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Molecular and Cellular Biology, January 2009, p. 172-186, Vol. 29, No. 1
0270-7306/09/$08.00+0     doi:10.1128/MCB.01038-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Chromatin Immunoprecipitation on Microarray Analysis of Smad2/3 Binding Sites Reveals Roles of ETS1 and TFAP2A in Transforming Growth Factor β Signaling{triangledown} ,{ddagger}

Daizo Koinuma,1 Shuichi Tsutsumi,2 Naoko Kamimura,2 Hirokazu Taniguchi,5 Keiji Miyazawa,3 Makoto Sunamura,4,{dagger} Takeshi Imamura,1 Kohei Miyazono,3* and Hiroyuki Aburatani2*

Department of Biochemistry, The Cancer Institute of the Japanese Foundation for Cancer Research (JFCR), Koto-ku, Tokyo 135-8550, Japan,1 Genome Science Division, Research Center for Advanced Science and Technology, University of Tokyo, Meguro-ku, Tokyo 153-8904, Japan,2 Department of Molecular Pathology, Graduate School of Medicine, University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan,3 Gastroenterological Surgery, Tohoku University School of Medicine, Sendai, Miyagi 980-8574, Japan,4 Department of Laboratory Medicine, National Cancer Center Hospital, Chuo-ku, Tokyo 104-0045, Japan5

Received 2 July 2008/ Returned for modification 14 August 2008/ Accepted 18 October 2008

The Smad2 and Smad3 (Smad2/3) proteins are principally involved in the transmission of transforming growth factor β (TGF-β) signaling from the plasma membrane to the nucleus. Many transcription factors have been shown to cooperate with the Smad2/3 proteins in regulating the transcription of target genes, enabling appropriate gene expression by cells. Here we identified 1,787 Smad2/3 binding sites in the promoter regions of over 25,500 genes by chromatin immunoprecipitation on microarray in HaCaT keratinocytes. Binding elements for the v-ets erythroblastosis virus E26 oncogene homolog (ETS) and transcription factor AP-2 (TFAP2) were significantly enriched in Smad2/3 binding sites, and knockdown of either ETS1 or TFAP2A resulted in overall alteration of TGF-β-induced transcription, suggesting general roles for ETS1 and TFAP2A in the transcription induced by TGF-β-Smad pathways. We identified novel Smad binding sites in the CDKN1A gene where Smad2/3 binding was regulated by ETS1 and TFAP2A. Moreover, we showed that small interfering RNAs for ETS1 and TFAP2A affected TGF-β-induced cytostasis. We also analyzed Smad2- or Smad3-specific target genes regulated by TGF-β and found that their specificity did not appear to be solely determined by the amounts of the Smad2/3 proteins bound to the promoters. These findings reveal novel regulatory mechanisms of Smad2/3-induced transcription and provide an essential resource for understanding their roles.

* Corresponding author. Mailing address for Kohei Miyazono: 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. Phone: 81 3 5841 3356. Fax: 81 3 5841 3354. E-mail: miyazono-ind{at}umin.ac.jp. Mailing address for Hiroyuki Aburatani: Genome Science Division, Research Center for Advanced Science and Technology, University of Tokyo, Meguro-ku, Tokyo 153-8904, Japan. Phone: 81 3 5452 5352. Fax: 81 3 5452 5355. E-mail: haburata-tky{at}umin.ac.jp

{triangledown} Published ahead of print on 27 October 2008.

{ddagger} Supplemental material for this article may be found at http://mcb.asm.org/.

{dagger} Present address: Cancer Surgery Section, Division of Surgery, Oncology, Reproductive Biology and Anaesthetics, Faculty of Medicine, Imperial College London, Hammersmith Hospital Campus, Du Cane Road, London W12 0NN, United Kingdom.

Molecular and Cellular Biology, January 2009, p. 172-186, Vol. 29, No. 1
0270-7306/09/$08.00+0     doi:10.1128/MCB.01038-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.



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