This study demonstrates the recruitment of AHR and associated cofactors to the CYP1A1 promoter in vivo. Ordered and cyclical association of AHR and coactivators led to histone acetylation, Pol II recruitment, and gene transcription. Furthermore, unlike results with other AHR antagonists or the active antagonism seen with steroid hormone receptors, DIM caused association of the AHR with the promoter and recruitment of coactivators. DIM antagonism of CYP1A1 induction appears to result from poor recruitment of critical HATs. This novel mechanism of antagonism may form the basis for the chemopreventative effects of DIM and related compounds.
AHR and cofactor recruitment seen here agrees with findings of previous reports. Rapid association of AHR with the XRE coincides with results of a study showing that green fluorescent protein-tagged AHR is mostly nuclear 15 min after treatment with an agonist and is forming nuclear foci by 30 min (8
). Each of the three p160 coactivators interacts with the mouse CYP1A1 promoter in vivo (1
), although ChIP was performed at a single time point after treatment, so it is not clear whether mouse AHR and cofactors also show cycles of association. Concomitant recruitment of p160 coactivators and p300 has been seen with the estrogen, androgen, and thyroid hormone receptors (31
). Transient, single-cycle association of p300 with the promoter is also a common factor among these receptors.
The results presented in Fig. appear to show that NCoA1, NCoA2, and NCoA3 are each present on the promoter simultaneously. However, since ChIP examines a population of cells, with only ~20% of the total promoters recovered following immunoprecipitation (Hestermann and Brown, unpublished results), these results do not demonstrate whether multiple members of the p160 family are recruited to the same promoter. Furthermore, with many XREs present, it is possible that individual AHR (or ARNT) proteins recruit separate p160s to different response elements on the same promoter. With DNA sheared to an average of 1 kb, ChIP lacks sufficient resolution to determine occupancy of individual, closely spaced response elements.
Ligand-induced transcription factor antagonists act through a variety of mechanisms. Ligands for both the estrogen (30
) and androgen (32
) receptors cause recruitment of corepressors and histone deacetylases, rather than that of coactivators and HATs, to promoters, leading to active repression of transcription. The glucocorticoid receptor suppresses NF-κB-mediated responses by inhibiting phosphorylation of the C-terminal domain, rather than recruitment, of Pol II (23
). Several flavone antagonists of the AHR block translocation to the nucleus in vivo and XRE binding in vitro (13
). In contrast, DIM was able to recruit AHR and some cofactors to the promoter but failed to bring about histone acetylation or Pol II recruitment. Taken together, the results indicate that these transcription factors are susceptible to interference at multiple steps in the transcriptional regulation pathway, presenting several opportunities for therapeutic intervention.
Our work suggests a novel mechanism of AHR antagonism. Dramatic differences between the transcriptional outcomes of exposure to AHR agonists and exposure to antagonists occur at a step after ligand binding, nuclear translocation, and p160 coactivator recruitment but before recruitment of HATs, histone acetylation, and recruitment of Pol II. Since local histone acetylation may be required for Pol II recruitment, it is most likely that failure to recruit critical HATs is the key difference between agonistic and antagonistic effects. Our data suggest that ligand-dependent changes in AHR conformation, either alone or with p160 coactivators, affect the stability of HAT, specifically CBP, binding. Failure to acetylate histone H4 reflects the role of CBP in specifically acetylating lysines 8 and 12 of H4 (21
). A lack of histone H4 acetylation has also been correlated with inhibition of CYP1A1 induction by NF-κB activation (15
Ligands like DIM that allow DNA binding and coactivator recruitment but inhibit CYP1A1 transcription likely interfere with carcinogenesis through parallel pathways. First, they inhibit tumor initiation by blocking CYP1A1 induction (and enzymatic activity [5
]) and thus preventing metabolism by CYP1A1 of procarcinogens into genotoxic forms. Second, proliferation of estrogen-dependent tumors is also inhibited because AHR interferes with estrogen receptor-mediated transcription and cell proliferation through competition for promoter binding sites (29
) and/or common cofactors (26
). Mechanism-based design of compounds that specifically block HAT recruitment by AHR in a gene- and/or target cell-specific manner may allow new cancer chemopreventative agents targeting AHR, analogous to the selective estrogen receptor modulators that have proven to be of great clinical value in the treatment of breast cancer.