Identification of a decidua-specific enhancer on the human prolactin gene with two critical activator protein 1 (AP-1) binding sites.
Deletion analysis of the human PRL promoter in endometrial stromal cells decidualized in vitro revealed a 536-bp enhancer located between nucleotide (nt) -2,040 to -1,505 in the 5'-flanking region. The 536-bp enhancer fragment ligated into a thymidine kinase (TK) promoter-luciferase reporter plasmid conferred enhancer activity in decidual-type cells but not nondecidual cells. DNase I footprint analysis of decidualized endometrial stromal cells revealed three protected regions, FP1-FP3. Transfection of overlapping 100-bp fragments of the 536-bp enhancer indicated that FP1 and FP3 each conferred enhancer activity. Gel shift assays indicated that both FP1 and FP3 bind activator protein 1 (AP-1), and JunD and Fra-2 are components of the AP-1 complex in decidual fibroblasts. Mutation of the AP-1 binding site in either FP1 or FP3 decreased enhancer activity by approximately 50%, while mutation of both sites almost completely abolished activity. Coexpression of the 536-bp enhancer and A-fos, a dominant negative to AP-1, decreased enhancer activity by approximately 70%. Conversely, coexpression of Fra-2 in combination with JunD or c-Jun and p300 increased enhancer activity 6- to 10-fold. Introduction of JunD and Fra-2 into nondecidual cells is sufficient to confer enhancer activity. JunD and Fra-2 protein expression was markedly increased in secretory phase endometrium and decidua of early pregnancy (high PRL content) compared with proliferative phase endometrium (no PRL). These investigations indicate that the 5'-flanking region of the human PRL gene contains a decidua-specific enhancer between nt -2,040/-1,505 and AP-1 binding sites within this enhancer region are critical for activity.
Division of Endocrinology, Children's Hospital Research Foundation, Cincinnati, Ohio 45229-3039, USA.
K
K
Watanabe
C A
CA
Kessler
C J
CJ
Bachurski
Y
Y
Kanda
B D
BD
Richardson
J
J
Stanek
S
S
Handwerger
A K
AK
Brar
D001665:Binding Sites; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000378:metabolism; D003656:Decidua / Q000502:physiology*; D003850:Deoxyribonuclease I; D004717:Endometrium / Q000166:cytology / Q000502:physiology; D004742:Enhancer Elements, Genetic; D005260:Female; D051921:Fos-Related Antigen-2; D006801:Humans; D011247:Pregnancy; D011388:Prolactin / Q000235:genetics*; D016755:Proto-Oncogene Proteins c-jun / Q000235:genetics / Q000378:metabolism; D020218:Response Elements; D017384:Sequence Deletion; D017154:Stromal Cells; D013937:Thymidine Kinase / Q000235:genetics; D018808:Transcription Factor AP-1 / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection
D004268:DNA-Binding Proteins; C497872:FOSL2 protein, human; D051921:Fos-Related Antigen-2; D016755:Proto-Oncogene Proteins c-jun; D018808:Transcription Factor AP-1; D014157:Transcription Factors; D011388:Prolactin; D013937:Thymidine Kinase; D003850:Deoxyribonuclease I