Cyclin D1 belongs to the core cell cycle machinery, and it is frequently overexpressed in human cancers1,2. 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 function3,4—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.
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We thank T. Liu, Y. Ndassa-Colday, J. Marto, R. Bronson, B. Smith, E. Jacobsen, M. Brown and members of the Brown laboratory for help at different stages of the project, M. Ewen for p-Babe-puro-Cyclin D1 and cyclin D1(K112E) plasmids, G. Seigel for R28 cells, T. Volkert, J. Love and E. Fox for help with arrays, P. White and O. Smirnova for help with BCBC arrays. This work was supported by grants R01 CA108420, P01 CA080111 and P01 CA109901 (to P.S.), HG3456 (to S.P.G.), R01 EYO9676 (to C.L.C.), HG004069 (to X.S.L.), Cancer Research UK, European Research Council Starting Grant, and an EMBO Young Investigator Award (all to D.T.O.). P.S. is a Leukemia and Lymphoma Society Scholar.
Author Contributions F.B. and P.S. designed the study, analysed the data and wrote the manuscript. F.B. performed the experiments with the help of co-authors as detailed below. S.J. performed protein purifications. J.E.E. performed and together with S.P.G. analysed and interpreted mass spectrometry analyses. C.A.M. and X.S.L. contributed biocomputational analyses, K.M. and C.L.C. contributed in vivo transduction of cyclin D1-null retinas with Notch1, A.M., G.M.F., M.F.C., D.T.O. and R.A.Y. contributed to analyses of ChIP-chip and gene expression data, J.O., Y.G., A.Z. and M.J. helped with the experiments. P.S. directed the study.
This file contains Supplementary Data, Supplementary Methods and Supplementary References. (PDF 214 kb)
This file contains Supplementary Figures 1-11 with Legends. (PDF 1256 kb)
This table shows peptides identified in four compartments in either experimental knock-in or wild-type samples. (XLS 17951 kb)
This table shows proteins identified in four compartments in either experimental knock-in or wild-type samples. (XLS 5025 kb)
This table shows proteins demonstrating significant (p<0.001) and substantial (fractional difference > 0.8) differences between experimental knock-in and wild-type samples. (XLS 384 kb)
This table contains a summary of cyclin D1-bound genomic regions identified in cyclin D1 ChIP-chip. (TXT 1186 kb)
tThis table contains cyclin D1 ChIP-chip data. (TXT 19937 kb)
This table contains the complete expression dataset for wild-type and cyclin D1-/- retinas. The expression data for 45,101 probesets from Affymetrix Mouse 430 2. microarrays was quantile normalized and analyzed for differential expression. (XLS 10012 kb)
This table contains the complete expression dataset for 14,498 genes that were compared to ChIP-chip binding data for cyclin D1. (XLS 3278 kb)
This table contains sequences of PCR primers. (XLS 20 kb)
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