CpG methylation represses the activity of the rat prolactin promoter in rat GH3 pituitary cell lines.
In the present report, we have investigated the role of DNA methylation on the binding and trans-acting properties of transcription factors involved in the regulation of the rat prolactin (rPRL) gene, specifically Pit-1. To this aim we took advantage of a model system composed of three GH3 rat pituitary tumor cell lines that greatly differed in the extent of rPRL gene methylation and in the level of rPRL gene expression. Northern blot analyses indicated that identical species of Pit-1 mRNA were present to similar extent in the three GH3 cell lines. Electrophoretic mobility shift assays further demonstrated that Pit-1 was present in nuclear extracts and displayed equal affinities to bind the 1P responsive element encompassing the -65 to -38 region of the rPRL promoter, whatever the GH3 cell line tested. These data suggested that differential expression of the rPRL gene among cell lines did not result from variable amounts of Pit-1. By combining in vitro methylation and transient transfection experiments with a rPRL promoter-driven CAT construct, we showed that extensive methylation at CpG sites abolished the expression of the reporter gene. Furthermore, in vivo competition assays demonstrated that CpG methylation inhibited gene expression by preventing the binding of transcription factors We propose that related mechanisms linked to DNA methylation might alter the activity of the endogenous PRL gene in the low expressing cell line.
Ngô
V M
VM
Groupe de Biologie de la Cellule Neuroendocrine, CNRS URA 1115, Collège de France, Paris.
Laverrière
J N
JN
Gourdji
D
D
D000818:Animals; D001374:Azacitidine / Q000494:pharmacology; D001483:Base Sequence; D015152:Blotting, Northern; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D004247:DNA / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000032:analysis / Q000378:metabolism / Q000502:physiology; D005786:Gene Expression Regulation; D008745:Methylation; D008969:Molecular Sequence Data; D010902:Pituitary Gland / Q000737:chemistry* / Q000166:cytology / Q000378:metabolism; D011388:Prolactin / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics*; D051381:Rats; D050817:Transcription Factor Pit-1; D014157:Transcription Factors / Q000032:analysis / Q000378:metabolism / Q000502:physiology; D014162:Transfection
D004268:DNA-Binding Proteins; C494186:Pou1f1 protein, rat; D050817:Transcription Factor Pit-1; D014157:Transcription Factors; D011388:Prolactin; D004247:DNA; D015500:Chloramphenicol O-Acetyltransferase; D001374:Azacitidine