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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.

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X-ChIP/qPCR analysis of six selected TRFs. TRF–TRF gene binding analysis was performed for six TRFs (TCF1, FOXA1, FOXA2, HNF4A, ONECUT1 and RXRA) by using the chromatin samples prepared from the siRNA-untreated HepG2 cells. Enrichment of the specific DNA fragments that are bound by a TRF is indicated by ΔCT (difference in the CT values observed for the ChIP samples with specific antibody and those observed without any antibody; see TRF binding assay by X-ChIP/qPCR in Materials and methods section for details). Error bars represent the SD between three separate experiments. Only the TRF genes exhibited the enrichment threshold (ΔCT >1.0, mean > 2 SD and P < 0.05) are shown. Black bars indicate autoregulatory edges.
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Figure 6: X-ChIP/qPCR analysis of six selected TRFs. TRF–TRF gene binding analysis was performed for six TRFs (TCF1, FOXA1, FOXA2, HNF4A, ONECUT1 and RXRA) by using the chromatin samples prepared from the siRNA-untreated HepG2 cells. Enrichment of the specific DNA fragments that are bound by a TRF is indicated by ΔCT (difference in the CT values observed for the ChIP samples with specific antibody and those observed without any antibody; see TRF binding assay by X-ChIP/qPCR in Materials and methods section for details). Error bars represent the SD between three separate experiments. Only the TRF genes exhibited the enrichment threshold (ΔCT >1.0, mean > 2 SD and P < 0.05) are shown. Black bars indicate autoregulatory edges.

Mentions: X-ChIP/qPCR analysis detected a total of 73 edges for physical TRF–TRF gene interactions besides their self-interactions (Figure 6). Interestingly, bindings of all TRFs examined to the upstream region of their own genes were demonstrated, indicating their autoregulation. Odom et al. also reported the transcriptional regulatory circuitry, in the human hepatocyte, which contained TCF1, FOXA2, HNF4A and ONECUT1 on the basis of TRF–TRF gene binding assays (21).Figure 6.


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)

X-ChIP/qPCR analysis of six selected TRFs. TRF–TRF gene binding analysis was performed for six TRFs (TCF1, FOXA1, FOXA2, HNF4A, ONECUT1 and RXRA) by using the chromatin samples prepared from the siRNA-untreated HepG2 cells. Enrichment of the specific DNA fragments that are bound by a TRF is indicated by ΔCT (difference in the CT values observed for the ChIP samples with specific antibody and those observed without any antibody; see TRF binding assay by X-ChIP/qPCR in Materials and methods section for details). Error bars represent the SD between three separate experiments. Only the TRF genes exhibited the enrichment threshold (ΔCT >1.0, mean > 2 SD and P < 0.05) are shown. Black bars indicate autoregulatory edges.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 6: X-ChIP/qPCR analysis of six selected TRFs. TRF–TRF gene binding analysis was performed for six TRFs (TCF1, FOXA1, FOXA2, HNF4A, ONECUT1 and RXRA) by using the chromatin samples prepared from the siRNA-untreated HepG2 cells. Enrichment of the specific DNA fragments that are bound by a TRF is indicated by ΔCT (difference in the CT values observed for the ChIP samples with specific antibody and those observed without any antibody; see TRF binding assay by X-ChIP/qPCR in Materials and methods section for details). Error bars represent the SD between three separate experiments. Only the TRF genes exhibited the enrichment threshold (ΔCT >1.0, mean > 2 SD and P < 0.05) are shown. Black bars indicate autoregulatory edges.
Mentions: X-ChIP/qPCR analysis detected a total of 73 edges for physical TRF–TRF gene interactions besides their self-interactions (Figure 6). Interestingly, bindings of all TRFs examined to the upstream region of their own genes were demonstrated, indicating their autoregulation. Odom et al. also reported the transcriptional regulatory circuitry, in the human hepatocyte, which contained TCF1, FOXA2, HNF4A and ONECUT1 on the basis of TRF–TRF gene binding assays (21).Figure 6.

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