Suppression of Prolactin Gene Expression in GH Cells Correlates with Site-Specific DNA Methylation

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Abstract
Prolactin- (PRL) producing and nonproducing subclones of the GH line of (rat) pituitary tumor cells have been compared to elucidate the regulatory mechanisms of PRL gene expression. Particular emphasis was placed on delineating the molecular basis of the suppressed state of the PRL gene in the prolactin-nonproducing (PRL-) GH subclone (GH12C1). We examined six methylatable cytosine residues (5,-CCGG- and 1,-GCGC-) within the 30-kb region of the PRL gene in these subclones. This analysis revealed that-CCGG-sequences of the transcribed region, and specifically, one in the fourth exon of the PRL gene, were heavily methylated in the PRL-, GH12C1 cells furthermore, the inhibition of PRL gene expression in GH12C1 was methylated in the PRL-, GH12C1 cells. Furthermore, the inhibition of PRL gene expression in GH12C1 was reversed by short-term treatment of the cells with a sublethal concentration of azacytidine (AzaC), an inhibitor of DNA methylation. The reversion of PRL gene expression by AzaC was correlated with the concurrent demethylation of the same -CCGG- sequences in the transcribed region of PRL gene. An inverse correlation between PRL gene expression and the level of methylation of the internal -C- residues in the specific -CCGG-sequence of the transcribed region of the PRL gene was demonstrated. The DNase I sensitivity of these regions of the PRL gene in PRL+, PRL-, and AzaC-treated cells was also consistent with an inverse relationship between methylation state, a higher order of structural modification, and gene expression. Consistent with our previously reported observations in vivo, we conclude that hypermethylation of a specific site in the fourth exon of the PRL gene is involved in the suppression of this gene, and that demethylation of the same -CCGG- sequence is permissive for the expression of this gene in GH12C1 cells.