with the polo box domain of the polo-like kinase Plk1. GRASP65 is phosphorylated in its C-terminal domain at four consensus sites by Cdk1-cyclin B, and mutation of these residues to alanine essentially abolishes both mitotic phosphorylation and Plk1 binding. Expression of the wild-type GRASP65 C-terminus but not the phosphorylation defective mutant in normal rat kidney cells causes a delay but not the block in mitotic entry expected if this were a true cell cycle checkpoint. These findings identify a Plk1-dependent signalling mechanism potentially linking Golgi structure and cell cycle control, but suggest that this may not be a cell cycle checkpoint in the classical sense (Preisinger, 2005).

Mitotic CDKs control the metaphase-anaphase transition and trigger spindle elongation

Mitotic cyclin-dependent kinases (CDKs) control entry into mitosis, but their role during mitotic progression is less well understood. This study characterizes the functions of CDK activity associated with the mitotic cyclins Clb1, Clb2, and Clb3. Clb-CDKs are important for the activation of the ubiquitin ligase Anaphase-Promoting Complex/Cyclosome (APC/C)-Cdc20 that triggers the metaphase-anaphase transition. Furthermore, an essential role is defined for Clb-CDK activity in anaphase spindle elongation. Thus, mitotic CDKs serve not only to initiate M phase, but are also needed continuously throughout mitosis to trigger key mitotic events such as APC/C activation and anaphase spindle elongation (Rahal, 2008).

It has been suggested that increasing amounts of CDKs establish consecutive cell cycle events, with low CDK levels promoting S phase and high levels of CDKs triggering M phase. The data indicate that increasing amounts of Clb-CDKs are also responsible for triggering consecutive mitotic events. Inactivation of CLB1 and CLB2 causes no delay in cell cycle progression prior to metaphase, whereas inactivation of CLB2 and CLB3 causes a modest 30-min delay. In contrast, inactivation of CLB1, CLB2, CLB3, and CLB4 causes cell cycle arrest in G2, with replicated DNA and unseparated SPBs. Furthermore, Clb-CDK activity rises as cells progress through mitosis. Based on these observations, it is proposed that higher amounts of mitotic CDK activity are needed for entry into anaphase than for entry into mitosis. This steady rise in mitotic CDK activity helps establish the order of events during early mitosis, with a lower threshold of Clb-CDK activity triggering entry into mitosis and a second, higher one triggering entry into anaphase. Finally, once anaphase entry has been initiated, Clb proteolysis causes a decline in Clb-CDK activity, triggering exit from mitosis (during which thresholds may also play a role. Interestingly, increasing amounts of mitotic CDK activity may also govern progression through early stages of mitosis in mammalian cells. Mitotic CDK activity rises as cells progress from G2 into metaphase. Furthermore, complete inactivation of Cdk1 by RNAi-based methods prevents entry into mitosis, whereas partial Cdk1 inactivation delays entry into anaphase. Therefore, requiring a steady rise in mitotic CDK activity for mitotic progression may be a general mechanism by which all eukaryotic cells ensure that chromosome segregation occurs only after chromosomes have condensed and a mitotic spindle has formed (Rahal, 2008).

Cdc2 and reactivation of transcription at the end of mitosis

Activation of HO in yeast involves recruitment of transcription factors in two waves. The first is triggered by inactivation of Cdk1 (Cdc2) at the end of mitosis, which promotes import into the nucleus of the Swi5 transcription factor. Swi5 recruits the Swi/Snf chromatin-remodeling complex, which then facilitates recruitment of the SAGA histone acetylase, which in turn permits the binding of the SBF transcription factor. SBF then recruits the SRB/mediator complex and this process occurs in the absence of Cdk1 activity. The second wave is triggered by reactivation of Cdk1, which leads to recruitment of PolII, TFIIB, and TFIIH. RNA polymerase is, therefore, recruited to HO in two steps and not as a holoenzyme. A similar sequence of events occurs at other SBF-regulated promoters, such as CLN1, CLN2, and PCL1 (Cosma, 2001).

Differential gene expression forms the basis for most differences in the behavior and properties of distinct cell types within a multicellular organism. This cell type diversification usually arises due to variations between cells in the abundance of DNA sequence-specific transcription factors (STFs). Such factors promote transcription either by recruiting general transcription factors (GTFs) to TATA boxes close to transcription initiation sites or by recruiting chromatin-remodeling factors like the Swi/Snf complex and the SAGA histone acetyl transferase, which alter a promoter's chromatin structure and thereby facilitate recruitment of other STFs and GTFs. Once established, changes in a promoter's chromatin structure are sometimes self-propagating and do not require the continued presence of STFs that initiated the change. Thus, the multitudinal differences between cell types in their patterns of gene expression arise through a complex interplay between the concentration of specific transcription factors within cells at the time of transcription and the history of each of its genes' chromatin structure. The latter is itself a legacy of specific transcription factors to which the cell has been exposed in the past and stochastic events that affect the propagation of chromatin structures induced by these factors (Cosma, 2001).

Recruitment of polymerase II is a crucial event in gene transcription. In yeast, 'core' polymerase consists of twelve subunits called Rpb1-Rpb12. The C-terminal domain (CTD) of the largest of these subunits, Rpb1, contains multiple copies of the sequence YSPTSPS, which is phosphorylated by several protein kinases, including Kin28 (the yeast equivalent to Cdk7), associated with TFIIH. PolII's CTD associates with an Srb/mediator complex, which is composed of a 'head' subcomplex containing Srb proteins and an middle/tail subcomplex containing proteins such as Rgr1. Because PolII can be isolated as a holoenzyme in which core polymerase is associated with the Srb/mediator complex, it is thought that the role of many if not most STFs is to recruit such an RNA polymerase holoenzyme to promoters (Cosma, 2001).

Transcription of protein-encoding genes requires, in addition to the PolII/Srb/mediator complex, several other general transcription factors: TFIID formed by TBP and TAFs (TBP-associated factors), TFIIA, TFIIB, TFIIE, TFIIF, and TFIIH. Recruitment of holoenzyme and GTFs to the TATA box to form a preinitiation complex (PIC) is thought to precede the activation of transcription. An important issue is whether holoenzyme and general transcription factors are recruited in a defined order or whether they are all recruited simultaneously, possibly as a preassembled 'complex of complexes'. Following initiation, a scaffold composed of activator, mediator, and some GTFs remains associated with the promoter and promotes formation of a functional reinitiation complex (Cosma, 2001 and references therein).

The precise roles of general transcription factors, activators, and coactivators have been extensively studied using in vitro systems in which they can be shown to stimulate or repress transcription. Such studies assess what factors are capable of, but cannot address their actual role at real promoters in vivo. To do this, it is necessary first to know where and when chromatin remodeling has taken place and where and when site-specific and general transcription factors have bound to the promoter in question. Such information can now be gathered by measuring the abundance of individual DNA sequences that can be immunoprecipitated using antibodies directed toward specific chromatin-bound factors. This technique, known as chromatin immunoprecipitation (chIP), can be combined with the use of genetic mutations to determine the interdependency of factor recruitment and thereby build a picture of the pathway by which transcription complexes are assembled on real promoters in vivo. A major limitation associated with this approach is the potential pleiotropy of mutations. This is especially problematic when the mutations in question affect general factors needed for transcription of most if not all PolII-dependent genes. Under these circumstances, it is difficult if not impossible to attribute dependency to direct effects unless phenotypes are measured within minutes of a factor's inactivation, which is often impossible to achieve. One solution to this problem is to study promoters at which events involving general transcription factors are controlled by specific physiological signals or by transcription factors whose realm of action is far more specific than that of general transcription factors. One promoter that is particularly suitable in this regard is that of the HO gene in yeast, which encodes an endonuclease that initiates mating-type switching in a lineage- and cell cycle-dependent manner in haploid yeast gametes (Cosma, 2001).

HO transcription is activated in late G1 of the cell cycle in only one of the two progeny produced at the previous cell division, in the so-called 'mother' cell that has just given birth to a bud, or 'daughter' cell. HO is activated in a stepwise fashion, which commences with the arrival during anaphase of the Swi5 transcription factor. Swi5 then recruits the Swi/Snf nucleosome-remodeling factor, which in turn recruits the SAGA histone acetyltransferase. Both events are aborted by the arrival within daughter nuclei but not within mother nuclei of a repressor called Ash1. The remodeling of nucleosomes on the HO promoter mediated by Swi/Snf and SAGA permits the binding of a second sequence-specific transcription factor called SBF, whose activity is also essential for HO transcription (Cosma, 2001).

The entry of Swi5 into nuclei during late anaphase/telophase (and hence all early events associated with the HO promoter's activation) is thought to be triggered by inactivation of the Cdk1 protein kinase, at least partly through the proteolysis of its B-type cyclin partners by a ubiquitin protein ligase called the anaphase-promoting complex (APC). This paper investigates events associated with the onset of transcription of four SBF-regulated genes, HO, CLN1, CLN2, and PCL1, since Cdk1 is reactivated through its association with the 'G1' Cln cyclins (e.g., Cln3) during late G1. Using chIP, the arrival on the HO promoter is measured of the large subunit of PolII, four subunits of the Srb/mediator complex, TFIIB, and Kin28, which is the protein kinase associated with TFIIH implicated in CTD phosphorylation and promoter clearance. Binding of SBF to HO, which is made possible by the prior remodeling of its chromatin structure by Swi/Snf and SAGA, is crucial for the subsequent recruitment of Srb/mediator, TFIIB, and Kin28. Surprisingly, the SRB/mediator complex is recruited to HO in the absence of Cdk1 activity, whereas PolII, TFIIB, and Kin28 are only recruited in its presence. These results suggest that RNA polymerase is recruited to SBF-regulated genes in two steps and not as a holoenzyme (Cosma, 2001).

Cyclin-dependent kinases regulate epigenetic gene silencing through phosphorylation of EZH2

The Polycomb group (PcG) protein, enhancer of zeste homologue 2 (EZH2), has an essential role in promoting histone H3 lysine 27 trimethylation (H3K27me3) and epigenetic gene silencing. This function of EZH2 is important for cell proliferation and inhibition of cell differentiation, and is implicated in cancer progression. This study demonstrates that under physiological conditions, cyclin-dependent kinase 1 (CDK1) and cyclin-dependent kinase 2 (CDK2) phosphorylate EZH2 at Thr 350 in an evolutionarily conserved motif. Phosphorylation of Thr 350 is important for recruitment of EZH2 and maintenance of H3K27me3 levels at EZH2-target loci. Blockage of Thr 350 phosphorylation not only diminishes the global effect of EZH2 on gene silencing, it also mitigates EZH2-mediated cell proliferation and migration. These results demonstrate that CDK-mediated phosphorylation is a key mechanism governing EZH2 function and that there is a link between the cell-cycle machinery and epigenetic gene silencing (Chen, 2010).

Enhancer of zeste homologue 2 (EZH2) is the catalytic subunit of Polycomb repressive complex 2 (PRC2) and catalyses the trimethylation of histone H3 on Lys 27 (H3K27), which represses gene transcription. EZH2 enhances cancer-cell invasiveness and regulates stem cell differentiation. This study demonstrates that EZH2 can be phosphorylated at Thr 487 through activation of cyclin-dependent kinase 1 (CDK1). The phosphorylation of EZH2 at Thr 487 disrupts EZH2 binding with the other PRC2 components SUZ12 and EED, and thereby inhibits EZH2 methyltransferase activity, resulting in inhibition of cancer-cell invasion. In human mesenchymal stem cells, activation of CDK1 promotes mesenchymal stem cell differentiation into osteoblasts through phosphorylation of EZH2 at Thr 487. These findings define a signalling link between CDK1 and EZH2 that may have an important role in diverse biological processes, including cancer-cell invasion and osteogenic differentiation of mesenchymal stem cells (Wei, 2011).

NURF facilitates the progesterone receptor-mediated recruitment of Cdk2/CyclinA, which is required for histone H1 displacement

Gene regulation by external signals requires access of transcription factors to DNA sequences of target genes, which is limited by the compaction of DNA in chromatin. Althought insight has been gained into how core histones and their modifications influence this process, the role of linker histones remains unclear. This study show that, within the first minute of progesterone action, a complex cooperation between different enzymes acting on chromatin mediates histone H1 displacement as a requisite for gene induction and cell proliferation. First, activated progesterone receptor (PR) recruits the chromatin remodeling complexes NURF and ASCOM (ASC-2 [activating signal cointegrator-2] complex) to hormone target genes. The trimethylation of histone H3 at Lys 4 by the MLL2/MLL3 subunits of ASCOM, enhanced by the hormone-induced displacement of the H3K4 demethylase KDM5B, stabilizes NURF binding. NURF facilitates the PR-mediated recruitment of Cdk2/CyclinA, which is required for histone H1 displacement. Cooperation of ATP-dependent remodeling, histone methylation, and kinase activation, followed by H1 displacement, is a prerequisite for the subsequent displacement of histone H2A/H2B catalyzed by PCAF and BAF. Chromatin immunoprecipitation (ChIP) and sequencing (ChIP-seq) and expression arrays show that H1 displacement is required for hormone induction of most hormone target genes, some of which are involved in cell proliferation (Vicent, 2011).

These results contribute to a better comprehension of the molecular mechanisms of gene induction by describing the very initial steps of hormonal promoter activation. The data reveal an unexpected complexity in the interactions between enzymatic activities implicated in preparing the chromatin for full access of transcription factors. Apart from previously described enzymatic activities, at least four complexes act 1 min after hormone addition. An ATP-dependent chromatin remodeling complex (NURF), a protein kinase complex (Cdk2/CyclinA), a histone lysine demethylase (JARID1B/KDM5B), and a histone lysine methylase (MLL2 or MLL3)-containing complex cooperate in the displacement of histone H1 from the promoter, an important early step in gene induction by progestins (Vicent, 2011).

It has been shown, in T47D-MTVL cells treated with hormone for 5 min, PR interacts with an exposed HRE on the surface of a nucleosome positioned over the MMTV promoter and recruits Brg1/Brm-containing BAF complexes. This study demonstrates that NURF interacts with PR, and that recruitment of the NURF complex in the first minute following hormone addition is a requisite for subsequent binding of BAF and activation of mammary tumor virus (MMTV) and other progesterone target genes. NURF is anchored at the promoter of progesterone target genes by an interaction with H3K4me3, likely generated by the MLL2/3 histone lysine methylases of the ASCOM complex. This is reminiscent of the role of hormone-induced acetylation of H3K14 in anchoring the BAF complex. At both phases in activation of the promoter, a histone tail modification stabilizes the binding of an ATP-dependent chromatin remodeling complex to the target promoters (Vicent, 2011).

Another similarity between the two subsequent cycles of promoter chromatin remodeling relates to the role of protein kinases. It was found previously that hormone-induced activation of the Src/Ras/Erk cascade leads to phosphorylation of PR at S294 and activation of Msk1, which is targeted to the promoter by PR and phosphorylates H3 at S10, contributing to the displacement of a repressive complex containing HP1γ. This study shows that, prior to this event, 1 min after hormone, PR interacts with a complex of Cdk2 and CyclinA that phosphorylates PR at S400, is recruited to the promoter, and phosphorylates histone H1, leading to its displacement. Thus, there are two similar and consecutive cycles essential for transcriptional activation of hormone-dependent genes, both involving the collaboration between protein kinases, histone-modifying enzymes, and ATP-dependent chromatin remodelers. Each of the remodeling complexes is anchored at the promoter by different epigenetic marks: H3K4me3 established by MLL2/3 anchors NURF, and H3K14ac established by PCAF anchors BAF. The final output of the first cycle is to decompact the chromatin fiber by displacing histone H1, and the outcome of the second cycle is to open the nucleosome by displacing H2A/H2B dimers (Vicent, 2011).

The chromatin remodeling complex NURF has been shown to be necessary for both transcription activation and repression in vivo. Most reports on the role of NURF in gene regulation come from studies in Drosophila, where NURF is involved in the activation of several genes, including the homeotic selector gene engrailed, ultrabithorax, ecdysone-responsive genes, and the roX noncoding RNA. These studies were complemented with mechanistic studies using recombinant Drosophila NURF complex. In contrast, little is known regarding the mechanism of action of NURF in human cells, except for reports on a role in neuronal physiology. It was found that, in T47D-MTVL human breast cancer cells, NURF is essential for efficient hormone-dependent activation of several PR target genes, and is recruited to the target promoters via an interaction with PR. The BAF complex is also recruited to the MMTV promoter within minutes after progestin treatment, but the kinetics of loading of both chromatin remodelers are different. NURF is recruited after 1 min of hormone treatment, while BAF is loaded only after 5 min and its recruitment depends on NURF action. These findings highlight the notion of transcription initiation as a process involving consecutive cycles of enzymatic chromatin remodeling, where each enzyme complex is necessary at a given time point and catalyzes a particular remodeling step. These results support the existence in T47D-MTVL cells of several pools of PR, associated with the different chromatin remodelers. How the coordinated action of each PR population on target promoters is orchestrated is not well understood, but phosphorylation of the receptor by different kinases and post-translational modifications of nucleosomal histones could provide possible mechanisms (Vicent, 2011).

Although H3K4me3 marks transcription start sites (TSSs) of virtually all active genes the role of this modification during MMTV activation has been questioned. This study shows that, in T47D-MTVL cells, the MLL2/3-containing complex ASCOM is recruited to a target promoter after 1 min of hormone and increases H3K4me3. Experiments with siRNA knockdown, ChIP, and peptide pull-down assays showed that H3K4me3 is critical for NURF anchoring at the promoter. The very early and transient appearance of the H3K4me3 mark could explain the apparent controversy with previously published studies. It was found that the H3K4me3 signal observed at the MMTV promoter is due to the concerted recruitment of the ASCOM complex and the localized displacement of the H3K4me3/2/1 demethylase KDM5B. Knockdown of KDM5B increased the basal and hormone-dependent activity of PR target genes and caused an increase in H3K4me3 levels at the promoters in the absence of hormone (Vicent, 2011).

The molecular mechanism underlying hormone-induced displacement of KDM5B is unclear. It has been reported that KDM5B forms a complex with histone deacetylases (HDACs). Ir was shown previously that an HP1γ-containing complex is bound to the MMTV promoter prior to induction, and is displaced by phosphorylation of H3S10 catalyzed by hormone-activated Msk1. However, in coimmunoprecipitation experiments, no interaction between KDM5B and HP1γ was detected. Recently, it has been reported that PARP1 can parylate and inactivate KDM5B catalytic activity. Since nuclear receptors are known to activate PARP1, it is possible that this pathway participates in the inactivation and displacement of KDM5B following progestin treatment (Vicent, 2011).

The PHD finger present in the BPTF subunit of NURF acts as a highly specialized methyl lysine-binding domain critical for NURF loading. H3S10ph and H3K14ac, two other post-translational modifications present in the MMTV promoter chromatin after hormone addition, increase the binding of the PHD domain to H3K4me3. Binding of BPTF to acetylated lysines could be expected, as the protein contains a bromodomain in its C terminus, but the interaction with H3 phosphopeptides was not predicted, as BPTF does not encompass a consensus 14-3-3-like domain. Regarding the role of the H3K9me3 signal in NURF recruitment, peptide pull-down experiments showed no interaction of NURF components with the H3K9me3 mark. Moreover, either knockdown or inhibition of the methyltransferase G9a increased the basal level of transcription in several target genes without affecting the fold induction after hormone. The same effect was observed when cells were depleted of HP1γ, indicating that the H3K9me3 signal anchors HP1γ at the target chromatin (Vicent, 2011).

The NURF complex is recruited after 1 min of hormone, decreased after 2 min, and is almost undetectable after 5 min. How NURF is released from target chromatin is still unknown. Binding of NURF correlates closely with H3K4me3, and therefore a decrease in the trimethylation of H3K4 would explain NURF displacement. It has been proposed that methylation of histone H3R2 by PRMT6 and methylation of H3K4 by MLLs are mutually exclusive. Moreover, H3R2 methylation has been reported to block the binding of effectors that harbor methyl-specific binding domains, including PHD domains, chromodomains, and Tudor domains. Thus, the presence of the H3R2me2 mark could cooperate in erasing the H3K4me3 signal from the promoters and in competing for NURF binding, thus triggering NURF displacement (Vicent, 2011).

MMTV minichromosomes reconstituted with Drosophila embryo extracts were used previously to address the role of histone H1. Histone H1 increases nucleosome spacing and compacts the chromatin, hinders access of general transcription factors to the MMTV promoter, and thus inhibits basal transcription. In the presence of bound PR, H1 is phosphorylated by Cdk2 and subsequently is removed from the promoter upon transcription initiation. The kinase Cdk2 is known to phosphorylate histone H1 in vivo, resulting in a more open chromatin structure by destabilizing H1-chromatin interactions. Histone H1 phosphorylation by Cdk2 has been associated with hormone-dependent transcriptional activation. This study found that NURF facilitates the access of Cdk2/CyclinA to target promoter chromatin, and this could explain its role in H1 displacement from the MMTV promoter and from 15 other PR-binding sites that also contain NURF and recruit Cdk2 after hormone treatment. Along with the general effect of Cdk2 inhibition on gene regulation by progestins, these results support a very general role of Cdk2/CyclinA in histone H1 eviction during the initial steps of hormonal chromatin remodeling (Vicent, 2011).

There is evidence for a direct interaction between PR and Cdk2, CyclinA, or cyclinE that could explain how Cdk2/CyclinA is recruited to the target promoters. In the T47D-MTVL breast cancer cell line, a hormone-independent association of PR with Cdk2 was found and recruitment of CyclinA to this complex upon hormone addition. Therefore, PR could recruit Cdk2/CyclinA to the target promoter upon hormone addition. It is not known whether NURF and Cdk2/CyclinA form a single ternary complex with PR, or rather are in two different PR-associated complexes. Although by coimmunoprecipitation interaction of PR with both complexes was detected after 1 min of hormone addition, a more in-depth analysis performed at 1-min intervals at 30°C revealed that NURF is recruited before Cdk2/CyclinA. These results suggest that NURF recruitment is required for Cdk2/CyclinA loading at target promoters, and support the existence of two independent complexes (Vicent, 2011).

Although H1 displacement takes place locally, it could have a long-range effect on chromatin decompaction, as demonstrated with in vitro assembled condensed chromatin. Displacement of histone H1 could be a prerequisite for all subsequent steps in remodeling, as SWI/SNF remodeling has been reported to be inhibited by the presence of histone H1. A connection between ISWI-containing remodeling machineries and histone H1 dynamics has been reported previously in Drosophila. In this system, ISWI promotes the association of the linker histone H1 with chromatin. Along these lines, it is still possible that NURF is also involved in later steps during hormone induction by helping histone H1 deposition back at the promoter (Vicent, 2011).

How H1 binding is regulated and leads to a more open chromatin structure remains unclear. Some models proposed that Cdk2-dependent H1 phosphorylation leads to the decondensation of chromatin during interphase by disrupting the association of HP1γ with the chromatin fiber. A hormone-dependent displacement of HP1γ from the MMTV promoter was observed without changes in H3K9me3 levels. Whether H1 and Hp1γ are interacting as part of a common repressive complex requires further studies but constitutes an attracting hypothesis. In contrast, PARP-1 possesses the ability to disrupt chromatin structure by PARylating histones (e.g., H1 and H2B) and a variety of nuclear proteins involved in gene regulation. Both PARP-1 and H1 compete for binding to nucleosomes and exhibit a reciprocal pattern of binding at actively transcribed promoters: H1 is depleted and PARP-1 is enriched. Other post-translational modifications of H1 have been proposed to influence its binding and function. Histone H1 is acetylated at Lys 26 in vivo and can be deacetylated by the NAD+-dependent HDAC SirT1, promoting the formation of repressive heterochromatin. This effect was accompanied by an enrichment of H1 at the promoter, and the spreading of heterochromatin marks like H3K9me3 and H4K20me1 throughout the coding region (Vicent, 2011).

Regarding the NURF-mediated changes in chromatin structure, analysis of nucleosome profiles obtained by MNase digestion before hormone treatment showed a preferential location of nucleosomes overlapping with NURF and PR sites that is less pronounced after hormone activation, indicating that chromatin remodeling is involved (Vicent, 2011).

Analysis of the hormone-regulated genes that are affected by depletion of NURF reveals many genes involved in cell cycle and cell proliferation, which could mediate the proliferative response of breast cancer cells to progestins. This may explain the inhibition of cell proliferation in response to progestins that was observed in T47D cells depleted of NURF. A similar inhibition of the proliferative response has been observed in cells depleted of Cdk2. These results indicate that histone H1 displacement may be a prerequisite for the effects of progestins on cell proliferation, and therefore the enzymes involved in this process would be novel targets for the pharmacological control of breast cancer cell proliferation (Vicent, 2011).

A model of the current view of the initial steps in progesterone activation of the MMTV promoter is presented. Although the different steps of remodeling are depicted as a linear time sequence, it cannot be excluded that some of these process occur in parallel and in different time sequences in different target promoters. The model reflects the average time sequence in the cell population. Briefly, after hormone induction, activated PR carrying Erk and Msk1 binds first to the exposed HRE1 on the surface of the MMTV promoter nucleosome in a process that does not require chromatin remodeling. Along with the activated PR kinases, the NURF and ASCOM complexes are recruited to the promoter chromatin in one or several complexes. The combined action of ASCOM recruitment and KDM5B displacement enhances H3K4me3 and stabilizes NURF at the promoter. Other modifications, such as H3S10phos and H3K14ac produced by Msk1 and PCAF, could also contribute to NURF anchoring. Once at the promoter, NURF remodels the nucleosome and facilitates the access of PR and the associated Cdk2/CyclinA kinase, which phosphorylates histone H1 and promotes its displacement, contributing to unfolding of the chromatin fiber. Although it was observed that H3S10 phosphorylation by Msk1 plays a role in HP1γ displacement, it is possible that phosphorylation of histone H1 also contributes to this process. H1-depleted nucleosomes constitute a suitable substrate for recruitment of PR-BAF complexes and further remodeling events catalyzed by BAF and PCAF. H3K14 acetylation by PCAF promotes BAF anchoring. BAF mediates ATP-dependent displacement of histones H2A/H2B, and thus facilitates binding of NF1. Bound NF1 stabilizes the open conformation of the H3/H4 tetramer particle that exposes the previously hidden HREs, allowing synergistic binding of further PR-BAF-kinase complexes and PCAF (Vicent, 2011).

Finally, given that NURF is also recruited to the promoter 30 min after hormone addition, when no H1 is present, it cannot be excluded that NURF catalyzes later steps in chromatin remodeling involving histones or nonhistone chromatin proteins. Indeed, the current results indicate that NURF can act on MMTV minichromosomes lacking histone H1. In this respect, it remains to be established whether NURF and BAF fulfill partly redundant functions, cooperate on the same promoter, or, rather, are mutually exclusive (Vicent, 2011).

Phosphorylation of the PRC2 component Ezh2 is cell cycle-regulated and up-regulates its binding to ncRNA

Ezh2 functions as a histone H3 Lys 27 (H3K27) methyltransferase when comprising the Polycomb-Repressive Complex 2 (PRC2). Trimethylation of H3K27 (H3K27me3) correlates with transcriptionally repressed chromatin. The means by which PRC2 targets specific chromatin regions is currently unclear, but noncoding RNAs (ncRNAs) have been shown to interact with PRC2 and may facilitate its recruitment to some target genes. This study shows that Ezh2 interacts with HOTAIR and Xist. Ezh2 is phosphorylated by cyclin-dependent kinase 1 (CDK1) at threonine residues 345 and 487 in a cell cycle-dependent manner. A phospho-mimic at residue 345 increased HOTAIR ncRNA binding to Ezh2, while the phospho-mimic at residue 487 was ineffectual. An Ezh2 domain comprising T345 was found to be important for binding to HOTAIR and the 5' end of Xist (Kaneko, 2010).

The results presented here demonstrate that PRC2 binding to HOTAIR (expressed from the HOXC cluster) and RepA (repeats found in Xist) ncRNAs is mediated through its Ezh2 component, and that phosphorylation of Ezh2-T345 up-regulates HOTAIR-binding activity. Given that phosphorylation at this site is cell cycle-regulated, it is speculated that PRC2 recruitment to chromatin, mediated through Ezh2 interaction with HOTAIR or RepA ncRNAs and presumably other ncRNAs, must be restricted to a tightly defined interval during the cell cycle (G2/M). Most importantly, the results establish that there are at least two populations of PRC2 complexes during the G2-M stages of the cell cycle. This is consistent with a model whereby PRC2 is recruited to specific genes to initiate repression as a function of its Ezh2 component being phosphorylated at T345, after which other PRC2 complexes then spread the repressing signature (H3K27me2/3). Of note, a recent study has also documented that human Ezh2 is phosphorylated at Thr 350 (murine T345) by CDK1, and, in agreement with the current findings, the report shows that this modification is ineffectual with respect to the integrity of the PRC2 complex and PRC2-mediated histone lysine methyltransferase activity. Instead, this study shows that mutant Ezh2 that is not subject to T350 phosphorylation results in down-regulated PRC2 recruitment, such that appropriate gene repression is thwarted. This report demonstrates that abrogation of this phosphorylation site within Ezh2 compromises Ezh2 interaction with ncRNAs, and this may bear directly on the mechanism by which PRC2 recruitment is impaired (Kaneko, 2010).

It has been demonstrated previously that the Eed component of PRC2 binds to trimethylated histone-repressive marks, but its binding to H3K27me3 in particular results in an allosteric effect that markedly increases the histone methyltransferase activity of its partner, Ezh2. Thus, PRC2 binding to the product of its activity increases its production of this mark. It is postulated that HOTAIR and RepA ncRNAs (and other ncRNAs) recruit PRC2 to initiate repression of target genes, and that this recruitment is enhanced by Ezh2 phosphorylation at T345. This proposed mechanism is consistent with only a small percentage of Ezh2 being phosphorylated at T345. If ncRNA species are responsible for targeting PRC2 to chromatin during G2/M, it is postulated that the recruited PRC2 would set the initial H3K27me3 mark. A larger number of PRC2 complexes, independent of their Ezh2 component being phosphorylated, would then propagate this mark upon their Eed component binding to the initial H3K27me3, with resultant allosteric activation of their Ezh2 activity (Kaneko, 2010).

An important question remaining is whether Ezh2 is the only component of PRC2 that binds to ncRNAs. These studies established that a 30-amino-acid domain of the PRC2-associated protein Jarid2 (see Drosophila Jarid2) also binds to ncRNAs. Additionally, a recent study suggested that the Suz12 subunit of PRC2 binds to nascent transcripts. It was postulated that this binding results in the halting of RNA polymerase II. Interestingly, it was postulated that Suz12 binding to nascent transcripts requires a unique stem-loop structure on the RNA. This structure is similar to the structure of RepA, and computer analysis of the amino acid sequence of Suz12 revealed a putative domain at its N terminus with a predicted RNA-binding domain. These observations collectively suggest that PRC2 recruitment to its target genes is mediated by ncRNA, different species of which likely bind to different PRC2 subunits. Whether specificity or affinity of PRC2 for its target genes is regulated by PRC2 binding through its component(s) to one family of ncRNA (specificity), or whether multiple subunits of PRC2 simultaneously bind different ncRNAs or domains within a ncRNA (affinity), remains to be established. Regardless, the studies described here are beginning to shed light on the role of ncRNAs in mediating the recruitment of mammalian PRC2 to its target genes (Kaneko, 2010).


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cdc2: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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