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V9: Cell cycle, CDKs and cancer Active CDK1-cyclin complexes phosphorylate more than 70 substrates during G2 and early mitosis to trigger e.g. - centrosome separation, - Golgi dynamics, - nuclear envelope breakdown and - chromosome condensation! In humans, the CDK family is composed of 13 members that interact with at least 29 cyclins or cyclin-related proteins. Today: role of CDKs during transcription + relation to cancer. Malumbres, Barbacid, Nature Rev. Cancer 9, 353 (2009) 9. Lecture WS 2010/11 Cellular Programs 1 Transcription by Polymerase II in Drosophila The 3 main phases of the transcription cycle are known as initiation, elongation and termination. During transcription initiation, a transcription-competent RNA polymerase complex forms at the promoter and the DNA template is aligned in the active site of the polymerase. The active site is where nucleotides are paired with the template and are joined processively during elongation to produce the RNA transcript. Termination of transcription involves release of the RNA transcript and the dissociation of the transcription complex from the DNA template. Saunders et al. Nat. Rev. Mol Cell Biol 7, 557 (2006) 9. Lecture WS 2010/11 Cellular Programs 2 Elongation Regulation of transcription occurs - at the level of RNA polymerase recruitment to the promoter and - at the level of elongation. RNA polymerase II (Pol II) transcription elongation is divided into 3 distinct stages: (1) promoter escape, (2) promoter-proximal pausing, and (3) productive elongation. Saunders et al. Nat. Rev. Mol Cell Biol 7, 557 (2006) 9. Lecture WS 2010/11 Cellular Programs 3 C-terminal tail of RNA Polymerase II The C-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II), Rpb1, consists of tandem heptapeptide repeats. This C-terminal domain distinguishes Pol II from the other two eukaryotic RNA polymerases. The number of repeats that exactly match the consensus sequence varies among species. Saunders et al. Nat. Rev. Mol Cell Biol 7, 557 (2006) 9. Lecture WS 2010/11 Cellular Programs 4 Modifications of C-terminal tail of Pol II The CTD can be modified by phosphorylation (mostly at Ser2 and Ser5), glycosylation, and cis/ trans isomerization of prolines. CDK8 phosphorylates the CTD at Ser5 and possibly Ser2. Peptidyl-prolyl isomerases (e.g. yeast Ess1 and mammalian PIN1) can alter the conformation of the CTD, and thereby regulate CTD phosphorylation and the binding of other protein factors to Pol II. Modification of the CTD is important for the coordination of transcription events. Different modification states of the CTD are characteristic of different transcriptional stages. Saunders et al. Nat. Rev. Mol Cell Biol 7, 557 (2006) 9. Lecture WS 2010/11 Cellular Programs 5 Role of C-terminal tail of Pol II during elongation During the transition from transcription initiation to elongation, Pol II changes from a hypophosphorylated form to a hyperphosphorylated form. The level of Ser5 phosphorylation peaks early in the transcription cycle and remains constant or decreases towards the 3‘ end of the gene. By contrast, Ser2 phosphorylation predominates in the gene body and towards the 3‘ end of the gene. Saunders et al. Nat. Rev. Mol Cell Biol 7, 557 (2006) 9. Lecture WS 2010/11 Cellular Programs 6 Transcription initiation Before transcription initiation, a pre-initiation complex (PIC) forms at the promoter, consisting of Pol and several general transcription factors (GTFs). The GTFs position Pol II near the transcription-start site (TSS) and dictate the precise location of transcription initiation. The general transcription factor TFIIH is needed for the structural remodelling of the PIC. 11-15 base pairs around the TSS are unwound to form an ‘open complex‘ that allows the single-stranded DNA template to enter the active site of Pol II. Saunders et al. Nat. Rev. Mol Cell Biol 7, 557 (2006) 9. Lecture WS 2010/11 Cellular Programs 7 Promoter escape: elongation stage 1 Productively elongating Pol II can transcribe the full length of a gene in a highly processive manner without dissociating from the template DNA or releasing the nascent RNA product. This is possible after promoter escape during which the polymerase breaks its contacts with promoter-sequence elements and at least some promoter-bound factors and simultaneously tightens its grip on the nascent RNA. Saunders et al. Nat. Rev. Mol Cell Biol 7, 557 (2006) 9. Lecture WS 2010/11 Cellular Programs 8 Promoter escape – formation of an early elongation complex The unwinding of promoter DNA to create a transcription bubble begins at a fixed position, ~20 base pairs downstream from the binding site of the TATA-box-binding protein (TBP). The upstream bubble edge (vertical dashed line) remains fixed until the completion of promoter escape, whereas the downstream edge expands together with transcription. The initially transcribing complex (ITC) cycles through several rounds of abortive initiation, releasing large amounts of 2–3-nucleotide-long RNA transcripts (red). Saunders et al. Nat. Rev. Mol Cell Biol 7, 557 (2006) 9. Lecture WS 2010/11 Cellular Programs 9 Escape commitment After synthesis of the first 4 nucleotides, the B-finger of TFIIB (orange) and a switch domain (dark blue oval) of Pol II (large blue oval) stabilize the short RNA, reducing abortive initiation. This transition to a metastable transcription complex is termed escape commitment. Saunders et al. Nat. Rev. Mol Cell Biol 7, 557 (2006) 9. Lecture WS 2010/11 Cellular Programs 10 Action of Polymerase II in Drosophila After 5 nucleotides are added, the nascent RNA collides with the B-finger of TFIIB, inducing stress within the ITC. This can cause increased abortive initiation, strong pausing, or transcript slippage, if the nucleotides at the 3′ end of the RNA–DNA hybrid interact weakly, and probably contributes to the rate-limiting step of promoter escape. Saunders et al. Nat. Rev. Mol Cell Biol 7, 557 (2006) 9. Lecture WS 2010/11 Cellular Programs 11 Promoter escape Stress from the growing transcription bubble and the production of a 7-nt-long RNA trigger collapse of the transcription bubble, providing the energy to remodel the transcription complex. The B-finger is ejected from the RNA-exit tunnel and TFIIB is released from the transcription complex. The RNA–DNA hybrid is at its full length of 8–9 base pairs and can make contacts with protein loops near the RNA-exit tunnel. Abortive initiation ceases, as does the need for ATP hydrolysis, and transcript slippage is markedly reduced, all indicating that the transcription complex has changed into an early elongation complex (EEC). Saunders et al. Nat. Rev. Mol Cell Biol 7, 557 (2006) 9. Lecture WS 2010/11 Cellular Programs 12 Transitioning to the pause region Following promoter escape, the RNA remains stably bound in the transcription complex, but has a tendency to undergo transcript slippage, backtracking and arrest until about +30. This phase is often accompanied by transcriptional pausing near the promoter. Progress depends on stimulation by appropriate signals. Consequently, this stage serves as a checkpoint for regulation. The details how this step is regulated are subject of very active current research. Saunders et al. Nat. Rev. Mol Cell Biol 7, 557 (2006) 9. Lecture WS 2010/11 Cellular Programs 13 Pol II binding profiles ChIP-chip assays were carried out with 2–4 h Toll10b embryos using antibodies that recognize both the initiating and the elongating forms of Pol II. y axis: enrichment ratios of Pol II. (a–d) Binding patterns across genes that are repressed in Toll10b embryos. All four genes show high Pol II signals near the transcription start sites. At some genes, such as tup (a), Pol II is tightly restricted to this region, whereas at other genes, including sog (c) and brk (d), Pol II is also detected at lower signals throughout the transcription unit. Zeitlinger et al. J. Nat. Gen. 39, 1513 (2007) 9. Lecture WS 2010/11 Cellular Programs 14 (e,f) Pol II is uniformly distributed across the transcription units of genes that are actively transcribed. The stumps gene (e) is specifically activated in mesodermal precursor cells, whereas RpL3 (f) is a highly expressed ribosomal gene. (g,h) No Pol II binding is found at many genes that are inactive during embryogenesis. The eyeless (ey) gene (g) is expressed during eye development at larval stages but not in the early embryo. Likewise, the torso (tor) gene (h) is active only during oogenesis but not in the early embryo. Zeitlinger et al. J. Nat. Gen. 39, 1513 (2007) 9. Lecture WS 2010/11 Cellular Programs 15 Cell cycle, CDKs and cancer Marcos Malumbres, Madrid Mariano Barbacid Malumbres, Barbacid, Nature Rev. Cancer 9, 353 (2009) 9. Lecture WS 2010/11 Cellular Programs 16 Timeline: cell cycle regulation and cancer Malumbres, Barbacid, Nature Rev. Cancer 9, 353 (2009) 9. Lecture WS 2010/11 Cellular Programs 17 Cell cycle, CDKs and cancer Three basic cell cycle defects are mediated by mis-regulation of CDKs: - unscheduled proliferation - genomic instability (GIN) - chromosomal instability (CIN) Tumour-associated mutations in human tumor cells often deregulate CDKcyclin complexes, leading either to continued proliferation or unscheduled reentry into the cell cycle. Do tumour cells use CDKs in the same way as „normal“ cells, or do they show different patterns of CDK activity? Indeed, some tumour cell lines display a selective dependence on interphase CDKs. Malumbres, Barbacid, Nature Rev. Cancer 9, 353 (2009) 9. Lecture WS 2010/11 Cellular Programs 18 Cell cycle models Malumbres, Barbacid, Nature Rev. Cancer 9, 353 (2009) 9. Lecture WS 2010/11 Cellular Programs 19 a The diagram depicts the S. cerevisiae cell cycle and 3 models of the mammalian cell cycle. In the currently accepted model based on biochemical evidence (classical' model), each of the main events that take place during interphase (G1, S and G2) is driven by unique CDKs bound to specific cyclins. The essential' cell cycle is based on genetic evidence indicating that CDK1 is sufficient to drive proliferation of all cell types up to mid gestation as well as during adult liver regeneration. The specialized' cell cycles are based on the unique requirements of specialized cell types for specific CDKs as indicated. b Unscheduled cell proliferation requires aberrant mitogenic signalling driven by either excessive exogenous signalling (for example, growth factors and nutrients) or endogenous oncogenic mutations. Other mutations affecting mitogenic breaks (for example, tumour suppressors and negative regulators) that decrease the threshold for mitogenic signalling also contribute to unscheduled proliferation. Oncogene-induced cell cycling provokes DNA replication stress that is sensed by the DNA replication checkpoints. Failure in this control mechanism results in an increased mutation rate and ultimately may lead to genomic instability. During mitosis, defects within the spindle assembly checkpoint induce deregulation of CDK1 activity that may result in abnormal chromosome segregation. All these defects converge in deregulation of CDK activity, eventually leading to tumour development. Malumbres, Barbacid, Nature Rev. Cancer 9, 353 (2009) 9. Lecture WS 2010/11 Cellular Programs 20 Effects of gene deletions during development Malumbres, Barbacid, Nature Rev. Cancer 9, 353 (2009) 9. Lecture WS 2010/11 Cellular Programs Mice lacking CDK2 and CK4 die at birth. 21 Figure legend The genotypes of the various CDK mutant strains used in this analysis are indicated. Wildtype CDKs are indicated in bold font. Ablated CDKs appear in normal font. The arrows indicate the extent to which each of the mouse strains develops with the indicated CDK content. Stop signs indicate the developmental stage at which the strains are no longer viable. The main defects responsible for loss of viability are also indicated. Briefly, mice expressing all interphase CDKs but not CDK1 do not progress beyond the two-cell embryo stage. Mice expressing CDK1 but no interphase CDKs, progress up to mid gestation (embryonic day (E)12.5–E13.5). Mice lacking CDK4 and CDK6 develop until late embryonic development (E16.5–E17.5), whereas those lacking CDK4 and CDK2 die at birth. Finally, mice lacking CDK6 and CDK2 develop to adulthood and have a normal life span. Except for embryos lacking CDK1, none of the mutant embryos or mice display cell cycle defects except in those cell types indicated in the yellow boxes. P, postnatal day. Malumbres, Barbacid, Nature Rev. Cancer 9, 353 (2009) 9. Lecture WS 2010/11 Cellular Programs 22 Key molecules involved in mitogenic progression In red: active molecules Malumbres, Barbacid, Nature Rev. Cancer 9, 353 (2009) 9. Lecture WS 2010/11 Cellular Programs 23 Figure legend The anaphase-promoting complex/cyclosome (APC/C)–CDC20 (cell division control 20) complex targets cyclin A (CycA) and NEK2 for degradation by ubiquitylation in a spindle assembly checkpoint (SAC)-independent manner. In the presence of unaligned chromosomes, separase is kept inactive by securin and CDK1–cyclin B. Under these conditions, sister chromatids are held together by cohesins. After complete bipolar attachment of chromosomes to the mitotic spindle, cyclin B and securin are also ubiquitylated by APC/C–CDC20, but in a SAC-dependent manner. Ubiquitin-dependent degradation of these proteins inhibits CDK1 leading to the activation of separase, which in turn cleaves cohesins and releases sister chromatids, hence facilitating the metaphase-toanaphase transition. APC/C–CDH1 also targets cyclin A and cyclin B to keep low levels of Cdk1 activity during mitotic exit and the following G1 phase. Other APC/C–CDH1 substrates involved in mitotic progression include CDC20, TPX2, forkhead box protein M1 (FOXM1), aurora kinase A (AURKA), AURKB and PLK1. Additional APC/C–CDH1 targets involved in the control of DNA replication, such as SKP2, CDC6 or geminin, are omitted for clarity. Tumours with chromosomal instability are characterized by molecular signatures in which most of these molecules (in bold) are overexpressed. Active proteins are represented by red boxes. Malumbres, Barbacid, Nature Rev. Cancer 9, 353 (2009) 9. Lecture WS 2010/11 Cellular Programs 24 Current clinical trials with CDK inhibitors Malumbres, Barbacid, Nature Rev. Cancer 9, 353 (2009) 9. Lecture WS 2010/11 Cellular Programs 25 CDK inhibitors in clinical tests CDK inhibitors are likely to produce certain toxicities by also affecting the proliferation of those cells that require specific interphase CDKs to maintain tissue homeostasis. The first generation of CDK inhibitors (e.g. flavopiridol, UCN-01) did not show significant clinical advantages. Some expected toxicities for CDK4 (diabetes), CDK2 (sterility) or CDK6 (mild anemia) inhibitors may be acceptable for adult cancer patients. On the other hand, small molecule against CDK1 and possibly against CDK7 may result in general toxicities. Therefore, promiscous CDK inhibitors often display high toxicities in early clinical trials. Malumbres, Barbacid, Nature Rev. Cancer 9, 353 (2009) 9. Lecture WS 2010/11 Cellular Programs 26