cyclin C

Intrinsic apoptosis requires mitochondrial outer membrane disruption triggered by recruitment, activation, and oligomerization of the Bcl-2 homology protein Bax. Following oxidative stress, we demonstrated that the transcriptional regulator cyclin C is released into the cytosol where it directs mitochondrial fragmentation and efficient apoptotic induction. This study reveals that cytoplasmic cyclin C is required for both normal Bax activation and its efficient mitochondrial localization. This activity appears direct as cyclin C coimmunoprecipitates with active Bax in stressed cells and binds recombinant Bax in vitro. In addition, stable cyclin C-Bax association requires the fission complex. Pharmacologically stimulating cyclin C nuclear release is sufficient for Bax association and their mitochondrial localization in the absence of any stress signals. However, these cells do not undergo cell death as Bax fails to oligomerize. These data support a model that cyclin C association defines an initial step in Bax mitochondrial recruitment and provides a physical connection between the fission and apoptotic factors. This strategy allows the cell to discriminate stress-induced fission able to recruit Bax from other types of mitochondrial divisions.

The steroid hormone ecdysone and its receptor (EcR) play critical roles in orchestrating developmental transitions in arthropods. However, the mechanism by which EcR integrates nutritional and developmental cues to correctly activate transcription remains poorly understood. Here, we show that EcR-dependent transcription, and thus, developmental timing in Drosophila, is regulated by CDK8 and its regulatory partner Cyclin C (CycC), and the level of CDK8 is affected by nutrient availability. We observed that cdk8 and cycC mutants resemble EcR mutants and EcR-target genes are systematically down-regulated in both mutants. Indeed, the ability of the EcR-Ultraspiracle (USP) heterodimer to bind to polytene chromosomes and the promoters of EcR target genes is also diminished. Mass spectrometry analysis of proteins that co-immunoprecipitate with EcR and USP identified multiple Mediator subunits, including CDK8 and CycC. Consistently, CDK8-CycC interacts with EcR-USP in vivo; in particular, CDK8 and Med14 can directly interact with the AF1 domain of EcR. These results suggest that CDK8-CycC may serve as transcriptional cofactors for EcR-dependent transcription. During the larval-pupal transition, the levels of CDK8 protein positively correlate with EcR and USP levels, but inversely correlate with the activity of sterol regulatory element binding protein (SREBP), the master regulator of intracellular lipid homeostasis. Likewise, starvation of early third instar larvae precociously increases the levels of CDK8, EcR and USP, yet down-regulates SREBP activity. Conversely, refeeding the starved larvae strongly reduces CDK8 levels but increases SREBP activity. Importantly, these changes correlate with the timing for the larval-pupal transition. Taken together,

Somatic mutations in exon 2 of the RNA polymerase II transcriptional Mediator subunit MED12 occur at very high frequency ($70%) in uterine leiomyomas. However, the influence of these mutations on Mediator function and the molecular basis for their tumorigenic potential remain unknown. To clarify the impact of these mutations, we used affinity-purification mass spectrometry to establish the global protein-protein interaction profiles for both wild-type and mutant MED12. We found that uterine leiomyoma-linked mutations in MED12 led to a highly specific decrease in its association with Cyclin C-CDK8/CDK19 and loss of Mediator-associated CDK activity. Mechanistically, this occurs through disruption of a MED12-Cyclin C binding interface that we also show is required for MED12-mediated stimulation of Cyclin C-dependent CDK8 kinase activity. These findings indicate that uterine leiomyoma-linked mutations in MED12 uncouple Cyclin C-CDK8/19 from core Mediator and further identify the MED12/Cyclin C interface as a prospective therapeutic target in CDK8-driven cancers.

In response to oxidative stress, cells decide whether to mount a survival or cell death response. The conserved cyclin C and its kinase partner Cdk8 play a key role in this decision. Both are members of the Cdk8 kinase module that, with Med12 and Med13, associate with the core mediator complex of RNA polymerase II. In S. cerevisiae, oxidative stress triggers Med13 destruction, which thereafter releases cyclin C into the cytoplasm. Cytoplasmic cyclin C associates with mitochondria where it induces hyper-fragmentation and regulated cell death. In this report, we show that residues 742-844 of Med13's 600 amino acid intrinsic disordered region (IDR) both directs cyclin C-Cdk8 association and serves as the degron that mediates ubiquitin ligase SCFGrr1-dependent destruction of Med13 following oxidative stress. Here, cyclin C-Cdk8 phosphorylation of Med13 most likely primes the phospho-degron for destruction. Next, pro-oxidant stimulation of the cell wall integrity pathway MAP Kinase S...

The steroid hormone ecdysone and its receptor (EcR) play critical roles in orchestrating developmental transitions in arthropods. However, the mechanism by which EcR integrates nutritional and developmental cues to correctly activate transcription remains poorly understood. Here, we show that EcR-dependent transcription, and thus, developmental timing in Drosophila, is regulated by CDK8 and its regulatory partner Cyclin C (CycC), and the level of CDK8 is affected by nutrient availability. We observed that cdk8 and cycC mutants resemble EcR mutants and EcR-target genes are systematically down-regulated in both mutants. Indeed, the ability of the EcR-Ultraspiracle (USP) heterodimer to bind to polytene chromosomes and the promoters of EcR target genes is also diminished. Mass spectrometry analysis of proteins that co-immunoprecipitate with EcR and USP identified multiple Mediator subunits, including CDK8 and CycC. Consistently, CDK8-CycC interacts with EcR-USP in vivo; in particular, CDK8 and Med14 can directly interact with the AF1 domain of EcR. These results suggest that CDK8-CycC may serve as transcriptional cofactors for EcR-dependent transcription. During the larval-pupal transition, the levels of CDK8 protein positively correlate with EcR and USP levels, but inversely correlate with the activity of sterol regulatory element binding protein (SREBP), the master regulator of intracellular lipid homeostasis. Likewise, starvation of early third instar larvae precociously increases the levels of CDK8, EcR and USP, yet down-regulates SREBP activity. Conversely, refeeding the starved larvae strongly reduces CDK8 levels but increases SREBP activity. Importantly, these changes correlate with the timing for the larval-pupal transition. Taken together,

As a partner of cyclin-dependent kinase (CDK) 3, Cyclin C controls cellular proliferation and, together with CDK8, represses gene transcription. In this study, we showed that the highly expressed Cyclin C gene is a direct target of the nuclear hormone all-trans retinoic acid (RA) in HEK293 human embryonal kidney cells. The RA receptor (RAR) γ associates with a Cyclin C promoter region containing two RAR binding sites. The Cyclin C gene also directly responds to the cAMP activator Forskolin via the transcription factor CREB1 (cAMP response element-binding protein 1), for which we identified four binding sites within the first 2250 bp of its promoter. RARγ and CREB1 show functional convergence via the corepressor NCoR1, which controls in particular the Forskolin response of Cyclin C. The histone deacetylases 1, 5, 6, 7 and 11 are involved in the basal expression of Cyclin C, but in HEK293 and MCF-7 human breast carcinoma cells the antiproliferative effects of the histone deacetylase inhibitor SAHA (suberoylanilide hydroxamic acid) are not mediated by Cyclin C. However, cell cycle progressing effects of all-trans RA and Forskolin are dependent on Cyclin C expression levels. This suggests that the primary regulation of Cyclin C by all-trans RA and Forskolin mediates some of the cell cycle control actions of these compounds.

Cyclin-dependent kinase 8 (CDK8) is a member of the CDK/Cyclin module of the mediator complex. A recent study reported that heterozygous missense CDK8 mutations cause a neurodevelopmental disorder in humans. The mechanistic basis of CDK8-related disorder has yet to be delineated. Here, we report 2 patients with de novo missense mutations within the kinase domain of CDK8 along with the results of in vitro and in vivo functional analyses using a zebrafish model. Patient 1 and Patient 2 had intellectual disabilities and congenital anomalies. Exome analyses showed that patient 1 had a heterozygous de novo missense p.G28A variant in the CDK8 (NM_001260.3) gene and patient 2 had a heterozygous de novo missense p.N156S variant in the CDK8 gene. We assessed the pathogenicity of these two variants using cultured-cells and zebrafish model. An in vitro kinase assay of human CDK8 showed that enzymes with a p.G28A or p.N156S substitution showed decreased kinase activity. An in vivo assays of zeb...

CYCLIN-DEPENDENT KINASE8 (CDK8) is a widely studied component of eukaryotic Mediator complexes. However, the biological and molecular functions of plant CDK8 are not well understood. Here, we provide evidence for regulatory functions of Arabidopsis thaliana CDK8 in defense and demonstrate its functional and molecular interactions with other Mediator and non-Mediator subunits. The cdk8 mutant exhibits enhanced resistance to Botrytis cinerea but susceptibility to Alternaria brassicicola. The contributions of CDK8 to the transcriptional activation of defensin gene PDF1.2 and its interaction with MEDIATOR COMPLEX SUBUNIT25 (MED25) implicate CDK8 in jasmonate-mediated defense. Moreover, CDK8 associates with the promoter of AGMATINE COUMAROYLTRANSFERASE to promote its transcription and regulate the biosynthesis of the defense-active secondary metabolites hydroxycinnamic acid amides. CDK8 also interacts with the transcription factor WAX INDUCER1, implying its additional role in cuticle dev...

S. and W.W. contributed molecular mechanistic analyses of cyclin C→Fbw7→ICN1 link, X.L. and H. v. B. contributed bone marrow transduction with ICN1 experiments, T.K. and H. v. B. performed analyses of thymocyte populations and studies of bone marrow chimeras. M.R.M. S.J., R.E.G. and D.C.L. discovered mutations in human patients which render cyclin C-CDK unable to phosphorylate Notch1, L.L. contributed to molecular studies and phosphorylation analyses, H.W. performed all analyses of ICN1 phosphorylation by cyclin C-CDK8, C-CDK19 and C-CDK3, B.K. and I.A. contributed in vivo mouse tumorigenesis studies, A.G. and A.T.L. contributed analyses of human TALL samples, A.O and J.W.H. performed analyses of endogenous ICN1 polyubiquitination, T.O. contributed shRNA analyses of mouse cells and analyses of pRB phosphorylation, Lu.B. and J.J.Z. helped with molecular in vivo analyses, C.A.M. analyzed gene expression data, M.J.K and J.C.A. performed immunostaining for ICN1, M.R. and J.C.A contributed analyses of anti-phospho ICN1 antibody, S.K. helped with analyses of human TALL , X.Z. and J.C.A. developed anti-phospho-ICN1 antibody, C.G.M. contributed DNA sequencing and copy number analyses of the cyclin C, CDK8, CDK19 and CDK3 genes in human T

In this study, we report a novel duplication causing North Carolina macular dystrophy (NCMD) identified applying whole genome sequencing performed on eight affected members of two presumed unrelated families mapping to the MCDR1 locus. In our families, the NCMD phenotype was associated with a 98.4 kb tandem duplication encompassing the entire CCNC and PRDM13 genes and a common DNase 1 hypersensitivity site. To study the impact of PRDM13 or CCNC dysregulation, we used the Drosophila eye development as a model. Knock-down and overexpression of CycC and CG13296, Drosophila orthologues of CCNC and PRDM13, respectively, were induced separately during eye development. In flies, eye development was not affected, while knocking down either CycC or CG13296 mutant models. Overexpression of CycC also had no effect. Strikingly, overexpression of CG13296 in Drosophila leads to a severe loss of the imaginal eye-antennal disc. This study demonstrated for the

Cancer is a global multidrug resistant calamity, demanding an urgent need to design a novel/potent anti cancer agent. CDK8, 3/cyclin C biosynthetic pathway plays a specific role in G 0 /G 1 /S phases of cell cycle. Cyclin C is identified as a potential anti cancer target candidate. In order to understand the mechanism of ligand binding and interaction between ligand and cyclin C, a 3D homology model for cyclin C is generated. The cyclin C binding groove can be checked by small ligand molecules leading to inhibition. Virtual screening of molecules from an online data base of ChemBank library throws light to arrive at possible inhibitors for cyclin C inhibition. The molecules with better docking scores and acceptable ADME properties were prioritised to obtain potential lead molecules as cyclin C inhibitors.

Van de Sompele et al. used multiomics profiling and in vitro and in vivo enhancer assays to dissect the regulatory mechanisms underlying North Carolina macular dystrophy, a rare autosomal-dominant disease affecting macular development. This study supports that this condition is a retinal enhanceropathy.

CDK8 and CDK19 Mediator kinases are transcriptional co-regulators implicated in several types of cancer. Small-molecule CDK8/19 inhibitors have recently entered or are entering clinical trials, starting with breast cancer and acute myeloid leukemia (AML). To identify other cancers where these novel drugs may provide benefit, we queried genomic and transcriptomic databases for potential impact of CDK8, CDK19, or their binding partner CCNC. sgRNA analysis of a panel of tumor cell lines showed that most tumor types represented in the panel, except for some central nervous system tumors, were not dependent on these genes. In contrast, analysis of clinical samples for alterations in these genes revealed a high frequency of gene amplification in two highly aggressive subtypes of prostate cancer and in some cancers of the GI tract, breast, bladder, and sarcomas. Analysis of survival correlations identified a group of cancers where CDK8 expression correlated with shorter survival (notably b...

Very late antigen-4 (VLA-4)/vascular cell adhesion molecule-1 (VCAM-1) are a pair of adhesion molecules mediating cell-cell interaction. The binding activity of each depends on its surface expression, yet integrin activity can also be modulated through inside-out signaling. However, the specific intracellular molecules involved in modulating integrin VLA-4 activation via inside-out signaling or in regulating VCAM-1 expression are poorly understood. We show here that constitutive coexpression of cyclin C and c-Myc in hematopoietic BAF-B03 cells induces homotypic cell adhesion, which results from enhanced VLA-4 ligand-binding activity and induced expression of VCAM-1. Furthermore, regulation of cell adhesion appears to be a feature unique to cyclin C, but not other G1 cyclins, E and D3, and its regulatory function is independent of CDK8 kinase activity. Our results provide a novel role for cyclin C and c-Myc in the regulation of cell adhesion through distinct mechanisms.

Mitochondria exist in an equilibrium between fragmented and fused states that shifts heavily toward fission in response to cellular damage. Nuclear-to-cytoplasmic cyclin C relocalization is essential for dynamin-related protein 1 (Drp1)–dependent mitochondrial fission in response to oxidative stress. This study finds that cyclin C directly interacts with the Drp1 GTPase domain, increases its affinity to GTP, and stimulates GTPase activity in vitro. In addition, the cyclin C domain that binds Drp1 is contained within the non–Cdk binding second cyclin box domain common to all cyclin family members. This interaction is important, as this domain is sufficient to induce mitochondrial fission when expressed in mouse embryonic fibroblasts in the absence of additional stress signals. Using gel filtration chromatography and negative stain electron microscopy, we found that cyclin C interaction changes the geometry of Drp1 oligomers in vitro. High–molecular weight low–GTPase activity oligomer...

In response to oxidative stress, cells decide whether to mount a survival or cell death response. The conserved cyclin C and its kinase partner Cdk8 play a key role in this decision. Both are members of the Cdk8 kinase module that, with Med12 and Med13, associate with the core mediator complex of RNA polymerase II. In S. cerevisiae, oxidative stress triggers Med13 destruction, which thereafter releases cyclin C into the cytoplasm. Cytoplasmic cyclin C associates with mitochondria where it induces hyper-fragmentation and regulated cell death. In this report, we show that residues 742-844 of Med13's 600 amino acid intrinsic disordered region (IDR) both directs cyclin C-Cdk8 association and serves as the degron that mediates ubiquitin ligase SCFGrr1-dependent destruction of Med13 following oxidative stress. Here, cyclin C-Cdk8 phosphorylation of Med13 most likely primes the phospho-degron for destruction. Next, pro-oxidant stimulation of the cell wall integrity pathway MAP Kinase S...

A number of mammalian multiprotein complexes containing homologs of Saccharomyces cerevisiae Mediator subunits have been described recently. High-molecular-mass complexes (1 to 2 MDa) sharing several subunits but apparently differing in others include the TRAP/SMCC, NAT, DRIP, ARC, and human Mediator complexes. Smaller multiprotein complexes (∼500 to 700 kDa), including the murine Mediator, CRSP, and PC2, have also been described that contain subsets of subunits of the larger complexes. To evaluate whether these different multiprotein complexes exist in vivo in a single form or in multiple different forms, HeLa cell nuclear extract was directly resolved over a Superose 6 gel filtration column. Immunoblotting of column fractions using antisera specific for several Mediator subunits revealed one major size class of high-molecular-mass (∼2-MDa) complexes containing multiple mammalian Mediator subunits. No peak was apparent at ∼500 to 700 kDa, indicating that either the smaller complexe...

The biological activity of two novel 14-epi-analogues of 1,25(OH)2D3, 19-nor-14-epi-23-yne-1,25(OH)2D3 (TX 522) and 19-nor-14,20-bisepi-23-yne-1,25(OH)2D3 (TX 527), is described. Both analogues were at least 10 times more potent than 1,25(OH)2D3 in inhibiting in vitro cell proliferation and had much lower in vivo calcemic effects than 1,25(OH)2D3. Treatment with 1,25(OH)2D3, TX 522, or TX 527 in vitro was accompanied by an accumulation of cells in the G1 phase of the cell cycle. Protein levels of cyclin C and cyclin D1 in in vitro cultures of MCF-7 cells were down-regulated to 50 and 30%, respectively, of control levels at 72 and 120 h after stimulation. Protein levels of p21 and p27 at 72 h were significantly enhanced by 1,25(OH)2D3 and TX 522 but surprisingly not by TX 527. The inability of TX 527 to up-regulate p21 seemed to be cell type specific because p21 was induced in other cell types. Diminished phosphorylation of the retinoblastoma protein after treatment with 1,25(OH)2D3,...

Steroid receptor coactivator 2 (SRC-2) is a coactivator that regulates nuclear receptor activity. We previously reported that SRC-2 protein is degraded through the action of cAMP-dependent protein kinase A (PKA) and cAMP response element binding protein (CREB). In the study presented here, we aimed to identify proteins that interact with and thereby regulate SRC-2. We isolated cyclin C (CCNC) as an interacting partner with the SRC-2 degradation domain aa 347-758 in a yeast two-hybrid assay and confirmed direct interaction in an in vitro assay. The protein level of SRC-2 was increased with CCNC overexpression in COS-1 cells and decreased with CCNC silencing in COS-1 and MCF-7 cells. In a pulse-chase assay, we further show that silencing of CCNC resulted in a different SRC-2 degradation pattern during the first 6 h after the pulse. Finally, we provide evidence that CCNC regulates expression of cell cycle genes upregulated by SRC-2. In conclusion, our results suggest that CCNC temporarily protects SRC-2 against degradation and this event is involved in the transcriptional regulation of SRC-2 cell cycle target genes.

Cyclin C was cloned as a growth-promoting G 1 cyclin, and was also shown to regulate gene transcription. Here we report that in vivo cyclin C acts as a haploinsufficient tumour suppressor, by controlling Notch1 oncogene levels. Cyclin C activates an 'orphan' CDK19 kinase, as well as CDK8 and CDK3. These cyclin-C-CDK complexes phosphorylate the Notch1 intracellular domain (ICN1) and promote ICN1 degradation. Genetic ablation of cyclin C blocks ICN1 phosphorylation in vivo, thereby elevating ICN1 levels in cyclin-C-knockout mice. Cyclin C ablation or heterozygosity collaborates with other oncogenic lesions and accelerates development of T-cell acute lymphoblastic leukaemia (T-ALL). Furthermore, the cyclin C encoding gene CCNC is heterozygously deleted in a significant fraction of human T-ALLs, and these tumours express reduced cyclin C levels. We also describe point mutations in human TALL that render cyclin-C-CDK unable to phosphorylate ICN1. Hence, tumour cells may develop different strategies to evade inhibition by cyclin C.

Cancer is a global multidrug resistant calamity, demanding an urgent need to design a novel/potent anti cancer agent. CDK8, 3/cyclin C biosynthetic pathway plays a specific role in G 0 /G 1 /S phases of cell cycle. Cyclin C is identified as a potential anti cancer target candidate. In order to understand the mechanism of ligand binding and interaction between ligand and cyclin C, a 3D homology model for cyclin C is generated. The cyclin C binding groove can be checked by small ligand molecules leading to inhibition. Virtual screening of molecules from an online data base of ChemBank library throws light to arrive at possible inhibitors for cyclin C inhibition. The molecules with better docking scores and acceptable ADME properties were prioritised to obtain potential lead molecules as cyclin C inhibitors.

The extent to which neurons proceed into the cell cycle and the mechanisms whereby cell cycle reentry leads to apoptosis vary in response to agonists. We previously showed upregulation of early G1 regulators in thrombin-treated neurons yet neurons did not proceed to S phase but to apoptosis. The objective of this study is to explore mechanisms which might prevent S phase entry and promote apoptosis in thrombin-treated neurons. Cultured rat brain neurons are exposed to thrombin (200 nM) for 30 min to 4.5 h and the expression of cyclin C, cyclin dependent kinases (cdk1, 2 ,3, 8) and the cell cycle inhibitor p27 assessed. Our data show a simultaneous decrease of both cyclin C and cdk3 proteins soon after thrombin treatment. The decrease in cyclin C also correlates with decreases in cdk1 and cdk2, at both mRNA and protein levels. There is no change in expression of cdk8 or the cell cycle inhibitor p27 in response to thrombin treatment. These results suggest that decreases in G1-S regulators cyclin C and cdks 3, cdk2 and cdk1 in response to thrombin could make conditions unfavorable for S phase entry and favor neuronal apoptosis.

Altered lipid metabolism underlies several major human diseases, including obesity and type 2 diabetes. However, lipid metabolism pathophysiology remains poorly understood at the molecular level. Insulin is the primary stimulator of hepatic lipogenesis through activation of the SREBP-1c transcription factor. Here we identified cyclin-dependent kinase 8 (CDK8) and its regulatory partner cyclin C (CycC) as negative regulators of the lipogenic pathway in Drosophila, mammalian hepatocytes, and mouse liver. The inhibitory effect of CDK8 and CycC on de novo lipogenesis was mediated through CDK8 phosphorylation of nuclear SREBP-1c at a conserved threonine residue. Phosphorylation by CDK8 enhanced SREBP-1c ubiquitination and protein degradation. Importantly, consistent with the physiologic regulation of lipid biosynthesis, CDK8 and CycC proteins were rapidly downregulated by feeding and insulin, resulting in decreased SREBP-1c phosphorylation. Moreover, overexpression of CycC efficiently suppressed insulin and feeding-induced lipogenic gene expression. Taken together, these results demonstrate that CDK8 and CycC function as evolutionarily conserved components of the insulin signaling pathway in regulating lipid homeostasis.

The mechanisms through which the oncoprotein c-Myc initiates locus-specific gene amplification are not understood. When analysing the initiation mechanism of c-Myc-dependent amplification of the mouse ribonucleotide reductase R2 (R2) gene, we observe c-Myc-dependent initiation of illegitimate DNA replication of the R2 gene. We demonstrate multiple simultaneous c-Myc-induced R2 replication forks, whereas R2 normally replicates with a single fork. In contrast,

Cyclin C, a putative G1cyclin, was originally isolated through its ability to complement aSaccharomyces cerevisiaestrain lacking the G1cyclin geneCLN1-3. Unlike cyclins D1 and E, the other two G1cyclins obtained by the same approach and subsequently shown to play important roles during the G1/S transition, there is thus far no evidence to support the hypothesis that cyclin C is indeed critical for the promotion of cell cycle progression. In BAF-B03 cells, an interleukin 3 (IL-3)-dependent murine pro-B-cell line, cyclin C gene mRNA was induced at the G1/S phase upon IL-3 stimulation and reached a maximal level in the S phase. Enforced expression of exogenous cyclin C in this cell line failed to alter its growth properties. In the present study, we examined whether cyclin C is capable of cooperating with the cytokine-responsive immediate-early gene products c-Myc and c-Fos in the promotion of cell proliferation. We found that cyclin C is able to cooperate functionally with c-Myc, but ...

Cyclin C, a putative G 1 cyclin, was originally isolated through its ability to complement a Saccharomyces cerevisiae strain lacking the G 1 cyclin gene CLN1-3. Unlike cyclins D1 and E, the other two G 1 cyclins obtained by the same approach and subsequently shown to play important roles during the G 1 /S transition, there is thus far no evidence to support the hypothesis that cyclin C is indeed critical for the promotion of cell cycle progression. In BAF-B03 cells, an interleukin 3 (IL-3)-dependent murine pro-B-cell line, cyclin C gene mRNA was induced at the G 1 /S phase upon IL-3 stimulation and reached a maximal level in the S phase. Enforced expression of exogenous cyclin C in this cell line failed to alter its growth properties. In the present study, we examined whether cyclin C is capable of cooperating with the cytokine-responsive immediate-early gene products c-Myc and c-Fos in the promotion of cell proliferation. We found that cyclin C is able to cooperate functionally with c-Myc, but not c-Fos, to induce both BAF-B03 cell proliferation in a cytokine-independent fashion and the formation of cell clusters. Furthermore, cyclin C was primarily responsible for the induction of cdc2 gene expression. Our data define a novel role for cyclin C in the regulation of both the G 1 /S and G 2 /M phases of the cell cycle, and this effect appears to be independent of the activity of CDK8 in the control of transcription.

Cyclin-dependent kinase (CDK) 8 associates with cyclin C (CycC) and belongs to the CDK module of the Mediator of transcription, together with MED12 and MED13. CDK8 is involved in the regulation of mRNA transcription and was identified as a potent oncogene in colon cancerogenesis. We have solved the 2.2-Å crystal structure of CDK8/CycC in complex with sorafenib, an anti-cancer drug of clinical relevance. The CDK8 structure reveals a unique CycC recognition helix that explains the specificity of the CDK8/CycC pair and discrimination among the highly promiscuous binding in the CDK/cyclin family. In contrast to all CDKs, the CDK8 activation loop appears not to be phosphorylated. Based on the structure, we discuss an alternate mode of CDK8 activation to the general CDK activation by T-loop phosphorylation. Sorafenib binds to the catalytic cleft of CDK8. It displays a deep pocket binding mode and is the first small molecule to induce a DFG-out conformation in the CDK family, which is actually DMG-out in CDK8.

In this study, we report a novel duplication causing North Carolina macular dystrophy (NCMD) identified applying whole genome sequencing performed on eight affected members of two presumed unrelated families mapping to the MCDR1 locus. In our families, the NCMD phenotype was associated with a 98.4 kb tandem duplication encompassing the entire CCNC and PRDM13 genes and a common DNase 1 hypersensitivity site. To study the impact of PRDM13 or CCNC dysregulation, we used the Drosophila eye development as a model. Knock-down and overexpression of CycC and CG13296, Drosophila orthologues of CCNC and PRDM13, respectively, were induced separately during eye development. In flies, eye development was not affected, while knocking down either CycC or CG13296 mutant models. Overexpression of CycC also had no effect. Strikingly, overexpression of CG13296 in Drosophila leads to a severe loss of the imaginal eye-antennal disc. This study demonstrated for the

In this study, we report a novel duplication causing North Carolina macular dystrophy (NCMD) identified applying whole genome sequencing performed on eight affected members of two presumed unrelated families mapping to the MCDR1 locus. In our families, the NCMD phenotype was associated with a 98.4 kb tandem duplication encompassing the entire CCNC and PRDM13 genes and a common DNase 1 hypersensitivity site. To study the impact of PRDM13 or CCNC dysregulation, we used the Drosophila eye development as a model. Knock-down and overexpression of CycC and CG13296, Drosophila orthologues of CCNC and PRDM13, respectively, were induced separately during eye development. In flies, eye development was not affected, while knocking down either CycC or CG13296 mutant models. Overexpression of CycC also had no effect. Strikingly, overexpression of CG13296 in Drosophila leads to a severe loss of the imaginal eye-antennal disc. This study demonstrated for the

It is commonly believed that neurons remain in G0 phase of the cell cycle indefinitely. Cell-cycle re-entry, however, is known to contribute to neuronal apoptosis. Moreover, recent evidence demonstrates the expression of cell-cycle proteins in differentiated neurons under physiological conditions. The functional roles of such expression remain unclear. Since DNA repair is generally attenuated by differentiation in most cell types,

Cyclin C was cloned as a growth-promoting G 1 cyclin, and was also shown to regulate gene transcription. Here we report that in vivo cyclin C acts as a haploinsufficient tumour suppressor, by controlling Notch1 oncogene levels. Cyclin C activates an 'orphan' CDK19 kinase, as well as CDK8 and CDK3. These cyclin-C-CDK complexes phosphorylate the Notch1 intracellular domain (ICN1) and promote ICN1 degradation. Genetic ablation of cyclin C blocks ICN1 phosphorylation in vivo, thereby elevating ICN1 levels in cyclin-C-knockout mice. Cyclin C ablation or heterozygosity collaborates with other oncogenic lesions and accelerates development of T-cell acute lymphoblastic leukaemia (T-ALL). Furthermore, the cyclin C encoding gene CCNC is heterozygously deleted in a significant fraction of human T-ALLs, and these tumours express reduced cyclin C levels. We also describe point mutations in human TALL that render cyclin-C-CDK unable to phosphorylate ICN1. Hence, tumour cells may develop different strategies to evade inhibition by cyclin C.

CYCLIN-DEPENDENT KINASE8 (CDK8) is a widely studied component of eukaryotic Mediator complexes. However, the biological and molecular functions of plant CDK8 are not well understood. Here, we provide evidence for regulatory functions of Arabidopsis thaliana CDK8 in defense and demonstrate its functional and molecular interactions with other Mediator and non-Mediator subunits. The cdk8 mutant exhibits enhanced resistance to Botrytis cinerea but susceptibility to Alternaria brassicicola. The contributions of CDK8 to the transcriptional activation of defensin gene PDF1.2 and its interaction with MEDIATOR COMPLEX SUBUNIT25 (MED25) implicate CDK8 in jasmonate-mediated defense. Moreover, CDK8 associates with the promoter of AGMATINE COUMAROYLTRANSFERASE to promote its transcription and regulate the biosynthesis of the defense-active secondary metabolites hydroxycinnamic acid amides. CDK8 also interacts with the transcription factor WAX INDUCER1, implying its additional role in cuticle dev...

In a differential gene expression profile, we showed previously that dynactin 1 (DCTN1), early growth response 3 (EGR3), acetyl-CoA transporter (ACATN), death receptor 5 (DR5), and cyclin C (CCNC) were prominently up-or downregulated in motor neurons of sporadic amyotrophic lateral sclerosis (ALS). In the present study, we examined the correlation between the expression levels of these genes and the levels of pathologic markers for motor neuron degeneration (i.e. cytoplasmic accumulation of phosphorylated neurofilament H [pNF-H] and ubiquitylated protein) and the numbers of residual motor neurons in 20 autopsies of patients with sporadic ALS. DCTN1 and EGR3 were widely downregulated, and the changes in gene expression were correlated to the number of residual motor neurons. In particular, DCTN1 was markedly downregulated in most residual motor neurons before the accumulation of pNF-H, even in cases with well-preserved motor neuron populations. ACATN, DR5, and CCNC were upregulated in subpopulations of residual motor neurons, and their expression levels were well correlated with the levels of pNF-H accumulation and the number of residual motor neurons. The expressions of DCTN1, EGR3, ACATN, and DR5 were all markedly altered before ubiquitylated protein accumulation. DCTN1 downregulation appears to be an early event before the appearance of neurodegeneration markers, whereas upregulations of DR5 and CCNC are relatively later phenomena associated with pathologic markers and leading to neuronal death. The sequence of motor neuronspecific gene expression changes in sporadic ALS can be beneficial information in developing appropriate therapeutic strategies for neurodegeneration.

In this study, we report a novel duplication causing North Carolina macular dystrophy (NCMD) identified applying whole genome sequencing performed on eight affected members of two presumed unrelated families mapping to the MCDR1 locus. In our families, the NCMD phenotype was associated with a 98.4 kb tandem duplication encompassing the entire CCNC and PRDM13 genes and a common DNase 1 hypersensitivity site. To study the impact of PRDM13 or CCNC dysregulation, we used the Drosophila eye development as a model. Knock-down and overexpression of CycC and CG13296, Drosophila orthologues of CCNC and PRDM13, respectively, were induced separately during eye development. In flies, eye development was not affected, while knocking down either CycC or CG13296 mutant models. Overexpression of CycC also had no effect. Strikingly, overexpression of CG13296 in Drosophila leads to a severe loss of the imaginal eye-antennal disc. This study demonstrated for the first time in an animal model that overexpression of PRDM13 alone causes a severe abnormal retinal development. It is noteworthy that mutations associated with this autosomal dominant foveal developmental disorder are frequently duplications always including an entire copy of PRDM13, or variants in one DNase 1 hypersensitivity site at this locus.

In this study, we report a novel duplication causing North Carolina macular dystrophy (NCMD) identified applying whole genome sequencing performed on eight affected members of two presumed unrelated families mapping to the MCDR1 locus. In our families, the NCMD phenotype was associated with a 98.4 kb tandem duplication encompassing the entire CCNC and PRDM13 genes and a common DNase 1 hypersensitivity site. To study the impact of PRDM13 or CCNC dysregulation, we used the Drosophila eye development as a model. Knock-down and overexpression of CycC and CG13296, Drosophila orthologues of CCNC and PRDM13, respectively, were induced separately during eye development. In flies, eye development was not affected, while knocking down either CycC or CG13296 mutant models. Overexpression of CycC also had no effect. Strikingly, overexpression of CG13296 in Drosophila leads to a severe loss of the imaginal eye-antennal disc. This study demonstrated for the first time in an animal model that overexpression of PRDM13 alone causes a severe abnormal retinal development. It is noteworthy that mutations associated with this autosomal dominant foveal developmental disorder are frequently duplications always including an entire copy of PRDM13, or variants in one DNase 1 hypersensitivity site at this locus.

Mediator is considered an enhancer of RNA-Polymerase II dependent transcription but its function and regulation in pluripotent mouse embryonic stem cells (mESCs) remains unresolved. One means of controlling the function of Mediator is provided by the binding of the Cdk8 module (Med12, Cdk8, Ccnc and Med13) to the core Mediator. Here we report that Med12 operates together with PRC1 to silence key developmental genes in pluripotency. At the molecular level, while PRC1 represses genes it is also required to assemble ncRNA containing Med12-Mediator complexes. In the course of cellular differentiation the H2A ubiquitin binding protein Zrf1 abrogates PRC1-Med12 binding and facilitates the association of Cdk8 with Mediator. This remodeling of Mediator-associated protein complexes converts Mediator from a transcriptional repressor to a transcriptional enhancer, which then mediates ncRNA-dependent activation of Polycomb target genes. Altogether, our data reveal how the interplay of PRC1, ncRNA and Mediator complexes controls pluripotency and cellular differentiation.

Mycosphaerella graminicola is an important wheat pathogen causing Septoria tritici blotch. To date, an efficient strategy to control M. graminicola has not been developed. More significantly, we have a limited understanding of the molecular mechanisms of M. graminicola pathogenicity. In this study, we attempted to characterize an MCC1-encoding c-type cyclin, a gene homologous to FCC1 in Fusarium verticillioides. Four independent MCC1 knock-out mutants were generated via Agrobacterium tumefaciens–mediated transformation. All of the MCC1 mutants showed consistent multiple phenotypes. Significant reductions in radial growth on potato dextrose agar (PDA) were observed in all of the MCC1 mutants. In addition, MCC1 gene-deletion mutants produced less aerial mycelium on PDA, showed delayed filamentous growth, had unusual hyphal swellings, produced more melanin, showed an increase in their stress tolerance response, and were reduced significantly in pathogenicity. These results indicate tha...

Cyclin C was cloned as a growth-promoting G 1 cyclin, and was also shown to regulate gene transcription. Here we report that in vivo cyclin C acts as a haploinsufficient tumour suppressor, by controlling Notch1 oncogene levels. Cyclin C activates an 'orphan' CDK19 kinase, as well as CDK8 and CDK3. These cyclin-C-CDK complexes phosphorylate the Notch1 intracellular domain (ICN1) and promote ICN1 degradation. Genetic ablation of cyclin C blocks ICN1 phosphorylation in vivo, thereby elevating ICN1 levels in cyclin-C-knockout mice. Cyclin C ablation or heterozygosity collaborates with other oncogenic lesions and accelerates development of T-cell acute lymphoblastic leukaemia (T-ALL). Furthermore, the cyclin C encoding gene CCNC is heterozygously deleted in a significant fraction of human T-ALLs, and these tumours express reduced cyclin C levels. We also describe point mutations in human TALL that render cyclin-C-CDK unable to phosphorylate ICN1. Hence, tumour cells may develop different strategies to evade inhibition by cyclin C.