Multiple steroidogenic factor 1 binding elements in the human steroidogenic acute regulatory protein gene 5'-flanking region are required for maximal promoter activity and cyclic AMP responsiveness.
A proximal element from the human StAR gene promoter, containing the sequence (-105)TATCCTTGAC(-95), was shown to confer responsiveness to 8-Br-cAMP in the presence of steroidogenic factor 1 (SF-1) when placed behind a minimal thymidine kinase promoter or an SV40 promoter and transfected into BeWo cells which normally lack StAR and SF-1. This element was also transactivated by SF-1 in a yeast one-hybrid system. The -105 to -95 sequence was protected by SF-1 in footprint analysis and a double-stranded oligonucleotide containing the element bound SF-1 specifically in electrophoretic mobility shift assays. Another SF-1-binding sequence 35 bp upstream of the transcription start site ((-42)CAGCCTTC(-35)) was identified in the DNase 1 footprint analysis and, when mutated, markedly reduced SF-1-dependent and 8-Br-cAMP-stimulated StAR promoter activity in BeWo cells. The two proximal SF-1 response elements were shown to be critical for StAR promoter function in human granulosalutein cells, which express SF-1 and respond to cAMP with increased transcription of the StAR gene. Mutation of either element substantially reduced basal and forskolin-stimulated promoter activity, although mutation of the -105 to -95 element had more pronounced effects. Mutation of a third, more distal, SF-1-binding site at -926 to -918 also reduced basal but not forskolin-stimulated promoter activity in the granulosa-lutein cells. These findings demonstrate that multiple SF-1 response elements are required for maximal StAR promoter activity and regulation by cAMP.
Sugawara
T
T
Center for Research on Reproduction and Women's Health and Department of Obstetrics and Gynecology, University of Pennsylvania Medical Center, Philadelphia 19104, USA.
Kiriakidou
M
M
McAllister
J M
JM
Kallen
C B
CB
Strauss
J F
JF
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D000242:Cyclic AMP / Q000378:metabolism*; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005260:Female; D051717:Fushi Tarazu Transcription Factors; D006107:Granulosa Cells / Q000737:chemistry; D006367:HeLa Cells; D018398:Homeodomain Proteins; D006801:Humans; D008184:Luteal Cells / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D018160:Receptors, Cytoplasmic and Nuclear; D011988:Receptors, Thyroid Hormone / Q000378:metabolism*; D054339:Steroidogenic Factor 1; D014157:Transcription Factors / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D016335:Zinc Fingers; D000098682:Steroidogenic Acute Regulatory Protein
D004268:DNA-Binding Proteins; D051717:Fushi Tarazu Transcription Factors; D018398:Homeodomain Proteins; D008565:Membrane Proteins; C515057:NR5A1 protein, human; D009363:Neoplasm Proteins; D010750:Phosphoproteins; D018160:Receptors, Cytoplasmic and Nuclear; D011988:Receptors, Thyroid Hormone; D054339:Steroidogenic Factor 1; D014157:Transcription Factors; D000098682:Steroidogenic Acute Regulatory Protein; D015124:8-Bromo Cyclic Adenosine Monophosphate; D000242:Cyclic AMP