TABLE 1

Oligonucleotides used for cloning, EMSA, and ChIP experiments

Primers for PCR cloning were designed based on the CYP2C19 sequence (GenBank accession number gi 27498938:1708014-1877575). Fragments for the reporter constructs were PCR-amplified from genomic DNA. Nonsense mutations in ERE half site I were introduced using gene tailor mutagenesis kit (Invitrogen). Mutated nucleotides in ERE half-site I are underlined. ERE half sites and mutations at ERE half sites are underlined. The ChIP primer pair generates a 305-bp PCR fragment including ERE half-site I.

PrimerSequence
Cloning primers
    CYP2C19_−1.6kb fw5′-CAGACGCGTATTGAGAGATTCCAAAGGGAT
    CYP2C19_ RV5′-CAGCTCGAGTGAAGCCTTCTCCTCTTGTTAA
Primers for mutagenesis
    CYP2C9_mut_ERE_I_forward5′-TTTATCTCTATCAGTGTTTTTAAGTCCTTTCA
    CYP2C9_mut_ERE_I_reverse5′-CACTGATAGAGATAAAAATAAAATGTCCTTTG
EMSA oligonucleotides
    2C19_wt_site I5′-ATCTCTATCAGTGGGTCAAAGTCCTTTCAG
    2C19_mut_site I5′-ATCTCTATCAGTGTTTTTAAGTCCTTTCAG
    ERE consensus5′-ACGGGTAGAGGTCACTGTGACCTCTACCCG
ChIP primers
    Forward5′-ACGGTGCATTGGAACCACTT
    Reverse5′-TGAAGCCTTCTCCTCTTG
  • 2C19_wt_site I, EMSA oligonucleotide containing the wild-type form at ERE half site I; 2C19_mut_site I, EMSA oligonucleotide carrying disruptive mutations at ERE half site I; ERE cons, oligonucleotide comprising a representative ERE consensus sequence shown to bind ERα (Higashi et al., 2007).