Characterization of an up-stream promoter directing extrapituitary expression of the human prolactin gene.
The human PRL gene is expressed outside pituitary lactotrophs in decidualized endometrium and lymphoid cells, but here the mRNA contains a 5'-untranslated region from an additional noncoding exon 1a. We have isolated a genomic DNA clone containing human PRL exon 1a with 2800 basepairs (bp) of 5'-flanking sequences. Sequencing locates exon 1a -5840 bp up-stream of the pituitary start site. To study its suspected regulatory function, various lengths of the 5'-flanking region were linked to the luciferase (Luc) reporter gene. Their ability to direct gene expression has been analyzed in transfection studies. The proximal 1620 bp of promoter sequence directed Luc expression in the T-lymphoid Jurkat cell line, and this was unaffected by 5'-deletion to the proximal 453 bp. However, further 5'-deletion to the most proximal 67 bp drastically reduced this activity by 90%. The exon 1a promoter was inactive in pituitary GH3 cells and HeLa cells; in contrast, the exon 1b pituitary promoter, active in GH3 cells, was inactive in Jurkat cells. DNase-I footprinting studies and further 5'- and 3'-deletion analysis identified factor-binding sites within an enhancer element located at -375/-212 bp, which contributed approximately 50% of the promoter activity.
Berwaer
M
M
Department of Medicine, University of Manchester, United Kingdom.
Martial
J A
JA
Davis
J R
JR
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D018076:DNA, Complementary / Q000302:isolation & purification; D004742:Enhancer Elements, Genetic; D005091:Exons; D005260:Female; D015870:Gene Expression; D005786:Gene Expression Regulation; D015698:Genomic Library; D006367:HeLa Cells; D006801:Humans; D008156:Luciferases / Q000096:biosynthesis; D008969:Molecular Sequence Data; D009928:Organ Specificity; D010920:Placenta / Q000378:metabolism; D010957:Plasmids; D011247:Pregnancy; D011388:Prolactin / Q000096:biosynthesis* / Q000235:genetics; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000096:biosynthesis; D012045:Regulatory Sequences, Nucleic Acid; D015183:Restriction Mapping; D017384:Sequence Deletion; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D018076:DNA, Complementary; D012333:RNA, Messenger; D011388:Prolactin; D004247:DNA; D008156:Luciferases