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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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Identification of HNF4α binding to CYP3A4, CYP2B6, MDR1, UGT1A1 and CYP4F12. Chromatin from cryopreserved human hepatocytes treated with vehicle (Control; 0.1% DMSO) or rifampicin (10 µM) for 3 h were immunoprecipitated with anti-HNF4α antibody, and the enrichment of HNF4α was determined by q-PCR. 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 7: Identification of HNF4α binding to CYP3A4, CYP2B6, MDR1, UGT1A1 and CYP4F12. Chromatin from cryopreserved human hepatocytes treated with vehicle (Control; 0.1% DMSO) or rifampicin (10 µM) for 3 h were immunoprecipitated with anti-HNF4α antibody, and the enrichment of HNF4α was determined by q-PCR. The error bars represent the SD from the mean of triplicate assays of an individual experiment, n = 3, *P < 0.05.

Mentions: Compared to the coactivators and corepressors that we assessed, HNF4α showed a significantly higher binding to the promoter elements of CYP3A4, CYP2B6, CYP4F12, UGT1A1 and MDR1 (Figures 7 and 8), but no increased binding of HNF4α was detectable after treatment of cells with rifampicin. In fact, the converse was true in that of the five genes evaluated, two (CYP3A4 and CYP4F12) showed a significantly decreased binding in the presence of rifampicin (Figure 7).Figure 7.


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)

Identification of HNF4α binding to CYP3A4, CYP2B6, MDR1, UGT1A1 and CYP4F12. Chromatin from cryopreserved human hepatocytes treated with vehicle (Control; 0.1% DMSO) or rifampicin (10 µM) for 3 h were immunoprecipitated with anti-HNF4α antibody, and the enrichment of HNF4α was determined by q-PCR. 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

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

Figure 7: Identification of HNF4α binding to CYP3A4, CYP2B6, MDR1, UGT1A1 and CYP4F12. Chromatin from cryopreserved human hepatocytes treated with vehicle (Control; 0.1% DMSO) or rifampicin (10 µM) for 3 h were immunoprecipitated with anti-HNF4α antibody, and the enrichment of HNF4α was determined by q-PCR. The error bars represent the SD from the mean of triplicate assays of an individual experiment, n = 3, *P < 0.05.
Mentions: Compared to the coactivators and corepressors that we assessed, HNF4α showed a significantly higher binding to the promoter elements of CYP3A4, CYP2B6, CYP4F12, UGT1A1 and MDR1 (Figures 7 and 8), but no increased binding of HNF4α was detectable after treatment of cells with rifampicin. In fact, the converse was true in that of the five genes evaluated, two (CYP3A4 and CYP4F12) showed a significantly decreased binding in the presence of rifampicin (Figure 7).Figure 7.

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