RESUMO
Cyclin D1 belongs to the core cell cycle machinery, and it is frequently overexpressed in human cancers. The full repertoire of cyclin D1 functions in normal development and oncogenesis is unclear at present. Here we developed Flag- and haemagglutinin-tagged cyclin D1 knock-in mouse strains that allowed a high-throughput mass spectrometry approach to search for cyclin D1-binding proteins in different mouse organs. In addition to cell cycle partners, we observed several proteins involved in transcription. Genome-wide location analyses (chromatin immunoprecipitation coupled to DNA microarray; ChIP-chip) showed that during mouse development cyclin D1 occupies promoters of abundantly expressed genes. In particular, we found that in developing mouse retinas-an organ that critically requires cyclin D1 function-cyclin D1 binds the upstream regulatory region of the Notch1 gene, where it serves to recruit CREB binding protein (CBP) histone acetyltransferase. Genetic ablation of cyclin D1 resulted in decreased CBP recruitment, decreased histone acetylation of the Notch1 promoter region, and led to decreased levels of the Notch1 transcript and protein in cyclin D1-null (Ccnd1(-/-)) retinas. Transduction of an activated allele of Notch1 into Ccnd1(-/-) retinas increased proliferation of retinal progenitor cells, indicating that upregulation of Notch1 signalling alleviates the phenotype of cyclin D1-deficiency. These studies show that in addition to its well-established cell cycle roles, cyclin D1 has an in vivo transcriptional function in mouse development. Our approach, which we term 'genetic-proteomic', can be used to study the in vivo function of essentially any protein.
Assuntos
Ciclina D1/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteômica , Transcrição Gênica , Alelos , Animais , Proteína de Ligação a CREB/metabolismo , Imunoprecipitação da Cromatina , Ciclina D1/deficiência , Ciclina D1/genética , Genoma/genética , Ensaios de Triagem em Larga Escala , Histona Acetiltransferases/metabolismo , Espectrometria de Massas , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas/genética , Ligação Proteica , Proteômica/métodos , Ratos , Receptor Notch1/genética , Receptor Notch1/metabolismo , Retina/citologia , Retina/embriologia , Retina/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismoRESUMO
Cyclin E is a component of the core cell cycle machinery, and it drives cell proliferation by regulating entry and progression of cells through the DNA synthesis phase. Cyclin E expression is normally restricted to proliferating cells. However, high levels of cyclin E are expressed in the adult brain. The function of cyclin E in quiescent, postmitotic nervous system remains unknown. Here we use a combination of in vivo quantitative proteomics and analyses of cyclin E knockout mice to demonstrate that in terminally differentiated neurons cyclin E forms complexes with Cdk5 and controls synapse function by restraining Cdk5 activity. Ablation of cyclin E led to a decreased number of synapses, reduced number and volume of dendritic spines, and resulted in impaired synaptic plasticity and memory formation in cyclin E-deficient animals. These results reveal a cell cycle-independent role for a core cell cycle protein, cyclin E, in synapse function and memory.