Comprehensive search for HNF-1beta-regulated genes in mouse hepatoma cells perturbed by transcription regulatory factor-targeted RNAi.
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.
Tanaka
Taku
T
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.
Tomaru
Yasuhiro
Y
Nomura
Yuki
Y
Miura
Hisashi
H
Suzuki
Masanori
M
Hayashizaki
Yoshihide
Y
D024506:5' Flanking Region / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D006528:Carcinoma, Hepatocellular / Q000235:genetics*; D045744:Cell Line, Tumor; D016384:Consensus Sequence / Q000235:genetics; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D051539:Hepatocyte Nuclear Factor 1-beta; D006801:Humans; D051379:Mice; D020411:Oligonucleotide Array Sequence Analysis; D011485:Protein Binding; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D020218:Response Elements / Q000235:genetics; D013379:Substrate Specificity; D013997:Time Factors; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism*
D004268:DNA-Binding Proteins; C496495:HNF1B protein, human; C496496:Hnf1b protein, mouse; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D014157:Transcription Factors; D051539:Hepatocyte Nuclear Factor 1-beta; D004247:DNA