RESUMO
The Cdk12/CycK complex promotes expression of a subset of RNA polymerase II genes, including those of the DNA damage response. CDK12 is among only nine genes with recurrent somatic mutations in high-grade serous ovarian carcinoma. However, the influence of these mutations on the Cdk12/CycK complex and their link to cancerogenesis remain ill-defined. Here, we show that most mutations prevent formation of the Cdk12/CycK complex, rendering the kinase inactive. By examining the mutations within the Cdk12/CycK structure, we find that they likely provoke structural rearrangements detrimental to Cdk12 activation. Our mRNA expression analysis of the patient samples containing the CDK12 mutations reveals coordinated downregulation of genes critical to the homologous recombination DNA repair pathway. Moreover, we establish that the Cdk12/CycK complex occupies these genes and promotes phosphorylation of RNA polymerase II at Ser2. Accordingly, we demonstrate that the mutant Cdk12 proteins fail to stimulate the faithful DNA double strand break repair via homologous recombination. Together, we provide the molecular basis of how mutated CDK12 ceases to function in ovarian carcinoma. We propose that CDK12 is a tumor suppressor of which the loss-of-function mutations may elicit defects in multiple DNA repair pathways, leading to genomic instability underlying the genesis of the cancer.
Assuntos
Quinases Ciclina-Dependentes/genética , Ciclinas/genética , Reparo do DNA/genética , Mutação , Sequência de Aminoácidos , Western Blotting , Linhagem Celular Tumoral , Quinases Ciclina-Dependentes/química , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/química , Ciclinas/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Ligação Proteica , Estrutura Terciária de Proteína , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de AminoácidosRESUMO
Gene delivery to, and expression in, the mouse brain is important for understanding gene functions in brain development and disease, or testing gene therapies. Here, we describe an approach to express a transgene in the mouse brain in a cell-type-specific manner. We use stereotaxic injection of a transgene-expressing adeno-associated virus into the mouse brain via the intracerebroventricular route. We demonstrate stable and sustained expression of the transgene in neurons of adult mouse brain, using a reporter gene driven by a neuron-specific promoter. This approach represents a rapid, simple, and cost-effective method for global gene expression in the mouse brain, in a cell-type-specific manner, without major surgical interventions. The described method represents a helpful resource for genetically engineering mice to express a therapeutic gene, for gene therapy studies, or to deliver genetic material for genome editing and developing knockout animal models.