Limits...
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.

Show MeSH

Related in: MedlinePlus

RNA Pol II binding at or near previously known and unknown PXR binding sites. Chromatin from vehicle or 10 µM rifampicin-treated hepatocytes (3 h) were immunoprecipitated with anti-Pol II antibodies, and binding of Pol II to the candidate PXR-binding sites or nearby gene sequences were determined by q-PCR. Sequences amplified by PCR were the same as for Figure 3. Signals were normalized for the β-actin housekeeping gene. 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
getmorefigures.php?uid=PMC2651806&req=5

Figure 4: RNA Pol II binding at or near previously known and unknown PXR binding sites. Chromatin from vehicle or 10 µM rifampicin-treated hepatocytes (3 h) were immunoprecipitated with anti-Pol II antibodies, and binding of Pol II to the candidate PXR-binding sites or nearby gene sequences were determined by q-PCR. Sequences amplified by PCR were the same as for Figure 3. Signals were normalized for the β-actin housekeeping gene. The error bars represent the SD from the mean of triplicate assays of an individual experiment, n = 3, *P < 0.05.

Mentions: In addition to assessing increased PXR-binding, we also determined whether increased PXR binding at the newly identified sites, as well as the previously identified literature controls, did result in increased binding of DNA-dependent RNA Pol II to these genes (Figure 4). Determinations were performed in a ChIP-type assay using an antibody against RNA Pol II (20). CYP4F12 showed a trend towards increased Pol II binding in the presence of rifampicin. However, ABCC4, UGT1A5, CARS, BMP1, TGIF2, BCL9L, CLG, GLDC and ABCB4 showed no change. As expected, both CYP3A4 and CYP2B6 showed a ∼2-fold increase in binding of Pol II (Figure 4). The increase observed for UGT1A1 and MDR1 was not significant.Figure 4.


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)

RNA Pol II binding at or near previously known and unknown PXR binding sites. Chromatin from vehicle or 10 µM rifampicin-treated hepatocytes (3 h) were immunoprecipitated with anti-Pol II antibodies, and binding of Pol II to the candidate PXR-binding sites or nearby gene sequences were determined by q-PCR. Sequences amplified by PCR were the same as for Figure 3. Signals were normalized for the β-actin housekeeping gene. 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 4: RNA Pol II binding at or near previously known and unknown PXR binding sites. Chromatin from vehicle or 10 µM rifampicin-treated hepatocytes (3 h) were immunoprecipitated with anti-Pol II antibodies, and binding of Pol II to the candidate PXR-binding sites or nearby gene sequences were determined by q-PCR. Sequences amplified by PCR were the same as for Figure 3. Signals were normalized for the β-actin housekeeping gene. The error bars represent the SD from the mean of triplicate assays of an individual experiment, n = 3, *P < 0.05.
Mentions: In addition to assessing increased PXR-binding, we also determined whether increased PXR binding at the newly identified sites, as well as the previously identified literature controls, did result in increased binding of DNA-dependent RNA Pol II to these genes (Figure 4). Determinations were performed in a ChIP-type assay using an antibody against RNA Pol II (20). CYP4F12 showed a trend towards increased Pol II binding in the presence of rifampicin. However, ABCC4, UGT1A5, CARS, BMP1, TGIF2, BCL9L, CLG, GLDC and ABCB4 showed no change. As expected, both CYP3A4 and CYP2B6 showed a ∼2-fold increase in binding of Pol II (Figure 4). The increase observed for UGT1A1 and MDR1 was not significant.Figure 4.

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