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Identification of pregnane-X receptor target genes and coactivator and corepressor binding to promoter elements in human hepatocytes.

Hariparsad N, Chu X, Yabut J, Labhart P, Hartley DP, Dai X, Evers R - Nucleic Acids Res. (2009)

Bottom Line: Genes, both previously known to be involved and not known to be involved in drug disposition, with PXR response elements (PXREs) located upstream, within or downstream from their potentially associated genes, were identified.We observed promoter-dependent patterns with respect to the binding of various coactivators and corepressors involved in the regulation of CYP4F12, CYP3A4, CYP2B6, UGT1A1 and P-glycoprotein.In conclusion, our findings indicate that PXR is involved in the regulation of CYP4F12 and that PXR along with SRC1 binds to a broad range of promoters but that many of these are not inducible by rifampicin.

View Article: PubMed Central - PubMed

Affiliation: Department of Drug Metabolism and Pharmacokinetics, Merck & Co, Rahway, NJ 07065, USA. niresh_hariparsad@merck.com

ABSTRACT
Chromatin immunoprecipitation (ChIP) studies were conducted in human hepatocytes treated with rifampicin in order to identify new pregnane-X receptor (PXR) target genes. Genes, both previously known to be involved and not known to be involved in drug disposition, with PXR response elements (PXREs) located upstream, within or downstream from their potentially associated genes, were identified. Validation experiments identified several new drug disposition genes with PXR binding sites. Of these, only CYP4F12 demonstrated increased binding in the presence of rifampicin. The role of PXR in the basal and inductive response of CYP4F12 was confirmed in hepatocytes in which PXR was silenced. We also assessed the association of PXR-coactivators and -corepressors with known and newly identified PXREs. Both PXR and the steroid receptor coactivator (SRC-1) were found to bind to PXREs in the absence of rifampicin, although binding was stronger after rifampicin treatment. We observed promoter-dependent patterns with respect to the binding of various coactivators and corepressors involved in the regulation of CYP4F12, CYP3A4, CYP2B6, UGT1A1 and P-glycoprotein. In conclusion, our findings indicate that PXR is involved in the regulation of CYP4F12 and that PXR along with SRC1 binds to a broad range of promoters but that many of these are not inducible by rifampicin.

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Comparison of CYP3A4-, CYP2B6-, CYP4F12-, MDR1- and UGT1A1-mRNA expression following 3 and 48 h treatment with rifampicin 10 µM. Cryopreserved human hepatocytes were treated with vehicle (Control; 0.1% DMSO) or 10 µM rifampicin for either 3 or 48 h. Thereafter, total RNA was isolated and subjected to TaqMan analysis as described in the Materials and methods section. Gene expression values were calculated based on the comparative ΔΔCt method and normalized to values obtained for 18S ribosomal RNA. The error bars represent the SD from the mean of triplicate assays of an individual experiment, n = 3, *P < 0.05.
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Figure 5: Comparison of CYP3A4-, CYP2B6-, CYP4F12-, MDR1- and UGT1A1-mRNA expression following 3 and 48 h treatment with rifampicin 10 µM. Cryopreserved human hepatocytes were treated with vehicle (Control; 0.1% DMSO) or 10 µM rifampicin for either 3 or 48 h. Thereafter, total RNA was isolated and subjected to TaqMan analysis as described in the Materials and methods section. Gene expression values were calculated based on the comparative ΔΔCt method and normalized to values obtained for 18S ribosomal RNA. The error bars represent the SD from the mean of triplicate assays of an individual experiment, n = 3, *P < 0.05.

Mentions: In order to study the correlation between Pol II binding by ChIP and mRNA levels, we also conducted quantitative mRNA analysis using TaqMan following treatment of hepatocytes with rifampicin for 3 or 48 h (Figure 5). While increases in mRNA expression were observed as early as 3 h, the expression was significantly higher after 48 h of drug treatment. CYP3A4, CYP2B6, CYP4F12, MDR1 and UGT1A1 demonstrated 4.8-, 5.6-, 1.9-, 2.7- and 2.1-fold higher levels of mRNA expression after 48 h compared to 3 h drug treatment. Differences in mRNA levels between genes did not correlate with the Pol II ChIP experiments. The antibody used in these analyses is against phospho-Ser 2 in the C-terminal domain of Pol II, which is the elongating form of the polymerase. The analysis was not done at the transcription start site of the genes of interest as it is known that Pol II may be bound at that area but not transcribing (22). Interestingly, the relative difference in mRNA accumulation after 3 and 48 h of treatment were most pronounced for CYP3A4 and CYP2B6.Figure 5.


Identification of pregnane-X receptor target genes and coactivator and corepressor binding to promoter elements in human hepatocytes.

Hariparsad N, Chu X, Yabut J, Labhart P, Hartley DP, Dai X, Evers R - Nucleic Acids Res. (2009)

Comparison of CYP3A4-, CYP2B6-, CYP4F12-, MDR1- and UGT1A1-mRNA expression following 3 and 48 h treatment with rifampicin 10 µM. Cryopreserved human hepatocytes were treated with vehicle (Control; 0.1% DMSO) or 10 µM rifampicin for either 3 or 48 h. Thereafter, total RNA was isolated and subjected to TaqMan analysis as described in the Materials and methods section. Gene expression values were calculated based on the comparative ΔΔCt method and normalized to values obtained for 18S ribosomal RNA. The error bars represent the SD from the mean of triplicate assays of an individual experiment, n = 3, *P < 0.05.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2651806&req=5

Figure 5: Comparison of CYP3A4-, CYP2B6-, CYP4F12-, MDR1- and UGT1A1-mRNA expression following 3 and 48 h treatment with rifampicin 10 µM. Cryopreserved human hepatocytes were treated with vehicle (Control; 0.1% DMSO) or 10 µM rifampicin for either 3 or 48 h. Thereafter, total RNA was isolated and subjected to TaqMan analysis as described in the Materials and methods section. Gene expression values were calculated based on the comparative ΔΔCt method and normalized to values obtained for 18S ribosomal RNA. The error bars represent the SD from the mean of triplicate assays of an individual experiment, n = 3, *P < 0.05.
Mentions: In order to study the correlation between Pol II binding by ChIP and mRNA levels, we also conducted quantitative mRNA analysis using TaqMan following treatment of hepatocytes with rifampicin for 3 or 48 h (Figure 5). While increases in mRNA expression were observed as early as 3 h, the expression was significantly higher after 48 h of drug treatment. CYP3A4, CYP2B6, CYP4F12, MDR1 and UGT1A1 demonstrated 4.8-, 5.6-, 1.9-, 2.7- and 2.1-fold higher levels of mRNA expression after 48 h compared to 3 h drug treatment. Differences in mRNA levels between genes did not correlate with the Pol II ChIP experiments. The antibody used in these analyses is against phospho-Ser 2 in the C-terminal domain of Pol II, which is the elongating form of the polymerase. The analysis was not done at the transcription start site of the genes of interest as it is known that Pol II may be bound at that area but not transcribing (22). Interestingly, the relative difference in mRNA accumulation after 3 and 48 h of treatment were most pronounced for CYP3A4 and CYP2B6.Figure 5.

Bottom Line: Genes, both previously known to be involved and not known to be involved in drug disposition, with PXR response elements (PXREs) located upstream, within or downstream from their potentially associated genes, were identified.We observed promoter-dependent patterns with respect to the binding of various coactivators and corepressors involved in the regulation of CYP4F12, CYP3A4, CYP2B6, UGT1A1 and P-glycoprotein.In conclusion, our findings indicate that PXR is involved in the regulation of CYP4F12 and that PXR along with SRC1 binds to a broad range of promoters but that many of these are not inducible by rifampicin.

View Article: PubMed Central - PubMed

Affiliation: Department of Drug Metabolism and Pharmacokinetics, Merck & Co, Rahway, NJ 07065, USA. niresh_hariparsad@merck.com

ABSTRACT
Chromatin immunoprecipitation (ChIP) studies were conducted in human hepatocytes treated with rifampicin in order to identify new pregnane-X receptor (PXR) target genes. Genes, both previously known to be involved and not known to be involved in drug disposition, with PXR response elements (PXREs) located upstream, within or downstream from their potentially associated genes, were identified. Validation experiments identified several new drug disposition genes with PXR binding sites. Of these, only CYP4F12 demonstrated increased binding in the presence of rifampicin. The role of PXR in the basal and inductive response of CYP4F12 was confirmed in hepatocytes in which PXR was silenced. We also assessed the association of PXR-coactivators and -corepressors with known and newly identified PXREs. Both PXR and the steroid receptor coactivator (SRC-1) were found to bind to PXREs in the absence of rifampicin, although binding was stronger after rifampicin treatment. We observed promoter-dependent patterns with respect to the binding of various coactivators and corepressors involved in the regulation of CYP4F12, CYP3A4, CYP2B6, UGT1A1 and P-glycoprotein. In conclusion, our findings indicate that PXR is involved in the regulation of CYP4F12 and that PXR along with SRC1 binds to a broad range of promoters but that many of these are not inducible by rifampicin.

Show MeSH
Related in: MedlinePlus