Author's personal copy Cell Cycle Regulatory Effects of Retinoic Acid and Forskolin Are Mediated by the Cyclin C Gene Katri M. Makkonen 1 †, Marjo Malinen 1 †, Antti Ropponen 2 , Sami Väisänen 1 and Carsten Carlberg 1,3 ⁎ 1 Department of Biosciences, University of Kuopio, FIN-70211 Kuopio, Finland 2 Department of Clinical Microbiology, University of Kuopio, FIN-70211 Kuopio, Finland 3 Life Sciences Research Unit, University of Luxembourg, L-1511 Luxembourg, Luxembourg Received 31 March 2009; received in revised form 30 July 2009; accepted 5 August 2009 Available online 14 August 2009 As a partner of cyclin-dependent kinase (CDK) 3, Cyclin C controls cellular proliferation and, together with CDK8, represses gene transcription. In this study, we showed that the highly expressed Cyclin C gene is a direct target of the nuclear hormone all-trans retinoic acid (RA) in HEK293 human embryonal kidney cells. The RA receptor (RAR) γ associates with a Cyclin C promoter region containing two RAR binding sites. The Cyclin C gene also directly responds to the cAMP activator Forskolin via the transcription factor CREB1 (cAMP response element-binding protein 1), for which we identified four binding sites within the first 2250 bp of its promoter. RARγ and CREB1 show functional convergence via the corepressor NCoR1, which controls in particular the Forskolin response of Cyclin C. The histone deacetylases 1, 5, 6, 7 and 11 are involved in the basal expression of Cyclin C, but in HEK293 and MCF-7 human breast carcinoma cells the antiprolifera- tive effects of the histone deacetylase inhibitor SAHA (suberoylanilide hydroxamic acid) are not mediated by Cyclin C. However, cell cycle progressing effects of all-trans RA and Forskolin are dependent on Cyclin C expression levels. This suggests that the primary regulation of Cyclin C by all-trans RA and Forskolin mediates some of the cell cycle control actions of these compounds. © 2009 Elsevier Ltd. All rights reserved. Edited by J. Karn Keywords: chromatin; all-trans retinoic acid; CREB signaling; Cyclin C; nuclear receptor Introduction Cyclins associate with specific cyclin-dependent kinases (CDKs) and play central roles in cell cycle regulation and transcription. 1 While CDK7/cyclin H and CDK9/cyclin T are parts of the general transcription factor TFIIH 2 and the transcription elongation factor pTEF-β, 3 respectively, Cyclin C associates with CDK8, Mediator protein 12 (MED12) and MED13 to form a module of the Mediator complex. 4 Via DNA looping, the Mediator complex forms a bridge between transcription factors and the basal transcriptional machinery; without the Cyclin C/CDK8/MED12/MED13 module it displays strong coactivator function, but with the module it turns to a repressor. 5 In the latter case, the RNA polymerase II (pol II) carboxy-terminal domain is phosphorylated prematurely, which prevents the formation of a transcription initiation complex. 6 Another role of Cyclin C, in complex with CDK3, is the regulation of the G 0 to G 1 transition of the cell cycle through specific phosphorylation of the retinoblastoma protein pRb. 7 The fact that the Cyclin *Corresponding author. Life Sciences Research Unit, Université du Luxembourg, 162A, Avenue de la Faïencerie, L-1511 Luxembourg, Luxembourg. E-mail address: carsten.carlberg@uni.lu. † K.M.K. and M.M. contributed equally to this work. Abbreviations used: CBP, CREB-binding protein; CDK, cyclin-dependent kinase; ChIP, chromatin immunoprecipitation; CRE, cAMP response element; CREB1, cAMP response element-binding protein 1; DMSO, dimethyl sulfoxide; DR, direct repeat; FACS, fluorescence-activated cell sorting; FBS, fetal bovine serum; HDAC, histone deacetylase; MED1, Mediator protein 1; NCoR1, nuclear corepressor 1; pPol II, phosphorylated RNA polymerase II; RA, retinoic acid; RAR, retinoic acid receptor; RARE, retinoic acid response element; RE, response element; RXR, retinoid X receptor; SAHA, suberoylanilide hydroxamic acid; siRNA, small interfering RNA; TSS, transcription start site. doi:10.1016/j.jmb.2009.08.024 J. Mol. Biol. (2009) 393, 261–271 Available online at www.sciencedirect.com 0022-2836/$ - see front matter © 2009 Elsevier Ltd. All rights reserved.