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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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Construction of the PXR-binding site library. Outline of the strategy for ChIP-cloning and filtering of sequences. ‘Filter’: short sequences (<40 bp), highly repetitive sequences and those with <90% identity to the genome were eliminated. ‘Selection’: 12 clusters with strong PXR binding (>5-fold binding relative to the negative control region) were selected for further testing. In addition, 29 tags were selected which were in the proximity of genes known to be involved in different metabolic processes including drug metabolism, or recently identified as PXR target genes by gene profiling in studies with the colon carcinoma cell line LS180 (Table 1) (2).
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Figure 2: Construction of the PXR-binding site library. Outline of the strategy for ChIP-cloning and filtering of sequences. ‘Filter’: short sequences (<40 bp), highly repetitive sequences and those with <90% identity to the genome were eliminated. ‘Selection’: 12 clusters with strong PXR binding (>5-fold binding relative to the negative control region) were selected for further testing. In addition, 29 tags were selected which were in the proximity of genes known to be involved in different metabolic processes including drug metabolism, or recently identified as PXR target genes by gene profiling in studies with the colon carcinoma cell line LS180 (Table 1) (2).

Mentions: A discovery approach for the identification of PXR binding sites in the human genome was initiated (see Materials and methods section). After chromatin precipitation with the anti-PXR antibody, ∼7300 tags representing anti-PXR immunoprecipitated DNA, which were derived from rifampicin-treated cells, was sequenced (Figure 2). Approximately 5000 of these sequences produced acceptable genomic alignments from which 32.7% (or about 1/3) did not map within 10 kb of any gene. The number of genes that had at least one tag alignment within 10 kb was 3745 (using the NCBI gene database). Of these 3745 genes, 500 had two or more tag alignments (Supplementary Table S2, Supplementary Data). For these 500 genes, there were a total of 1244 aligns (within ±10 kb), of which 877 (70.5%) mapped inside genes, 190 (15.3%) upstream and 177 (14.2%) downstream. Our analysis included all of these tags since PXRE sites may be important wherever they lie within the gene and do not necessarily need to be located in the 5′ UTR. Because ChIP fragments were <600 bp, 1200 was set as the maximal distance between two tags representing the same binding site, such that ∼500 tags generated 182 clusters consisting of two or more distinct alignments within 1200 bp). Genes identified as known to contain PXR binding sites in their promoter regions were CYP3A4 and MDR1. As expected, the lack of identification of additional known PXR target genes indicated that the screen had not reached saturation.Figure 2.


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)

Construction of the PXR-binding site library. Outline of the strategy for ChIP-cloning and filtering of sequences. ‘Filter’: short sequences (<40 bp), highly repetitive sequences and those with <90% identity to the genome were eliminated. ‘Selection’: 12 clusters with strong PXR binding (>5-fold binding relative to the negative control region) were selected for further testing. In addition, 29 tags were selected which were in the proximity of genes known to be involved in different metabolic processes including drug metabolism, or recently identified as PXR target genes by gene profiling in studies with the colon carcinoma cell line LS180 (Table 1) (2).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 2: Construction of the PXR-binding site library. Outline of the strategy for ChIP-cloning and filtering of sequences. ‘Filter’: short sequences (<40 bp), highly repetitive sequences and those with <90% identity to the genome were eliminated. ‘Selection’: 12 clusters with strong PXR binding (>5-fold binding relative to the negative control region) were selected for further testing. In addition, 29 tags were selected which were in the proximity of genes known to be involved in different metabolic processes including drug metabolism, or recently identified as PXR target genes by gene profiling in studies with the colon carcinoma cell line LS180 (Table 1) (2).
Mentions: A discovery approach for the identification of PXR binding sites in the human genome was initiated (see Materials and methods section). After chromatin precipitation with the anti-PXR antibody, ∼7300 tags representing anti-PXR immunoprecipitated DNA, which were derived from rifampicin-treated cells, was sequenced (Figure 2). Approximately 5000 of these sequences produced acceptable genomic alignments from which 32.7% (or about 1/3) did not map within 10 kb of any gene. The number of genes that had at least one tag alignment within 10 kb was 3745 (using the NCBI gene database). Of these 3745 genes, 500 had two or more tag alignments (Supplementary Table S2, Supplementary Data). For these 500 genes, there were a total of 1244 aligns (within ±10 kb), of which 877 (70.5%) mapped inside genes, 190 (15.3%) upstream and 177 (14.2%) downstream. Our analysis included all of these tags since PXRE sites may be important wherever they lie within the gene and do not necessarily need to be located in the 5′ UTR. Because ChIP fragments were <600 bp, 1200 was set as the maximal distance between two tags representing the same binding site, such that ∼500 tags generated 182 clusters consisting of two or more distinct alignments within 1200 bp). Genes identified as known to contain PXR binding sites in their promoter regions were CYP3A4 and MDR1. As expected, the lack of identification of additional known PXR target genes indicated that the screen had not reached saturation.Figure 2.

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