RESUMO
One of the most intriguing features of cell-cycle control is that, although there are multiple cyclin-dependent kinases (CDKs) in higher eukaryotes, a single CDK is responsible for both G1-S and G2-M in yeasts. By leveraging a rapid conditional silencing system in human cell lines, we confirm that CDK1 assumes the role of G1-S CDK in the absence of CDK2. Unexpectedly, CDK1 deficiency does not prevent mitotic entry. Nonetheless, inadequate phosphorylation of mitotic substrates by noncanonical cyclin B-CDK2 complexes does not allow progression beyond metaphase and underscores deleterious late mitotic events, including the uncoupling of anaphase A and B and cytokinesis. Elevation of CDK2 to a level similar to CDK1 overcomes the mitotic defects caused by CDK1 deficiency, indicating that the relatively low concentration of CDK2 accounts for the defective anaphase. Collectively, these results reveal that the difference between G2-M and G1-S CDKs in human cells is essentially quantitative.
Assuntos
Proteína Quinase CDC2/metabolismo , Proliferação de Células , Mitose , Epitélio Pigmentado da Retina/enzimologia , Neoplasias do Colo do Útero/enzimologia , Proteína Quinase CDC2/genética , Ciclina B/genética , Ciclina B/metabolismo , Quinase 2 Dependente de Ciclina/genética , Quinase 2 Dependente de Ciclina/metabolismo , Feminino , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Células HeLa , Humanos , Fosforilação , Transdução de Sinais , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/patologiaRESUMO
Loss-of-function analysis is one of the major arsenals we have for understanding gene functions in mammalian cells. For analysis of essential genes, the major challenge is to develop simple methodologies for tight and rapid inducible gene inactivation. One approach involves CRISPR-Cas9-mediated disruption of the endogenous locus in conjunction with the expression of a rescue construct, which can subsequently be turned off to produce a gene inactivation effect. Here we describe the development of a set of Sleeping Beauty transposon-based vectors for expressing auxin-inducible degron (AID)-tagged genes under the regulation of a tetracycline-controlled promoter. The dual transcriptional and degron-mediated post-translational regulation allows rapid and tight silencing of protein expression in mammalian cells. We demonstrated that both non-essential and essential genes could be targeted in human cell lines using a one-step transfection method. Moreover, multiple genes could be simultaneously or sequentially targeted, allowing inducible inactivation of multiple genes. These resources enable highly efficient generation of conditional gene silencing cell lines to facilitate functional studies of essential genes.
