Transcriptional Cyclin Research Articles

Hog1 targets Whi5 and Msa1 transcription factors to downregulate cyclin expression upon stress.

Yeast cells have developed complex mechanisms to cope with extracellular insults. An increase in external osmolarity leads to activation of the stress-activated protein kinase Hog1, which is the main regulator of adaptive responses, such as gene expression and cell cycle progression, that are essential for cellular survival. Upon osmostress, the G1-to-S transition is regulated by Hog1 through stabilization of the cyclin-dependent kinase inhibitor Sic1 and the downregulation of G1 cyclin expression by an unclear mechanism. Here, we show that Hog1 interacts with and phosphorylates components of the core cell cycle transcriptional machinery such as Whi5 and the coregulator Msa1. Phosphorylation of these two transcriptional regulators by Hog1 is essential for inhibition of G1 cyclin expression, for control of cell morphogenesis, and for maximal cell survival upon stress. The control of both Whi5 and Msa1 by Hog1 also revealed the necessity for proper coordination of budding and DNA replication. Thus, Hog1 regulates G1 cyclin transcription upon osmostress to ensure coherent passage through Start.

Murine norovirus replication induces G0/G1 cell cycle arrest in asynchronously growing cells.

Many viruses replicate most efficiently in specific phases of the cell cycle, establishing or exploiting favorable conditions for viral replication, although little is known about the relationship between caliciviruses and the cell cycle. Microarray and Western blot analysis of murine norovirus 1 (MNV-1)-infected cells showed changes in cyclin transcript and protein levels indicative of a G1 phase arrest. Cell cycle analysis confirmed that MNV-1 infection caused a prolonging of the G1 phase and an accumulation of cells in the G0/G1 phase. The accumulation in G0/G1 phase was caused by a reduction in cell cycle progression through the G1/S restriction point, with MNV-1-infected cells released from a G1 arrest showing reduced cell cycle progression compared to mock-infected cells. MNV-1 replication was compared in populations of cells synchronized into specific cell cycle phases and in asynchronously growing cells. Cells actively progressing through the G1 phase had a 2-fold or higher increase in virus progeny and capsid protein expression over cells in other phases of the cell cycle or in unsynchronized populations. These findings suggest that MNV-1 infection leads to prolonging of the G1 phase and a reduction in S phase entry in host cells, establishing favorable conditions for viral protein production and viral replication. There is limited information on the interactions between noroviruses and the cell cycle, and this observation of increased replication in the G1 phase may be representative of other members of the Caliciviridae. Noroviruses have proven recalcitrant to growth in cell culture, limiting our understanding of the interaction between these viruses and the infected cell. In this study, we used the cell-culturable MNV-1 to show that infection of murine macrophages affects the G1/S cell cycle phase transition, leading to an arrest in cell cycle progression and an accumulation of cells in the G0/G1 phase. Furthermore, we show that MNV replication is enhanced in the G1 phase compared to other stages of the cell cycle. Manipulating the cell cycle or adapting to cell cycle responses of the host cell is a mechanism to enhance virus replication. To the best of our knowledge, this is the first report of a norovirus interacting with the host cell cycle and exploiting the favorable conditions of the G0/G1 phase for RNA virus replication.

Cyclin E-CDK2 Protein Phosphorylates Plant Homeodomain Finger Protein 8 (PHF8) and Regulates Its Function in the Cell Cycle

Cyclin E-CDK2 is a key regulator in G1/S transition. Previously, we identified a number of CDK2-interacting proteins, including PHF8 (plant homeodomain finger protein 8). In this report, we confirmed that PHF8 is a novel cyclin E-CDK2 substrate. By taking the approach of mass spectrometry, we identified that PHF8 Ser-844 is phosphorylated by cyclin E-CDK2. Immunoblotting analysis indicated that WT PHF8 demethylates histone H3K9me2 more efficiently than the cyclin E-CDK2 phosphorylation-deficient PHF8-S844A mutant. Furthermore, flow cytometry analysis showed that WT PHF8 promotes S phase progression more robustly than PHF8-S844A. Real-time PCR results demonstrated that PHF8 increases transcription of cyclin E, E2F3, and E2F7 to significantly higher levels compared with PHF8-S844A. Further analysis by ChIP assay indicated that PHF8 binds to the cyclin E promoter stronger than PHF8-S844A and reduces the H3K9me2 level at the cyclin E promoter more efficiently than PHF8-S844A. In addition, we found that cyclin E-CDK2-mediated phosphorylation of PHF8 Ser-844 promotes PHF8-dependent rRNA transcription in luciferase reporter assays and real-time PCR. Taken together, these results indicate that cyclin E-CDK2 phosphorylates PHF8 to stimulate its demethylase activity to promote rRNA transcription and cell cycle progression.

Whole Genome Sequence Analysis Suggests Intratumoral Heterogeneity in Dissemination of Breast Cancer to Lymph Nodes

BackgroundIntratumoral heterogeneity may help drive resistance to targeted therapies in cancer. In breast cancer, the presence of nodal metastases is a key indicator of poorer overall survival. The aim of this study was to identify somatic genetic alterations in early dissemination of breast cancer by whole genome next generation sequencing (NGS) of a primary breast tumor, a matched locally-involved axillary lymph node and healthy normal DNA from blood.MethodsWhole genome NGS was performed on 12 µg (range 11.1–13.3 µg) of DNA isolated from fresh-frozen primary breast tumor, axillary lymph node and peripheral blood following the DNA nanoball sequencing protocol. Single nucleotide variants, insertions, deletions, and substitutions were identified through a bioinformatic pipeline and compared to CIN25, a key set of genes associated with tumor metastasis.ResultsWhole genome sequencing revealed overlapping variants between the tumor and node, but also variants that were unique to each. Novel mutations unique to the node included those found in two CIN25 targets, TGIF2 and CCNB2, which are related to transcription cyclin activity and chromosomal stability, respectively, and a unique frameshift in PDS5B, which is required for accurate sister chromatid segregation during cell division. We also identified dominant clonal variants that progressed from tumor to node, including SNVs in TP53 and ARAP3, which mediates rearrangements to the cytoskeleton and cell shape, and an insertion in TOP2A, the expression of which is significantly associated with tumor proliferation and can segregate breast cancers by outcome.ConclusionThis case study provides preliminary evidence that primary tumor and early nodal metastasis have largely overlapping somatic genetic alterations. There were very few mutations unique to the involved node. However, significant conclusions regarding early dissemination needs analysis of a larger number of patient samples.

Epidemiological and Clinical Characteristics of Colorectal Cancer in Hypothyroidism

Introduction: Colorectal cancer (CRC) is the third most common cancer and the third leading cause of cancer deaths in the United States. Overall, CRC incidence and mortality rates are higher in men and highest in African Americans, followed by Caucasians. Few studies demonstrated a preventive role of thyroid hormone (TH) in the development and invasiveness of CRC. Methods: In a retrospective cohort, we identified 48 patients with CRC and hypothyroidism (cases). The patients were sex-matched with 174 patients who had CRC without hypothyroidism (controls). Data were collected for depth of tumor invasion, lymphovascular invasion, tumor location, staging (TNM), histologic type, level of differentiation, and positive-to-total lymph node ratio. Results: Females constituted 83.3% of cases. Mean age at diagnosis in case and control was (74.7±11.05 years, 76.3±12.28 years, respectively; p=-0.25). The other demographic variables were similar in 2 groups except race, with the patients in the cases being overwhelmingly Caucasian (91.6% versus 54.0% in control; p<0.001). In the unadjusted regressions, patients with hypothyroidism were more likely to have deep tumor invasions (OR 0.874; 95% CI -0.01, 1.76). In the adjusted regressions, Hispanic patients with hypothyroidism had significantly lower odds of high stage than Caucasian patients (OR -7.007; 95% CI -13.47, -0.55). The point estimates for hypothyroidism and the interaction with age at diagnosis for the TNM stage (OR 10.222; 95% CI 0.91, 19.53; OR -0.138; 95% CI -0.26, -0.02, respectively) and for lymphovascular invasion, (OR 8.618; 95% CI 0.09, 17.15; OR -0.120; 95% CI -0.23, -0.01, respectively), imply that at age 74 years or younger at diagnosis of CRC, hypothyroidism raises the odds of having higher TNM stage and lymphovascular invasion. Conclusion: Cyclin D1 is an oncogenic cyclin, which is frequently overexpressed in CRC and Wnt, signaling pathway is involved in development sporadic CRC. TH-bound thyroid receptors (TRs) inhibit the transcription of cyclin D though the Tcf/Lef-1 site, which is positively regulated by Wnt signaling pathway. In patients with hypothyroidism, colorectal tumor exhibit reduced TRB1 expression. This may be the basis for why our study demonstrated that female Caucasian patients with hypothyroidism are more likely to have worse tumor characteristics of CRC at age less than 74 years.

Never in mitosis gene A-related kinase 6 promotes cell proliferation of hepatocellular carcinoma via cyclin B modulation.

Never in mitosis gene A-related kinase (Nek) 6 is a recently identified Nek that is required for mitotic cell cycle progression; however, the role and mechanism of Nek6 activity during hepatocarcinogenesis is not well known. The aim of this study was to investigate the potential roles and internal mechanism of Nek6 in hepatocellular carcinoma (HCC) development. In the present study, Nek6 was found to be overexpressed in HCC samples and cell lines by florescent real-time quantitative polymerase chain reaction, immunohistochemistry and western blot analysis. Furthermore, it was evidenced to contribute to oncogenesis and progression. The ectopic overexpression of Nek6 promoted cell proliferation and colony formation, whereas gene silencing of Nek6 inhibited these phenotypes, as documented in Huh7, PLC/PRF/5, Hep3B and HepG2 HCC cell lines. Mechanistic analyses indicated that Nek6 regulates the transcription of cyclin B through cdc2 activation, and promotes the accumulation of G0/G1-phase cells. In conclusion, the findings of the current study suggested that Nek6 contributes to the oncogenic potential of HCC, and may present as a potential therapeutic target in this disease.

A data-driven, mathematical model of mammalian cell cycle regulation.

Few of >150 published cell cycle modeling efforts use significant levels of data for tuning and validation. This reflects the difficultly to generate correlated quantitative data, and it points out a critical uncertainty in modeling efforts. To develop a data-driven model of cell cycle regulation, we used contiguous, dynamic measurements over two time scales (minutes and hours) calculated from static multiparametric cytometry data. The approach provided expression profiles of cyclin A2, cyclin B1, and phospho-S10-histone H3. The model was built by integrating and modifying two previously published models such that the model outputs for cyclins A and B fit cyclin expression measurements and the activation of B cyclin/Cdk1 coincided with phosphorylation of histone H3. The model depends on Cdh1-regulated cyclin degradation during G1, regulation of B cyclin/Cdk1 activity by cyclin A/Cdk via Wee1, and transcriptional control of the mitotic cyclins that reflects some of the current literature. We introduced autocatalytic transcription of E2F, E2F regulated transcription of cyclin B, Cdc20/Cdh1 mediated E2F degradation, enhanced transcription of mitotic cyclins during late S/early G2 phase, and the sustained synthesis of cyclin B during mitosis. These features produced a model with good correlation between state variable output and real measurements. Since the method of data generation is extensible, this model can be continually modified based on new correlated, quantitative data.

Effect of acetyltransferase inhibitor Garcinol on estrogen-promoted proliferation of breast cancer cell line MCF-7 and the related mechanism

Objective To investigate the effect of acetyltransferase inhibitor Garcinol on estrogen-induced proliferation,cell cycle promotion and apoptosis inhibition of human breast cancer MCF-7 cells and the related mechanism.Methods The effect of Garcinol on 17β-estradiol(17β-E2)-induced proliferation of MCF-7 cells was investigated using CCK-8 assay,and the optimal concentration of Garcinol was determine according to the inhibition rate.The cell cycle and apoptosis were analyzed by flow cytometry;the nuclear translocation of NF-κB/p65was examined by immune cell fluorescence.RT-PCR was used to determine the expressions of cyclin D1,Bcl-2 and Bcl-xLmRNA in MCF-7cells;and the expressions of acH3,ac-H4,NF-κB/ac-p65,cyclin D1,Bcl-2,and Bcl-xLprotein were determined by Western blotting analysis.Results Acetyltransferase inhibitor Garcinol inhibited 17β-E2-induced proliferation of MCF-7cells,arrested MCF-7cell cycle at G0/G1 phase,and increased the cell apoptosis(P0.01).17β-E2 increased the expressions of ac-H3,ac-H4 and NF-κB/ac-p65 protein in MCF-7cells(P0.01,P0.05,P0.01),while Garcinol significantly inhibited the increase of ac-H3and NF-κB/ac-p65(P0.01)but not ac-H4(P0.05).17β-E2-induced nuclear translocation of NF-κB/p65in MCF-7cells was also significantly inhibited by Garcinol(P0.01).Garcinol also significantly inhibited 17β-E2-induced transcription and protein expression of cyclin D1,Bcl-2and Bcl-xL mRNA in MCF-7 cells(P0.05).Conclusion 17β-E2-induced proliferation and apoptosisinhibition of MCF-7 cells are associated with elevated acetylation level of histone and nonhistone NF-κB/p65,and acetyltransferase inhibitor Garcinol may inhibit the effect of 17β-E2 by decreasing acetylation,probably through decreasing acp65 expression of NF-κB pathway,and subsequently down-regulating cyclin D1,Bcl-2,and Bcl-xL.

Autonomous Cycling in a Shared Cytoplasm

Localization of cyclin transcripts enables nuclei sharing a common cytoplasm to divide autonomously.

Identification and Functional Characterisation of CRK12:CYC9, a Novel Cyclin-Dependent Kinase (CDK)-Cyclin Complex in Trypanosoma brucei.

The protozoan parasite, Trypanosoma brucei, is spread by the tsetse fly and causes trypanosomiasis in humans and animals. Both the life cycle and cell cycle of the parasite are complex. Trypanosomes have eleven cdc2-related kinases (CRKs) and ten cyclins, an unusually large number for a single celled organism. To date, relatively little is known about the function of many of the CRKs and cyclins, and only CRK3 has previously been shown to be cyclin-dependent in vivo. Here we report the identification of a previously uncharacterised CRK:cyclin complex between CRK12 and the putative transcriptional cyclin, CYC9. CRK12:CYC9 interact to form an active protein kinase complex in procyclic and bloodstream T. brucei. Both CRK12 and CYC9 are essential for the proliferation of bloodstream trypanosomes in vitro, and we show that CRK12 is also essential for survival of T. brucei in a mouse model, providing genetic validation of CRK12:CYC9 as a novel drug target for trypanosomiasis. Further, functional characterisation of CRK12 and CYC9 using RNA interference reveals roles for these proteins in endocytosis and cytokinesis, respectively.

Protein Aggregation Behavior Regulates Cyclin Transcript Localization and Cell-Cycle Control

Little is known about the active positioning of transcripts outside of embryogenesis or highly polarized cells. We show here that a specific G1 cyclin transcript is highly clustered in the cytoplasm of large multinucleate cells. This heterogeneous cyclin transcript localization results from aggregation of an RNA-binding protein, and deletion of a polyglutamine stretch in this protein results in random transcript localization. These multinucleate cells are remarkable in that nuclei cycle asynchronously despite sharing a common cytoplasm. Notably, randomization of cyclin transcript localization significantly diminishes nucleus-to-nucleus differences in the number of mRNAs and synchronizes cell-cycle timing. Thus, nonrandom cyclin transcript localization is important for cell-cycle timing control and arises due to polyQ-dependent behavior of an RNA-binding protein. There is a widespread association between polyQ expansions and RNA-binding motifs, suggesting that this is a broadly exploited mechanism to produce spatially variable transcripts and heterogeneous cell behaviors. PAPERCLIP:

Assessing the contribution of HRPT2 to the pathogenesis of jaw fibrous dysplasia, ossifying fibroma, and osteosarcoma

To investigate HRPT2 in jaw ossifying fibroma (OF), fibrous dysplasia (FD), and osteosarcoma (OS). We combined microsatellite loss of heterozygosity (LOH), HRPT2 sequence alterations at the mRNA level by reverse-transcription polymerase chain reaction (PCR), cDNA sequencing, and quantitative PCR (qPCR) and immunohistochemistry (IHC) in a total of 19 OF, 15 FD, and 9 OS. Because HRPT2 (parafibromin) interacts with cyclin D1, we investigated cyclin D1 expression with the use of qPCR and IHC. LOH was detected in 3/5 FD, 6/9 OF, and 2/2 OS heterozygous samples. LOH was not associated with decreased mRNA levels or HRPT2 protein expression except for 1 OF which harbored an inactivating mutation. However, this tumor did not display altered transcription or protein levels of HRPT2 nor cyclin compared with the other OF. The contribution of HRPT2 inactivation to the pathogenesis of OF, FD, and OS is marginal at best and may be limited to progression rather than tumor initiation.

Two transcripts and the expression profiles of cyclin A in ovary of the mud crab, Scylla paramamosain

Two transcripts of cyclin A (SPCyA) were identified and characterized from ovaries of the mud crab, Scylla paramamosain. The sequences of the two transcripts contained the same open reading frame and 5′ untranslated region (UTR), but differed in 3′ UTR. The polyadenylation signals “AATAAA” were found in the different sites, which might generate alternative polyadenalation and thus the different length of the transcripts. The dendrogram, based on comparison of cyclin A proteins in different species, agreed with the classic taxonomic structure. Reverse transcription-PCR (RT-PCR) showed that the SPCyA mRNA was expressed at the highest level in the ovary. Real-time RT-PCR revealed that the amount of SPCyA mRNA in the ovary increased from the first stage to the fourth stage, then declined at the fifth stage (p < 0.05). It implies that SPCyA is closely related to oogonial proliferation (mitosis) and oocyte meiosis.

The multi‐kinase inhibitor TG02 overcomes signalling activation by survival factors to deplete MCL1 and XIAP and induce cell death in primary acute myeloid leukaemia cells

The novel multi-kinase inhibitor TG02 has selectivity against cell cycle and transcriptional cyclin dependent kinases (CDKs) as well as fms-like tyrosine kinase receptor-3 (FLT3). Inhibition of transcriptional CDKs preferentially depletes short-lived proteins such as MCL1. We evaluated the in vitro toxicity of TG02 to primary acute myeloid leukaemia (AML) cells in the presence of survival signalling pathway activation by cytokines and fibronectin. One hundred nanomolar TG02 induced a median decrease of 40% in bulk cell survival and 43% in the CD34(+) CD38(-) CD123(+) subset. A 90% inhibitory concentration of 500nmol/l indicated that TG02 toxicity is not halted by protective cell cycle arrest. Samples with FLT3 internal tandem duplication were not preferentially targeted. By flow cytometry, TG02 treatment caused loss of RNA Polymerase II serine 2 phosphorylation in patient samples, which correlated strongly with BAX activation (R(2) =0·89), suggesting these as potential biomarkers for clinical studies. MCL1 and XIAP expression also decreased. Repeated brief exposure to TG02 in MOLM-13 cells did not result in compensatory up-regulation of survival protein expression. In conclusion, TG02 is potently cytotoxic towards CD34(+) CD38(-) CD123(+) and bulk AML cells, despite protective signalling pathway activation. This antitumour activity is most likely mediated by dephosphorylation of RNA Polymerase II leading to depletion of survival molecules such as MCL1 and XIAP, with subsequent BAX activation and apoptosis.

A chemical‐genetic screen to unravel the genetic network of CDC28/CDK1 links ubiquitin and Rad6–Bre1 to cell cycle progression

Cyclin‐dependent kinases (CDKs) control the eukaryotic cell cycle, and a single CDK, Cdk1 (also known as Cdc28), is necessary and sufficient for cell cycle regulation in the budding yeast Saccharomyces cerevisiae. Cdk1 regulates cell cycle‐dependent processes such as transcription, DNA replication and repair, and chromosome segregation. To gain further insight into the functions of Cdk1, we performed a high‐throughput chemical‐genetic array (CGA) screen aimed at unraveling the genetic network of CDK1. We identified 107 genes that strongly genetically interact with CDK1. Although these genes serve multiple cellular functions, genes involved in cell cycle regulation, transcription, and chromosome metabolism were overrepresented. DOA1, which is involved in maintaining free ubiquitin levels, as well as the RAD6–BRE1 pathway, which is involved in transcription, displayed particularly strong genetic interactions with CDK1. We discovered that DOA1 is important for cell cycle entry by supplying ubiquitin. Furthermore, we found that the RAD6–BRE1 pathway functions downstream of DOA1/ubiquitin but upstream of CDK1, by promoting transcription of cyclins. These results link cellular ubiquitin levels and the Rad6–Bre1 pathway to cell cycle progression.

3.6-KB mouse cyclin C promoter fragment is predominantly active in the testis

Cyclin C is a highly conserved protein that regulates cell-cycle, messenger RNA transcription and cell adhesion. Recently published studies demonstrate that this protein is an essential player during early embryonic development of multicellular eukaryotes as well. In order to understand better its complex function at the level of tissues or organs, spatial expression characteristics of cyclin C and regulatory components of its expression are needed to be determined. In vitro studies on human cells suggested that approximately the first 3 kilobases of the cyclin C promoter might contain all the regulatory elements that might mimic transcription of cyclin C. To test the hypothesis, we generated reporter transgenic lines where the first 3.6-kilobase region of mouse cyclin C promoter fragment drives the transcription of a marker gene. Messenger RNA levels of the marker gene and cyclin C isoforms were measured in nine organs with reverse transcription coupled quantitative realtime polymerase chain reaction and their expression patterns were compared. The marker gene is predominantly transcribed in testes and does not follow the transcriptional regulation of the examined cyclin C isoforms. Thus, the isolated promoter fragment alone is not sufficient for the complete physiological modulation of cyclin C RNA levels, however, it is capable of enhancing testicular transcription which can be exploited in future applications.

Regulation of a Myb Transcription Factor by Cyclin-dependent Kinase 2 in Giardia lamblia

The protozoan Giardia lamblia parasitizes the human small intestine to cause diseases. It undergoes differentiation into infectious cysts by responding to intestinal stimulation. How the activated signal transduction pathways relate to encystation stimulation remain largely unknown. During encystation, genes encoding cyst wall proteins (CWPs) are coordinately up-regulated by a Myb2 transcription factor. Because cell differentiation is linked to cell cycle regulation, we tried to understand the role of cell cycle regulators, cyclin-dependent kinases (Cdks), in encystation. We found that the recombinant Myb2 was phosphorylated by Cdk-associated complexes and the levels of phosphorylation increased significantly during encystation. We have identified a putative cdk gene (cdk2) by searching the Giardia genome database. Cdk2 was found to localize in the cytoplasm with higher expression during encystation. Interestingly, overexpression of Cdk2 resulted in a significant increase of the levels of cwp gene expression and cyst formation. In addition, the Cdk2-associated complexes can phosphorylate Myb2 and the levels of phosphorylation increased significantly during encystation. Mutations of important catalytic residues of Cdk2 resulted in a significant decrease of kinase activity and ability of inducing cyst formation. Addition of a Cdk inhibitor, purvalanol A, significantly decreased the Cdk2 kinase activity and the levels of cwp gene expression and cyst formation. Our results suggest that the Cdk2 pathway may be involved in phosphorylation of Myb2, leading to activation of the Myb2 function and up-regulation of cwp genes during encystation. The results provide insights into the use of Cdk inhibitory drugs in disruption of Giardia differentiation into cysts.

The Gain of Function of p53 Mutant p53S in Promoting Tumorigenesis by Cross-talking with H-RasV12

The loss of wild type p53 tumor suppressive function and oncogenic gain-of-function of p53 mutants have been showing important implications in tumorigenesis. The p53N236S (p53N239S in human, p53S) mutation has been shown to lose wild type p53 function by yeast assay. However, its gain of function is still not clear. By gel shift assay, we showed that mutant p53S had lost its DNA binding ability to its target promoters. Further real-time PCR data confirmed that p53S had lost the function of regulating the transcription of p21 Cip1/Waf1, cyclin G, PUMA, and Bax in response to 10Gy irradiation. These data confirmed the loss of function of p53S in mammalian cells. By xenograft assay, we showed that the p53S per se was not oncogenic enough to form tumor, however, cooperating with H-RasV12, p53S could dramatically promote tumorigenesis in p53 null MEFs. Further study showed that co-expression of p53S and H-RasV12 could increase the expression level of H-RasV12 and partially eliminate the elevation of stress response proteins such as Chk2, γ-H2AX, Hsp70, Rb, p16Ink4a caused by either p53S or H-RasV12. These data suggested that p53S cross-talked with H-RasV12 and reduced the cellular stress response to oncogenic signals, which facilitated the cell growth and tumorigenesis. Together these data provided the molecular basis for the cooperation of p53S and H-RasV12 and revealed the gain of function of p53S in cross-talking with H-RasV12. This study revealed an important aspect of gain of function for p53 mutant, therefore might shed light on the clinical strategy in targeting p53 mutant.

A chemical-genetic screen to unravel the genetic network of CDC28/CDK1 links ubiquitin and Rad6–Bre1 to cell cycle progression

Cyclin-dependent kinases (CDKs) control the eukaryotic cell cycle, and a single CDK, Cdc28 (also known as Cdk1), is necessary and sufficient for cell cycle regulation in the budding yeast Saccharomyces cerevisiae. Cdc28 regulates cell cycle-dependent processes such as transcription, DNA replication and repair, and chromosome segregation. To gain further insight into the functions of Cdc28, we performed a high-throughput chemical-genetic array (CGA) screen aimed at unraveling the genetic network of CDC28. We identified 107 genes that strongly genetically interact with CDC28. Although these genes serve multiple cellular functions, genes involved in cell cycle regulation, transcription, and chromosome metabolism were overrepresented. DOA1, which is involved in maintaining free ubiquitin levels, as well as the RAD6-BRE1 pathway, which is involved in transcription, displayed particularly strong genetic interactions with CDC28. We discovered that DOA1 is important for cell cycle entry by supplying ubiquitin. Furthermore, we found that the RAD6-BRE1 pathway functions downstream of DOA1/ubiquitin but upstream of CDC28, by promoting transcription of cyclins. These results link cellular ubiquitin levels and the Rad6-Bre1 pathway to cell cycle progression.

NF-Y binds to both G1- and G2-specific cyclin promoters; a possible role in linking CDK2/Cyclin A to CDK1/Cyclin B

We previously reported that CDK2/Cyclin A can phosphorylate and activate the transcription factor NF-Y. In this study, we investigated a potential regulatory role for NF-Y in the transcription of Cyclin A and other cell cycle regulatory genes. Gel-shift assays demonstrate that NF-Y binds to CCAAT sequences in the Cyclin A promoter, as well as to those in the promoters of cell cycle G2 regulators such as CDC2, Cyclin B and CDC25C. Furthermore, expression of Cyclin A increases NF-Y's affinity for CCAAT sequences in the CDC2 promoter; however, Cyclin A's induction of CDC2 transcription is antagonized by p21, an inhibitor of CDK2/Cyclin A. These results suggest a model wherein NF-Y binds to and activates transcription from the Cyclin A promoter, increasing cellular levels of Cyclin A/CDK2 and potentiating NF-Y's capacity for transcriptional transactivation, and imply a positive feedback loop between NF-Y and Cyclin A/CDK2. Our findings are additionally indicative of a role for Cyclin A in activating Cyclin B/CDK1 through promoting NF-Y dependent transcription of Cyclin B and CDC2; NF-Y mediated crosstalk may therefore help to orchestrate cell-cycle progression.