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

Taku Tanaka | Zendy; Yasuhiro Tomaru | Zendy; Yuki Nomura | Zendy; Hisashi Miura | Zendy; Masanori Suzuki | Zendy; Yoshihide Hayashizaki | Zendy

Open Access

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

Author(s) -

Taku Tanaka,

Yasuhiro Tomaru,

Yuki Nomura,

Hisashi Miura,

Masanori Suzuki,

Yoshihide Hayashizaki

Publication year - 2004

Publication title -

nucleic acids research

Language(s) - English

Resource type - Journals

SCImago Journal Rank - 9.008

H-Index - 537

eISSN - 1362-4954

pISSN - 0305-1048

DOI - 10.1093/nar/gkh597

Subject(s) - biology , gene , rna interference , gene silencing , transcription factor , complementary dna , hepatocyte nuclear factors , genetics , microbiology and biotechnology , regulatory sequence , gene expression , microarray analysis techniques , rna

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-1beta (HNF-1beta) 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-1beta. The upstream regions of the top 26 downregulated genes were searched for the HNF-1beta 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-1beta-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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