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

Tomaru Y, Nakanishi M, Miura H, Kimura Y, Ohkawa H, Ohta Y, Hayashizaki Y, Suzuki M - Nucleic Acids Res. (2009)

Bottom Line: This approach focusing on several liver-enriched TRF families, each of which consists of structurally homologous members, revealed many significant regulatory relationships.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.

View Article: PubMed Central - PubMed

Affiliation: OMICS Sciences Center (OSC), RIKEN Yokohama Institute 1-7-22 Suehiro-Cho, Japan.

ABSTRACT
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.

Show MeSH

Related in: MedlinePlus

RNAi knockdown of 19 TRF genes in HepG2 cells. Knockdown ratio was calculated according to the ΔΔCT method (23) and the averages of the ΔΔCT values in four biological replicates are indicated with SD (error bar).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2651797&req=5

Figure 1: RNAi knockdown of 19 TRF genes in HepG2 cells. Knockdown ratio was calculated according to the ΔΔCT method (23) and the averages of the ΔΔCT values in four biological replicates are indicated with SD (error bar).

Mentions: Chemically modified siRNAs (Stealth siRNAs; Supplementary Table 1) were used for RNAi knockdown of HepG2 TRF genes. Although we attempted knockdowns of CEBPB and RARB, no highly effective siRNAs against these TRF genes were obtained. Therefore, we investigated perturbation of expressions of the 21 TRF genes by siRNA-mediated knockdown of the 19 TRF genes in the present Matrix RNAi analysis. The siRNA-induced changes in expression levels of the 21 TRF genes were quantified by qRT-PCR with specific primer sets (Supplementary Table 2). Ten nanomolar siRNAs were sufficient to cause more than 70% knockdown against all TRF genes but RARA gene (60.3% knockdown) (Figure 1).Figure 1.


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

Tomaru Y, Nakanishi M, Miura H, Kimura Y, Ohkawa H, Ohta Y, Hayashizaki Y, Suzuki M - Nucleic Acids Res. (2009)

RNAi knockdown of 19 TRF genes in HepG2 cells. Knockdown ratio was calculated according to the ΔΔCT method (23) and the averages of the ΔΔCT values in four biological replicates are indicated with SD (error bar).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2651797&req=5

Figure 1: RNAi knockdown of 19 TRF genes in HepG2 cells. Knockdown ratio was calculated according to the ΔΔCT method (23) and the averages of the ΔΔCT values in four biological replicates are indicated with SD (error bar).
Mentions: Chemically modified siRNAs (Stealth siRNAs; Supplementary Table 1) were used for RNAi knockdown of HepG2 TRF genes. Although we attempted knockdowns of CEBPB and RARB, no highly effective siRNAs against these TRF genes were obtained. Therefore, we investigated perturbation of expressions of the 21 TRF genes by siRNA-mediated knockdown of the 19 TRF genes in the present Matrix RNAi analysis. The siRNA-induced changes in expression levels of the 21 TRF genes were quantified by qRT-PCR with specific primer sets (Supplementary Table 2). Ten nanomolar siRNAs were sufficient to cause more than 70% knockdown against all TRF genes but RARA gene (60.3% knockdown) (Figure 1).Figure 1.

Bottom Line: This approach focusing on several liver-enriched TRF families, each of which consists of structurally homologous members, revealed many significant regulatory relationships.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.

View Article: PubMed Central - PubMed

Affiliation: OMICS Sciences Center (OSC), RIKEN Yokohama Institute 1-7-22 Suehiro-Cho, Japan.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus