RESUMO
The RNA polymerase II (RNAPII) transcription cycle is regulated at every stage by a network of cyclin-dependent protein kinases (CDKs) and protein phosphatases. Progression of RNAPII from initiation to termination is marked by changing patterns of phosphorylation on the highly repetitive carboxy-terminal domain (CTD) of RPB1, its largest subunit, suggesting the existence of a CTD code. In parallel, the conserved transcription elongation factor SPT5, large subunit of the DRB sensitivity-inducing factor (DSIF), undergoes spatiotemporally regulated changes in phosphorylation state that may be directly linked to the transitions between transcription-cycle phases. Here we review insights gained from recent structural, biochemical, and genetic analyses of human SPT5, which suggest that two of its phosphorylated regions perform distinct functions at different points in transcription. Phosphorylation within a flexible, RNA-binding linker promotes release from the promoter-proximal pause-frequently a rate-limiting step in gene expression-whereas modifications in a repetitive carboxy-terminal region are thought to favor processive elongation, and are removed just prior to termination. Phosphorylations in both motifs depend on CDK9, catalytic subunit of positive transcription elongation factor b (P-TEFb); their different timing of accumulation on chromatin and function during the transcription cycle might reflect their removal by different phosphatases, different kinetics of phosphorylation by CDK9, or both. Perturbations of SPT5 regulation have profound impacts on viability and development in model organisms through largely unknown mechanisms, while enzymes that modify SPT5 have emerged as potential therapeutic targets in cancer; elucidating a putative SPT5 code is therefore a high priority.
RESUMO
The RNA polymerase II (RNAPII) transcription cycle is regulated throughout its duration by reversible protein phosphorylation. The elongation factor SPT5 contains two regions targeted by cyclin-dependent kinase 9 (CDK9) and previously implicated in promoter-proximal pausing and termination: the linker between KOWx-4 and KOW5 domains and carboxy-terminal repeat (CTR) 1, respectively. Here we show that phosphorylations in the KOWx-4/5 linker, CTR1 and a third region, CTR2, coordinately control pause release, elongation speed and RNA processing. Pausing was increased by mutations preventing CTR1 or CTR2 phosphorylation, but attenuated when both CTRs were mutated. Whereas mutating CTR1 alone slowed elongation and repressed nascent transcription, simultaneous mutation of CTR2 partially reversed both effects. Nevertheless, mutating both CTRs led to aberrant splicing, dysregulated termination and diminished steady-state mRNA levels, and impaired cell proliferation more severely than did either single-CTR mutation. Therefore, tripartite SPT5 phosphorylation times pause release and regulates RNAPII elongation rates positively and negatively to ensure productive transcription and cell viability.
RESUMO
Cyclin-dependent kinase 7 (Cdk7) is required in cell-cycle and transcriptional regulation owing to its function as both a CDK-activating kinase (CAK) and part of transcription factor TFIIH. Cdk7 forms active complexes by associating with Cyclin H and Mat1, and is regulated by two phosphorylations in the activation segment (T loop): the canonical activating modification at T170 and another at S164. Here we report the crystal structure of the human Cdk7/Cyclin H/Mat1 complex containing both T-loop phosphorylations. Whereas pT170 coordinates basic residues conserved in other CDKs, pS164 nucleates an arginine network unique to the ternary Cdk7 complex, involving all three subunits. We identify differential dependencies of kinase activity and substrate recognition on the individual phosphorylations. CAK function is unaffected by T-loop phosphorylation, whereas activity towards non-CDK substrates is increased several-fold by T170 phosphorylation. Moreover, dual T-loop phosphorylation stimulates multisite phosphorylation of the RNA polymerase II (RNAPII) carboxy-terminal domain (CTD) and SPT5 carboxy-terminal repeat (CTR) region. In human cells, Cdk7 activation is a two-step process wherein S164 phosphorylation precedes, and may prime, T170 phosphorylation. Thus, dual T-loop phosphorylation can regulate Cdk7 through multiple mechanisms, with pS164 supporting tripartite complex formation and possibly influencing processivity, while pT170 enhances activity towards key transcriptional substrates.
Assuntos
Quinase Ativadora de Quinase Dependente de Ciclina , Quinases Ciclina-Dependentes , Fosforilação , Humanos , Quinases Ciclina-Dependentes/metabolismo , Quinases Ciclina-Dependentes/química , Quinases Ciclina-Dependentes/genética , Ciclina H/metabolismo , Ciclina H/química , Ciclina H/genética , Cristalografia por Raios X , RNA Polimerase II/metabolismo , RNA Polimerase II/química , Fator de Transcrição TFIIH/metabolismo , Fator de Transcrição TFIIH/química , Fator de Transcrição TFIIH/genética , Modelos Moleculares , Fatores de Transcrição/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/genética , Domínios Proteicos , Proteínas de Ciclo CelularRESUMO
Cyclin-dependent kinase 7 (Cdk7) occupies a central position in cell-cycle and transcriptional regulation owing to its function as both a CDK-activating kinase (CAK) and part of the general transcription factor TFIIH. Cdk7 forms an active complex upon association with Cyclin H and Mat1, and its catalytic activity is regulated by two phosphorylations in the activation segment (T loop): the canonical activating modification at T170 and another at S164. Here we report the crystal structure of the fully activated human Cdk7/Cyclin H/Mat1 complex containing both T-loop phosphorylations. Whereas pT170 coordinates a set of basic residues conserved in other CDKs, pS164 nucleates an arginine network involving all three subunits that is unique to the ternary Cdk7 complex. We identify differential dependencies of kinase activity and substrate recognition on individual phosphorylations within the Cdk7 T loop. The CAK function of Cdk7 is not affected by T-loop phosphorylation, whereas activity towards non-CDK substrates is increased several-fold by phosphorylation at T170. Moreover, dual T-loop phosphorylation at both T170 and S164 stimulates multi-site phosphorylation of transcriptional substrates-the RNA polymerase II (RNAPII) carboxy-terminal domain (CTD) and the SPT5 carboxy-terminal repeat (CTR) region. In human cells, Cdk7-regulatory phosphorylation is a two-step process in which phosphorylation of S164 precedes, and may prime, T170 phosphorylation. Thus, dual T-loop phosphorylation can regulate Cdk7 through multiple mechanisms, with pS164 supporting tripartite complex formation and possibly influencing Cdk7 processivity, while the canonical pT170 enhances kinase activity towards critical substrates involved in transcription.
RESUMO
A cyclin-dependent kinase triggers degradation of prematurely terminated RNAs.
Assuntos
Proteína Quinase CDC2 , Neoplasias , Proteínas Supressoras de Tumor , Humanos , Neoplasias/genética , RNA/genética , Proteínas Supressoras de Tumor/genética , Proteína Quinase CDC2/genética , Proteína Quinase CDC2/metabolismoRESUMO
Co-transcriptional histone modifications are a ubiquitous feature of RNA polymerase II (RNAPII) transcription, with profound but incompletely understood effects on gene expression. Unlike the covalent marks found at promoters, which are thought to be instructive for transcriptional activation, these modifications occur in gene bodies as a result of transcription, which has made elucidation of their functions challenging. Here we review recent insights into the regulation and roles of two such modifications: monoubiquitylation of histone H2B at lysine 120 (H2Bub1) and methylation of histone H3 at lysine 36 (H3K36me). Both H2Bub1 and H3K36me are enriched in the coding regions of transcribed genes, with highly overlapping distributions, but they were thought to work largely independently. We highlight our recent demonstration that, as was previously shown for H3K36me, H2Bub1 signals to the histone deacetylase (HDAC) complex Rpd3S/Clr6-CII, and that Rpd3S/Clr6-CII and H2Bub1 function in the same pathway to repress aberrant antisense transcription initiating within gene coding regions. Moreover, both of these histone modification pathways are influenced by protein phosphorylation catalyzed by the cyclin-dependent kinases (CDKs) that regulate RNAPII elongation, chiefly Cdk9. Therefore, H2Bub1 and H3K36me are more tightly linked than previously thought, sharing both upstream regulatory inputs and downstream effectors. Moreover, these newfound connections suggest extensive, bidirectional signaling between RNAPII elongation complexes and chromatin-modifying enzymes, which helps to determine transcriptional outputs and should be a focus for future investigation.
Assuntos
Código das Histonas , Histonas/metabolismo , Humanos , Lisina/metabolismo , Metilação , UbiquitinaçãoRESUMO
The transcription cycle of RNA polymerase II (RNAPII) is governed at multiple points by opposing actions of cyclin-dependent kinases (CDKs) and protein phosphatases, in a process with similarities to the cell division cycle. While important roles of the kinases have been established, phosphatases have emerged more slowly as key players in transcription, and large gaps remain in understanding of their precise functions and targets. Much of the earlier work focused on the roles and regulation of sui generis and often atypical phosphatases-FCP1, Rtr1/RPAP2, and SSU72-with seemingly dedicated functions in RNAPII transcription. Decisive roles in the transcription cycle have now been uncovered for members of the major phosphoprotein phosphatase (PPP) family, including PP1, PP2A, and PP4-abundant enzymes with pleiotropic roles in cellular signaling pathways. These phosphatases appear to act principally at the transitions between transcription cycle phases, ensuring fine control of elongation and termination. Much is still unknown, however, about the division of labor among the PPP family members, and their possible regulation by or of the transcriptional kinases. CDKs active in transcription have recently drawn attention as potential therapeutic targets in cancer and other diseases, raising the prospect that the phosphatases might also present opportunities for new drug development. Here we review the current knowledge and outstanding questions about phosphatases in the context of the RNAPII transcription cycle.
Assuntos
Fosfoproteínas Fosfatases/metabolismo , RNA Polimerase II/genética , Transcrição Gênica/genética , Animais , Sistemas de Liberação de Medicamentos , Humanos , Fosfoproteínas Fosfatases/genéticaRESUMO
Reversible phosphorylation of Pol II and accessory factors helps order the transcription cycle. Here, we define two kinase-phosphatase switches that operate at different points in human transcription. Cdk9/cyclin T1 (P-TEFb) catalyzes inhibitory phosphorylation of PP1 and PP4 complexes that localize to 3' and 5' ends of genes, respectively, and have overlapping but distinct specificities for Cdk9-dependent phosphorylations of Spt5, a factor instrumental in promoter-proximal pausing and elongation-rate control. PP1 dephosphorylates an Spt5 carboxy-terminal repeat (CTR), but not Spt5-Ser666, a site between Kyrpides-Ouzounis-Woese (KOW) motifs 4 and 5, whereas PP4 can target both sites. In vivo, Spt5-CTR phosphorylation decreases as transcription complexes pass the cleavage and polyadenylation signal (CPS) and increases upon PP1 depletion, consistent with a PP1 function in termination first uncovered in yeast. Depletion of PP4-complex subunits increases phosphorylation of both Ser666 and the CTR, and promotes redistribution of promoter-proximally paused Pol II into gene bodies. These results suggest that switches comprising Cdk9 and either PP4 or PP1 govern pause release and the elongation-termination transition, respectively.
Assuntos
Quinase 9 Dependente de Ciclina/metabolismo , RNA Polimerase II/metabolismo , Transcrição Gênica/fisiologia , Quinase 9 Dependente de Ciclina/antagonistas & inibidores , Células HCT116 , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilação , Fator B de Elongação Transcricional Positiva/metabolismo , Interferência de RNA , RNA Polimerase II/genética , Receptores de Neuropeptídeo Y/metabolismo , Fatores de Elongação da Transcrição/genética , Fatores de Elongação da Transcrição/metabolismoRESUMO
Mono-ubiquitylation of histone H2B (H2Bub1) and phosphorylation of elongation factor Spt5 by cyclin-dependent kinase 9 (Cdk9) occur during transcription by RNA polymerase II (RNAPII), and are mutually dependent in fission yeast. It remained unclear whether Cdk9 and H2Bub1 cooperate to regulate the expression of individual genes. Here, we show that Cdk9 inhibition or H2Bub1 loss induces intragenic antisense transcription of â¼10% of fission yeast genes, with each perturbation affecting largely distinct subsets; ablation of both pathways de-represses antisense transcription of over half the genome. H2Bub1 and phospho-Spt5 have similar genome-wide distributions; both modifications are enriched, and directly proportional to each other, in coding regions, and decrease abruptly around the cleavage and polyadenylation signal (CPS). Cdk9-dependence of antisense suppression at specific genes correlates with high H2Bub1 occupancy, and with promoter-proximal RNAPII pausing. Genetic interactions link Cdk9, H2Bub1 and the histone deacetylase Clr6-CII, while combined Cdk9 inhibition and H2Bub1 loss impair Clr6-CII recruitment to chromatin and lead to decreased occupancy and increased acetylation of histones within gene coding regions. These results uncover novel interactions between co-transcriptional histone modification pathways, which link regulation of RNAPII transcription elongation to suppression of aberrant initiation.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Quinase 9 Dependente de Ciclina/metabolismo , Histonas/metabolismo , RNA Polimerase II/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/genética , Elongação da Transcrição Genética , Fosforilação , Fatores de Elongação da Transcrição/metabolismo , UbiquitinaçãoRESUMO
Largely non-overlapping sets of cyclin-dependent kinases (CDKs) regulate cell division and RNA polymerase II (Pol II)-dependent transcription. Here we review the molecular mechanisms by which specific CDKs are thought to act at discrete steps in the transcription cycle and describe the recent emergence of transcriptional CDKs as promising drug targets in cancer. We emphasize recent advances in understanding the transcriptional CDK network that were facilitated by development and deployment of small-molecule inhibitors with increased selectivity for individual CDKs. Unexpectedly, several of these compounds have also shown selectivity in killing cancer cells, despite the seemingly universal involvement of their target CDKs during transcription in all cells. Finally, we describe remaining and emerging challenges in defining functions of individual CDKs in transcription and co-transcriptional processes and in leveraging CDK inhibition for therapeutic purposes.
Assuntos
Antineoplásicos/farmacologia , Quinases Ciclina-Dependentes/antagonistas & inibidores , Regulação Neoplásica da Expressão Gênica , Neoplasias/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , RNA Polimerase II/genética , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Antineoplásicos/química , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/genética , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , Modelos Animais de Doenças , Humanos , Neoplasias/enzimologia , Neoplasias/genética , Neoplasias/patologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteínas Quinases/química , Proteólise , RNA Polimerase II/antagonistas & inibidores , RNA Polimerase II/metabolismo , Transdução de Sinais , Bibliotecas de Moléculas Pequenas/química , Transcrição GênicaRESUMO
In this issue of Molecular Cell, Cossa et al. (2020) uncover the basis for a dependency of tumor cells with deregulated MYC on the kinase NUAK1, which acts through PP1 and PNUTS to ensure that splicing keeps up with MYC-driven transcription.
Assuntos
Spliceossomos , Proteína Fosfatase 1RESUMO
Histone H2B monoubiquitylation (H2Bub1) is tightly linked to RNA polymerase II transcription elongation, and is also directly implicated in DNA replication and repair. Loss of H2Bub1 is associated with defects in cell cycle progression, but how these are related to its various functions, and the underlying mechanisms involved, is not understood. Here we describe a role for H2Bub1 in the regulation of replication-dependent histone genes in the fission yeast Schizosaccharomyces pombe H2Bub1 activates histone genes indirectly by suppressing antisense transcription of ams2+ -a gene encoding a GATA-type transcription factor that activates histone genes and is required for assembly of centromeric chromatin. Mutants lacking the ubiquitylation site in H2B or the H2B-specific E3 ubiquitin ligase Brl2 had elevated levels of ams2+ antisense transcripts and reduced Ams2 protein levels. These defects were reversed upon inhibition of Cdk9-an ortholog of the kinase component of positive transcription elongation factor b (P-TEFb)-indicating that they likely resulted from aberrant transcription elongation. Reduced Cdk9 activity also partially rescued chromosome segregation phenotypes of H2Bub1 mutants. In a genome-wide analysis, loss of H2Bub1 led to increased antisense transcripts at over 500 protein-coding genes in H2Bub1 mutants; for a subset of these, including several genes involved in chromosome segregation and chromatin assembly, antisense derepression was Cdk9-dependent. Our results highlight antisense suppression as a key feature of cell cycle-dependent gene regulation by H2Bub1, and suggest that aberrant transcription elongation may underlie the effects of H2Bub1 loss on cell cycle progression.
Assuntos
Fatores de Transcrição GATA/genética , Regulação Fúngica da Expressão Gênica , Histonas/metabolismo , RNA Antissenso/genética , Proteínas de Schizosaccharomyces pombe/genética , Ubiquitinação , Segregação de Cromossomos , Quinase 9 Dependente de Ciclina/genética , Quinase 9 Dependente de Ciclina/metabolismo , Fatores de Transcrição GATA/metabolismo , Schizosaccharomyces , Proteínas de Schizosaccharomyces pombe/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
The transcription cycle of RNA polymerase II (Pol II) is regulated by a set of cyclin-dependent kinases (CDKs). Cdk7, associated with the transcription initiation factor TFIIH, is both an effector CDK that phosphorylates Pol II and other targets within the transcriptional machinery, and a CDK-activating kinase (CAK) for at least one other essential CDK involved in transcription. Recent studies have illuminated Cdk7 functions that are executed throughout the Pol II transcription cycle, from promoter clearance and promoter-proximal pausing, to co-transcriptional chromatin modification in gene bodies, to mRNA 3´-end formation and termination. Cdk7 has also emerged as a target of small-molecule inhibitors that show promise in the treatment of cancer and inflammation. The challenges now are to identify the relevant targets of Cdk7 at each step of the transcription cycle, and to understand how heightened dependence on an essential CDK emerges in cancer, and might be exploited therapeutically.
Assuntos
Antineoplásicos/farmacologia , Quinases Ciclina-Dependentes/metabolismo , Descoberta de Drogas , Neoplasias/tratamento farmacológico , Neoplasias/genética , Transcrição Gênica/genética , Animais , Quinases Ciclina-Dependentes/antagonistas & inibidores , Humanos , Neoplasias/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Transcrição Gênica/efeitos dos fármacos , Quinase Ativadora de Quinase Dependente de CiclinaRESUMO
The end of the RNA polymerase II (Pol II) transcription cycle is strictly regulated to prevent interference between neighbouring genes and to safeguard transcriptome integrity 1 . The accumulation of Pol II downstream of the cleavage and polyadenylation signal can facilitate the recruitment of factors involved in mRNA 3'-end formation and termination 2 , but how this sequence is initiated remains unclear. In a chemical-genetic screen, human protein phosphatase 1 (PP1) isoforms were identified as substrates of positive transcription elongation factor b (P-TEFb), also known as the cyclin-dependent kinase 9 (Cdk9)-cyclin T1 (CycT1) complex 3 . Here we show that Cdk9 and PP1 govern phosphorylation of the conserved elongation factor Spt5 in the fission yeast Schizosaccharomyces pombe. Cdk9 phosphorylates both Spt5 and a negative regulatory site on the PP1 isoform Dis2 4 . Sites targeted by Cdk9 in the Spt5 carboxy-terminal domain can be dephosphorylated by Dis2 in vitro, and dis2 mutations retard Spt5 dephosphorylation after inhibition of Cdk9 in vivo. Chromatin immunoprecipitation and sequencing analysis indicates that Spt5 is dephosphorylated as transcription complexes traverse the cleavage and polyadenylation signal, concomitant with the accumulation of Pol II phosphorylated at residue Ser2 of the carboxy-terminal domain consensus heptad repeat 5 . A conditionally lethal Dis2-inactivating mutation attenuates the drop in Spt5 phosphorylation on chromatin, promotes transcription beyond the normal termination zone (as detected by precision run-on transcription and sequencing 6 ) and is genetically suppressed by the ablation of Cdk9 target sites in Spt5. These results suggest that the transition of Pol II from elongation to termination coincides with a Dis2-dependent reversal of Cdk9 signalling-a switch that is analogous to a Cdk1-PP1 circuit that controls mitotic progression 4 .
Assuntos
Quinase 9 Dependente de Ciclina/metabolismo , Fosfoproteínas Fosfatases/metabolismo , RNA Polimerase II/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/enzimologia , Schizosaccharomyces/genética , Elongação da Transcrição Genética , Terminação da Transcrição Genética , Sequência de Aminoácidos , Quinase 9 Dependente de Ciclina/química , Humanos , Mitose , Fosfoproteínas Fosfatases/química , Fosforilação , RNA Polimerase II/química , Schizosaccharomyces/citologia , Proteínas de Schizosaccharomyces pombe/química , Transdução de Sinais , Fatores de Elongação da Transcrição/química , Fatores de Elongação da Transcrição/metabolismoRESUMO
Post-translational modifications of the transcription elongation complex provide mechanisms to fine-tune gene expression, yet their specific impacts on RNA polymerase II regulation remain difficult to ascertain. Here, in Schizosaccharomyces pombe, we examine the role of Cdk9, and related Mcs6/Cdk7 and Lsk1/Cdk12 kinases, on transcription at base-pair resolution with Precision Run-On sequencing (PRO-seq). Within a minute of Cdk9 inhibition, phosphorylation of Pol II-associated factor, Spt5 is undetectable. The effects of Cdk9 inhibition are more severe than inhibition of Cdk7 and Cdk12, resulting in a shift of Pol II toward the transcription start site (TSS). A time course of Cdk9 inhibition reveals that early transcribing Pol II can escape promoter-proximal regions, but with a severely reduced elongation rate of only ~400 bp/min. Our results in fission yeast suggest the existence of a conserved global regulatory checkpoint that requires Cdk9 kinase activity.
Assuntos
Quinase 9 Dependente de Ciclina/genética , Genes cdc , Regiões Promotoras Genéticas , RNA Polimerase II/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/enzimologia , Fatores de Elongação da Transcrição/metabolismo , Região 3'-Flanqueadora , Região 5'-Flanqueadora , Pareamento de Bases , Fosforilação , Processamento de Proteína Pós-Traducional , Proteínas de Schizosaccharomyces pombe/genética , Sítio de Iniciação de Transcrição , Fatores de Elongação da Transcrição/genéticaRESUMO
Dependence on glycolysis under aerobic conditions, a frequent metabolic derangement in cancer cells, suggests a therapeutic opportunity. Now, through chemical genetics, CDK8, a kinase associated with the Mediator transcriptional coactivator complex, has emerged as an upstream inducer of glycolysis and a possible target for anticancer drug discovery.
Assuntos
Quinase 8 Dependente de Ciclina , Glicólise , FosforilaçãoRESUMO
The glucocorticoid (GC) receptor (GR) suppresses inflammation by activating anti-inflammatory and repressing pro-inflammatory genes. GR-interacting protein-1 (GRIP1) is a GR corepressor in macrophages, however, whether GRIP1 mediates GR-activated transcription, and what dictates its coactivator versus corepressor properties is unknown. Here we report that GRIP1 loss in macrophages attenuates glucocorticoid induction of several anti-inflammatory targets, and that GC treatment of quiescent macrophages globally directs GRIP1 toward GR binding sites dominated by palindromic GC response elements (GRE), suggesting a non-redundant GRIP1 function as a GR coactivator. Interestingly, GRIP1 is phosphorylated at an N-terminal serine cluster by cyclin-dependent kinase-9 (CDK9), which is recruited into GC-induced GR:GRIP1:CDK9 hetero-complexes, producing distinct GRE-specific GRIP1 phospho-isoforms. Phosphorylation potentiates GRIP1 coactivator but, remarkably, not its corepressor properties. Consistently, phospho-GRIP1 and CDK9 are not detected at GR transrepression sites near pro-inflammatory genes. Thus, GR restricts actions of its own coregulator via CDK9-mediated phosphorylation to a subset of anti-inflammatory genes.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Transporte/metabolismo , Quinase 9 Dependente de Ciclina/metabolismo , Glucocorticoides/metabolismo , Macrófagos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Sítios de Ligação/genética , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/genética , Linhagem Celular , Células Cultivadas , Dexametasona/farmacologia , Técnicas de Silenciamento de Genes , Glucocorticoides/farmacologia , Humanos , Inflamação/genética , Inflamação/metabolismo , Macrófagos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Biológicos , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Fosforilação , Receptores de Glucocorticoides/metabolismo , Elementos de Resposta , Ativação TranscricionalRESUMO
Cdk7, the CDK-activating kinase and transcription factor IIH component, is a target of inhibitors that kill cancer cells by exploiting tumor-specific transcriptional dependencies. However, whereas selective inhibition of analog-sensitive (AS) Cdk7 in colon cancer-derived cells arrests division and disrupts transcription, it does not by itself trigger apoptosis efficiently. Here, we show that p53 activation by 5-fluorouracil or nutlin-3 synergizes with a reversible Cdk7as inhibitor to induce cell death. Synthetic lethality was recapitulated with covalent inhibitors of wild-type Cdk7, THZ1, or the more selective YKL-1-116. The effects were allele specific; a CDK7as mutation conferred both sensitivity to bulky adenine analogs and resistance to covalent inhibitors. Non-transformed colon epithelial cells were resistant to these combinations, as were cancer-derived cells with p53-inactivating mutations. Apoptosis was dependent on death receptor DR5, a p53 transcriptional target whose expression was refractory to Cdk7 inhibition. Therefore, p53 activation induces transcriptional dependency to sensitize cancer cells to Cdk7 inhibition.
Assuntos
Antineoplásicos/farmacologia , Quinases Ciclina-Dependentes/antagonistas & inibidores , Fenilenodiaminas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Apoptose , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , Resistencia a Medicamentos Antineoplásicos , Fluoruracila/farmacologia , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Humanos , Imidazóis/farmacologia , Piperazinas/farmacologia , Ativação Transcricional , Proteína Supressora de Tumor p53/genética , Quinase Ativadora de Quinase Dependente de CiclinaRESUMO
Hematopoietic transitions that accompany fetal development, such as erythroid globin chain switching, play important roles in normal physiology and disease development. In the megakaryocyte lineage, human fetal progenitors do not execute the adult morphogenesis program of enlargement, polyploidization, and proplatelet formation. Although these defects decline with gestational stage, they remain sufficiently severe at birth to predispose newborns to thrombocytopenia. These defects may also contribute to inferior platelet recovery after cord blood stem cell transplantation and may underlie inefficient platelet production by megakaryocytes derived from pluripotent stem cells. In this study, comparison of neonatal versus adult human progenitors has identified a blockade in the specialized positive transcription elongation factor b (P-TEFb) activation mechanism that is known to drive adult megakaryocyte morphogenesis. This blockade resulted from neonatal-specific expression of an oncofetal RNA-binding protein, IGF2BP3, which prevented the destabilization of the nuclear RNA 7SK, a process normally associated with adult megakaryocytic P-TEFb activation. Knockdown of IGF2BP3 sufficed to confer both phenotypic and molecular features of adult-type cells on neonatal megakaryocytes. Pharmacologic inhibition of IGF2BP3 expression via bromodomain and extraterminal domain (BET) inhibition also elicited adult features in neonatal megakaryocytes. These results identify IGF2BP3 as a human ontogenic master switch that restricts megakaryocyte development by modulating a lineage-specific P-TEFb activation mechanism, revealing potential strategies toward enhancing platelet production.