Transcriptional role of cyclin D1 in development revealed by a genetic–proteomic screen

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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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Figure 1: Proteomic analyses of cyclin D1-associated proteins.
Figure 2: Analyses of cyclin D1 interaction with the mouse genome.
Figure 3: Analyses of cyclin D1 transcriptional function in retina.
Figure 4: In vivo and molecular analyses of the cyclin D1–Notch1 connection.

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Gene Expression Omnibus

Data deposits

The complete ChIP-chip and expression datasets have been submitted to the online data repository Gene Expression Omnibus (GEO;, under accession GSE13636.


  1. 1

    Malumbres, M. & Barbacid, M. Cell cycle, CDKs and cancer: a changing paradigm. Nature Rev. Cancer 9, 153–166 (2009)

  2. 2

    Sherr, C. J. & Roberts, J. M. CDK inhibitors: positive and negative regulators of G1-phase progression. Genes Dev. 13, 1501–1512 (1999)

  3. 3

    Fantl, V., Stamp, G., Andrews, A., Rosewell, I. & Dickson, C. Mice lacking cyclin D1 are small and show defects in eye and mammary gland development. Genes Dev. 9, 2364–2372 (1995)

  4. 4

    Sicinski, P. et al. Cyclin D1 provides a link between development and oncogenesis in the retina and breast. Cell 82, 621–630 (1995)

  5. 5

    Yu, Q., Geng, Y. & Sicinski, P. Specific protection against breast cancers by cyclin D1 ablation. Nature 411, 1017–1021 (2001)

  6. 6

    Coqueret, O. Linking cyclins to transcriptional control. Gene 299, 35–55 (2002)

  7. 7

    McMahon, C., Suthiphongchai, T., DiRenzo, J. & Ewen, M. E. P/CAF associates with cyclin D1 and potentiates its activation of the estrogen receptor. Proc. Natl Acad. Sci. USA 96, 5382–5387 (1999)

  8. 8

    Fu, M. et al. Cyclin D1 inhibits peroxisome proliferator-activated receptor gamma-mediated adipogenesis through histone deacetylase recruitment. J. Biol. Chem. 280, 16934–16941 (2005)

  9. 9

    Adnane, J., Shao, Z. & Robbins, P. D. Cyclin D1 associates with the TBP-associated factor TAFII250 to regulate Sp1-mediated transcription. Oncogene 18, 239–247 (1999)

  10. 10

    Inoue, K. & Sherr, C. J. Gene expression and cell cycle arrest mediated by transcription factor DMP1 is antagonized by D-type cyclins through a cyclin-dependent-kinase-independent mechanism. Mol. Cell. Biol. 18, 1590–1600 (1998)

  11. 11

    Zwijsen, R. M., Buckle, R. S., Hijmans, E. M., Loomans, C. J. & Bernards, R. Ligand-independent recruitment of steroid receptor coactivators to estrogen receptor by cyclin D1. Genes Dev. 12, 3488–3498 (1998)

  12. 12

    Marson, A. et al. Foxp3 occupancy and regulation of key target genes during T-cell stimulation. Nature 445, 931–935 (2007)

  13. 13

    Blackshaw, S. et al. Genomic analysis of mouse retinal development. PLoS Biol. 2, e247 (2004)

  14. 14

    Saxonov, S., Berg, P. & Brutlag, D. L. A genome-wide analysis of CpG dinucleotides in the human genome distinguishes two distinct classes of promoters. Proc. Natl Acad. Sci. USA 103, 1412–1417 (2006)

  15. 15

    Alexson, T. O., Hitoshi, S., Coles, B. L., Bernstein, A. & van der Kooy, D. Notch signaling is required to maintain all neural stem cell populations–irrespective of spatial or temporal niche. Dev. Neurosci. 28, 34–48 (2006)

  16. 16

    Jadhav, A. P., Cho, S. H. & Cepko, C. L. Notch activity permits retinal cells to progress through multiple progenitor states and acquire a stem cell property. Proc. Natl Acad. Sci. USA 103, 18998–19003 (2006)

  17. 17

    Flora, A., Garcia, J. J., Thaller, C. & Zoghbi, H. Y. The E-protein Tcf4 interacts with Math1 to regulate differentiation of a specific subset of neuronal progenitors. Proc. Natl Acad. Sci. USA 104, 15382–15387 (2007)

  18. 18

    Heine, P., Dohle, E., Bumsted-O’Brien, K., Engelkamp, D. & Schulte, D. Evidence for an evolutionary conserved role of homothorax/Meis1/2 during vertebrate retina development. Development 135, 805–811 (2008)

  19. 19

    Yeung, S. C. & Yip, H. K. Developmental expression patterns and localization of DNA-binding protein inhibitor (Id3) in the mouse retina. Neuroreport 16, 673–676 (2005)

  20. 20

    Jadhav, A. P., Mason, H. A. & Cepko, C. L. Notch 1 inhibits photoreceptor production in the developing mammalian retina. Development 133, 913–923 (2006)

  21. 21

    Das, G., Choi, Y., Sicinski, P. & Levine, E. M. Cyclin D1 fine-tunes the neurogenic output of embryonic retinal progenitor cells. Neural Dev. 4, 15 (2009)

  22. 22

    Hatakeyama, J. & Kageyama, R. Retinal cell fate determination and bHLH factors. Semin. Cell Dev. Biol. 15, 83–89 (2004)

  23. 23

    Yaron, O., Farhy, C., Marquardt, T., Applebury, M. & Ashery-Padan, R. Notch1 functions to suppress cone-photoreceptor fate specification in the developing mouse retina. Development 133, 1367–1378 (2006)

  24. 24

    Fu, M. et al. Cyclin D1 represses p300 transactivation through a CDK-independent mechanism. J. Biol. Chem. 280, 29728–29742 (2005)

  25. 25

    Ratineau, C., Petry, M. W., Mutoh, H. & Leiter, A. B. Cyclin D1 represses the basic helix-loop-helix transcription factor, BETA2/NeuroD. J. Biol. Chem. 277, 8847–8853 (2002)

  26. 26

    McManus, K. J. & Hendzel, M. J. Quantitative analysis of CBP- and P300-induced histone acetylations in vivo using native chromatin. Mol. Cell. Biol. 23, 7611–7627 (2003)

  27. 27

    Musgrove, E. A. Cyclins: roles in mitogenic signaling and oncogenic transformation. Growth Factors 24, 13–19 (2006)

  28. 28

    Ronchini, C. & Capobianco, A. J. Induction of cyclin D1 transcription and CDK2 activity by Notchic: implication for cell cycle disruption in transformation by Notchic . Mol. Cell. Biol. 21, 5925–5934 (2001)

  29. 29

    Lindsay, J. et al. ErbB2 induces Notch1 activity and function in breast cancer cells. Clin. Transl. Sci. 1, 107–115 (2008)

  30. 30

    Seigel, G. M. Establishment of an E1A-immortalized retinal cell culture. In Vitro Cell. Dev. Biol. Anim. 32, 66–68 (1996)

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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.

Author information

Correspondence to Piotr Sicinski.

Supplementary information

Supplementary information

This file contains Supplementary Data, Supplementary Methods and Supplementary References. (PDF 214 kb)

Supplementary Figures

This file contains Supplementary Figures 1-11 with Legends. (PDF 1256 kb)

Supplementary Table 1

This table shows peptides identified in four compartments in either experimental knock-in or wild-type samples. (XLS 17951 kb)

Supplementary Table 2

This table shows proteins identified in four compartments in either experimental knock-in or wild-type samples. (XLS 5025 kb)

Supplementary Table 3

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)

Supplementary Table 4

This table contains a summary of cyclin D1-bound genomic regions identified in cyclin D1 ChIP-chip. (TXT 1186 kb)

Supplementary Table 5

tThis table contains cyclin D1 ChIP-chip data. (TXT 19937 kb)

Supplementary Table 6

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)

Supplementary Table 7

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)

Supplementary Table 8

This table contains sequences of PCR primers. (XLS 20 kb)

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