The aryl hydrocarbon receptor nuclear translocator (ARNT) is the dimerization partner of a growing family of structurally related transcription regulators. Responses to a diverse array of signals, including hypoxia and environmental pollutants, are mediated by the ARNT protein dimerized with other bHLH-PAS proteins (18
). These heterodimers bind distinct DNA response elements in the upstream regulatory regions of specific target genes. One such partner, the aryl hydrocarbon receptor, only binds ARNT after it has been activated by a wide range of chemical carcinogens, most notably halogenated and polycyclic aromatic hydrocarbons. The induction of CYP1A1
provides a model system for the study of ARNT-mediated transcriptional processes. The characteristics of the ARNT-coactivator interactions that we have defined here may thus be applicable to many or all of the ARNT-dependent transcription systems besides those mediated by the AHR/ARNT dimer.
The principal mechanism by which polycyclic aromatic hydrocarbons cause cancer is understood in broad terms. Polycyclic aromatic hydrocarbons induce CYP1A1
via AHR. These cytochrome P450s then metabolize the polycyclic aromatic hydrocarbons to electrophilic derivatives that can mutate DNA, thereby activating proto-oncogenes or inactivating tumor suppressor genes (21
). Unlike polycyclic aromatic hydrocarbons, TCDD is not genotoxic but acts as a tumor promoter. However, it is the most carcinogenic compound ever tested (24
). Furthermore, TCDD has a wide range of effects besides carcinogenicity, including teratogenesis, suppression of the immune system, adverse effects on the reproductive system, and modulation of various hormonal effects. Most if not all of these effects depend upon ARNT as well as AHR (reviewed in references 17
). This wide range of toxic responses suggests that the AHRC signaling pathway may interact with other signal transduction pathways. One way this may occur is via direct protein-protein interactions between AHR and ARNT and a common pool of modulatory proteins, such as SRC-1, NCoA-2, and p/CIP studied here.
In the present study, we investigated the role of the NCoA/SRC/p160 family of transcriptional coactivators in the modulation of AHRC-mediated gene regulation. We demonstrated the ability of SRC-1, NCoA-2, and p/CIP to coactivate transcription of TCDD-responsive genes. Furthermore, with a single-cell microinjection reporter assay, we demonstrated the requirement for SRC-1 and p/CIP for AHRC-dependent gene activation. We also demonstrate by coimmunoprecipitation experiments that ARNT can interact with SRC-1 and NCoA-2, but not p/CIP. This is in contrast to the situation with AHR, which can interact with all three NCoA/SRC/p160 coactivators. The interaction of ARNT with SRC-1 and NCoA2 was confirmed by reporter gene analysis in mammalian cells and colocalization experiments in mammalian cells. The latter type of experiment also confirmed the interaction between AHR and SRC-1.
We also showed that the endogenous coactivators associate with AHRC-regulated chromatin in a TCDD-dependent manner in vivo. Finally, we identified the regions within SRC-1 that are responsible for direct interactions with AHR and ARNT and the region within ARNT responsible for interaction with SRC-1. These represent important steps in our understanding of the role that coactivators play in gene regulation by the AHRC. Previously, it was proposed that SRC-1 does not interact with ARNT, based on the observations that deletion of the ARNT TAD had little effect on SRC-1-mediated coactivation of AHRC-dependent transcription and that anti-ARNT antibodies poorly immunoprecipitated 35
S-labeled SRC-1 in vitro (29
). We cannot fully reconcile the discrepancies between these data and ours except to note that that our studies focused on the amino-terminal helix 2 domain of ARNT.
In most of our assays, AHR interacted with SRC-1 in a ligand-independent fashion. It is possible that the unliganded AHR does genuinely interact with SRC-1 in vivo. Alternatively, the absence of a ligand effect may be an artifact of our experimental systems, particularly when AHR was overexpressed, perhaps being due to the absence or insufficiency of XAP2, p23, or other chaperone proteins that are associated with AHR in its unliganded state and which modulate its folding and functionality.
Activator-coactivator interactions appear to be extremely weak compared to interactions within activator homo- and heterodimers, so much so that studies demonstrating a direct interaction between the endogenous components are rare. Previous studies concerning interactions of CBP and the NCoA/SRC/p160 family of coactivators with nuclear hormone receptors have relied on coexpression experiments and in vitro pulldown assays to demonstrate interaction (8
). The interactions we observed between ARNT and SRC-1 and NCoA-2 were not negated by ARNT's dimerization with ligand-bound AHR (Fig. ), consistent with the notion that SRC-1 and NCoA-2 play a role in gene regulation by the AHRC. Furthermore, negation of activity of a xenobiotic compound-responsive element-driven reporter by anti-SRC-1 and anti-p/CIP IgG suggests that these coactivators are part of complexes that are absolutely required for AHRC-dependent transcription (Fig. ).
The chromatin immunoprecipitation assays presented here demonstrate in an unambiguous fashion that endogenous SRC-1, NCoA-2, and p/CIP are recruited to transcriptionally active CYP1A1
chromatin in a TCDD-dependent fashion. Combined, these experiments are the first demonstration that endogenous P160 coactivator protein is recruited for a functional role during activated transcription by the AHRC. Further studies on the kinetics of these phenomena would potentially help elucidate the mechanisms by which these activators and coactivators direct tissue- and target-specific gene activation. Interestingly, coinjection of CBP expression vector with p/CIP expression vector was not required to rescue reporter gene activity after injection with anti-p/CIP IgG (Fig. ). This is in direct contrast with the results obtained with a retinoic acid receptor-responsive reporter (51
), implying that the ARHC recruits a different p/CIP-containing complex or that it recruits the p/CIP-CBP complex for a different function. Not surprisingly, this is consistent with the notion that ARNT is responsible for recruitment of CBP (28
) and does not appear to interact with p/CIP, whereas AHR can.
Studies with enhanced green fluorescent protein and rhodamine immunofluorescence indicate that ARNT and SRC-1 colocalize in the nucleus. The punctate redistribution of mARNT-EGFP within the nucleus after overexpression of SRC-1 indicates a direct interaction between the two proteins in vivo. This evidence is bolstered by the colocalization of SRC-1 in the same bodies (Fig. ). Furthermore, these foci do not appear to be nucleoli, but may be promyelocytic leukemia protein (PML) bodies (31
). This punctate distribution is very similar to the observations made with GFP-HIF-1α after overexpression of SRC-1 and TIF2 (6
) and GFP-AHR after overexpression of RIP140 (30
). Furthermore, Voegel and colleagues have reported that TIF2 (NCoA-2) is present in similar dot-like structures within the nucleus (52
It has been suggested that these TIF2-containing dot-like structures are PML bodies (37
). These bodies have also been shown to contain p300/CBP, which is known to interact with ARNT (32
) and retinoblastoma protein, which is known to interact with AHR (1
). Furthermore, these bodies have been associated with RNA polymerase II activity, the production of nascent RNA, and the modulation of multiple hormone signaling pathways (12
). Therefore, it appears that SRC-1 directs ARNT to multimeric transcriptional complexes, and its ability to modulate AHRC-dependent gene transcription may not therefore be dependent on its intrinsic histone acetyltransferase activity or its transactivation function. This may signify a novel mechanism of action for SRC-1 with regard to gene regulation.
We undertook studies to determine the domains within SRC-1 and within AHR and ARNT required for their mutual interactions. Studies in yeast cells and in in vitro GST pulldown assays indicate that amino acids 763 to 1033 define the ARNT interaction domain on SRC-1, a region encompassing the CBP interaction site (see Fig. ). This domain contains two signature LXXLL motifs (where L is leucine and X is any amino acid) that have been shown to be critical for interaction with CBP, and other similar motifs within SRC-1 are responsible for interaction with nuclear hormone receptors (11
). However, deletion of amino acids 763 to 895 of the mSRC-1 protein strongly diminished its ability to interact with the mARNTΔQ bait in the yeast two-hybrid system. The amino acid sequences deleted do not contain LXXLL motifs, suggesting that these LXXLL motifs may not be critical for mSRC-1/mARNT interaction.
Surprisingly, two-hybrid and GST pulldown assays demonstrated that amino acids 896 to 1200 of SRC-1 define the AHR interaction domain (Fig. ). No other flanking fragment of SRC-1 that does not overlap this region, in particular SRC-1763-1100
, was capable of interacting with AHR. Thus, the interaction domain on SRC-1 for AHR is distinct from that for ARNT. This raises the possibility that one molecule of SRC-1 is capable of interacting with each subunit of the AHRC heterodimer. However, we have no direct data that would give insight into the stoichiometry of this interaction. Interestingly, a previous study has demonstrated that SRC-1 interacts with AHR through AHR's TAD, in particular the Q-rich region (30
). Determination of the exact amino acid residues in each protein responsible for this interaction will provide a powerful means with which to study cross talk between the ARNT- and AHR-dependent pathways and other signal transduction pathways.
Studies to determine the SRC-1 interaction domain within mARNT revealed an absolute requirement for helix 2. This would appear to be a rare case of a transcription factor that can recruit a coactivator with motifs outside of its TAD and may signify a novel mode of action for SRC-1. Our example is not without precedent. The recently described LXXLL coactivator CIA modulates endoplasmic reticulum-dependent signaling in an AF-2 independent fashion (46
), and more recently, Elferink and colleagues have demonstrated that enhancement of AHRC-dependent gene transcription is modulated via a direct interaction between retinoblastoma protein and the PAS B domain of AHR (14
). Furthermore, like the interaction between HIF-1α and SRC-1, ours is another example of two bHLH-PAS proteins that do not require both bHLH-PAS domains to interact (6
The interactions between ARNT and SRC-1 and NCoA-2 were not negated by ARNT's dimerization with ligand-bound AHR (Fig. ), consistent with the notion that SRC-1 and NCoA-2 play a role in gene regulation by the AHRC. The fact that ARNT and SRC-1 interact in a nonclassical fashion for bHLH-PAS proteins may allow for AHR/ARNT dimerization when ARNT is associated with SRC-1, despite the fact that ARNT's helix 2 is required for its interaction with both AHR and SRC-1. Much of our work attempted to characterize the direct interaction between SRC-1 and ARNT. We do not rule out that SRC-1 may modulate ARNT's action in an indirect fashion. It has been established that SRC-1 can form complexes with CBP (26
), and an interaction between CBP and ARNT's TAD has been reported (28
). Therefore, it is possible that a myriad number of proteins may affect ARNT-dependent transcriptional processes through indirect mechanisms of interaction.
In conclusion, we have demonstrated that AHR and ARNT can interact with the NCoA/SRC-1/p160 transcriptional coactivator proteins. Furthermore, the coactivator proteins enhance TCDD-dependent transcription. We have identified domains within SRC-1 and ARNT and SRC-1 and AHR that are critical for their mutual interactions. ARNT is the dimerization partner of numerous bHLH-PAS proteins, including HIF-1α, EPAS1, SIM1, and SIM2. Our observations regarding the interactions between the bHLH-PAS coactivators and ARNT may apply when ARNT interacts with these other proteins as well as AHR. The continuation of these studies should culminate in a more realistic model of gene activation in response to chemical carcinogens and provide insight into several phenomena, such as cross talk between signal transduction pathways and the pleiotropic effects of AHRC activation.