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Nucleic Acids Research Advance Access published online on January 7, 2009
Nucleic Acids Research, doi:10.1093/nar/gkn1028
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© 2009 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Methods Online

Identification of an inter-transcription factor regulatory network in human hepatoma cells by Matrix RNAi

Yasuhiro Tomaru1,2, Misato Nakanishi1,2, Hisashi Miura1,2, Yasumasa Kimura1, Hiroki Ohkawa1,2, Yusuke Ohta1,2, Yoshihide Hayashizaki1,2,3 and Masanori Suzuki1,2,*

1OMICS Sciences Center (OSC), RIKEN Yokohama Institute 1-7-22 Suehiro-Cho, 2Division of Functional Genomics, Supramolecular Biology, International Graduate School of Arts and Sciences, Yokohama City University, 1-7-29 Suehiro-Cho, Tsurumi-Ku, Yokohama, Kanagawa 230-0045 and 3Genome Science Laboratory, Discovery and Research Institute, RIKEN Wako Main Campus, 2-1 Hirosawa, Wako, 351-0198, Japan

* To whom correspondence should be addressed. Tel: + 81 45 508 7241; Fax: + 81 45 508 7370; Email: msuzuki{at}gsc.riken.jp or msuzuki{at}tsurumi.yokohama-cu.ac.jp

Received October 10, 2008. Revised December 5, 2008. Accepted December 10, 2008.

Transcriptional regulation by transcriptional regulatory factors (TRFs) of their target TRF genes is central to the control of gene expression. To study a static multi-tiered inter-TRF regulatory network in the human hepatoma cells, we have applied a Matrix RNAi approach in which siRNA knockdown and quantitative RT-PCR are used in combination on the same set of TRFs to determine their interdependencies. This approach focusing on several liver-enriched TRF families, each of which consists of structurally homologous members, revealed many significant regulatory relationships. These include the cross-talks between hepatocyte nuclear factors (HNFs) and the other TRF groups such as CCAAT/enhancer-binding proteins (CEBPs), retinoic acid receptors (RARs), retinoid receptors (RXRs) and RAR-related orphan receptors (RORs), which play key regulatory functions in human hepatocytes and liver. In addition, various multi-component regulatory motifs, which make up the complex inter-TRF regulatory network, were identified. A large part of the regulatory edges identified by the Matrix RNAi approach could be confirmed by chromatin immunoprecipitation. The resultant significant edges enabled us to depict the inter-TRF TRN forming an apparent regulatory hierarchy of (FOXA1, RXRA) -> TCF1 -> (HNF4A, ONECUT1) -> (RORC, CEBPA) as the main streamline.


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O. Wallerman, M. Motallebipour, S. Enroth, K. Patra, M. S. R. Bysani, J. Komorowski, and C. Wadelius
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