Received for publication April 12, 2007.
Revised September 21, 2007.
Accepted for publication September 21, 2007.

Analysis of the transcriptional regulation and molecular function of the Aryl Hydrocarbon Receptor Repressor in human cell lines

Abstract

The aryl hydrocarbon receptor repressor (AhRR) is a member of the AhR signaling cascade, which mediates dioxin toxicity and is involved in regulation of cell growth and differentiation. The AhRR was described as a feedback modulator which counteracts AhR-dependent gene expression. We investigated the molecular mechanisms of transcriptional regulation of the human AhRR by cloning its regulatory DNA region located in intron I of the AhRR. By means of reporter gene analyses and generation of deletion variants, we identified a functional, 3-methylcholanthrene-sensitive XRE site. Chromatin immunoprecipitation analyses revealed that the AhRR binds to this XRE, displaying an autoregulatory loop of AhRR expression. In addition we show that an adjacent GC-box is of functional relevance for AhRR transcription, since blocking of this GC-box resulted in a decrease of constitutive and inducible AhRR gene activity. The differences in constitutive AhRR mRNA level observed in HepG2, primary fibroblast and HeLa cells are directly correlated with CYP1A1 inducibility. We show that the non-responsiveness of high AhRR-expressing cells towards AhR-agonists is associated with a constitutive binding of the AhRR to XRE sites of CYP1A1. Treatment with the histone deacetylase (HDAC) inhibitor sodium butyrate restored the responsiveness of CYP1A1 in these cell lines, due to the dissociation of AhRR from the XREs. Furthermore, transient AhRR mRNA-silencing in untreated HeLa cells was accompanied by an increase of basal CYP1A1 expression, pointing to a constitutive role of the AhRR in regulation of CYP1A1. The functional relevance of the AhRR in high AhRR-expressing primary fibroblasts is discussed.

Key words: Ah receptor, ARNT, CYP expression, CYP1A, DNA binding, gene regulation, polycyclic aromatic hydrocarbons

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