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Published online 17 May 2004

Nucleic Acids Research, 2004, Vol. 32, No. 9 2740-2750
© 2004 Oxford University Press

Comprehensive search for HNF-1ß-regulated genes in mouse hepatoma cells perturbed by transcription regulatory factor-targeted RNAi

Taku Tanaka1,2, Yasuhiro Tomaru2,3, Yuki Nomura1,2, Hisashi Miura1,2, Masanori Suzuki*,1,2 and Yoshihide Hayashizaki1,2,3

1 Division of Genomics, Science of Biological Supramolecular Systems, Graduate School of Integrated Science, Yokohama City University, 1-7-29 Suehiro-Cho, Tusurumi-Ku, Yokohama, Kanagawa 230-0045, Japan, 2 Laboratory of Genome Exploration Research Group, RIKEN Genomic Sciences Center (GSC), RIKEN Yokohama Institute 1-7-22 Suehiro-Cho, Tsurumi-Ku, Yokohama, Kanagawa 230-0045, Japan and 3 Institute of Basic Medical Sciences, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba, Ibaraki 305-8577, Japan

*To whom correspondence should be addressed. Tel: +81 045 508 7241; Fax: +81 045 508 7370; Email: msuzuki{at}gsc.riken.go.jp

Received December 24, 2003; Revised March 19, 2004; Accepted April 19, 2004

The identification of genes targeted by a specific transcription regulatory factor (TRF) is essential to our understanding of the regulatory mechanism of gene expression. We constructed a system for the comprehensive identification of genes directly regulated by a TRF. It includes a combination of perturbation of gene expression by RNA interference (RNAi) of the TRF, cDNA microarray analysis, computer searches for the putative TRF recognition sequences, and in vivo and in vitro TRF–DNA binding assays. Endogenous hepatocyte nuclear factor-1ß (HNF-1ß) mRNA was efficiently degraded by transfection of mouse hepatoma cells with short interfering RNAs. Expression profile analysis with 20 K mouse cDNA microarrays detected 243 genes whose expression levels were decreased by >50% upon RNAi of HNF-1ß. The upstream regions of the top 26 downregulated genes were searched for the HNF-1ß consensus recognition sequences leading to the extraction of 13 candidate genes. Finally, TRF–DNA binding assays identified five novel as well as three known HNF-1ß-regulated genes. In combination with quantitative real-time RT–PCR, the present system revealed the existence of a more expanded regulatory network among seven HNF family members, demonstrating its practicability to identify the TRF network as well as genes directly regulated by a specific TRF.


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