The Active Form of Human Aryl Hydrocarbon Receptor (AHR) Repressor Lacks Exon 8, and Its Pro185 and Ala185 Variants Repress both AHR and Hypoxia-Inducible Factor

The aryl hydrocarbon receptor (AHR) repressor (AHRR) inhibits AHR-mediated transcription and has been associated with reproductive dysfunction and tumorigenesis in humans. Previous studies have characterized the repressor function of AHRRs from mice and fish, but the human AHRR ortholog (AHRR₇₁₅) appeared to be nonfunctional in vitro. Here, we report a novel human AHRR cDNA (AHRRΔ8) that lacks exon 8 of AHRR₇₁₅. AHRRΔ8 was the predominant AHRR form expressed in human tissues and cell lines. AHRRΔ8 effectively repressed AHR-dependent transactivation, whereas AHRR₇₁₅ was much less active. Similarly, AHRRΔ8, but not AHRR₇₁₅, formed a complex with AHR nuclear translocator (ARNT). Repression of AHR by AHRRΔ8 was not relieved by overexpression of ARNT or AHR coactivators, suggesting that competition for these cofactors is not the mechanism of repression. AHRRΔ8 interacted weakly with AHR but did not inhibit its nuclear translocation. In a survey of transcription factor specificity, AHRRΔ8 did not repress the nuclear receptor pregnane X receptor or estrogen receptor α but did repress hypoxia-inducible factor (HIF)-dependent signaling. AHRRΔ8-Pro¹⁸⁵ and -Ala¹⁸⁵ variants, which have been linked to human reproductive disorders, both were capable of repressing AHR or HIF. Together, these results identify AHRRΔ8 as the active form of human AHRR and reveal novel aspects of its function and specificity as a repressor.The aryl hydrocarbon receptor (AHR) repressor (AHRR) is a basic-helix-loop-helix/Per-AHR nuclear translocator (ARNT)-Sim (bHLH-PAS) protein discovered because of its similarity to the AHR, a ligand-activated transcription factor involved in the response to synthetic aromatic hydrocarbons (48). The AHR and AHRR form a negative regulatory loop that is evolutionarily conserved in vertebrates (32); expression of AHRR is regulated by the AHR, and AHRR acts as a transcriptional repressor of AHR function (1, 32, 48). Like the AHR, AHRR can dimerize with the ARNT, and the AHRR-ARNT complex can bind to AHR-responsive enhancer elements (AHREs). Repression occurs through competition between AHR and AHRR for binding to AHREs (14, 48) as well as through additional mechanisms that do not involve competition for ARNT and are independent of AHRE binding by AHRR (14).The biological and toxicological functions of AHRR are not well understood, but recent findings suggest that AHRR is involved in human reproductive physiology and in the regulation of cell growth (reviewed in references 20 and 22). A human AHRR (hAHRR) Ala185Pro polymorphism has been associated with altered reproductive development and infertility in men (16, 46, 59, 64) and endometriosis in women (19, 35, 62, 65), but the functional properties of the polymorphic variants have never been assessed. AHRR overexpression inhibits the growth of human tumor cells in culture (30, 56, 68). Conversely, knockdown of AHRR expression enhances cell growth and confers resistance to apoptosis; consistent with this, the AHRR gene has been found to be silenced by hypermethylation in a variety of human cancers (71). Based on these and other findings, the AHRR has been proposed to function as a tumor suppressor gene (22, 71).In order to assess the functions of AHRR and its polymorphic variants and their relationship to human disease, it is important to understand the nature of the transcripts and proteins encoded by the AHRR gene, as well as their expression in human tissues and cell lines. An hAHRR cDNA identified in a large-scale screen of cDNAs from brain (50) encodes a protein of 715 amino acids (aa) (referred to here as AHRR₇₁₅). The human AHRR gene encoding this protein has been reported to contain 12 exons, the first of which is noncoding (8, 16). Our initial functional analysis of this protein suggested that, unlike AHRRs from mouse, frog, and fish (15, 32, 48, 70), human AHRR₇₁₅ was not an effective repressor of AHR function in vitro. In phylogenetic analyses involving amino acid sequence alignments of multiple vertebrate AHRRs, we identified an 18-aa segment of AHRR₇₁₅ that was absent from all other AHRRs. We therefore hypothesized the existence of an alternative hAHRR form lacking this segment and also hypothesized that this alternative form might exhibit characteristic repressor function.Here, we report the identification and cloning of a novel hAHRR cDNA that lacks exon 8 of the original AHRR clone. This new AHRR (AHRRΔ8) is the predominant form expressed in multiple human tissues and human tumor cell lines. We compare the functions of the two AHRR splice variants and provide the first functional mechanistic assessment of the hAHRR Pro185Ala polymorphic variants that have been associated with increased susceptibility to reproductive dysfunction in human populations. We also show that competition for ARNT or AHR coactivators is not involved in the mechanism of AHR repression and that human AHRRΔ8 (hAHRRΔ8) is capable of repressing hypoxia-inducible factor (HIF)-dependent signaling.