Transcriptional regulation of prolactin gene expression by thyroid hormone--alternate suppression and stimulation in different GH cell lines.
Transient expression experiments, using chimeric plasmids containing 3000 base pairs of PRL 5'-flanking sequences linked to the bacterial chloramphenicol acetyl transferase structural gene, demonstrate that L-T3 can inhibit (GH1 cells) or stimulate (GH4C1 cells) chloramphenicol acetyl transferase activity. Deletion experiments have defined the region necessary for these effects to sequences between -176 and -11 of the PRL gene. This region seems to contain the sequences necessary both for basal expression and for L-T3 regulation. Gel mobility shift experiments revealed that proteins extracted from GH1 and GH4C1 cell nuclei but not rat-2 fibroblasts interact with the PRL gene from -176 to +75. DNase I footprinting studies reveal two footprints which are the same in all pituitary derived cells tested. These footprints are not seen in rat-2 fibroblasts. Neither of these footprints likely represents binding of the L-T3-receptor since extracts from cells containing very low levels of receptor form footprints identical to those from cells with an abundance of receptors. These results suggest that different trans-acting factors, not identifiable by conventional footprinting techniques, are present in these cell lines which account for their opposite responses to L-T3. The regulation of PRL gene expression by L-T3 is unique in that both stimulation and suppression can be demonstrated using a single hormone-gene system. This should allow us to answer fundamental questions regarding the molecular switch between stimulation and suppression of gene expression by hormones.
Stanley
F
F
Department of Medicine, New York University Medical Center, New York 10016.
D000818:Animals; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D004247:DNA / Q000032:analysis / Q000235:genetics; D003850:Deoxyribonuclease I / Q000032:analysis / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D005786:Gene Expression Regulation / Q000187:drug effects*; D010957:Plasmids; D011388:Prolactin / Q000235:genetics*; D011485:Protein Binding; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D051381:Rats; D013489:Suppression, Genetic; D013963:Thyroid Hormones / Q000494:pharmacology*; D014158:Transcription, Genetic
D012333:RNA, Messenger; D013963:Thyroid Hormones; D011388:Prolactin; D004247:DNA; D015500:Chloramphenicol O-Acetyltransferase; D003850:Deoxyribonuclease I