SILENCING OF CYP1A1 EXPRESSION IN RABBITS BY DNA METHYLATION

Unlike most experimental animals, treatment of adult rabbits with 3-me thylcholanthrene (MC) does not induce the expression of the CYP1A1 gen e. In this study, we show that DNA methylation plays one of the key ro les in the suppression of CYP1A1 gene expression. S1 nuclease protecti on assay showed that the induction of CYP1A1 mRNA by MC occurred in ra bbit kidney RK13 cells but not in rabbit lung R9ab cells, while aryl h ydrocarbon receptor (AhR) and AhR nuclear translocator (Arnt) mRNAs we re expressed in both cells at similar levels. Interestingly, the treat ment of R9ab cells with a DNA demethylating agent, 5-aza-2'-deoxycitid ine, resulted in the induction of the expression of the CYP1A1 gene by MC, The results indicate that DNA methylation is one of the factors i nvolved in the loss of the MC-induced expression of the CYP1A1 gene. T hus, it seemed that the binding of the AhR/Arnt complex to the xenobio tic-responsive element (XRE) was inhibited by the hypermethylation of CpG dinucleotides within an XRE core sequence (5'-CGTG-3'). To explore this possibility, we compared the methylation status of XRE in R9ab c ells with that in RK13 cells, A bisulfite sequence analysis using geno mic DNAs from R9ab cells showed that the CpG site within XRE was highl y methylated on both coding and non-coding strands. Ln contrast to thi s result, the hypomethylation of XRE was seen in RK13 cells. To examin e whether or not the binding of the AhR/Arnt heterodimer to XRE is aff ected by the methylation status of XRE, a gel shift assay using a meth ylated XRE as a probe was carried out. As expected, the AhR/Arnt compl ex could not bind to the methylated XRE. From these results, we conclu de that the cell type specific transcription of the rabbit CYP1A1 gene is caused by DNA methylation. (C) 1998 Academic Press.