Abstract
The retinoblastoma protein (RB) restricts cell cycle gene expression and entry into the cell cycle. The RB-related protein p130 forms the DREAM (DP, RB-like, E2F, and MuvB) complex and contributes to repression of cell cycle-dependent genes during quiescence. Although both RB and DREAM bind and repress an overlapping set of E2F-dependent gene promoters, it remains unclear whether they cooperate to restrict cell cycle entry. To test the specific contributions of RB and DREAM, we generated RB and p130 knockout cells in primary human fibroblasts. Knockout of both p130 and RB yielded higher levels of cell cycle gene expression in G0 and G1 cells compared to cells with knockout of RB alone, indicating a role for DREAM and RB in repression of cell cycle genes. We observed that RB had a dominant role in E2F-dependent gene repression during mid to late G1 while DREAM activity was more prominent during G0 and early G1. Cyclin D–Cyclin-Dependent Kinase 4 (CDK4)-dependent phosphorylation of p130 occurred during early G1, and led to the release of p130 and MuvB from E2F4 and decreased p130 and MuvB binding to cell cycle promoters. Specific inhibition of CDK4 activity by palbociclib blocked DREAM complex disassembly during cell cycle entry. In addition, sensitivity to CDK4 inhibition was dependent on RB and an intact DREAM complex in both normal cells as well as in palbociclib-sensitive cancer cell lines. Although RB knockout cells were partially resistant to CDK4 inhibition, RB and p130 double knockout cells were significantly more resistant to palbociclib treatment. These results indicate that DREAM cooperates with RB in repressing E2F-dependent gene expression and cell cycle entry and supports a role for DREAM as a therapeutic target in cancer.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 50 print issues and online access
$259.00 per year
only $5.18 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Litovchick L, Sadasivam S, Florens L, Zhu X, Swanson SK, Velmurugan S, et al. Evolutionarily conserved multisubunit RBL2/p130 and E2F4 protein complex represses human cell cycle-dependent genes in quiescence. Mol Cell. 2007;26:539–51.
Pilkinton M, Sandoval R, Colamonici OR. Mammalian Mip/LIN-9 interacts with either thep107, p130/E2F4 repressor complex or B-Myb in a cell cycle-phase-dependent context distinct from the Drosophila dREAM complex. Oncogene. 2007;26:7535–43.
Sadasivam S, Duan S, DeCaprio JA. The MuvB complex sequentially recruits B-Myb and FoxM1 to promote mitotic gene expression. Genes Dev. 2012;26:474–89.
Schmit F, Korenjak M, Mannefeld M, Schmitt K, Franke C, von Eyss B, et al. LINC, a human complex that is related to pRB-containing complexes in invertebrates regulates the expression of G2/M genes. Cell Cycle Georget Tex. 2007;6:1903–13.
Osterloh L, von Eyss B, Schmit F, Rein L, Hübner D, Samans B, et al. The human synMuv-like protein LIN-9 is required for transcription of G2/M genes and for entry into mitosis. EMBO J. 2007;26:144–57.
Araki K, Nakajima Y, Eto K, Ikeda M-A. Distinct recruitment of E2F family members to specific E2F-binding sites mediates activation and repression of the E2F1 promoter. Oncogene. 2003;22:7632–41.
Marceau AH, Felthousen JG, Goetsch PD, Iness AN, Lee H-W, Tripathi SM, et al. Structural basis for LIN54 recognition of CHR elements in cell cycle-regulated promoters. Nat Commun. 2016;7:12301.
Muller GA, Quaas M, Schumann M, Krause E, Padi M, Fischer M, et al. The CHR promoter element controls cell cycle-dependent gene transcription and binds the DREAM and MMB complexes. Nucleic Acids Res. 2012;40:1561–78.
Muller GA, Wintsche A, Stangner K, Prohaska SJ, Stadler PF, Engeland K. The CHR site: definition and genome-wide identification of a cell cycle transcriptional element. Nucleic Acids Res. 2014;42:10331–50.
Schmit F, Cremer S, Gaubatz S. LIN54 is an essential core subunit of the DREAM/LINC complex that binds to the cdc2 promoter in a sequence-specific manner: LIN54 is an essential core subunit of the DREAM/LINC complex. FEBS J. 2009;276:5703–16.
Guiley KZ, Liban TJ, Felthousen JG, Ramanan P, Litovchick L, Rubin SM. Structural mechanisms of DREAM complex assembly and regulation. Genes Dev. 2015;29:961–74.
Takahashi Y, Rayman JB, Dynlacht BD. Analysis of promoter binding by the E2F and pRB families in vivo: distinct E2F proteins mediate activation and repression. Genes Dev. 2000;14:804–16.
Fischer M, Müller GA. Cell cycle transcription control: DREAM/MuvB and RB-E2F complexes. Crit Rev Biochem Mol Biol 2017;52:638–62.
Dick FA, Rubin SM. Molecular mechanisms underlying RB protein function. Nat Rev Mol Cell Biol. 2013;14:297–306.
Narasimha AM, Kaulich M, Shapiro GS, Choi YJ, Sicinski P, Dowdy SF. Cyclin D activates the Rb tumor suppressor by mono-phosphorylation. eLife. 2014;3:e02872.
Hitomi M, Stacey DW. Cyclin D1 production in cycling cells depends on ras in a cell-cycle-specific manner. Curr Biol. 1999;9:1075–84.
Brown JR, Nigh E, Lee RJ, Ye H, Thompson MA, Saudou F, et al. Fos family members induce cell cycle entry by activating cyclin D1. Mol Cell Biol. 1998;18:5609–19.
Lew DJ, Dulić V, Reed SI. Isolation of three novel human cyclins by rescue of G1 cyclin (Cln) function in yeast. Cell. 1991;66:1197–206.
Ohtsubo M, Roberts JM. Cyclin-dependent regulation of G1 in mammalian fibroblasts. Science. 1993;259:1908–12.
Burke JR, Liban TJ, Restrepo T, Lee H-W, Rubin SM. Multiple mechanisms for E2F binding inhibition by phosphorylation of the retinoblastoma protein C-terminal domain. J Mol Biol. 2014;426:245–55.
Canhoto AJ, Chestukhin A, Litovchick L, DeCaprio JA. Phosphorylation of the retinoblastoma-related protein p130 in growth-arrested cells. Oncogene. 2000;19:5116–22.
Farkas T, Hansen K, Holm K, Lukas J, Bartek J. Distinct phosphorylation events regulate p130- and p107-mediated repression of E2F-4. J Biol Chem. 2002;277:26741–52.
Burkhart DL, Sage J. Cellular mechanisms of tumour suppression by the retinoblastoma gene. Nat Rev Cancer. 2008;8:671–82.
Sherr CJ, Beach D, Shapiro GI. Targeting CDK4 and CDK6: from discovery to therapy. Cancer Discov. 2016;6:353–67.
Toogood PL, Harvey PJ, Repine JT, Sheehan DJ, VanderWel SN, Zhou H, et al. Discovery of a potent and selective inhibitor of cyclin-dependent kinase 4/6. J Med Chem. 2005;48:2388–406.
Davis PK, Ho A, Dowdy SF. Biological methods for cell-cycle synchronization of mammalian cells. Biotechniques. 2001;30:1322–6. 1328, 1330–11
Tobey RA, Valdez JG, Crissman HA. Synchronization of human diploid fibroblasts at multiple stages of the cell cycle. Exp Cell Res. 1988;179:400–16.
Bhattacharya S, Garriga J, Calbó J, Yong T, Haines DS, Graña X. SKP2 associates with p130 and accelerates p130 ubiquitylation and degradation in human cells. Oncogene. 2003;22:2443–51.
Hansen K, Farkas T, Lukas J, Holm K, Rönnstrand L, Bartek J. Phosphorylation-dependent and -independent functions of p130 cooperate to evoke a sustained G1 block. EMBO J. 2001;20:422–32.
Gaubatz S, Lees JA, Lindeman GJ, Livingston DM. E2F4 is exported from the nucleus in a CRM1-dependent manner. Mol Cell Biol. 2001;21:1384–92.
Fry DW, Harvey PJ, Keller PR, Elliott WL, Meade M, Trachet E, et al. Specific inhibition of cyclin-dependent kinase 4/6 by PD 0332991 and associated antitumor activity in human tumor xenografts. Mol Cancer Ther. 2004;3:1427–38.
Jorda R, Hendrychová D, Voller J, Řezníčková E, Gucký T, Kryštof V. How selective are pharmacological inhibitors of cell-cycle-regulating cyclin-dependent kinases? J Med Chem. 2018;61:9105–20.
Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F. Genome engineering using the CRISPR-Cas9 system. Nat Protoc. 2013;8:2281–308.
Chicas A, Wang X, Zhang C, McCurrach M, Zhao Z, Mert O, et al. Dissecting the unique role of the retinoblastoma tumor suppressor during cellular senescence. Cancer Cell. 2010;17:376–87.
Fischer M, Grossmann P, Padi M, DeCaprio JA. Integration of TP53, DREAM, MMB-FOXM1 and RB-E2F target gene analyses identifies cell cycle gene regulatory networks. Nucleic Acids Res. 2016;44:6070–86.
Barretina J, Caponigro G, Stransky N, Venkatesan K, Margolin AA, Kim S, et al. The Cancer Cell Line Encyclopedia enables predictive modelling of anticancer drug sensitivity. Nature. 2012;483:603–7.
Aka JA, Adjo Aka J, Lin S-X. Comparison of functional proteomic analyses of human breast cancer cell lines T47D and MCF7. PLoS ONE. 2012;7:e31532.
Finn RS, Crown JP, Lang I, Boer K, Bondarenko IM, Kulyk SO, et al. The cyclin-dependent kinase 4/6 inhibitor palbociclib in combination with letrozole versus letrozole alone as first-line treatment of oestrogen receptor-positive, HER2-negative, advanced breast cancer (PALOMA-1/TRIO-18): a randomised phase 2 study. Lancet Oncol. 2015;16:25–35.
Odajima J, Saini S, Jung P, Ndassa-Colday Y, Ficaro S, Geng Y, et al. Proteomic landscape of tissue-specific cyclin E functions in vivo. PLoS Genet. 2016;12:e1006429.
Mages CF, Wintsche A, Bernhart SH, Müller GA. The DREAM complex through its subunit Lin37 cooperates with Rb to initiate quiescence. eLife. 2017;6. https://doi.org/10.7554/eLife.26876.
Trimarchi JM, Lees JA. Sibling rivalry in the E2F family. Nat Rev Mol Cell Biol. 2002;3:11–20.
Liban TJ, Thwaites MJ, Dick FA, Rubin SM. Structural conservation and E2F binding specificity within the retinoblastoma pocket protein family. J Mol Biol. 2016;428:3960–71.
Ho VM, Schaffer BE, Karnezis AN, Park KS, Sage J. The retinoblastoma gene Rb and its family member p130 suppress lung adenocarcinoma induced by oncogenic K-Ras. Oncogene. 2009;28:1393–9.
Schaffer BE, Park K-S, Yiu G, Conklin JF, Lin C, Burkhart DL, et al. Loss of p130 accelerates tumor development in a mouse model for human small-cell lung carcinoma. Cancer Res. 2010;70:3877–83.
Lara MF, García-Escudero R, Ruiz S, Santos M, Moral M, Martínez-Cruz AB, et al. Gene profiling approaches help to define the specific functions of retinoblastoma family in epidermis. Mol Carcinog. 2008;47:209–21.
Mayhew CN, Bosco EE, Fox SR, Okaya T, Tarapore P, Schwemberger SJ, et al. Liver-specific pRB loss results in ectopic cell cycle entry and aberrant ploidy. Cancer Res. 2005;65:4568–77.
Ruiz S, Santos M, Segrelles C, Leis H, Jorcano JL, Berns A, et al. Unique and overlapping functions of pRb and p107 in the control of proliferation and differentiation in epidermis. Dev Camb Engl. 2004;131:2737–48.
Corona SP, Generali D. Abemaciclib: a CDK4/6 inhibitor for the treatment of HR+/HER2– advanced breast cancer. Drug Des Devel Ther. 2018;12:321–30.
Ortiz AB, Garcia D, Vicente Y, Palka M, Bellas C, Martin P. Prognostic significance of cyclin D1 protein expression and gene amplification in invasive breast carcinoma. PLoS ONE. 2017;12:e0188068.
Tang B, Li Y, Qi G, Yuan S, Wang Z, Yu S, et al. Clinicopathological significance of CDKN2A promoter hypermethylation frequency with pancreatic cancer. Sci Rep. 2015;5:13563.
Hesbacher S, Pfitzer L, Wiedorfer K, Angermeyer S, Borst A, Haferkamp S, et al. RB1 is the crucial target of the Merkel cell polyomavirus Large T antigen in Merkel cell carcinoma cells. Oncotarget. 2016;7:32956–68.
Nor Rashid N, Yong ZL, Yusof R, Watson RJ. HPV 16E7 and 48E7 proteins use different mechanisms to target p130 to overcome cell cycle block. Virol J. 2016;13. https://doi.org/10.1186/s12985-015-0460-8.
Boichuk S, Parry JA, Makielski KR, Litovchick L, Baron JL, Zewe JP, et al. The DREAM complex mediates GIST cell quiescence and is a novel therapeutic target to enhance imatinib-induced apoptosis. Cancer Res. 2013;73:5120–9.
MacDonald J, Ramos-Valdes Y, Perampalam P, Litovchick L, DiMattia GE, Dick FA. A systematic analysis of negative growth control implicates the DREAM complex in cancer cell dormancy. Mol Cancer Res MCR. 2017;15:371–81.
George J, Lim JS, Jang SJ, Cun Y, Ozretić L, Kong G, et al. Comprehensive genomic profiles of small cell lung cancer. Nature. 2015;524:47–53.
Sanjana NE, Shalem O, Zhang F. Improved vectors and genome-wide libraries for CRISPR screening. Nat Methods. 2014;11:783–4.
Acknowledgements
We thank Viktor Huang and Christian Berrios for providing de-identified neo-natal foreskin specimens. We thank William G. Kaelin Jr. (DFCI) for reagents, and Nicole Persky and Cory Johannessen (Broad Institute) for personal communication regarding CDK4 mutant alleles. We thank Donglim Esther Park for RT-qPCR primers for JAG1, MDM4, and CK1α. We thank the Dana-Farber Flow Cytometry Core. We thank members of the DeCaprio laboratory for critical reading of this manuscript.
Funding
This work was supported by US Public Health Service grants R01CA63113, R01CA173023, and P01CA050661 to JAD; F31CA220800 to AES; K08CA222657 to MGO; and a Harvard Landry Biology Consortium Fellowship to AES.
Author information
Authors and Affiliations
Contributions
Conceptualization, AES and JAD; Methodology, AES and JAD; Investigation, AES, MGO, and HEN; Resources, AES, MGO, and HEN; Writing—original draft, AES; Writing—review and editing, AES, MGO, and JAD; Supervision, JAD; Funding acquisition, AES and JAD.
Corresponding author
Ethics declarations
Conflict of interest
JAD has received honoraria for participation in an advisory board from Merck & Co., Inc. JAD has received research funding from Constellation Pharmaceuticals, Inc. The remaining authors declare that they have no conflict of interest.
Additional information
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary information
Rights and permissions
About this article
Cite this article
Schade, A.E., Oser, M.G., Nicholson, H.E. et al. Cyclin D–CDK4 relieves cooperative repression of proliferation and cell cycle gene expression by DREAM and RB. Oncogene 38, 4962–4976 (2019). https://doi.org/10.1038/s41388-019-0767-9
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/s41388-019-0767-9
This article is cited by
-
Cell cycle control in cancer
Nature Reviews Molecular Cell Biology (2022)
-
The MuvB complex binds and stabilizes nucleosomes downstream of the transcription start site of cell-cycle dependent genes
Nature Communications (2022)
-
Structure and function of MuvB complexes
Oncogene (2022)
-
Licorice extract inhibits growth of non-small cell lung cancer by down-regulating CDK4-Cyclin D1 complex and increasing CD8+ T cell infiltration
Cancer Cell International (2021)
-
Implication of human endogenous retrovirus W family envelope in hepatocellular carcinoma promotes MEK/ERK-mediated metastatic invasiveness and doxorubicin resistance
Cell Death Discovery (2021)